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How does 2-mercaptoethanol lead to shift of the band to a higher molecular weight?

How does 2-mercaptoethanol lead to shift of the band to a higher molecular weight?


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I have a project to isolate a protein with biological properties from a plant. The purified protein forms four bands with similar molecular weight on SDS-PAGE (30-35 kDa) in the presence of 5 % 2-mercaptoethanol, and two bands with lower MW (27-30 kDa) when 2-mercaptoethanol is omitted.

How can these results be explained? What is the easiest and lowest cost method to separate closely related proteins (if that's what these are, and not just subunits of a single protein)?


2-mercaptoethanol (2ME) reduces the disulphide bonds in proteins. If disulphide bonds are connecting two polypeptide chains ("intermolecular") then 2ME would cause them to separate and therefore instead of a higher molecular weight (MW) band you would get one or more lower MW bands.

However, if there are disulphide bonds in the same polypeptide chain, they may cause the chain to fold up. This results in the polypeptide chain to effectively have a smaller hydrodynamic radius compared to the fully denatured chain. In such cases reducing the disulphide bonds using 2ME would cause the band to shift to a higher MW.

What is the easiest and lowest cost method to separate closely related proteins

This is a different question altogether and there are several techniques depending on how "dissimilar" or "similar" the closely related proteins are. 2D PAGE (with isoelectric focussing) is one technique that can offer higher resolution compared to a normal SDS-PAGE. There are many other chromatographic methods.


Troubleshooting: Why does the Observed Protein Molecular Weight Differ from the Calculated One?

The first WB step is sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), followed by protein transfer on a membrane and subsequent detection with specific antibodies. Because the SDS-PAGE is conducted in denaturing conditions, proteins migrate according to their molecular weights irrespective of their secondary/tertiary structure, charge or protein–protein interactions. This means that smaller proteins migrate faster than larger ones.

The predicted molecular weight (MW) of the protein is the sum of the molecular weights of all protein amino acids. It is easy to calculate, e.g., using the free online ExPASy tool. Often the calculated MW is different from that observed on the WB. Here we try to summarize the most common reasons for why this may occur (Figure 1).

Unusual or unexpected size of WB bands
1. Signal peptide (and a pro-peptide) gets cleaved off

Many proteins that undergo transport through the secretory pathway have signal peptides of 15–35 aa. length located predominantly at their N-termini. They are often cleaved by various proteases during their subcellular transport. This results in the mature protein running at a lower than predicted molecular weight. The presence of signal peptides can be predicted by various online tools or based on previously published data. They are usually well annotated in protein databases, e.g., UniProt. Additionally, a subset of proteins has pro-peptides – protein domains that are present in protein precursors. Protein precursors need to be processed by proteases in order to engender a functional product (without pro-peptide).

Figure 2: PINK1 (23274-1-AP) is a mitochondrial serine/threonine-protein kinase that protects cells from stress-induced mitochondrial dysfunction. The precursor of PINK1 (65 kDa) is synthesized in the cytosol and is imported into the outer membrane of mitochondria. PINK1 is further transferred into the inner membrane where it is cleaved into a 52 kDa mature form.

Caspases, a family of endoproteases, are critical players in cell regulatory networks controlling inflammation and cell death. Caspase 3 (19677-1-AP) exists as an inactive proenzyme form of 32 kDa (p32), which upon apoptotic signaling gets cleaved into two active subunits (p19/17 and p12) that assemble into a functional tetrameric enzyme (PMID: 7596430).

Proteins of the matrix metalloproteinase (MMP) family are involved in the breakdown of the extracellular matrix in many physiological processes, including embryonic development, reproduction, tissue remodeling, and disease processes such as arthritis or metastasis. Most MMPs are secreted as inactive proproteins that are activated when cleaved by extracellular proteinases. The inactive pro-MMP9 (10375-2-AP) is 92 kDa. It gets sequentially cleaved by MMP3 into a processed form of 68 kDa through an intermediate form – 78/82 kDa (PMID: 1371271). Besides, MMP9 can also exist as a dimer of 180 kDa (PMID: 7492685).

2. Posttranslational modifications (PTMs)
A) Glycosylation and glycanation

The majority of proteins that are synthetized on ribosomes associated with the endoplasmic reticulum undergo glycosylation. That means a covalent attachment of sugar moieties is added to the polypeptide chain. The two most common types of glycosylation in Eukaryotes are N-linked glycosylation – to asparagine, and O-linked glycosylation – to serine and threonine. Extensive glycosylation adds additional molecular weight, not included in the original protein sequence, which makes proteins migrate slower.

Figure 3: Programmed cell death ligand 1 (PD-L1, CD274, or B7-H1) (66248-1-Ig) is a type I transmembrane protein, acting as a key regulator of the adaptive immune response. Full-length PD-L1 MW is 33 kDa. The signal peptide is cleaved off during protein transport to the plasma membrane and the protein is heavily N-glycosylated with an apparent molecular weight of 45–70 kDa with the major glycosylated form of 45–50 kDa (PMID: 27572267).

Please note: An enzymatic deglycosylation is a commonly used experimental technique to verify whether a studied protein is glycosylated. Prior to WB, the protein sample is incubated with an enzyme that is able to remove parts or full glycan chains. WB protein species from the digested sample are then compared with the undigested sample, and any observed shift in molecular weight indicates protein glycosylation. One commonly used enzyme is PNGase F it removes N-linked glycans by cleaving the bond between the innermost N-Acetylglucosamine of the glycan chain and the asparagine residue.

CD133, also known as PROM1 (prominin-1) (18470-1-AP), is a transmembrane glycoprotein with an NH2-terminal extracellular domain, five transmembrane loops, and a cytoplasmic tail. CD133 is a highly glycosylated protein with an apparent molecular weight of 115-120 kDa. After lysates treatment with PNGase F, CD133 shifts to a protein with an MW of 75–85 kDa. That corresponds to the calculated molecular weight of deglycosylated CD133 (PMID: 23150174).

Proteoglycans are a special case group of glycoproteins. They are extracellular matrix proteins with long unbranched glycosaminoglycan chains covalently attached to the amino peptide chain core. Usually, the molecular weight of the sugar group is even larger than the protein component.

Figure 4: Decorin (14667-1-AP) is a member of the small leucine-rich proteoglycan family of proteins. Decorin precursor forms a range of 43–47 kDa MW. It contains a cleavable N-terminal peptide signal and can also be glycosylated. The attachment of glycosaminoglycans (chondroitin sulfate or dermatan sulfate) to decorin occurs in the Golgi apparatus prior to secretion of the mature glycanted form from cells.

B) Phosphorylation

One of the most common posttranslational modifications is protein phosphorylation. It takes place on serine, threonine, and tyrosine residues. Phosphorylation regulates protein function, its enzymatic activity, protein–protein interactions, and protein localization. Phosphorylation is catalyzed by phosphatases and can be reversible – phosphorylated proteins can be dephosphorylated by protein dephosphatases. The addition of a single phosphoryl group adds +/- 1 kDa to the MW, which is often beyond the resolution of the standard SDS-PAGE. However, multiple phosphorylation sites can lead to more prominent MW changes.

Figure 5: The serine/threonine-protein kinase AKT plays a role in many cellular processes. Survival factors can suppress apoptosis in a transcription-independent manner by activating the serine/threonine kinase AKT1, which then phosphorylates and inactivates components of the apoptotic machinery. (60203-2-Ig detects all the AKT members with or without phosphorylation, 66444-1-Ig detects the phospho-Ser473 of AKT1 and phospho-S474 of AKT2/phospho-Ser472 of AKT3.)

C) Ubiquitination

Protein ubiquitination means a covalent ubiquitin is added to lysine, cysteine, serine, threonine, or directly to the protein N-terminus. Ubiquitin is a small (+/-8.6 kDa) protein expressed across almost all tissue types. Ubiquitination is an enzymatic reaction catalyzed by a three-enzyme cascade (E1, E2, and E3). That provides substrate specificity and activation, conjugation, and ligation steps. Proteins can be monoubiquitinated (with one ubiquitin molecule) or polyubiquitinated. Polyubiquitination takes place when additional ubiquitin molecules are added to the initial ubiquitin molecule. Ubiquitination via the proteome can mark proteins for degradation. It is also important for cellular signaling, the internalization of membrane proteins , and the development and regulation of transcription. Ubiquitin can be removed from proteins by deubiquitinating enzymes, which lowers their MW.

Figure 6: Ubiquitin B (UBB) (10201-2-AP), a member of the ubiquitin family, is required for ATP-dependent, non-lysosomal intracellular protein degradation of abnormal proteins and normal proteins with a rapid turnover. This gene consists of three direct repeats of the ubiquitin coding sequence with no spacer sequence.

3. Protein complexes

WB SDS-PAGE is performed in reducing conditions. That means that the majority of the protein complexes composed of proteins linked via non-covalent bonds disassociates during sample preparation and electrophoresis, and (individual) proteins run as monomers. However, some proteins remain partially or fully present in homo- or hetero-meric complexes, even in the presence of SDS and β-Mercaptoethanol. In this case, their observed molecular weight can be substantially higher than the predicted, calculated monomeric form. Some proteins, especially transmembrane proteins and proteins with hydrophobic domains, can aggregate during cell lysis as they are released from their native protein complexes and lipid membranes. These aggregates have high molecular weights and may not represent interactions that occur in their native states.

Please note: 20% β-Mercaptoethanol (or 100 mM DTT) for the 4X SDS sample buffer might help to remove unspecific bands due to dissociation of the protein complex.

Figure 7: NQO1 (11451-1-AP) is an enzyme that serves as a quinone reductase together with conjugation reactions of the hydroquinones involved in detoxification pathways as well as in biosynthetic processes such as the vitamin K-dependent gamma-carboxylation of glutamate residues in prothrombin synthesis. NQO1 has three isoforms of 26, 27, and 31 kDa MW, and the formation of homodimers (66-70 kDa) is needed for its enzymatic activity.

Mlx-interacting protein (MLXIP, also known as MONDOA) (13614-1-AP) acts as a transcription factor by forming a heterodimer with MLX protein. This complex binds to and activates transcription from CACGTG E boxes, playing a role in the transcriptional activation of the glycolytic target and glucose-responsive gene regulation. MLXIP has three isoforms: 110, 57, and 69 kDa, and the molecular weight of the MLXIP-MLX heterodimer is 130 kDa.

4. Protein isoforms

Many proteins encoded by a single gene exist in more than one sequence variant – called protein isoforms. They arise from alternative splicing during mRNA maturation. Selected exons and introns can be included/excluded from the final mRNA product. Included additional protein-coding sequences can reflect in higher MW protein products. On the other hand, the addition of sequences can introduce alternative (premature) stop codons, leading to proteins of lower molecular weights. Some proteins have multiple translation start sites, which gives rise to isoforms with different N-termini. Protein isoforms can have a different half-life and subcellular localization. They can interact with different subsets of proteins, form distinctive protein complexes as well as have different, even opposite, functions.

Figure 8: GLS, also known as GLS1 and KIAA0838, belongs to the glutaminase family. Three isoforms of GLS, named KGA, GAM, and GAC, vary in their MW and tissue expression patterns (PMID: 11015561). KGA, kidney-type glutaminase, has an MW of 65 kDa. GAC, glutaminase C, is 58 kDa, being a product of gene splicing that results in loss of the C-terminal domain that is present in KGA. GAM is the shortest isoform with no catalytic activity and comes into being from the inclusion of intron 2 and premature stop codon. (12855-1-AP detects KGA and GAC isoforms, 20170-1-AP is specific to KGA, 23549-1-AP is specific to all three (KGA, GAM, GAC) isoforms of GLS, and 19958-1-AP is specific to GAC.)

PARD3 (also known as ASIP, Par3, or Bazooka) is one of the PARD family proteins involved in asymmetric cell division and polarized growth. PARD3 has multiple splice isoforms with three main ones: 100 kDa, 150 kDa, and 180 kDa. (11085-1-AP recognizes all three main isoforms of PARD3.)

5. Technical obstacles
A) Antibody cross-reactivity

It is possible for the selected antibody to recognize not only its target protein but also cross-react nonspecifically with other proteins in the analyzed sample. To avoid this issue we might set up an appropriate controls panel and protocol optimization.

Suggested controls:

- a purified target protein

- a lysate from a cell line known to express the target protein

- a lysate from a cell line overexpressing the target protein.

- lysates from cell lines with lower expression of the target protein

- lysates from cell lines with the target protein knocked down (e.g., by siRNA or shRNA) or knocked out (e.g., by CRISPR).

Experimental optimization:
  • Extraction buffers (e.g., RIPA buffer)
  • Blocking buffers (e.g., 5% skimmed milk, casein, or BSA)
  • Incubation and washing times (e.g., overnight at 4C or 1.5h at RT)
  • Secondary antibodies used for detection (e.g., dilution factor)
  • Membranes types (nitrocellulose vs. PVDF).
B) Unspecific proteolytic cleavage and protein degradation

Proteins can undergo unspecific proteolytic digestion if the protein sample is not correctly handled. Proteases released during cell lysis or tissue extraction can cause protein fragmentation, resulting in fragments of lower molecular weights. Some proteins are more susceptible to degradation than others. The choice of cell/tissue lysis buffers and lysis conditions, along with supplementation with protease inhibitors, are vital elements for efficient protein extraction.

Summary table

Observed molecular weight

Potential causes

Higher than expected

1. Posttranslational modifications.

2. Antibody is detecting a protein isoform with a longer sequence.

Lower than expected

1. Cleavage of signal peptide.

2. Antibody is detecting a protein isoform with a shorter sequence.

3. Unspecific protein cleavage.

More than one band observed

2. One protein product but with different posttranslational modifications.

3. Antibody is detecting protein with and without pro-peptide.

5. Antibody cross-reactivity, potentially due to homology of the immunogen sequence.


House Dust Mite Der p 1 Downregulates Defenses of the Lung by Inactivating Elastase Inhibitors

House dust mites (HDM) are the most common source of aeroallergens and in genetic susceptible individuals can cause symptoms ranging from atopic dermatitis to bronchial asthma. Der p 1, a major target of the human immune responses to HDM, through its enzymatic properties can modulate the adaptive immune system by the cleavage of CD23 and CD25. The consequences of this would be to promote allergic inflammatory responses. Furthermore, by disrupting epithelial tight junctions Der p 1 facilitates the transport of allergen across the epithelium. Here, we report that Der p 1 has additional effects on the innate defense mechanisms of the lung, by inactivating in vitro and ex vivo the elastase inhibitors human (h) α1-proteinase inhibitor (h-A1-Pi), mouse (m-), (but not human [h])-SLPI and h-elafin. We confirm that Der p 1 contain both cysteine and serine proteinases, and extend this finding to demonstrate for the first time that h-elafin is particularly sensitive to the biological activity of the latter. Because these elastase inhibitors have antimicrobial, as well as antielastase activity, our results suggest that inactivation of these innate components of the lung defense system by Der p 1 may increase the susceptibility of patients with allergic inflammation to infection.

Asthma is a chronic obstructive disease of the lower airways characterized by intermittent exacerbations of reversible airways obstruction, airways inflammation, and bronchial hyperreactivity (1). The chronic immune response in asthma and other atopic diseases is characterized by a predominately Th2 T cell response, associated with interleukin (IL)-4, IL-5, IL-13, and IgE production (1). Although a third of all cases of asthma can be characterized as intrinsic nonallergic, where the eliciting agent is unknown (2), it is clear that in most cases of asthma (extrinsic) the eliciting factor is of allergen origin, with a strong atopic component (3).

Prevalence of asthma varies in studies between 5 and 25% (1), and has doubled in the industrialized world over the past 20 yr (4). Factors implicated in this increase in prevalence include altered indoor environment such as warmer housing, increased use of broad spectrum antibiotics with altered bacterial infection profiles, dietary changes, and increased aeroallergen exposure.

Exposure to a number of aeroallergens has been shown to contribute to both immediate hypersensitivity and chronic asthma, among these the common indoor allergens produced by the house dust mite (HDM). In human atopic disease, inflammatory responses to the group I antigens of Dermatophagoides pteronyssinus and Dermatophagoides farinae (Der p 1 and Der f 1) are well documented (5–6). The Der p 1 cysteine protease is a 25-kD glycoprotein present in significant quantities in HDM fecal pellets, and is suggested to have a digestive role in the gut of the mite. A number of recent studies have demonstrated that Der p 1 is capable of cleaving human proteins with potentially immunomodulatory effects including α1-proteinase inhibitor (A1-Pi), (7), CD23 (the human low-affinity IgE receptor) (8), CD25 (the α subunit of the human IL-2 receptor) (9), and tight junctions of bronchial epithelium, leading to increased bronchial epithelial permeability (10). In addition to A1-Pi (11–12), the lung also secretes the mucosal/alarm antiproteases secretory leukocyte proteinase inhibitor (SLPI) and elafin, which all contribute significantly to not only antielastase activity but also to activation of innate immunity (13–16). For example, using an adenovirus-overexpression strategy, we have recently reported that elafin has chemotactic activities for neutrophils and protects the lung in a Pseudomonas aeruginosa model of acute inflammation (17–18).

Accordingly, we sought to investigate in the present study whether or not Der p 1 was able to inactivate these important antiproteases and consequently could shift the “elastase–antielastase balance” in favor of a proinflammatory environment to facilitate both the initiation and the maintenance of the asthmatic response.

We have demonstrated here that all three antiproteases studied can potentially be degraded by Der p 1 in vitro and ex vivo. Furthermore, we have investigated in detail the enzymologic properties of Der p 1 against synthetic substrates and selective inhibitors to clarify the nature of the proteases that inactivate innate antiprotease defences. As a consequence, we have uncovered that in addition to the well-characterized cysteine proteinase present in Der p 1, a co-purifying serine protease is extremely active against human elafin.

Human (h) A1-Pi was purchased from Sigma (Poole, Dorset, UK) and human (h) synthetic elafin was manufactured by Albachem (Edinburgh, UK) as described previously (19). Recombinant human (h) SLPI was purchased from R&D Systems (Minneapolis, MN) and recombinant murine (m)-SLPI was a gift from Dr. C. Wright (Amgen, Thousand Oaks, CA). Human neutrophil elastase (HNE) was obtained from Elastin Products (Owensville, MO). E-64 (L-trans-epoxysuccinyl-leucyl-amido[4-guanidino]butane Sigma) is an active site directed irreversible inhibitor of cysteine proteases which inhibits Der p 1 (20).

Human bronchoalveolar lavage fluid (BALF) from six patients with established acute respiratory distress syndrome (21) was a generous gift from Dr. S. Donnelly. The samples were pooled and incubated with Der p 1, as described below.

Sixty grams of house dust mite powder (gift from Dr. T. Wayne, University of Perth, Perth, Australia) was dissolved into 1 liter of Dulbecco's phosphate-buffered saline (PBS) (calcium and magnesium-free) containing 0.5M NaCl, 0.01% (wt/wt) sodium azide, pH 7.4. Mouse anti–Der p1 monoclonal antibody 4C1 (Indoor Biotechnologies, Clwyd, UK) was coupled to 5 g of Cyanogen-activated Sepharose 4B (Amersham Biosciences UK Limited, Pollard's Wood, Buck, UK), suspended, and washed in an affinity column according to manufacturer's instructions. One hundred milliliters of Der p 1 solution was applied to the column at a flow rate of 20 ml/h. The column was washed with 400 ml PBS 0.5 M NaCl and elution performed at 20 ml/h using 5 mM glycine in 50% ethylene glycol pH 10.0. Fractions were assayed for absorbance at 280 nm, and were extensively dialyzed for 18 h against 10 liters PBS. These fractions will be denoted CS-Der p 1 for the rest of the study. Further purification was performed on half of the dialysate by applying it to an affinity column containing 100 mg of soybean trypsin inhibitor (SBTI) coupled to 5 g of Cyanogen-activated Sepharose 4B (Pharmacia Biotech), according to the manufacturer's instructions. Fractions were collected and reassayed for absorbance at 280 nm to collect serine-protease depleted Der p 1 and re-dialysed extensively against PBS. Der p 1, which had been serine protease depleted by this process, was denoted C-Der p 1 for the rest of the study. The protein concentration of both C/CS-Der p 1 was assessed using a bicinchoninic acid plate assay (Pierce and Warriner Ltd., Chester, Cheshire, UK). Purity of the preparations was assessed using migration on 12% SDS-PAGE analysis (gels stained with using Biorad Silver Stain). Samples were stored in aliquots at –20°C.

A range of synthetic fluorescent substrates (Novabiochem, Nottingham, UK) were used to analyze Der p 1 substrate selectivity, and included Cbz-Arg-Arg-AMC, H-Arg-AMC, Cbz-Phe-arg-AMC, Boc-Gln-Ala-Arg-AMC, Tosyl-Gly-Pro-Arg- AMC, H-Pro-Phe-Arg-AMC, Suc-Ala-Ala-Pro-Phe-AMC (22) and Suc-Gly-Pro-Leu-Gly-Pro-AMC (23). They were dissolved in dimethyl sulfoxide (DMSO) to generate a 10-mM stock and hydrolysis of each substrate (10 μM final) by 6 μg of CS-Derp1 was performed in triplicate in 0.5 M Tris-HCl (pH 8.0) containing 5 mM cysteine and 2% DMSO. Hydrolysis was measured by monitoring the release of AMC every 10 s for 2 min, using a Hoeffer TK 100 mini-fluorimeter (excitation wavelength of 365 nm and emission detection at 465 nm, San Francisco, CA). Substrate concentrations were kept constant by allowing no more than 5% hydrolysis. The rate of hydrolysis was determined by linear regression analysis using the software program SIGMA PLOT, version 4.00. The concentration of AMC was determined from the regression line: [AMC] = (y − c)/m, where y is the fluorescent units released from hydrolysis, c is the intercept, and m is the slope of the regression line.

The pH profile of purified CS-Derp1 against the synthetic substrates Boc-Gln-Ala-Arg-AMC and Tosyl-Gly-Pro-Arg-AMC was determined in the following buffers:

0.1 M citric acid/sodium citrate buffer (pH 3–6), 0.1M phosphate buffer (pH 6–8) and 0.5 M Tris-HCl (pH 8–9.5). All reactions were performed in the presence of 5 mM cysteine.

The rate constant (Km) of CS-Der p 1 for synthetic substrates was determined by monitoring the initial rates of hydrolysis at the optimum pH for each substrate. The range of substrate concentrations used for Boc-Gln-Ala-Arg-AMC, Tosyl-Gly-pro-Arg-AMC, and Cbz-Phe-Arg-AMC were 0–600 μM, 0–10 μM, and 0–100 μM, respectively. The initial rates of hydrolysis (Vo) were plotted against the differing substrate concentrations [S] to ensure that curvature was seen. The data were also represented as a straight line using the Hanes-Woolf plot it was then applied to the Michaelis-Menten rate equation as described below using a nonlinear regression analysis program, in SIGMA PLOT version 4.00, to obtain the Km and maximum initial velocity (Vmax).

CS-Der p 1 and C-Der p 1 enzymatic activities were further characterized by differential inhibition by using E64 and APMSF, inhibitors of cysteinyl and serine proteinases, respectively. CS-Der p 1 and C-Der p 1 were preincubated with 10 μM E64 or 100 μM APMSF before assaying residual activity as described above using either Boc-Gln-Ala-Arg-AMC (10 μM final in 0.1 M phosphate buffer pH 6.0) or Tosyl-Gly-Pro-Arg-AMC (10 μM final in 0.5 M Tris-HCl pH 8.0). All buffers contained 5 mM cysteine.

Protein samples were submitted to SDS-PAGE and/or analyzed by Western Blot analysis as described (24). Briefly, after electrophoretic transfer onto Hybond nitrocellulose membranes (Amersham, Bucks, UK), the membranes were probed sequentially with anti–h-elafin rabbit IgG (1:1,000 dilution, 1 h incubation at room temperature) and horseradish peroxidase–conjugated goat anti-rabbit IgG (1:2,500 dilution, 20 min at room temperature Dako, Ely, Cambridgeshire, UK). Western blots were developed by enhanced chemiluminescence (ECL kit Amersham).

50 mM Tris 0.5 M NaCl 0.1% Triton pH 8 buffer was added to defined quantities of HNE on a 96-well ELISA plate (Linbro Flow Laboratories, McLean, VA) to a volume of 200 μl. Fifty microliters of synthetic elastase substrate N-methoxysuccinyl-ala-ala-pro-val-p-nitroanilide (Sigma) was added to each well, and cleavage of substrate was monitored through change in absorbance (at 405 nm), measured specrophotometrically, at defined time points, using a Dynex MRX II plate reader (Dynatech, Billinghurst, UK).

Anti-HNE activity of test solutions (either purified proteins or BALF) was assessed by addition of the solution to the HNE/buffer mix, maintaining a total volume of 200 μl. The plate was then incubated for 15 min at 37°C before addition of substrate as described above. It was demonstrated that Der p 1 fractions, dithiotreitol (DTT) and L-cysteine, alone or in combination, did not influence the rate of HNE cleavage of substrate (data not shown).

We first sought to establish the enzymatic specificities of our preparation of CS-Der p 1, after purification from the 4C1 affinity chromatography column. Several synthetic substrates were used as listed in Table 1

TABLE 1 Substrate specificity of CS-Der p 1

Substrate specificity of CS-Der p 1 was analyzed using a number of synthetic fluorogenic substrates (10 μM final concentration). All assays were carried out in 0.5 M Tris-HCl pH 8.0 containing 5 mM cysteine. Results are expressed as nM of 7-amino-4-methylcoumarin (AMC) released/s/mg ± SD.

The affinity of these proteases was further studied in Figure 1

Figure 1. Michaelis-Menten and Hanes-Woolf plots for the hydrolysis of Boc-Gln-Ala-Arg-AMC, Tosyl-Gly-Pro-Arg-AMC and Cbz-Phe-Arg-AMC by CS-Der p 1. The curves in A, C, and E represent the initial rate of hydrolysis (V0) of Boc-Gln-Ala-Arg-AMC, Tosyl-Gly-Pro-Arg-AMC, and Cbz-Phe-Arg-AMC by CS-Der p 1. The corresponding Hanes-Woolf plots are shown in B, D, and F. The Km and Vmax were determined by fitting the data to the hyperbolic form of the rate equation in the nonlinear regression program of SIGMAPLOT 4.00.

Figure 2. Inhibitory profiles of CS-Der p 1 (A, B) and C-Der p 1 (C) activities by E64 and APMSF. CS-Der p 1 and C-Der p 1 were differentially inhibited by preincubation with 10 μM E64 and 100 μM APMSF before assaying residual activity using either Boc-Gln-Ala-Arg-AMC (A, C) or Tosyl-Gly-Pro-Arg-AMC (B) at pH 6.0 or pH 8.0, respectively. All buffers contained 5 mM cysteine. Results are expressed as initial rates of hydrolysis (pM/s) measured over a 2-min period. ND, not detected.

Having established that CS-Der p 1 contained both serine protease and cysteine protease components, we segregated both activities by submitting the CS-Der p 1 fraction to SBTI-affinity chromatography.

The resulting purified C-Der p 1 was then tested on both Tosyl-Gly-Pro-Arg-AMC and Boc-Gln-Ala-Arg-AMC. We found that C-Der p 1 was active against Boc-Gln-Ala-Arg-AMC and only E-64 abolished that activity ( Figure 2C ). There was no recordable activity against Tosyl-Gly-Pro-Arg-AMC (data not shown), establishing that indeed, C-Der p 1 only contained the cysteine proteinase activity.

Having demonstrated enzymatically that CS-Der p 1 contained both cysteine and serine protease activities (Table 1, and Figures 1 , 2A , and 2B ), whereas C-Der p 1 only contained the former ( Figure 2C ), the activity of both fractions on the lung elastase inhibitors h-A1-Pi, h-elafin, h-SLPI, and m-SLPI was investigated.

The interaction of Der p 1 (CS and C) with h-A1-Pi was analyzed by SDS-PAGE: Figure 3A

Figure 3. Inactivation of h-A1-Pi by Der p 1. (A) Degradation of h-A1-Pi by Der p 1 (SDS-PAGE analysis). h-A1-Pi (2 μg) was incubated with C/CS-Der p 1 (2 h at 37°C), with or without L-Cys (20 mM) and E-64 (0.4 mM). A molar ratio Der p 1/h-A1-Pi of 2.0:1 was chosen after previous pilot experiments (not shown). Samples were diluted with loading buffer (SDS and 2-mercaptoethanol), and SDS-PAGE analysis was performed (12% gel). Lane 1, h-A1-Pi alone lane 2, h-A1-Pi + L-Cys lane 3, h-A1-Pi + CS-Der p 1 + L-Cys lane 4, h-A1-Pi + CS-Der p 1 + L-Cys + E-64 lane 5, h-A1-Pi + C-Der p 1 + L-Cys lane 6, h-A1-Pi + C-Der p 1 + L-Cys + E-64 standards molecular weights (kD) are indicated by arrows. (B) Functional inactivation of h-A1-Pi by Der p 1 as measured by residual HNE activity. h-A1-Pi was incubated (0, 1, 2, 4 h) at 37°C with Der p 1(C/CS) and L-Cys (20 mM), with or without E-64 (0.4 mM). A molar ratio Der p 1/h-A1-Pi of 2.0:1 was chosen on the basis of the gel analysis (A). At the end of the incubation, the mixture was added to purified HNE (25 ng) for a further 15-min incubation at 37°C, before adding the HNE substrate N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide. After 15 min, the absorbance was read at 405 nm. Results are expressed as % of HNE activity, where HNE was incubated with the control buffer alone (50 mM Tris 0.5 M NaCl, 0.1% Triton X, pH 8.0). Squares, C-Der p 1 + E-64 diamonds, CS-Der p 1 + E-64 circles, C-Der p 1 triangles, CS-Der p 1.

The same Der p 1/A1-Pi ratio was used for the functional assay where the residual anti-HNE activity of h-A1-Pi was measured ( Figure 3B ).

At all time points (0, 1, 2, 4 h), the results are expressed as percentage of residual HNE activity, as compared with HNE incubated with buffer alone. Mirroring the results from the SDS-PAGE analysis, we observed that both CS-Der p 1 and C-Der p 1 functionally inactivated h-A1-Pi (as determined by increased HNE activity over time). Incubation of Der p 1 with E-64 prevented h-A1-Pi inactivation because HNE activity remained minimal throughout the experiment, again demonstrating that indeed, the cysteine proteinase of Der p 1 is responsible for the activity. It was also demonstrated that Der p 1 and L-cys, alone or in combination, did not influence HNE activity (not shown).

The interaction between Der p 1 and h-elafin was analyzed as described above, using SDS-PAGE and Western blot analysis ( Figure 4A )

Figure 4. Inactivation of h-elafin by Der p 1. (A) Cleavage of elafin by Der p 1 (SDS-PAGE/Western Blot analysis). Top: 0.5 μg of h-elafin was incubated with C-Der p 1 (2 h at 37°C) and L-Cys (20 mM). Different molar ratios C-Der p 1/elafin were used: 0.5:1 (lane 4), 0.1:1 (lane 5), 0.05:1 (lane 6), 0.01:1 (lane 7), and 0.005:1 (lane 8). h-Elafin was also incubated alone (lane 1), with L-Cys 20 mM (lane 2), or with C-Der p 1 in the absence of L-Cys (lane 3). Samples were diluted with loading buffer (SDS and 2-mercaptoethanol) and SDS-PAGE analysis was performed (15% gel). Following electroblotting, membranes were successively probed with IgG anti-h-elafin antibodies (1/1,000 dilution, 1 h) and goat anti-rabbit IgG (1/5,000 dilution, 20 min) coupled to horseradish peroxidase. Blots were revealed using the ECL reagent (Amersham). Bottom: As above, except that CS-Der p 1 was used in place of C-Der p 1. CS-Der p1/elafin molar ratio used: 0.5:1 (lane 4), 0.1:1 (lane 5), 0.05:1 (lane 6), 0.01:1 (lane 7), and 0.005:1 (lane 8). Elafin was also incubated alone (lane 1), with L-Cys 20 mM (lane 2), or with CS-Der p1 in the absence of L-Cys (lane 3, molar ratio CS-Der p 1/elafin = 1:1). (B) Functional inactivation of h-elafin by Der p 1 as measured by residual HNE activity. h-elafin was incubated (0, 1, 2, 4 h) at 37°C with Der p 1(C/CS) and L-Cys (20 mM), with or without E-64 (0.4 mM). A molar ratio Der p 1/h-elafin of 0.3:1 was chosen on the basis of the gel analysis (A). At the end of the incubation, h-elafin residual anti-HNE activity was measured as described in Figure 3 . Squares, C-Der p 1 + E-64 diamonds, CS-Der p 1 + E-64 circles, C-Der p 1 triangles, CS-Der p 1.

Cleavage of h-elafin was obtained at a CS-Der p 1/h-elafin ratio of 0.5:1 ( Figure 4A , bottom, lane 4). Decreasing the ratio resulted in the gradual preservation of h-elafin integrity ( Figure 4A , bottom, lanes 4–8). L-Cys alone did not influence h-elafin migration (lane 2). Very significantly, the interaction between CS-Der p 1 and h-elafin in the absence of L-Cys still induced h-elafin degradation ( Figure 4A , bottom, lane 3), suggesting that the h-elafin degrading activity is at least partly due to the serine protease contained in CS-Der p 1.

Expectedly, C-Der p 1, which only has cysteine proteinase activity, needed activation with L-Cys to be able to degrade h-elafin ( Figure 4A , top, lanes 4–8, compared with lane 3, without L-Cys). Taken together, the data presented here suggest that the serine and cysteine activities of Der p 1 are both able to degrade h-elafin.

For the functional assay ( Figure 4B ), an “intermediate” molar ratio Der p 1/h-elafin of 0.3:1 was chosen on the basis that a ratio of 0.5:1 cleaved most of the native elafin molecule after 2 h ( Figure 4A , lane 4), whereas a ratio of 0.1:1 was less efficient ( Figure 4A , lane 5).

The results reveal that both CS-Der p 1 and C-Der p 1 functionally inactivated h-elafin. Incubation of CS-Der p 1 with E-64 failed to prevent h-elafin inactivation, supporting the Western blot analysis results that indeed the serine protease contributes very significantly to the h-elafin–degrading activity in the CS fraction. Nevertheless, C-Der p 1, which only has cysteine proteinase activity, lost its h-elafin–inactivating activity upon incubation with E-64, suggesting that both serine and cysteine proteases have the capacity to inactivate h-elafin.

Figure 5. Der p 1/h-SLPI interactions. (A) Incubation of C-Der p 1 with native h-SLPI (SDS-PAGE analysis). Two micrograms of h-SLPI was incubated (2 h at 37°C) with C-Der p 1 and L-Cys (20 mM), with (lane 4) or without (all other lanes) E-64. Two different molar ratios C-Der p 1/h-SLPI were used: 0.5:1 (lanes 3–5) and 1 (lane 6). h-SLPI was also incubated alone (lane 1) or with L-Cys (lane 2). Samples were then analyzed by SDS-PAGE (15% gel) as explained above. (B) Incubation of CS-Der p 1 with native h-SLPI (SDS-PAGE analysis). CS-Der p 1/h-SLPI molar ratios: 0.5:1 (lanes 3–5), 1:1 (lane 6), and 0.25:1 (lane 7). Lane 1, h-SLPI alone lane 2, h-SLPI + L-Cys lane 3, h-SLPI + CS-Der p 1 lane 4, h-SLPI + CS-Der p 1 + E-64 lane 5, h-SLPI + CS-Der p 1 + L-Cys lanes 6 and 7, as lane 5 (but different molar ratios, see above). N.B.: The band marked with an asterisk is a contaminant noted in some R&D h-SLPI preparations. The numbers 7.2 and 32 represent the molecular weight (kD) of standards. (C) Incubation of CS/C-Der p 1 with native or denatured h-SLPI (without or with incubation with DTT) (SDS-PAGE analysis). Left: no DTT preincubation: 2 μg of h-SLPI was incubated (2 h at 37°C) with C/CS-Der p 1and L-Cys (20 mM), with or without E-64. The molar ratio C/S-Der p 1/h-SLPI was 0.5:1. Lane 1, h-SLPI alone lane 2, h-SLPI + L-Cys + C-Der p 1 lane 3, h-SLPI + C-Der p 1 + L-Cys + E-64 lane 4, h-SLPI + CS-Der p 1 + L-Cys lane 5, h-SLPI + CS-Der p 1 + L-Cys + E-64. Samples were then analyzed by SDS-PAGE (15% gel) as above. Right: DTT preincubation: 2 μg of h-SLPI was preincubated for 30 min at 37°C with 2 mM DTT. The h-SLPI mixture was then incubated (2 h at 37°C) with the same molecules as above. Lanes: as above. N.B.: The R&D contaminant in h-SLPI, although present, is less notable here. The numbers 7.2 and 32 represent, as above, molecular weight standards (kD).

Using a C/CS-Der p 1/SLPI molar ratio of 0.5:1, the preincubation of h-SLPI with 2 mM DTT rendered it susceptible to further cleavage by both C-Der p 1 (compare Figure 5C , left and right, lane 2) and CS-Der p 1 (compare Figure 5C , left and right, lane 4). At the dilutions of DTT used in the experiment, we checked that DTT itself was not affecting the activity of Der p 1 (not shown). Interestingly, both C-Der p 1 ( Figure 5C , right, lane 3) and CS-Der p 1 activities ( Figure 5C , right, lane 5) were abolished to a similar degree with E-64, suggesting that it is the cysteine protease of Der p 1, which plays a role in the degradation of DTT-treated h-SLPI.

Contrary to h-SLPI ( Figure 5 ), m-SLPI was very sensitive to CS-Der p 1 inactivation ( Figure 6 )

Figure 6. Degradation of native m-SLPI by Der p 1 (SDS-PAGE analysis). Two micrograms of m-SLPI was incubated (2 h at 37°C) with or without CS-Der p 1 and with or without L-Cys (20 mM). A molar ratio CS-Der p 1/m-SLPI of 0.5:1 was chosen from previous pilot experiments. Lane 1, m-SLPI alone lane 2, m-SLPI + CS-Der p 1 lane 3, as lane 2 + L-Cys lane 4, m-SLPI + L-Cys lane 5, CS-Der p 1 alone. Samples were diluted and submitted to SDS-PAGE analysis (15% acrylamide) as above. N.B.: m-SLPI runs as a doublet. Standards molecular weights (kD) are indicated by arrows.

Because the three human elastase inhibitors A1-Pi, elafin, and SLPI studied above are the only three major elastase inhibitors so far identified in the lung, we were prompted to test if Der p 1 was able to inactivate the elastase-inhibitory capacity in human BALF. Because patients with established acute respiratory distress syndrome (ARDS) have a very high level of active antiproteases in their BALF (21), we chose to study a pool of six BAL samples.

As demonstrated in our previous study (21), we show here that untreated BALF had a marked anti-HNE activity over the time course of the experiment, with HNE inhibition ranging between 85 and 98%. Incubation of BALF with L-Cys alone did not change this activity. By contrast, both C-Der p 1 and CS-Der p 1, when incubated with L-Cys, degraded the anti-HNE activity of this pool of BALFs ( Figure 7 )

Figure 7. Inactivation of human bronchoalveolar lavage fluid (BALF) samples with Der p 1. Twenty microliters of BALF from patients with established ARDS (see M aterials and M ethods ) were incubated (0, 1, 2, 4 h) with 7.5 μg (1.2 μM) Der p 1 (C/CS) and L-Cys (20 mM), with or without E-64 (0.4 mM), in a final volume of 250 μl. Following the incubation, 10 μL of the incubation mixture was added to 10 ng HNE in microplate wells, and after a further 15-min incubation at 37°C, the HNE substrate was added as explained above. Squares, BALF alone diamonds, BALF + L-Cys circles, BALF + CS-Der p 1 + L-Cys + E-64 triangles, BALF + CS-Der p 1 + L-Cys squares with plus signs, BALF + C-Der p 1 + L-Cys.

Der p 1 is one of the commonest aeroallergens associated with atopic asthma (25–26). A number of recent studies have implicated the cysteine protease activity of Der p 1 in the pathogenesis of asthma (7–10). We have in the present article explored further issues relating to the enzymatic properties of Der p 1 in relation to its activity on the lung defense molecules A1-Pi, SLPI, and elafin.

The main protein present in conventionally affinity-purified Der p 1, which we have named CS-Der p 1 here, is undoubtedly a cysteine proteinase, as evidenced by silver-stained gel purity and N-terminal sequencing (7, 27). However, previous work suggested that there is a minor serine protease contaminant in that preparation, which was removed with an additional SBTI-affinity chromatography step (28–30). Our results confirm and extend these findings by showing, using a wide variety of synthetic substrates, that indeed cysteine and protease activities coexist in CS-Der p 1, and that the SBTI additional step removed the serine protease contaminant, because the remaining protein (C-Der p 1) was only able to degrade the cysteine protease substrate ( Figure 2 ). However, we have been unable to purify and identify the serine protease component, probably because of its very high affinity for SBTI, which makes it very difficult to elute from the column (A. Brown, unpublished data).

Because of the importance of the elastase inhibitors A1-Pi, elafin, and SLPI as key molecules in the defense of the lung (13), we were prompted to determine whether or not CS-Der p 1 and C-Der p 1 were able to biochemically and functionally inactivate these molecules.

In accordance with our previous study (7), we found that CS-Der p 1 could inactivate h-A1-Pi, and further established here that it is the cysteine proteinase of Der p 1 that is responsible for the h-A1-Pi–degrading activity, because CS- and C-Der p 1 have similar activities. In contrast, for h-elafin, we found that the serine protease had the most potent inactivating activity, as illustrated by the fact that even in the presence of an inactive cysteine protease (without L-Cys) h-elafin was almost completely degraded. Furthermore, when the cysteine protease was inhibited by E-64, the activity of h-elafin was still degraded to the same extent as when no E-64 was added. These findings suggest that the serine protease, albeit probably present as a minor contaminant in the CS-Der p 1 fraction, has a much greater affinity for h-elafin than the cysteine protease.

Interestingly, h-SLPI and m-SLPI were not equally sensitive to Der p 1. Indeed, m-SLPI was much more sensitive than h-SLPI (compare Figures 5 and 6 ), with the latter only becoming susceptible to cleavage by Der p 1 after pretreatment with DTT, which disrupts the molecule by reduction of its internal disulfide bonds. This difference in sensitivity is notable in view of the significant homologies between m-SLPI and h-SLPI (58% overall) (31) and between human elafin (which is sensitive to Der p 1) and human SLPI (which is not, 47% homology) (13). It is interesting that the three molecules found in the present study as being sensitive to Der p 1 (h-elafin, h-A1-Pi, and m-SLPI) have an alanine at or near the antiprotease-reactive site (at positions P1, P4, and P4, respectively) whereas there is no alanine in h-SLPI. Indeed, when Der p 1 cleavage sites on h-A1-Pi were studied (7), the enzymes were shown to cleave on either side of the alanine residue. In addition, m-SLPI has an alanine-arginine motif in the P3-P4 position, also present in the cysteine proteinase synthetic substrate Boc-Gln-Ala-Arg-AMC used in this study.

Regardless of the mechanism of Der p 1 differential inhibition of lung antiproteases, our in vitro experiments performed on the isolated human proteins SLPI, A1-Pi, and elafin revealed that the latter two molecules were mostly susceptible to the cysteine and serine proteinases present in Der p 1, respectively, whereas the in vitro resistance of h-SLPI to Der p 1 would suggest that the former may not be a significant Der p 1 substrate in vivo.

To determine the importance of these findings ex vivo in human lung samples, we obtained BALF from patients with established ARDS, with the knowledge that these patients would have high levels of antiproteases (21) and would provide a good marker for lung sensitivity to Der p 1. Indeed, incubation of BALF fluid with Der p 1 of either preparation (C/CS-Der p 1) reveals that both proteases lead to a marked loss of antielastase activity in the fluid. This finding is in keeping with our data, which show the high susceptibility of human antielastases A1-Pi and elafin to cleavage by the mite proteases. Because concentrations in excess of 3 ng/ml of Der p 1 have been detected in the BAL of allergic individuals (32) and the antiproteases studied here are also present in ng/ml in BAL (21), similar molar ratios of Der p 1:antiproteases as the ones used in vitro are likely to be present in vivo, making our findings pathophysiologically relevant. The studies presented here add to our knowledge on the biological activities of Der p 1 and the role of its enzymatic functions on the development and persistence of asthma by compromising the antiprotease defences of the lung. In addition, Der p 1 has a direct effect on some of the components of the adaptive immune system, including CD23 on B cells and CD25 on T cells, which would increase IgE synthesis by disrupting a negative feedback signal and by favoring a Th1 to Th2 shift by affecting interferon-γ responses, respectively (8, 9, 20, 27). The consequence of this would be to promote allergic inflammatory responses. Furthermore, Der p 1 has destructive activities on structural cells in that it disrupts epithelial cells junctions (10), which facilitates transport of the allergen across the epithelium.

Overall, these findings have potential implications at at least three levels. First, they suggest that following exposure of the bronchial mucosa to HDM fecal pellet, the resultant solubilized mite proteases can access the lung interstitium, promote Th2 responses, and skew the elastase/antielastase balance toward elastase and thus a proinflammatory state. Indeed, elastase, in addition of cleaving a variety of substrates within the lung (33), can upregulate the potent neutrophil chemokine IL-8 (34–35), with ensuing neutrophilic inflammation, which in extreme instances could trigger the onset of fatal asthma (36–40).

Second, the HNE inhibitor h-A1-Pi can inhibit protease-mediated airway hyperresponsiveness (AHR) in an allergic model (41). It therefore follows that inactivation of A1-Pi by Der p 1 may be deleterious by facilitating AHR.

Third, A1-Pi, SLPI, and elafin possess properties in addition to their antielastase activity. They can function as antimicrobials, either directly or indirectly (15–16, 18–19, 42) and, at least for elafin and SLPI, can “prime” the innate immune system (17, 43). Thus, inactivation of these additional innate immunity functions may contribute to the infectious exacerbations found in patients with asthma (44–45).

In addition, our report suggests for the first time that the serine protease of Der p 1 may also be an important therapeutic target and identifies elafin, a key molecule in lung defense as an important substrate for this protease (13–14, 46).

Further work will be needed to establish in vivo whether a dual blockade of the cysteine and serine proteases of Der p 1 will be beneficial to patients with HDM-induced allergic inflammation, by modulating type 2 responses and/or by restoring lung antimicrobial functions.


RESULTS

SLC25A46 associates with several OM proteins, particularly components in mitochondrial dynamics

In a previous study, we found that rare cases of PCH were likely caused by a deletion in exon 1 (causing gene disruption) or a point mutation in exon 8 in mitochondrial OM protein SLC25A46 (leucine at position 341 is replaced with proline, designated L341P (Wan et al., 2016) SLC25A46 L341P was not stable, and the mitochondria displayed hyperfused mitochondria and other aberrant morphologies (Wan et al., 2016). To investigate the potential function of SLC25A46, we performed immunoprecipitations and mass spectrometry analysis on mitochondria isolated from a stable HEK293T cell line expressing wild-type (WT) SLC25A46 tagged with a 2× hemagglutinin (HA) epitope at the N-terminus. A tag placed at either the N- or the C-terminus did not interfere with insertion into the mitochondrial OM (Supplemental Figure S1A Abrams et al., 2015 Janer et al., 2016 Wan et al., 2016). After lysis in 1% digitonin, SLC25A46 mostly interacted with proteins involved in fission and fusion such as MFN1/2 or OPA1 and with components of the MICOS complex (Supplemental Table S1). A subset of these potential interaction partners was confirmed by a variety of approaches. First, we solubilized mitochondria that contained SLC25A46 with an N-terminal HA tag in 1% digitonin and used glycerol gradients to examine the distribution of mitochondrial proteins (Figure 1A). SLC25A46 WT mostly migrated near the center of the gradient in fractions 7–13. Migration of OM proteins MFN1 and MTCH2 was also enriched in similar fractions as the SLC25A46 WT protein. Instead, MIC60, MIC27, and MIC19 of the MICOS complex and TOMM40 migrated in heavier fractions (Bohnert et al., 2015 Friedman et al., 2015). Additional controls included PNPase and YME1L of the intermembrane space, TIMM23 in the IM, and matrix-localized PreP and LRP130, which all showed a different distribution pattern than SLC25A46. To assess SLC25A46 and MFN1 binding, analysis of coimmunoprecipitation (coIP) fractions 9, 11, and 13 revealed that a subset of MFN1 and MFN2 indeed interacted with SLC25A46 (Figure 1B). The interactions between SLC25A46 and MFN1/MFN2 were specific because OM protein MID51 did not copurify with SLC25A46 WT or L341P in similar coIP experiments (Figure 2A). We also used the glycerol gradient system to probe the assembly of SLC25A46 L341P from a similar cell line—a stable HEK293T cell line expressing 2xHA-SLC25A46 L341P. In contrast, SLC25A46 L341P was less abundant and sedimented in fractions 17–23, near the bottom of the gradient (Figure 1A and Supplemental Figure S1B). Complexes of TOMM40, MFN1 and MIC60 were not altered in mitochondria with SLC25A46 L341P (Supplemental Figure S1B).

FIGURE 1: SLC25A46 comigrates with mitochondrial dynamics and MICOS proteins. (A) Glycerol gradient density centrifugation of mitochondrial extracts generated from a stable cell line expressing 2xHA-SLC25A46 WT. Mitochondria were solubilized in 1% digitonin, and lysates were loaded on a linear 10–50% glycerol gradient and centrifuged overnight. Fractions 1–23 were analyzed with the indicated antibodies. A similar sample was included, and the HA antibody was used to determine SLC25A46 L341P assembly. SM, starting material. (B) Immunoprecipitation (IP) with the HA antibody of 2xHA-SLC25A46 WT from the indicated fractions (1, 9, 11,13) of the glycerol gradient density centrifugation in A. Input samples correspond to 10% of each fraction. Antibodies against HA, MFN1, and MFN2 were used. (C) Mitochondria isolated form HEK293T cells stably expressing 2xHA-SLC25A46 WT or L341P were solubilized with digitonin, and mitochondrial extracts were resolved by two-dimensional BN-PAGE. Antibodies against MFN1, TOMM40, PreP, and HA were used for immunoblotting. For MFN1, TOMM40, and PreP, the analysis from WT mitochondria is shown, and the complexes migrated identically in mitochondria from the SLC25A46 L341P cell line. (▪) The MFN1-SLC25A46 complex. (D, E) Mitochondria were isolated from LAN5 cells and then lysed in 1% digitonin. Mitochondrial extracts were resolved by one- (D) or two- (E) dimensional BN-PAGE antibodies for endogenous SLC25A46, MIC19, MIC60, MFN2, and MTCH2 were included. In D, shorter and longer exposures for SLC25A46 are shown to highlight the larger complex that comigrates with the MICOS complex. (▪) The MFN1-SLC25A46 complex (▴) the MICOS-SLC25A46 complex (*) the MTCH2-SLC25A46 complex.

FIGURE 2: SLC25A46 interacts with proteins important for mitochondrial biogenesis and morphology. (A) IP with the HA antibody from whole-cell extracts of HEK293T or stable HEK293T cells expressing 2xHA-SLC25A46 WT or 2xHA-SLC25 L341P. Cells were cross-linked with dithiobis(succinimidyl propionate) before lysis. Samples were analyzed with the indicated antibodies. Input samples represent 2% of the material used for the IP. Short (S) and long (L) forms of OPA1 are marked. (B) Steady-state level of SLC25A46 interaction partners in HCT116 WT or SLC25A46 knockout (SLC25A46 − / ) cells was determined. Mitochondria from each cell line were isolated, solubilized, separated by SDS–PAGE, and analyzed with immunoblotting. Three different single-cell knockout clones designated 1.7, 4.1, and 4.3, using two different gRNAs. The asterisk marks a nonspecific band of the antibody. (C) Steady-state levels of MFN1 and MFN2 in HEK293 T-REx Flp-In WT, and SLC25A46 -/- cells were measured in three independent experiments. Mitochondria from each cell line were isolated, solubilized, separated by SDS–PAGE, and analyzed with immunoblotting. TOMM40 was included as a loading control. (D) Densitometric analysis of C. Results are shown as mean ± SEM of three independent experiments. t test, p < 0.05. (E) Analysis of MFN1 and MFN2 expression in HEK293 T-REx Flp-In WT and SLC25A46 –/– cells by qPCR (mean ± SEM, n = 3). (F) Knockout of SLC25A46 does not alter the phospholipid composition of mitochondria. Phospholipids were extracted from 0.75 mg of mitochondria in the cell lines as in B, separated by thin-layer chromatography, and visualized using molybdenum blue staining. Scanned images were analyzed using Quantity 1 software, and the relative abundance of each phospholipid was calculated as percentage of the total phospholipid in each sample (mean ± SEM, n = 3). CL, cardiolipin PA, phosphatidic acid PC, phosphatidylcholine PE, phosphatidylethanolamine PI, phosphatidylinositol PG, phosphatidylglycerol PS, phosphatidylserine.

Using blue-native (BN) gels, we also investigated the migration of SLC25A46 WT and L341P, as well as that of partner proteins. The majority of SCL25A46 WT was at a molecular mass of 70–200 kDa, but larger complexes were also identified (Figure 1C). In contrast, the majority of SCL25A46 L341P migrated at a larger size, >600 kDa, but SLC25A46 L341P could be detected throughout the gel. In mitochondria from LAN5 neuronal cells (Figure 1D), endogenous SLC25A46 migrated in similar-sized complexes as in HEK293T cells expressing HA-SCL25A46. MFN1, MFN2, and MTCH2 also comigrated with SLC25A46 (Figure 1, C–E MFN1/2–SLC25A46 complex [▪] and MTCH2–SLC25A46 complex [▴]), and a small amount of SLC25A46 was detected in the larger complex that comigrated with MIC19 and MIC60 (Figure 1, D and E MICOS–SLC25A46 complex is marked with an asterisk). TOMM40 did not comigrate with SLC25A46 (Figure 1C). Complexes of TOMM40, MFN1, and MIC60 were not altered in mitochondria with SLC25A46 L341P (Supplemental Figure S1B). Thus the BN-gel assay supports that mutation L341P in SLC25A46 alters its complex formation. Moreover, the WT SLC25A46 comigrated with dynamics proteins MFN1 and MFN2 in addition to MTCH2, and a small amount of SLC25A46 comigrated with the MICOS complex. In sum, SLC25A46 associates with several proteins and may function as a scaffold for assembly of proteins involved in mitochondrial ultrastructure.

Because the interactions between SLC25A46 and protein partners may be transient or weak, we performed intracellular cross-linking followed by cell solubilization and coIP assays (Figure 2A). This seemed to trap interactions because SLC25A46 coprecipitated with OPA1 (short and long forms DeVay et al., 2009), MFN1, MFN2, and MICOS components MIC60 and MIC19. This agrees with our previous study (Wan et al., 2016) and recent publications that SLC25A46 is a component similar to yeast Ugo1, which maintains mitochondrial morphology (Abrams et al., 2015 Janer et al., 2016). In addition, TOMM40 interacted with SLC25A46, which may reflect a role for TOMM40 in SLC25A46 assembly. SCL25A46 L341P also bound to similar proteins but showed an increased binding with TOMM40, MTCH2, and MULAN, an E3 ubiquitin ligase that functions in turnover of OM proteins (Braschi et al., 2009 Ambivero et al., 2014 Yun et al., 2014). However, SLC25A46 did not display strong binding to MIC27. To confirm specificity of SLC25A46 interactions with OM proteins, we found that MID51 did not copurify with SLC25A46 (Figure 2A). This immunoprecipitation experiment under cross-linking conditions supports that SLC25A46 interactions with partner proteins may be transient and require a method such as cross-linking for stabilization.

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system was used to knock out SLC25A46 from the HCT116 cell line. SLC25A46 was successfully deleted, and three representative monoclonal cell lines (designated 1.7, 4.1, 4.3 SLC25A46 − / − ) were chosen for additional characterization. The analysis of steady-state levels of partner proteins identified in Figure 2A was tested by immuno­blotting (Figure 2B). The abundance of most of these proteins was not altered, indicating that SLC25A46 is not a bona fide partner protein with the MICOS complex. However, we observed a slight increase in the levels of mitochondrial dynamics proteins MFN1 and MFN2 (Figure 2B). We also observed increased steady-state levels of MFN1 and MFN2 in mitochondria when SLC25A46 was knocked out in the cell line HEK293 T-REx Flp-In (Figure 2, C and D). Specifically, the abundance of MFN1/2 increased ∼1.6- and 2-fold, respectively (Figure 2D). To analyze whether these elevated levels were caused by enhanced protein import, we imported radiolabeled MFN1-13myc and MFN2-20myc (Chen et al., 2003) into isolated mitochondria from WT and SLC25A46 − / − HEK293 T-REx Flp-In cells and analyzed the import by SDS–PAGE and BN-PAGE (Supplemental Figure S2, A and B). The presence of the additional myc tags on the C-terminus of MFN1 and MFN2 does not interfere with function or assembly (Chen et al., 2003) but results in increased molecular weight (Supplemental Figure S2, A and B). Imported MFN1 and MFN2 were recovered in the pellet fraction after alkali extraction. For both proteins, we did not observe any changes in protein import. We also included control import reactions that lacked mitochondria to confirm that MFN1 and MFN2 did not nonspecifically fraction in the pellet after alkali extraction MFN1 and MFN2 were not recovered in the pellet fraction in the absence of mitochondria (Supplemental Figure S2A). We also did not note any differences in MFN1 and MFN2 expression in HEK293 T-REx Flp-In SLC25A46 knockout cells using quantitative PCR (qPCR Figure 2E). Thus the knockout of SLC25A46 stabilizes MFN1 and MFN2 on mitochondria because of decreased degradation of MFN1 and MFN2.

SLC25A46 does not function in mitochondrial phospholipid biogenesis and trafficking pathways

Because the MICOS complex has been implicated in lipid biogenesis and trafficking and SLC25A46 yeast homologue Ugo1 was believed to regulate the local lipid composition of the OM (Hoppins et al., 2009 Anton et al., 2011), we assessed whether mitochondrial lipid levels were altered in SLC25A46 − / − cells. Total phospholipid content in mitochondria was analyzed, and no differences were detected (Supplemental Figure S3A). The lipid profile was assessed using thin-layer chromatography (Supplemental Figure S3B), and the individual phospholipids were quantitated (Figure 2F). The profile of the individual lipids was not different in the SLC25A46 − / − cell lines than in WT HCT116 cells. Thus SLC25A46 does not play a direct role in lipid biogenesis or lipid trafficking.

The abundance of MFN1 and MFN2 is increased in cells lacking SLC25A46

Because we observed higher steady-state levels of MFN1 and MFN2 in cells lacking SLC25A46, we investigated the assembly of these proteins. Because SLC25A46 is highly expressed in neuronal tissue and mutant SLC25A46 impairs neuronal function (Haitina et al., 2006 Abrams et al., 2015 Wan et al., 2016), we used short hairpin RNAs (shRNAs) to knock down SLC25A46 in LAN5 cells (Figure 3, A and B). As observed in HCT116 and HEK293 T-REx Flp-In SLC25A46 − / − cells, the steady-state levels of MFN1 and MFN2 were slightly elevated after SLC25A46 knockdown (Figure 3A). This led to an elevated abundance of MFN1 and MFN2 complexes in cells lacking SCL25A46 when isolated mitochondria were solubilized in digitonin and separated by BN-PAGE (Figure 3B). Again, we did not observe any marked changes in the expression of transcripts for MFN1 and MFN2 (Figure 3C). We obtained similar results in HCT116 SLC25A46 − / − cells (Supplemental Figure S4).

FIGURE 3: SLC25A46 levels affect the steady-state levels and assembly of MFN1 and MFN2. (A) The oligomerization of MFN1 and MFN2 was determined in isolated mitochondria from LAN5 cell lines in which SLC25A46 was knocked down with different shRNA constructs after doxycycline induction using BN-PAGE and immunoblotting. (B) The steady-state levels of the indicated proteins were determined using the same mitochondrial extracts loaded on BN-PAGE in A. (C) Analysis of MFN1 and MFN2 expression in LAN5 cells in which SLC25A46 was knocked down with a specific shRNA construct by qPCR (mean ± SEM, n = 3). (D) The mitochondrial network in LAN5 cell lines in which SLC25A46 was knocked down after doxycycline induction was investigated by immunofluorescence staining using an antibody against the matrix protein Mortalin or OM protein TOMM20. 4′,6-Diamidino-2-phenylindole was used to stain the nucleus. Scale bar, 10 μm. (E) Quantification of the mitochondrial network from D. For each experiment, 100 cells were counted (mean ± SEM, n = 3). (F) Live cells were quantitated using a trypan blue assay in the LAN5 and HEK294 T-REx Flp-In lines. An equal number of cells from each line was plated, and then live cells were counted after 4 d. The expression of SLC25A46 in LAN5 was knocked down for 2 wk before the experiment. Data represent mean ± SEM. (n = 3). (G) As in A, but the oligomerization of MFN1 and MFN2 was analyzed in HEK293 T-REx Flp-In cells lacking SLC25A46. SLC25A46 knockout was rescued by expressing 2xHA-SLC25A46 WT or L341P for 48 h after induction with 10 ng/ml doxycycline. (H) Lysates from G were separated by SDS–PAGE and the steady-state levels of indicated proteins investigated by immunoblot. 2xHA-SLC25A46 and endogenous SLC25A46 were detected with anti-SLC25A46 a longer exposure to detect HA-SLC25A46 L341P of the panel was included. A panel with anti-HA was also included to detect expressed HA-SLC25A46 WT and L341P.

To determine whether the enhanced oligomerization states of MFN1 and MFN2 lead to hyperfused mitochondria, we knocked down SLC25A46 in LAN5 cells by RNA interference (RNAi) and visualized mitochondria by immunofluorescence staining using an antibody against the matrix protein Mortalin and the OM protein TOMM20. As expected, mitochondria appeared elongated and hyperfused in cells with SLC25A46 knock down, as reported previously (Figure 3D Abrams et al., 2015 Janer et al., 2016 Wan et al., 2016). To confirm that the mitochondrial network was indeed hyperfused in SLC25A46-knockdown cells, we quantitated the mitochondrial network (Figure 3E). In cells that lacked SLC25A46, the network consisted of ∼65% hyperfused and ∼30% normal mitochondria. In contrast, the control cells had a network with ∼70% normal and ∼25% hyperfused mitochondria. Thus the mitochondrial network in LAN5 cells that lack SLC25A46 is aberrant, marked with a hyperfused network.

MFN1 and MFN2 oligomerization can be confirmed by cross-linking with a cysteine–cysteine cross-linker, which yields high–molecular weight complexes in SDS gels. In SLC25A46 − / − HCT116 or LAN5 SLC25A46 knockdown cells, an increased abundance of MFN1 and MFN2 oligomers was detected by cross-linking (Supplemental Figure S5, A and B). Our results support the observation that decreased SLC25A46 expression leads to hyperfused mitochondria (Figure 3, D and E Abrams et al., 2015 Wan et al., 2016), which correlates with increased steady-state levels and oligomerization of MFN1 and MFN2 complexes in hyperfused mitochondria.

Because hyperfused mitochondria might influence cell viability, we analyzed the cell growth of HEK293 T-REx Flp-In SLC25A46 − / − and LAN5 cells in which SLC25A46 expression was silenced by a specific shRNA (Figure 3F). However, changes in cell viability were not detected in these lines (Figure 3F). Our results contrast with a recent study by Janer et al. (2016) in which fibroblast lines derived from a patient or knocked down for SLC25A46 showed increased cell senescence.

Expression of SLC25A46 L341P does not rescue elevated MFN1 and MFN2 levels in cells lacking SLC25A46

Next we analyzed whether mutant SLC25A46 L341P is able to rescue the phenotype in SLC25A46 knockout cells because our previous studies indicated that the L341P mutation destabilized SLC25A46, causing hyperfused mitochondria and lethal PCH (Wan et al., 2016). SLC25A46 was deleted in HEK 293 T-REx Flp-In cells, and doxycycline-inducible 2xHA-SLC25A46 WT or L341P was introduced. Isolated mitochondria from these cell lines were subjected to SDS- and BN-PAGE as in Figure 3, A and B. Again, MFN1 and MFN2 showed increased steady-state levels and oligomerization when SLC25A46 was knocked out (Figure 3, G and H). Whereas the induction of 2xHA-SLC25A46 WT rescued the knockout, the induction of 2xHA-SLC25A46 L341P failed and instead increased steady-state levels, and oligomerization of MFN1 and MFN2 prevailed (Figure 3, G and H). Note that expression of the HA-SLC25A46 proteins was confirmed by immunoblotting with an anti-HA antibody. In addition, anti-SLC25A46 was used to detect expression of endogenous SLC25A46 and HA-tagged SLC25A46 because 2xHA-SLC25A46 L341P is rapidly degraded, this panel was overexposed to verify that 2xHA-SLC25A46 L341P was present (Figure 3H). Thus cells expressing SLC25A46 L341P have a phenotype identical to that of SLC25A46 knockout.

SLC25A46 L341P is not stable

Because rapid turnover of a specific mitochondrial OM protein is an unexpected molecular basis for a disease, we investigated this degradation pathway in detail. We transiently transfected HEK293T cell lines with constructs that expressed SLC25A46 WT or L341P mutant protein containing a 2xHA epitope at the N-terminus. We treated cells with cycloheximide to block cytosolic translation and removed aliquots in a time-course chase (Figure 4A). SLC25A46 was detected with an anti-HA antibody. Mitochondrial OM protein TOMM40 was included as a control. The abundance of SLC25A46 was quantitated by densitometry (Figure 4B). Whereas TOMM40 and WT SLC25A46 were stable during the chase, SLC25A46 L341P was not detected, except when the blot was overexposed relative to WT SLC25A46. Note that the apparent increase in abundance of SLC25A46 at the 40- and 60-min time points is unexpected but likely indicative of the normal variability associated with the stable protein in these types of experiments we observed this previously (Hwang et al., 2007). To determine whether different amino acid substitutions were tolerated, changes including a basic (arginine, R), acidic (glutamate, E), and conservative hydrophobic (valine, V) substitution were made to SLC25A46 at position 341, and the stability was tested as in Figure 4A (Figure 4C). Again, the arginine and glutamate substitutions markedly reduced stability, similar to the proline mutation, and the conservative valine substitution also decreased the stability of SLC25A46, but the protein was detectable upon a shorter exposure of the film. Therefore the mutation at position 341 strongly decreases the stability of SLC25A46.

FIGURE 4: SLC25A46 L341P is very unstable in cultured cells and does not reach its native conformation. (A) The half-life of WT SLC25A46 or SLC25A46 L341P was determined by cycloheximide (CHX)-chase experiments. HEK293T cells were transiently transfected with constructs for 2xHA-SLC25A46 WT or 2xHA-SLC25A46 L341P. At 24 h posttransfection, CHX was added, and samples were taken at the indicated time points. The mitochondria were isolated and analyzed with antibodies against the HA tag and control TOMM40. (B) Densitometry of immunoblots from A. (C) As in A, constructs were generated with the indicated mutations at position 341. (D) As in A, constructs were generated with the indicated mutations at positions 341, 333, and 340. (E) Radiolabeled 2xHA-SLC25A46 WT, L341P, and R340C were imported for 10 min into mitochondria isolated from HEK293 T-REx Flp-In cells. After import, half of the sample was treated with 1.25 µg/ml trypsin for 15 min on ice, and inserted proteins were recovered in the pellet after alkali extraction. As a control, the other half was incubated for 15 min on ice, followed by alkali extraction. All samples were resolved by SDS–PAGE and visualized by autoradiography. Arrows labeled 1–3 indicate different cleavage products that were prominent in the panel that was exposed for a longer time.

Mutations in SLC25A46 were also identified in patients with optic atrophy spectrum disorder (Abrams et al., 2015). These mutations were associated with defects in mitochondrial morphology but the fate of mutant SLC25A46 protein was not determined. Two mutations, P333L and R340C, were near L341P and localized to the putative fifth membrane-spanning domain, based on homology with mitochondrial carriers (Wan et al., 2016). As in Figure 4C, the abundance of these proteins was determined using cycloheximide-chase studies (Figure 4D). SLC25A46 R340C was detected, but L341P and P333L variants were identified only when the blot was exposed for a longer time. The patient with the P333L mutant died at 15 wk of life, whereas the patient with the R340C mutation was 51 yr old at the time of the study (Abrams et al., 2015). Thus rapid turnover of SLC25A46 during development is likely associated with the early onset of death.

To determine whether the rapid degradation of SLC24A46 L341P was a result of the mutant protein failing to reach its native conformation in the OM after import, we imported radiolabeled SLC25A46 WT, L341P, and R340C into isolated mitochondria and treated half of the sample with a low amount of trypsin to perform limited proteolysis. Membrane insertion was confirmed by alkali extraction (Figure 4E). In the absence of trypsin treatment, all three proteins were imported to the same extent. Comparison of the cleavage products after limited proteolysis showed that the three proteins had differing cleavage patterns. Three prominent degradation products were detected and labeled on the panel with the longer exposure. SLC25A46 WT and R340C had a prominent cleavage product at position 2 and minor product at position 1, with the exception that R340C had less of these products than WT. In contrast, SLC25A46 L341P had an additional cleavage product at position 3 that was more prominent than for WT and R340C. Thus we conclude that SLC25A46 L341P is imported normally but does not reach the native conformation, resulting in rapid degradation. That SLC25A46 L341P does not fold into its final conformation is also supported by the observation that SLC25A46 L341P displayed a different distribution pattern in the glycerol gradients and BN-PAGE than did WT SLC25A46 (Figure 1, A and C).

SLC25A46 L341P is polyubiquitylated and degraded by the proteasome

The strong interaction of SLC25A46 L341P with MULAN implies that mutant SLC25A46 may be rapidly degraded by the UPS (Figure 2A). However, it has been suggested that mitochondrial OM proteins are turned over only by mitophagy with the ultimate removal of the entire organelle (Ling and Jarvis, 2013). Therefore we in­vestigated the turnover process in more detail. Cells expressing SLC25A46 L341P were treated with the two proteasome inhibitors MG132 and bortezomib and with the late-phase autophagy inhibitor bafilomycin A1 (Figure 5A). Because MG132 at a 10-fold higher concentration can also inhibit the proteolytic calpains and cathespins (Tsubuki et al., 1996 Kisselev and Goldberg, 2001), bortezomib, which is proteasome specific, was also used (Moore et al., 2008). The steady-state level of SLC25A46 L341P was increased only when the cells were treated with MG132 or bortezomib (Figure 5A). Proteins that are degraded by the proteasome are typically ubiquitylated, resulting in a decreased mobility in SDS gels. A fraction of SLC25A46 L341P displayed reduced mobility after proteasome inhibition, suggesting ubiquitylation (Figure 5A the monoubiquitylated fraction is marked with an asterisk). In contrast, SLC25A46 L341P was not stabilized upon bafilomycin A1 treatment, although the autophagy marker LC3-II accumulated, indicating autophagy was indeed inhibited (Figure 5A). When we analyzed mitochondria from HEK293T cells that were transiently transfected with HA-SLC25A46 WT or L341P mutant, we found that MG132 treatment again markedly stabilized HA-SLC25A46 L341P, whereas HA-SLC25A46 WT was only slightly stabilized (Figure 5B). Moreover, a fraction of the SLC25A46 WT and L341P from MG132-treated cells also showed decreased mobility after increased exposure, supporting ubiquitylation of the proteins (Figure 5B). In HeLa cells transiently transfected with HA-tagged SLC25A46 WT or L341P, mutant SLC25A46 was barely detectable (Supplemental Figure S6) and localized to mitochondria and the cytosol however, no large aggregates were observed. In comparison, SLC25A46 WT was abundant and exclusively localized to mitochondria. When the cells were treated with MG132, SLC25A46 L341P and WT accumulated on mitochondria and in the cytosol. We conclude that SLC25A46 L341P does not form large aggregates but is imported into mitochondria and then is quickly retrotranslocated to the cytosol due to instability. WT SLC25A46 follows a similar pathway but with slower kinetics.

FIGURE 5: SLC25A46 L341P is polyubiquitylated and degraded by the proteasome. (A) HEK293 T-REx Flp-In cells expressing 2xHA-SLC25A46 L341P were treated with MG132, bortezomib, bafilomycin A1, or DMSO (0.1%) for 6 h. Cells were lysed, and total extracts were analyzed by SDS–PAGE. Antibodies against LC3 and ubiquitin were included as controls to verify inhibition of autophagy and protein degradation, respectively. TOMM40 served as loading control. The asterisk marks monoubiquitylated 2xHA-SLC25A46 L341P. (B) SLC25A46 L341P accumulates on mitochondria upon proteasome inhibition. HEK293T cells were transfected as in A and then treated with 10 μM MG132 (M) or control DMSO (D) for 6 h. Mitochondria were isolated and lysed HA-tagged SLC25A46 L341P was detected by immunoblotting. (C) Ubiquitylated SLC25A46 WT or L341P was immunoprecipitated under denaturing conditions with a Flag antibody from cells transiently expressing HA-ubiquitin-GFP and 3xFlag-SLC25A46 WT or L341P. Ubiquitin was detected with an anti-HA antibody. GFP cleaved from HA-ubiquitin served as transfection control, and TOMM40 was included as a loading control. (D) The ratio between ubiquitylated and nonubiquitylated SLC25A46 WT and L341P was determined after densitometric analysis of both proteins pools of C. Mean ± SEM n = 3. (E) Proteasome inhibition blocks the rapid degradation of SLC25A46 L341P in mitochondria. HEK293T were transiently transfected with constructs for 2xHA-SLC25A46 WT or L341P for 24 h and then harvested before (0) and 15 or 30 min after the addition of cycloheximide. Mitochondria were isolated and analyzed by SDS–PAGE and immunoblotting. To block proteasome-dependent degradation, 25 μM MG132 was added at the time as cycloheximide addition.

To confirm SLC25A46 ubiquitylation, we transiently transfected cells with Flag-tagged SLC25A46 WT or L341P, as well as with a construct for HA-tagged ubiquitin-GFP (and as a control, an empty vector Figure 5C). SLC25A46 from lysates was immunoprecipitated with anti-Flag followed by immunoblotting with anti-HA. As expected, SLC25A46 was ubiquitylated, with the mutant L341P possessing greater modification (Figure 5, C and D).

To verify the UPS involvement in the turnover of SLC25A46, were subjected cells transiently transfected with SLC25A46 WT or L341P to a cycloheximide chase in the presence of the MG132 to assess SLC25A46 stability (Figure 5E). SLC25A46 WT was stable, whereas SLC25A46 L341P was present at lower levels, but degradation was detected upon longer exposure to film. The addition of MG132 stabilized the SLC25A46 L341P, confirming that the proteasome degraded mutant L341P. TOMM40 was stable and included as a loading control. Thus SCL25A46 L341P in the mitochondrial OM is extensively ubiquitylated and degraded by the proteasome, independently of mitophagy.

MULAN and MARCH5 coordinately ubiquitylate SLC25A46 L341P, and P97 mediates the degradation of SLC25A46

We determined the components that mediate SLC25A46 turnover by taking advantage of inhibitors, mutants, and RNAi silencing. When HEK293T cells stably expressing SLC25A46 L341P were treated with the proteasome inhibitor MG132 (M) or P97 inhibitor NMS873 (N Magnaghi et al., 2013), coIP assays revealed a robust interaction of SCL25A46 L341P with MULAN and a weaker interaction with P97 (Figure 6A). A strong interaction with MARCH5 was not detected, because MARCH5 failed to coimmunoprecipitate with SCL25A46 L341P (Figure 6A). Note that immunoblots with the antibody for endogenous MARCH5 yield some background, and a nonspecific band that migrates at a higher molecular weight than MARCH5 was detected (marked with an asterisk in Figure 6A).

FIGURE 6: Rapid turnover of SLC25A46 L341P strongly depends on P97. (A) SLC25A46 binds to P97, MARCH5, and MULAN. Stable HEK293T cells expressing 2xHA-SLC25A46 L341P were treated with DMSO (vehicle control), 25 μM MG132 (M), or 5 μM NMS873 (N) for 3 h before cell lysis. Cells were lysed in digitonin, and SLC25A46 was immunoprecipitated from whole-cell extracts with anti-HA antibody. Antibodies against HA, P97, MULAN, and matrix control LRP130 were used for immunoblotting. Input samples correspond to 2% of the amount of lysate used for the IP. The asterisk marks an unspecific signal in the IP lanes for MARCH5. (B) Inhibition of P97 alters degradation of SLC25A46 L341P. The same cells as in A were treated with DMSO and 5 or 10 μM NMS873 in combination with CHX. At the indicated times, cells were harvested and mitochondria were isolated. SLC25A46 L341P degradation was detected by immunoblotting, and TOMM40 was included as a loading control. The asterisk marks polyubiquitylated species. (C) HEK293T cells stably expressing doxycycline (Doxy)-inducible shRNA targeting P97 were transfected with a construct for 2xHA-SLC25A46 L341P. After 24 h, cells were treated with CHX, and samples were removed at the indicated times. Cells were separated into a mitochondrial pellet and a postmitochondrial supernatant equal amounts of protein from each fraction were analyzed by SDS–PAGE and immunoblotting. Doxycycline treatment was started 24 h before transfection and maintained during the analysis. The asterisk marks monoubiquitylated SLC25A46. (D) The half-life of SLC25A46 L341P was determined in HEK293T cells transiently expressing 2xHA-SLC25A46 L341P in combination with WT P97, active-site-mutant P97-QQ, or empty vector. Cells were treated with CHX and analyzed as described in C. The asterisk marks monoubiquitylated SLC25A46.

We followed this investigation with cycloheximide-chase experiments to stop cytosolic translation and investigated SLC25A46 L341P stability under conditions that decreased proteolysis (Figure 6, B–D). Aliquots were removed before cycloheximide treatment and 15 and 30 min posttreatment, and mitochondria lysates were subjected to immunoblotting. Treatment with NMS873 to inhibit P97 before the cycloheximide chase resulted in SLC25A46 L341P accumulating in large–molecular mass complexes (marked with an asterisk), indicating an accumulation of polyubiquitylated SLC25A46 L341P mutant on mitochondria (Figure 6B). We do not expect that this fraction of SLC25A46 (marked with an asterisk) is a nonspecific aggregate because 1) the high–molecular mass form of SLC25A46 was not detected when the cells were mock treated with dimethyl sulfoxide (DMSO) 2) the samples were prepared for SDS–PAGE with a sample buffer that contained SDS, resulting in the expected solubilization of SLC25A46 and 3) SLC25A46 prepared in SDS-containing sample buffer is not observed in the stacking gel, and the SLC25A46 fraction at the top of the gel (marked with an asterisk) entered into the separating gel. P97 protein expression was decreased using a doxycycline-inducible shRNA system (Figure 6C). Decrease in P97 protein levels correlated with increased stability of SLC25A46 L341P on mitochondria and a slight increase in the stability of a cytosolic pool. This supports a pathway in which SLC25A46 is first inserted into the mitochondrial OM and then subsequently degraded. In addition, expression of the active-site P97-QQ mutant mirrored the shRNA induction (Figure 6D), in which SLC25A46 L341P was stabilized on the mitochondrial membrane moreover, a monoubiquitylated form of SLC25A46 L341P (marked with an asterisk) was detected, and a fraction of the P97-QQ mutant associated with the SLC25A46 mutant on mitochondria. In contrast, overexpression of active P97 did not increase accumulation of mutant SLC25A46 (Figure 6D). Note that in Figure 6C with anti-P97, a fraction of the P97 pool was detected in the mitochondrial fraction, which likely reflects a pool of P97 that might be engaged in SLC25A46 degradation. Similarly, a small fraction of the P97-QQ mutant was identified in the mitochondrial fraction this pool may be larger because the anti-histidine antibody is not as robust as the P97 antibody.

We manipulated MULAN expression. Overexpressing MULAN resulted in increased monoubiquitylation of SLC25A46 L341P (marked with an asterisk). However, “ligase-dead” MULAN H319A increased SLC25A46 L341P stability but lacked ubiquitylation (Zhang et al., 2008 Figure 7A). Initially, we anticipated that overexpression of WT MULAN would result in increased turnover of SLC25A46 L341P, similar to the conditions of the empty vector control. However, overexpression of MULAN is likely not well tolerated by cells because reports indicate that overexpression of WT MULAN inhibits cell growth and causes cell death (Zhang et al., 2008 Bae et al., 2012). Therefore we interpret this result as indicating that increased abundance of active MULAN causes increased degradation of key proteins required for cell survival, such as Akt (Bae et al., 2012), and long-term MULAN overexpression leads to apoptosis via activation of caspases (Zhang et al., 2008). Indeed, long-term overexpression of MULAN leads to cell death in these experiments. We conclude that under apoptotic conditions, the quality control pathway for proteins such as SLC25A46 L341P is not working efficiently and therefore SLC25A46 turnover is not as robust as in the case of expression of the empty vector. In addition, a similar result was found with the endoplasmic reticulum (ER) ubiquitin ligase HRD1. Overexpression of the mutant HRD1 failed to degrade nonsecreted immunoglobulin κ light chain (Ig κ LC), but overexpression of WT HRD1 also inhibited degradation of nonsecreted Ig κ LC (Okuda-Shimizu and Hendershot, 2007). Thus the results with HRD1 mirror those with overexpression of WT MULAN.

FIGURE 7: MARCH5 and MULAN mediate SLC25A46 L341P ubiquitylation and degradation. (A) Increased half-life of SLC25A46 L341P by overexpressing the RING-finger mutant MULAN H319A. A stable cell line expressing 2xHA-SLC25A46 L341P was transiently transfected with an empty vector, WT MULAN, or MULAN H319A and treated with CHX for indicated time points. Mitochondria were isolated, and MULAN was detected with an anti-myc antibody. The asterisk marks monoubiquitylated SLC25A46. (B) As in A, with the exception that the cells were transiently transfected with WT MARCH5 or the RING-mutant MARCH5 H43W. (C) HEK293T cells stably expressing Doxy-inducible control shRNA or a shRNA targeting MULAN were transfected with constructs for control shRNA or shRNA targeting MARCH5 and 2xHA-SLC25A46 L341P. Doxycycline (1 μg/ml) was added during transfection. After 48 h, CHX was added, and cells were harvested just before CHX addition (0) and at 15 and 30 min. Mitochondria were isolated and analyzed by SDS–PAGE and immunoblotting. (D) As in C, but cells were transfected with 3xFlag-SLC25A46 L341P and HA-Ubiquitin-GFP. After treatment of the cells with 25 μM MG132 for 3 h, HA-tagged ubiquitylated proteins were immunoprecipitated under denaturing conditions with anti-HA. Ubiquitylated 3xFlag-SLC25A46 L341P was detected with an anti-Flag antibody. The asterisk marks polyubiquitylated species. TOMM40 is a loading control. (E) Determination of SLC25A46 L341P abundance in HEK293 T-REx Flp-In WT, MARCH5, MULAN, or MARCH5/MULAN knockout cells. Cells were transfected with a 2xHA-SLC25A46 L341P construct mitochondria from the cell lines were isolated, solubilized, separated by SDS–PAGE, and analyzed by immunoblotting. PreP was included as a loading control. (F) As in E, with CHX treatment for the indicated times. Cells were harvested at each time point, and mitochondria were isolated. SLC25A46 L341P and endogenous SLC25A46 were detected by immunoblotting, and TOMM40 was included as a loading control. (G) Under physiological expression of WT SLC25A46, the levels of MFN1, MNF2, and dimers are kept constant so as not to disturb the fine balance between fission and fusion, which is important for development and survival (left). However, when the gene SLC25A46 is disrupted by deletions or the gene product destabilized by point mutations like L341P, which is quickly degraded by OM–associated degradation, MFN1 and MFN2 dimers accumulate on the OM and shift the balance towards fusion. This leads to pontocerebellar hypoplasia and early death (right).

We used a similar approach with manipulation of MARCH5 expression. Transient transfection of WT MARCH5 resulted in degradation of SLC25A46 L341P similar to the transfection with an empty vector (Figure 7B). In contrast, transfection with the ring-domain mutant MARCH5 H43W resulted in stabilization of SLC25A46 L341P to a minor extent (Karbowski et al., 2007).

Using the shRNA approach to decrease MULAN and MARCH5 expression, we found that SLC25A46 L341P was markedly stabilized when the expression of both was reduced (Figure 7C). To confirm ubiquitylation of SLC25A46 L341P specifically by MULAN and MARCH5, we combined Flag-tagged SLC25A46 L341P and HA-ubiquitin–green fluorescent protein (GFP) transfection with the MULAN and MARCH5 RNAi treatment as in Figure 5C. Again, knockdown of both MULAN and MARCH5 shifted the pool of polyubiquitylated SLC25A46 L341P to a cohort that was less ubiquitylated (Figure 7D, asterisk). As in Figure 6B, we do not expect the fraction of Flag-SLC25A46 L341P that migrates at a high molecular mass (asterisk) to be a nonspecific aggregate because this fraction was not detected in the sample treated with the control shRNA. In addition, on the basis of the assay, we first immunoprecipitated proteins that were HA-ubiquitylated and then blotted for Flag-SLC25A46 L341P with anti-Flag. Therefore the fraction of Flag-SLC25A46 L341P that migrates at a high molecular mass (asterisk) is polyubiquitylated.

To verify the role of MULAN and MARCH5 on the degradation of SLC25A46 L341P, we generated HEK293 T-REx Flp-In MULAN − / − , MARCH5 − / − , and MULAN − / − /MARCH5 − / − cell lines and analyzed the abundance of the SLC25A46 L341P when transiently transfected (Figure 7E). Again, only in the absence of MARCH5 and MULAN were SLC25A46 L341P steady-state levels increased in comparison to the single E3 ligase knockouts. As observed in the shRNA approach, SLC25A46 L341P was stabilized markedly when both E3 ligases were knocked out in the HEK293 T-REx Flp-In cell lines (Figure 7F). Single knockout of MULAN or MARCH5 had little effect on the half-life of SLC25A46.

The Cullin RING ligase family does not ubiquitylate SLC25A46 L341P

Another class of E3 ubiquitin ligases that may play a role in degrading SLC25A46 L341P is the Cullin RING ligase (CRL) family (Supplemental Figure S7). Previously SLC25A46 was identified as a potential CRL3 substrate (Emanuele et al., 2011). The small molecule MLN4924, which inhibits the NEDD8-activating enzyme and thereby prevents the activation of Cullin RING ligases, was added to cells before the cycloheximide chase (Soucy et al., 2009). SLC25A46 L341P was not stabilized under these conditions. However, the inhibitor was active because it prevented the neddylation of Cul4a by Nedd8 (Brownell et al., 2010). Thus, of the ubiquitin ligases, MARCH5 and MULAN coordinate SLC25A46 degradation and seem to have redundant functions.


Toxic Oligomers Contain α-Sheet Structure, Not β-Sheet Structure

The secondary structure of Aβ was then evaluated as a function of time by circular dichroism (CD) (Fig. 1C). Consistent with the ThT profile, aggregation initiated from an unstructured random coil conformation (0 h) and proceeded to β-sheet structure in the course of aggregation (48 h and beyond). Interestingly, the curve lifted and flattened in the lag phase before β-sheet formation (shown for 8–36 h). Thus, the hexamer and dodecamer oligomers populated during the lag phase do not contain measurable conventional secondary structure, while the higher-molecular-weight late species (48 h and beyond) contain β-structure consistent with the inference from the ThT-binding assay. The featureless CD spectra of the intermediate oligomers (in both size and time) are similar to what we proposed and confirmed experimentally for α-sheet structure (29, 38 ⇓ –40). Designed α-sheet peptides (denoted as AP#, for alternating peptide) produce a “null signal” due to their unique structural characteristics that lead to cancellation of the CD signal, as shown in Fig. 1C for the α-sheet design AP407, which is distinct from random coil, α-helical, and β-sheet CD spectra (29, 38 ⇓ –40). Contrary to our results, many assume that Aβ soluble oligomers adopt β-sheet structure in the lag phase, but a number of other studies report CD spectra very similar to the α-sheet spectra provided here (17, 31, 41, 42). While similar spectra were obtained, they were not recognized to be α-sheet due to its novelty, as model compounds are critical to the assignment of spectra. Here we used our synthetic α-sheet designs, such as AP407, for that purpose, but to do so, further characterization of the structure was necessary.

To obtain more detailed structural information for our α-sheet designs, 2D NMR experiments of AP407 were performed, which resulted in 455 distinct nuclear Overhauser effect interactions (NOEs) between protons for this 23-residue peptide (Dataset S1), allowing for the calculation and testing of structural models of AP407 (SI Appendix, Fig. S4A). The chemical shifts from NMR and the NMR-derived structural ensemble are in excellent agreement (SI Appendix, Fig. S4B). The secondary chemical shifts, which are often used to determine regions of secondary structure, are consistent with α-helical structure while the coupling constants reflecting the Φ dihedral angles are not (SI Appendix, Fig. S4C). Instead, the coupling constants are indicative of β-sheet or extended structure. Thus, the NMR results point to both α-helix and extended sheet structure, as we would expect for an α-sheet comprised of local helical (Φ,Ψ) values of alternating chirality (SI Appendix, Fig. S1) forming a hairpin sheet structure. The NOEs provide further support for this conclusion. Sequential HN-HN NOEs expected for α-sheet structure were observed, while the standard main-chain NOE patterns expected for α-helical and β-sheet structures were not present (SI Appendix, Fig. S4 D and E), which is consistent with the flat CD spectrum for AP407 (Fig. 1C, with one of the NMR structures provided in the Inset). In an earlier study, we obtained NOEs for two other α-sheet designs, but not a sufficient number to calculate a structure (38). Here, however, we obtained a greater number of NOEs —including side-chain NOEs—by using a more constrained design with a disulfide bond linking the α-strands. One hundred percent of the 455 NOEs are satisfied by the ensemble presented in SI Appendix, Fig. S4. As is generally the case with small peptides, however, AP407 retains conformational flexibility, as is particularly evident in the turn region due to alternative side-chain packing (SI Appendix, Fig. S4A).

Microfluidic modulation spectroscopy (MMS) was used to further characterize the structural characteristics of Aβ during aggregation. MMS measures peptide absorption spectra by optically scanning across the amide I band, which reflects a combination of patterns of hydrogen bonding, dipole–dipole interactions, and the geometric orientations throughout the peptide. As this is a new technique and α-sheet is a nonstandard structure, we used synthetic designed α-sheet hairpins as model compounds to help interpret the spectra, along with controls for other conventional secondary structures: designed α-sheet hairpins (AP5, AP90, AP407, and AP421), β-sheet (P411), and α-helical (PSMα1) peptides (sequences and color mapping provided in SI Appendix, Table S2). Each class of peptide produced distinctive spectral features, as shown in the second derivative plots of the amide I region (Fig. 1D). This is further illustrated by subtracting the AP407 α-sheet peptide spectrum from the other samples, highlighting the similarities between the different α-sheet peptides with the largest difference at 1,680 nm, which we surmise is due to the improved dipole alignment from stabilization of the α-sheet structure in the hairpin due to the disulfide cross-link (Fig. 1E). Moreover, this band was predicted to be dominant for nonsolvated α-sheet structure (43) and confirmed experimentally by conventional Fourier-transform infrared spectroscopy (FTIR) of dry films (29, 38, 39).

After establishing the spectral features of the model compounds, we analyzed the most toxic Aβ sample (24 h) and found its spectrum to be consistent with α-sheet and distinct from β-sheet and α-helix (Fig. 1 D and E). In fact, the Aβ oligomer spectrum nicely overlays the other α-sheet spectra. With increasing aggregation time, the Aβ spectrum shifted and was most similar to our β-sheet control (P411) rather than any of our α-sheet designs (120 h, Fig. 1 D and E), but note the shift in the 120-h Aβ sample relative to the monomeric β-hairpin P411. Such shifts are routinely seen between conventional β-structure and fibrils by FTIR (44). Consistent with the ThT and CD results, the α-sheet structure preceded β-sheet formation. In addition, it was recently found that fibrils of a fragment of a variant of the amyloidogenic protein transthyretin contain spectroscopic signals of both α-sheet and β-sheet structures by FTIR, providing further support for the presence of α-sheet in amyloid systems and the possibility of coexistence of an α- and β-sheet (27, 45).


FAQs - Autophagy and LC3

Atg4 cleaves pro-LC3 to form LC3-I which then gets conjugated to PE (by Atg7) for the generation of LC3-II. The latter gets recruited to the autophagosomal membrane for helping membrane elongation. ATG7 also mediates ATG5-ATG12-ATG16 complex formation and the latter along with LC3-II is highly critical for autophagosome formation. Adaptor protein p62/SQSTM1 binds to ubiquitinated proteins and LC3-II for mediating autophagy via localizing into autophagic compartments, transporting ubiquitinated proteins and organelles for degradation.

Several molecular markers of autophagy have been studied to date but the conversion of LC3-I to LC3-II via phosphatidylethanolamine (PE) conjugation has been accepted as the gold standard for autophagosome formation. p62/SQSTM1 is also important since it is a substrate for LC3 which facilitates selective degradation during autophagy. Some of the major proteins involved in autophagy signaling are:

Some of the major proteins involved in autophagy signaling

What is LC3 and how is it related to or different from Atg8?
LC3 was originally identified as a subunit of microtubule-associated proteins 1A and 1B (MAP1LC3) and was subsequently found to have similarities to the yeast protein Atg8 (also called Apg8, Aut7 or Cvt5). The mammalian homologues of yeast are subdivided into two major subfamilies: MAP1LC3/LC3 (LC3A, LC3B and LC3C) and GABARAP (GABARAP, GABARAPL1 and GABARAPL2/GATE-16). Both LC3 and GABARAP are expressed as precursor proteins which undergo cleavage followed by lipidation/PE-conjugation to generate LC3-II and GABARAP–PE respectively. Besides GABARAPL2, it has been documented that all mammalian Atg8 homologues play a role in autophagosome biogenesis. Furthermore, because of unique features in the distribution of their molecular surface charges, it has been suggested that LC3 and GABARAP recognize distinct sets of cargoes for selective autophagy. Return to FAQs

What is the difference between LC3A, LC3B and LC3C, or LC3-I and LC3-II?
LC3 is a soluble protein with a molecular mass of ∼17 kDa and is distributed ubiquitously in eukaryotes. It is expressed as the splice variants LC3A, LC3B, and LC3C which display unique tissue distribution. All LC3 isoforms undergo post-translational modifications, especially PE conjugation (lipidation) during autophagy. Upon autophagic signal, the cytosolic form of LC3 (LC3-I) is conjugated to PE to form LC3-PE conjugate (LC3-II), which is recruited to the autophagosomal membranes. Return to FAQs

WB analysis of HeLa (1), HeLa + Chlorquine/CQ (2), SHSY5Y (3), SHSY5Y +CQ (4), A431 (5), A431 +CQ (6) and Ntera2 (7) using rabbit polyclonal LC3 antibody at 2 ug/ml concentration.

ICC/IF of CQ treated HeLa cells using LC3B (NBP2-46892) and Tubulin (NB100-690) antibodies with detection via Dylight 488 (green) and Dylight 550 (red) labelled secondaries respectively. Nuclei were counterstained with DAPI (blue).

Is it possible to distinguish LC3-I and LC3-II pools in immunostaining assays with two different fluorescent labelled antibodies?
No, because the main difference between LC3-I and LC3-II is their lipidation status (the conjugated PE moiety), all commercially available LC3 antibodies are known to detect both forms. To our knowledge, antibodies detecting only one isoform do not exist. Return to FAQs

When doing Western blot of LC3, what percentage gel should be selected for its effective separation?
The predicted molecular weight of LC3 is

17kDa and the processed forms LC3-I/II show up between 14-18kDa in WB analysis. For an effective separation of the two forms, we recommend using a 16% or a 4-20% gradient gel. If running a 16% gel, ensure the gel is not over-run. The LC3 protein may run off the gel due to its fast mobility/low molecular weight. Using a lower gel percentage, loading high sample amounts, and running the gel at high voltage may result in inappropriate separation/over-lapping of LC3-I and LC3-II bands, which makes it very difficult to distinguish between the two target bands and complicates data interpretation. Return to FAQs

Are there any tips for the transfer step in Western blot assay of LC3?
After running step, equilibrate the gel in transfer buffer properly to remove the entire running buffer from the gel. Importantly, ensure not to add even traces of SDS to the transfer buffer. Some researchers find it better to use PVDF membrane compared to nitrocellulose membrane for LC3 detection. When choosing a membrane, a pore size of 0.2 um is recommended. Because LC3 is a significantly small protein, membranes with a pore size of 0.45 um should not be used (LC3 may cross through the membrane). Voltage /current for the transfer should be kept low and an extended transfer should also be avoided to prevent the target protein from crossing the membrane. After performing the transfer, one should use Amido black or Ponceau S staining to visulize protein transfer and to ensure that the proteins have actually transferred in the low molecular weight region (15-20kDa range). Return to FAQs

What is the best blocking buffer for LC3 Western blot assay?
In our antibody validation assays, the conditions we have had the most success with are 5% non-fat dry milk in TBST as the blocking buffer. We suggest at least 1 hour of blocking on a slow shaker at room temperature. The blocking buffer should be prepared fresh because old blocking buffer (with potential microbial contamination) may lead to problems such as high background or the appearance of large dots on the blot. Return to FAQs

What positive control may I use for Western blot of LC3?
Novus offers ready to use HeLa Chloroquine Treated / Untreated Cell Lysate (NBP2-49689) and Neuro2a Chloroquine Treated / Untreated Cell Lysate (NBP2-49688) which are highly recommended positive controls for WB assay of LC3. Overexpression lysates of LC3 such as NBP2-04906, or a total cell lysate from serum starved cells depicting excessive vacuolization are other options for use as positive control when performing LC3 WB analysis. Return to FAQs

- Neuro2A cells (mouse neuroblastoma) were treated with (+) and without (-) 50 uM Chloroquine overnight. Approximately 10 ug of each whole cell lysates in 1x Laemmli sample buffer (NBP2-49688) was separated on a gradient gel by SDS-PAGE, transferred to 0.2 um PVDF membrane and blocked in 5% non-fat milk in TBST. The membrane was probed with 1 ug/ml anti-LC3 antibody (NB100-2220) and 1 ug/ml anti-alpha tubulin (NB100-690), and detected with the appropriate secondary antibodies using chemiluminescence.

– HeLa cells (human cervical carcinoma) were treated with (+) and without (-) 50 uM Chloroquine overnight. Approximately 10 ug of each whole cell lysates in 1x Laemmli sample buffer (NBP2-49689) was separated on a gradient gel by SDS-PAGE, transferred to 0.2 um PVDF membrane and blocked in 5% non-fat milk in TBST. The membrane was probed with 1 ug/ml anti-LC3 (NB100-2220) and 1 ug/ml anti-alpha tubulin (NB100-690), and detected with the appropriate secondary antibodies using chemiluminescence.

How long should the cells be serum starved for inducing autophagy?
Depending upon the cell type, serum starvation may take from a few hours to up to two days to induce autophagy. However, subjecting the cells to media such as Earle's Balanced Salt Solution or Hank's Balanced Salt Solution can induce autophagy in time points ranging from a few minutes to a couple of hours. The key would be to observe the cells for morphological changes, and the appearance of a large number of vacuoles in the cells is a good indicator of induction of autophagic death. Return to FAQs

I do not see LC3-II band in my blot. How do I fix this problem?
LC3-II expression correlates with autophagy induction and will only be detected when significant autophagic activity is present in the samples being tested. Before producing lysates from cultured cells, be sure to look for morphological changes which are characteristics of autophagic death (especially the excessive vacuolization). If working on tissues, it should be noted that autophagic cells are cleared more rapidly in vivo, and inclusion of autophagic flux blockage-based positive control (such as NBP2-49688 or NBP2-49689) can be helpful in the analysis of the extent of autophagic activity in the tissues under investigation. Return to FAQs

My control samples also show very high levels of LC3II. What does it signify?
Some cell lines may have higher basal levels of autophagy than others and in those cases, the control sample will show high levels of LC3-II. Excessive starvation before treatments of interest may also lead to the detection of high levels of LC3-II in control samples. Return to FAQs

In Western blot analysis, I can visualize the loading control bands, but I do not see any bands for LC3. Is there something wrong with my primary antibody?
It can be a problem related to the primary antibody as well, but before drawing that conclusion, one may consider some other potential reasons also. Prior to making the lysates from cultured cells, make sure to look at the morphology of the cells to confirm if autophagy is being induced or not (excessive vacuolization is a good indicator of autophagy). Use 4-20% gel for separation and do not run the gel at a high voltage. The pore size of the transfer membrane should be

0.2 um, and the transfer buffer should not contain SDS. After transfer, use Amido black or Ponceau S staining to see if you have proteins transferred in the low molecular weight region (15-20kDa range). In order to provide optimum temperature/time for antigen-antibody interaction, perform the primary incubation at room temperature for a couple of hours followed by overnight incubation at 4°C. Return to FAQs

Besides LC3-I/II bands, I see a signal above

40kDa also in my samples. Is it a non-specific band or potentially a dimer of LC3?
In literature, the LC3 homolog GABARAP has been shown to assume a dimeric conformation which is required for it to bind to microtubules and GABA receptors (Nymann-Andersen et al. 2002). Baisamy et al. 2009 demonstrated that FLAG- and GFP-tagged LC3 are able to co-immunoprecipitate from HEK-293 cell lysates, suggesting that LC3 can form oligomers inside cells. They further proposed that in this configuration, LC3 binding could promote a conformational change that impacts the Rho-GEF activity of AKAP-Lbc. Therefore, we believe that in LC3 blots, the bands that run above 40 kDa position might be originating from the potential dimeric/oligomeric forms of LC3. Return to FAQs

ATG5 Antibody [NB110-53818] WB analysis of lysates from wild-type mouse embryonic stem/ES cells (atg+/+) and the ATG5 knockout mouse's ES cells (negative control atg-/-) showing specific band of Atg5-Atg12 conjugate at 56 kDa position.

p62/SQSTM1 Antibody [NBP1-48320] HeLa cells were treated with (+) or without (-) 50 uM of Chloroquine for 24 hours and the whole cell lysates were analyzed in WB using anti-p62/SQSMT1 and anti-alpha tubulin (NB100-690 loading control).

Where can I get autophagy inhibitors or inducers?
Tocris (a bio-techne brand) is an established name in the field of bioactive small molecules and offers several different inhibitors/inducers of autophagy:

Does Novus offer any peptides for the induction of autophagy?
Yes, we offer Tat-D11 peptide (NBP2-49888) which is useful for the in vitro and in vivo induction of autophagy. Structurally, Tat-D11 is a shorter version of Tat-Beclin 1 which was engineered by Shoji-Kawata et al 2013 as a peptide composed of the autophagy-inducing region of Beclin 1 fused to the HIV-Tat protein. Mechanistically, these peptides induce autophagy through interaction with the negative regulator of autophagy GAPR-1/GLIPR2. Notably, in comparison to Tat-Beclin 1, Tat-D11 increases the induction of autophagosomes and autolysosomes by over five fold. Return to FAQs

Fluorescent microscopy analysis of GFP-puncta in HeLa cells expressing GFP-LC3B treated with scrambled peptide Tat-L11S (NBP2-49887) or peptide Tat-D11 (NBP2-49888).

For immunocytochemistry/immunofluorescence (ICC/IF) of LC3, how should I fix my cells? Do I need to permeabilize cells?
In our QC validation testing of LC3 antibodies, we routinely fix various cells in 4% paraformaldehyde/PFA (i.e. 10% buffered formalin) for 10 minutes at room temperature. Yes, permeabilization is a required step because LC3 localizes to the cytosol and membranes of the autophagosomes. In our lab, we use 0.1-0.5% triton X100 for 10 minutes to permeabilize paraformaldehyde fixed cells. Importantly, permeabilization step is not required when fixing the cells in methanol for 5 minutes since methanol itself acts a permeabilizing agent. Return to FAQs

Do I need to include an antigen retrieval step while performing IHC-P of LC3?
Antigen retrieval is generally a required step when the tissues are fixed in 4% PFA (i.e. 10% buffered formalin) for more than 4-6 hours. For routine IHC-P methods, fixation time varies from 6-24 hours and over fixation should be avoided as it may lead to excessive crosslinking of the proteins in the fixed tissues. Over fixed tissues will require more rigorous antigen retrieval which can lead to development of false signal. In our lab, we routinely use 10 mM sodium citrate (pH 6.0) buffer based method of heat induced antigen retrieval, wherein we incubate the slides in the buffer at a sub-boiling temperature for 10 minutes followed by cooling of slides (while in retrieval buffer) for 30 minutes at room temperature. Return to FAQs

What staining pattern should I expect in IHC-P assay of LC3?
LC3-I is found in the cytosol whereas LC3-II localizes to the autophagosomes, and in IHC-P, a diffused cytoplasmic staining (LC3-I) may be observed with punctate staining (LC3-II) in cells undergoing autophagic death. For reference images on LC3 IHC staining, we suggest checking publications such as Parajuli and MacMillan-Crow 2013, Kang et al. 2013, Guo et al. 2011, Shintaku 2011, and more listed on the Reviews and Publications section of datasheets of our LC3 Antibodies. Return to FAQs

I see a diffused staining of LC3 in IHC-P. How do I confirm if it is LC3 and not non-specific background?
Diffuse to punctate pattern of staining should be expected for LC3, and in order to confirm the specificity of immuno-reactivity, we suggest the following controls in IHC-P assay: Negative control (no primary, no secondary), Secondary only negative control (no primary added), and Peptide block control (Peptide Competition Protocol). Depending upon the outcome of the staining in test/control samples, additional measures may be taken by following appropriate troubleshooting suggestions from our Support by Application page. Return to FAQs

For LC3 IHC-P staining, is it normal to obtain a signal in the nucleus of the cells?
The majority of research publications suggests that the LC3-I form is cytosolic whereas the LC3-II form localizes to the autophagosomal membranes of cells undergoing autophagic death. There are a few research reports, however, that shows the nuclear localization of LC3. For additional information on localization /the biological relevance of nuclear pool of LC3, we strongly recommend reviewing the following publications – Hasui et al. 2011, Drake et al. 2010, Martinez-Lopezand et al. 2013, and Karim et al. 2007. From the assay point of view, it is helpful to check fixation time since inappropriate/under-fixation may lead to dispersal of LC3 into the cellular nuclei of dissected tissues or cultured cells. Optimizing the permeablization recipes/timing and the concentration or incubation time of primary antibody may also improve localization and sensitivity. Return to FAQs

Are there any other resources or guidelines on tips for working with LC3?
Yes, one of the best resources is the recently documented "Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)" (Klionsky et al. 2016). We also recommend "How to interpret LC3 immunoblotting" by Mizushima and Yoshimori 2007, and "Autophagy: assays and artifacts" by Barth et al. 2010. Return to FAQs


Results

Sustained TCR-mediated signaling correlates with proliferation, whereas transient signaling parallels with unresponsiveness

Peripheral human T cells were stimulated using sAbs or iAbs. These stimuli induce markedly different activation kinetics and cellular responses (Figure 1). Stimulation with sAbs resulted in a strong and transient induction of global tyrosine phosphorylation, as well as of ZAP70, LAT, and PLCγ-1. In contrast, when primary human T cells were treated with iAbs, global tyrosine phosphorylation and the phosphorylation of ZAP70, LAT and PLCγ-1 were weak, but sustained (Figure 1A, B). Additionally, phosphorylation of TCRζ was greatly and rapidly enhanced upon sAbs. In contrast, iAbs stimulation induces only weak TCRζ phosphorylation (Figure 1C). Interestingly, the phosphorylation kinetics of PAG/Cbp, a transmembrane adaptor protein running at about 70 KDa which is dephosphorylated upon TCR stimulation [9], is comparable under both stimulation conditions (Figure 1A). We next analyzed the signaling kinetics of the Erk cascade. Surprisingly, we found that Erk was very strongly activated under both conditions of stimulation. However, the activation induced by iAbs was sustained and lasted up to 90 minutes whereas, upon stimulation with sAbs Erk activation was transient, peaked at 1–5 minutes, and rapidly declined thereafter (Figure 1D). Thus, despite the weak activation of proximal signaling molecules, iAbs are capable of inducing strong and prolonged activation of downstream signaling pathways.

Analysis of the signaling signature and functional effects of sAbs and iAbs stimulation. A-D) Purified human T cells were treated with either soluble (sAbs) or immobilized (iAbs) CD3xCD28 mAbs for the indicated time periods. Total cell lysates (A, B, and D) or TCRζ immunoprecipitates C) were prepared and analyzed by Western blotting using the indicated Abs. One representative immunoblot of at least 3 independent experiments is shown. The phosphorylation of TCRζ was quantified using the 1D ImageQuant software and the values were normalized to the corresponding total TCRζ signal. Data represent the mean of the phosphorylation levels shown as arbitrary units ± SEM of 3 independent experiments. E) T cells were labeled with CFSE and stimulated as indicated. Proliferation was assessed after 72h by analyzing CFSE content on a FACS Calibur. One representative experiment of three independent experiments is shown.

It is generally accepted that transient signals triggered by soluble antibodies cannot induce productive T-cell responses. Indeed, we and others have demonstrated that human peripheral T cells, mouse OT-I transgenic T cells, and cytotoxic T-lymphocyte clones are not activated and do not differentiate upon stimulation with antibodies cross-linked in suspension [7, 8, 10]. Here, we have stimulated primary human T cells with either sAbs or iAbs and analyzed their functional responses. Treatment with sAbs failed to induce T-cell proliferation (Figure 1E). Conversely, Figure 1E shows that treatment of T cells with iAbs led to a strong proliferative response.

Transient signaling is regulated via negative regulatory feedbacks

The data presented above, show that sAbs induced a rapid, but transient TCR-mediated signaling kinetics, which cannot induce productive T-cell response, whereas stimulation with iAbs resulted in a sustained activation of a variety of signaling molecules and led to proliferation. These data indicate that there may be different regulatory mechanisms induced upon sAbs vs. iAbs stimulation. Thus, we next investigated how TCR-mediated signaling is differentially regulated under the two conditions. We hypothesized that a fast internalization of the available TCR molecules upon stimulation with sAbs could provide an explanation for the rapid termination of TCR-mediated signaling. Therefore, we compared the expression levels of the TCR after stimulation with either sAbs or iAbs by flow cytometry. Figure 2A shows that sAbs induce a slow rate of TCR downregulation, which became evident after 30 minutes of stimulation. It is important to note that the majority of the signaling molecules that we have tested reverted to the dephosphorylated/inactive state already 15 minutes after sAbs stimulation (Figure 1). Therefore, termination of TCR-mediated signaling occurs before TCR internalization. On the other hand, the data presented in Figure 2A show that stimulation with iAbs does not reduce, but rather slightly increases TCR levels. This is likely due to the fact that Abs bound to a solid matrix limit TCR internalization, but do not interfere with its transport to the plasma membrane. Moreover, we have previously shown that sustained TCR-mediated signaling and proliferation can occur under conditions of stimulation inducing TCR downregulation [7]. Thus, on the basis of these observations, we exclude that TCR internalization induced by sAbs is the cause of transient signaling.

sAbs, but not iAbs, induce inhibitory feedback loops. Purified human T cells were treated with either soluble (sAbs) or immobilized (iAbs) CD3xCD28 mAbs for the indicated time points. A) Measurement of TCR internalization. The expression of the TCR was assessed by PE-conjugated anti-TCRαβ mAb staining analysis by flow cytometry. Data represent the % of the mean fluorescence intensity (MFI) of the TCR expression relative to time 0 of 4 independent experiments. B) The phosphorylation of c-Cbl on Y 731 and Dok2 on Y 351 was determined by Western blotting. The phosphorylated c-Cbl and Dok2 bands were quantified using the 1D ImageQuant software and the values were normalized to the corresponding β-actin signal. Data represent the mean of the phosphorylation levels shown as arbitrary units ± SEM of at least 4 independent experiments. C) The expression of ZAP70 was determined by Western blotting. Equal loading is shown by reprobing immunoblots with antibodies specific for β-actin. ZAP70 bands were quantified as above. Data represent the mean of the expression levels shown as arbitrary units ± SEM of 6 independent experiments. D) Tyrosine phosphorylation of Fyn and Lck in the activation loop was determined by Western blotting using the pSrc (Y 416 ) antibody. Bands were quantified using the 1D ImageQuant software and values were normalized to the corresponding total Fyn and Lck signals. Data on the graph represent the mean of the phosphorylation levels shown as arbitrary units ± SEM of 4 independent experiments. Asterisks (*) indicate the Ig heavy chain. Statistical analysis ** P<0.01 ns, not statistically significant.

Having ruled out this possibility, we next focused on the analysis of feedback regulation events, which have been shown to play a crucial role in T-cell activation [11–13]. Proximal negative feedback loops can be activated by the TCR signalosome and can regulate the amplitude, the duration, and the specificity of the signal (reviewed in Acuto et al. [13]). We asked the question of whether the stimulation with sAbs induced the activation of negative regulatory molecules that may terminate signaling, thus resulting in the transient signal observed above. Among the many inhibitory molecules organizing negative regulatory circuits, we decided to focus on c-Cbl, an E3 ubiquitin ligase belonging to the CBL family, and the adaptor protein Dok2, which regulate TCR-mediated signaling through two different mechanisms. Whereas members of the CBL family are involved in the downregulation of signaling molecules via ubiquitination [14], Dok2 and its homolog Dok1 inhibit the activation of signaling pathways by competing for binding to SH2 domains or by recruiting other negative regulators, such as SHIP1 and RasGAP, to the TCR signalosome [15]. The activity of both c-Cbl and Dok2 have been reported to be regulated by tyrosine phosphorylation [15–17] and can be easily monitored by using anti-c-Cbl and anti-Dok2 phosphospecific antibodies, respectively. Figure 2B shows that upon sAbs stimulation, T cells very rapidly and strongly phosphorylated both c-Cbl and Dok2, whereas, treatment of human T cells with iAbs resulted only in a very weak phosphorylation of both molecules.

c-Cbl targets many signaling molecules for degradation, including ZAP70 [18]. Thus, we next tested whether sAbs, in addition to inducing strong c-Cbl phosphorylation, would also induce ZAP70 ubiquitination and degradation. We have previously shown in mouse OT-I T cells that ubiquitination of ZAP70 results in the appearance of ZAP70 bands displaying retarded migration in SDS-PAGE [7]. We checked whether stimulation with soluble CD3xCD28 Abs also resulted in the appearance of ZAP70 bands running at a higher molecular weight in primary human T cells and we found that activation/phosphorylation of c-Cbl upon stimulation with sAbs indeed correlates with retarded ZAP70 migration (Figure 2C). Additionally, the data presented in Figure 2C suggest that stimulation with sAbs also induced ZAP70 degradation. Conversely, stimulation with iAbs did not significantly induce either c-Cbl phosphorylation or retarded migration and degradation of ZAP70 (Figure 2C). Thus, it appears that stimulation with sAbs activates inhibitory feedback loops that may be responsible for terminating TCR-mediated signaling.

In addition to inducing a strong tyrosine phosphorylation of c-Cbl and Dok2, stimulation with sAbs also results in a strong phosphorylation of TCR proximal signaling molecules including TCRζ, ZAP70, and LAT (Figure 1B, 1C). Therefore, we investigated whether sAbs induce a stronger activation of the tyrosine kinases Lck and Fyn compared to iAbs. We immunoprecipitated TCRζ and assessed the level of active Lck and Fyn associated with the TCR. As shown in Figure 2D, sAbs stimulation significantly enhances the level of Lck and Fyn phosphorylated on the activation loop, which is believed to be a sign of an active enzyme. Conversely, this significant increase in Lck and Fyn phosphorylation is not observed upon iAbs stimulation. Hence, the data suggest that, in marked contrast to iAbs, sAbs stimulation enhances Lck and Fyn activation. We postulate that the enhanced activation of Lck and Fyn may result in a stronger tyrosine phosphorylation of downstream molecules (including negative regulators, such as c-Cbl and Dok2), which might imbalance TCR-mediated signaling, thus dampening T-cell activation.

Sustained activation is regulated by positive feedback loops

We next investigated whether positive feedback loops may be triggered by iAbs, thus leading to sustained activation of TCR-mediated signaling. In particular, we explored the regulatory circuit involving Lck phosphorylation by activated Erk [11]. This model is based on observations showing that the Erk-mediated phosphorylation of Lck on serine 59 alters Lck mobility and the ability of the SH2 domain of Lck to bind phosphotyrosines [19–21]. Stefanova et al. further demonstrated that Erk-mediated phosphorylation of Lck prevents SHP-1 binding, thus interfering with SHP-1-mediated Lck inactivation [11]. According to this model, active Erk would feedback to Lck to sustain signaling. To assess whether stimulation with iAbs triggers this Erk-mediated positive feedback loop, T cells were stimulated with iAbs and sAbs and the phosphorylation of Lck on S 59 was detected by the appearance of a new Lck band running at 59 kDa by Western blot [11]. As shown in Figure 3A and B, stimulation of T cells with iAbs clearly resulted in the formation of p59 Lck (up to 50% of total Lck), whereas this shift in the molecular weight of Lck was barely detectable upon sAbs treatment.

iAbs induce an Erk-mediated positive feedback loop. A) Purified human T cells were treated with either soluble (sAbs) or immobilized (iAbs) CD3xCD28 mAbs for the indicated time points. Lck expression was detected in cell lysates by anti-Lck immunoblotting. B) Bands corresponding to p56 or p59 Lck were quantified as described in Figure 2. Data represent the ratio of the levels of p56 and p59, and total (p56+p59) Lck shown as arbitrary units ± SEM of 5 independent experiments. C) Purified human T cells were treated with immobilized (iAbs) CD3xCD28 mAbs for the indicated time periods in the presence or absence of MEK Inhibitor I or U0126. Samples were analyzed by Western blotting using the Abs indicated. One representative immunoblot of 4 independent experiments is shown. D) J.CaM1.6 cells were transfected with various constructs carrying different mutations (S42A, S42D, S59A, S59D, S42A/S59A). After transfection, cells were either left unstimulated or stimulated with iAbs for 45 min. Samples were analyzed by Western blotting using the indicated Abs. One representative experiment of five independent experiments is shown.

To demonstrate that the appearance of p59 Lck indeed depends on Erk-mediated phosphorylation, T cells were stimulated with iAbs for 30 min in the presence or absence of U0126 or MEK Inhibitor I, inhibitors of the Erk activator MEK. This treatment has previously been shown to abolish the conversion of Lck to the p59 form [11]. In agreement with these observations, we also found that treatment of iAbs-stimulated T cells with U0126 or MEK Inhibitor I completely abolished both Erk activation and the shift of Lck to the p59 form (Figure 3C). We next tested whether the molecular shift of Lck upon iAbs stimulation is indeed induced by phosphorylation of S 59 . To assess this issue, we took advantage of Lck constructs carrying S to D and S to A mutations at this position, which mimic constitutive phosphorylation or prevent phosphorylation, respectively. We used the following mutants S59D, S59A, S42D, S42A, and S42A/S59A, which were expressed in the Lck-deficient Jurkat T-cell line J.CaM1.6. As shown in Figure 3D, mutations of S 42 do not affect the mobility shift of Lck either in unstimulated or iAbs stimulated cells. Conversely, the S59D mutation results in a constitutive shift to p59 Lck, thus indicating that phosphorylation on this site plays a major role in the regulation of Lck mobility. Accordingly, the S59A substitution, which results in a non-phosphorylatable mutant, prevents the generation of the 59 kDa form of Lck upon iAbs stimulation (Figure 3D). In summary, these data demonstrate that Erk-mediated phosphorylation of Lck at S 59 results in its retarded mobility on SDS-PAGE.

To check whether the inhibition of Erk-mediated Lck phosphorylation also resulted in a reduction of its activity, we investigated phosphorylation levels of downstream signaling molecules that are substrates of Lck, such as the tyrosine kinase ZAP70 and the adaptor protein LAT whose phosphorylation depends on ZAP70. T cells were stimulated for 30 min with iAbs. Subsequently, Erk activity was blocked by the addition of the MEK inhibitor U0126. The data presented in (Figure 4A, B) show that the phosphorylation of both ZAP70 and LAT is reduced upon MEK inhibition, thus indicating that Erk-mediated Lck phosphorylation may enhance its response. Conversely, treatment of sAbs-stimulated T cells with the MEK inhibitor reduced Erk phosphorylation, as expected, but not ZAP70 or LAT phosphorylation (Figure 4C, D).

An Erk-Lck feedback loop regulates TCR-mediated signaling. A) Purified human T cells were treated with iAbs alone for 30 min and then either DMSO or the MEK inhibitor U0126 was added and incubated for an additional 30 to 60 min. Samples were analyzed by Western blotting using the indicated Abs. B) Bands in A) were quantified and the values were normalized as described. Graphs show the mean of the phosphorylation levels of Erk1/2, ZAP70, and LAT or the level of p59 Lck as arbitrary units ± SEM of 4 independent experiments. C) Purified human T cells were preincubated either in the presence of DMSO or the MEK inhibitor U0126 and subsequently stimulated with sAbs for the indicated time points. Samples were analyzed by Western blotting using the indicated Abs. D) Bands in C) were quantified as described above and the data from at least two independent experiments are shown.

Collectively, these data suggest that stimulation with iAbs activates an Erk-mediated positive feedback loop which is required for proper T-cell response and proliferation. Importantly, the regulatory circuit induced by iAbs seems to mimic a previously described mechanism that is induced in T cells upon physiological stimulation [11].

Enhancement of Src kinases phosphorylation converts sustained into transient signal

The data presented above suggest that sAbs and iAbs induce qualitatively different signals and feedback regulation which are translated into distinct cellular responses. How the cell senses the quality of the signal is not yet fully understood. Our data suggest that sAbs induce stronger Src kinases activation and a stronger tyrosine phosphorylation pattern compared to iAbs stimulation (Figure 1). These observations may suggest that Src kinases are involved in deciphering the nature of the signal. To test the contribution of Lck, the major Src kinase in T cells, in the regulation of signaling dynamics, we suppressed its expression by RNAi in Jurkat T cells and evaluated the effects on Erk activation. Figure 5A shows that cells expressing low amount of Lck displayed prolonged Erk1/2 activation. These observations are in line with previous studies showing that knockdown of Lck in Jurkat and primary human T cells prolonged Erk phosphorylation and transcriptional activation [22, 23].

Lck phosphorylation correlates with decreased T-cell activation. A) Jurkat T cells were transfected with Lck siRNA duplex or siRNA control (Ctrl) and cultured for 48 h. Subsequently, cells were stimulated with soluble CD3 mAbs (clone OKT3) for the indicated times. Cell lysates were analyzed by immunoblotting using the indicated Abs. Immunoblot verifying Lck downregulation is shown. One representative experiment of three independent experiments is shown. B)-D) Purified human CD4 + T cells were treated with immobilized (iAbs) CD3xCD28 mAbs in the presence or absence of cross-linked CD4 mAb as indicated. B) The phosphorylation levels of Erk1/2 and Src kinases were determined by Western blotting. The phospho-specific bands were quantified using the 1D ImageQuant software and the values were normalized to the corresponding β-actin signal. Data on the graph represent the mean of the phosphorylation levels shown as arbitrary units ± SEM of 3 independent experiments. C) 24h after stimulation, the activation of CD4 + T cells was analyzed by staining with CD69 and flow cytometry. Data on the graph represent the mean of the expression levels shown as arbitrary units ± SEM of 3 independent experiments. D) CD4 + T cells were labeled with CFSE and stimulated as indicated. Proliferation was assed after 72h by analyzing CFSE content on a LSRFortessa. One representative experiment of 3 independent experiments is shown.

We next decided to investigate whether strong phosphorylation of Lck and Fyn may convert a sustained into a transient signal. To this aim, CD4 + primary human T cells were stimulated with iAbs for a short time period and subsequently CD4 was cross-linked using soluble anti-CD4 mAbs. It is known that CD4 crosslinking results in trans-phosphorylation of Lck, thus strongly enhancing its activity. As presented in Figure 5B, CD4 crosslinking indeed resulted in a strong induction of Lck phosphorylation measured using an anti-pY 416 Src antibody. Most importantly, enhanced Lck phosphorylation paralleled with a significant reduction in Erk phosphorylation (Figure 5B). Accordingly, we found that also CD69 expression and proliferation were strongly reduced upon CD4 crosslinking (Figure 5C, D). These data suggest that strong Src-family kinase activity may result in the activation of inhibitory signals suppressing T-cell activation.

In summary, we have shown that stimulation with iAbs induces different feedback regulation than sAbs treatment (Figure 6). sAbs lead to strong and rapid activation of Src kinases and subsequently to the phosphorylation of inhibitory molecules (e.g. c-Cbl, Dok2), which terminate signaling. On the other hand, iAbs induce only slight increase in kinase activity and an Erk-Lck positive feedback loop, which may be required to prevent rapid Lck dephosphorylation by SHP-1 or other phosphatases, and therefore lead to sustained activation.

Feedback regulation of TCR-mediated signaling. sAbs stimulation triggers strong phosphorylation of Src kinases, such as Lck, and leads to strong activation of downstream signaling pathways. In addition to the activation of positive regulators, sAbs also induce inhibitory molecules (c-Cbl, Dok2), which might imbalance TCR-mediated signaling, thus rapidly terminating T-cell activation (left side). On the other hand, iAbs stimulation results in the activation of an Erk-Lck positive feedback loop, which is required to sustain signaling (right side).


Discussion

Assignment of the separated AEC fractions

Based on the protein determination by SDS-PAGE and mass spectrometry the AEC fractions 2 to 4 could be assigned to PSII and PSI. Although Fraction 1 contained protein subunits of PSII, the low amounts of protein, but high concentrations of pigments, in this fraction makes it unlikely that Fraction 1 consists of specific pigment protein complexes. The dominance of PSII protein subunits in Fraction 2 argues for the presence of a high amount of PSII core complexes in this fraction. Fraction 3 contained protein subunits of both PSII and PSI and seems to represent a fraction with a mixed population of PSII and PSI core complexes. Fraction 4, on the other hand, was characterized by a lower number of PSII proteins compared to Fractions 2 and 3 but still contained the three PSI subunits which were typically observed in the present study. This argues for a higher concentration of PSI core complexes in Fraction 4. Fraction 5, which represented the main peak of the AEC chromatogram, was characterized by a strong enrichment of FCP proteins and thus most likely represents the peripheral FCP complexes of T. pseudonana. The enrichment of PSII core complexes in Fraction 2 and PSI in Fraction 4 was in line with the spectroscopic characterization of these fractions. Fraction 2 contained Chl a molecules absorbing at shorter wavelengths in the red part of the spectrum which are typical for PSII. Fraction 4, on the other hand, was characterized by the presence of longer-wavelength absorbing and fluorescence emitting Chl a molecules typical for PSI. Fraction 5 contained Chl a molecules which were absorbing at shorter wavelengths in the red part of the spectrum which is in line with the presence of FCP complexes in this fraction. The high fluorescence emission of Fraction 5 in the long-wavelength region is most likely caused by a strong aggregation of the FCPs by the high salt concentration needed for elution like in the experiments of Schaller et al. [30] who used Mg 2+ ions to aggregate the FCP complexes. In some cases a short wavelength emission was observed for Fraction 5. In this case it is reasonable to believe that the FCP complexes in Fraction 5 showed a weaker aggregation. Differences in the aggregation state of the FCP complexes in Fraction 5 may have been caused by slight differences in the solubilisation conditions of the thylakoid membranes, which, in general, could not be isolated with such a high reproducibility as e.g. spinach thylakoids. Fraction 5 showed high Fx per Chl a and Fx per DD ratios which is typical for FCP complexes with a primary light-harvesting function. Although Fraction 5 contained the largest part of the FCP complexes of T. pseudonana, FCP complexes were also present in Fractions 2 to 4. However, the higher β-carotene concentrations of Fractions 2 to 4 indicate that the PSI and PSII core complexes and not the FCP complexes were enriched in these fractions. The presence of FCP complexes in the isolated PSII core complexes observed in the present study is in line with studies of Nagao et al. [26, 28]. In these studies thylakoid membranes of the centric diatom C. gracilis were solubilized with Triton X-100 and oxygen-evolving PSII core complexes were isolated by differential centrifugation [26]. These FCP containing PSII preparations could then be further purified by anion exchange chromatography [28]. Like in our present AEC separation Ikeda et al. [17, 18] isolated PSI core complexes with associated FCP complexes from the centric diatoms C. gracilis and T. pseudonana with the help of sucrose gradient centrifugation and size exclusion chromatography [17] or sucrose gradient centrifugation in combination with AEC [18]. The isolation procedures led to the purification of PSI core complexes with two different FCP complexes which were termed FCPI-1 and FCPI-2. FCPI-2 seems to be tightly associated with the PSI core complex while FCPI-1 is lost after a more severe detergent treatment. Ikeda et al. [18] proposed that the FCPI-2 complex mediates the excitation energy transfer between the more peripheral FCPI-1 and the PSI core.

According to the recent data of Gundermann et al. [16] who purified the FCP complexes of the centric diatom C. meneghiniana with a combination of AEC and sucrose density gradient centrifugation it is possible that during the AEC separation described in the present study a co-elution of FCPa complexes and PSII and PSI core complexes has taken place. The AEC elution profile presented by Gundermann et al. [16] shows a pronounced peak at high salt concentrations which has been assigned to the FCPb complex. Additional smaller peaks were eluted from the column at lower salt concentrations and have been characterized as different subtypes of the so called FCPa complexes. While the FCPb peak at high salt concentrations most likely corresponds to Fraction 5 of the present AEC separation the smaller FCPa peaks exhibit retention times which are comparable with the retention times of the PSII and PSI fractions, i.e. Fractions 2 to 4, of our protein separation. The possible co-elution of the FCPa complexes and the PSII/PSI fractions makes it difficult to decide if the FCPs found in the present PSII and PSI fractions represent antenna proteins which are tightly associated with the photosystem core complexes or if these proteins are subunits of the different FCPa complexes of T. pseudonana.

Separation of the pigment protein complexes of T. pseudonana with the method presented in this study was compared to the separation of the photosynthetic pigment proteins of spinach (Additional file 2A). Separation of the spinach pigment proteins led to the appearance of one major and several minor peaks. The major peak, which eluted at 12–15 mL, showed pronounced Chl a and Chl b maxima in the blue and red part of the spectrum and could be unequivocally assigned to the LHCII (Additional file 3). The short retention time of the LHCII indicates that the major light-harvesting complex of higher plants exhibits a rather low negative net charge and thus could be eluted from the AEC column with low salt concentrations. The peripheral FCP of T. pseudonana, on the other hand, was characterized by the longest retention time of the separated diatom pigment protein complexes and only eluted at high concentrations of NaCl, which indicates a high negative charge of the FCP complexes. Comparing the LHCII and the peripheral FCP complexes it is possible that the exposed regions of the proteins, which interact with the positively charged matrix of the AEC column, show differences in their negative charge. It is also possible that differences in the oligomerization state of the light-harvesting complexes lead to the different negative net charges and thus the different retention times. The LHCII of higher plants is usually isolated as trimeric LHCII. Higher oligomeric states of FCP complexes are typical for the centric diatoms like C. meneghiniana or the diatom used in the present study, T. pseudonana. In these algae the FCPb complexes, which represent the last protein fraction in the purification of FCP complexes by AEC [16], and thus are comparable to Fraction 5 of the present separation, seem to be composed of FCP nonamers while FCPa complexes show a trimeric structure [3, 13, 27]. The increased negative surface charge of FCPs may be seen in conjunction with the high concentration of the negatively charged lipid SQDG in the thylakoid membranes of diatoms [22]. Pronounced repulsion between FCPs and SQDG may lead to the separation of PSI into SQDG enriched outer thylakoid membrane regions and PSII and the peripheral FCP into the inner membrane lamellae composed of mainly MGDG. Such a separation of the photosystem has been proposed by Lepetit et al. [22] and has recently been supported by the data of Bina et al. [4] and Flori et al. [10].

Applicability of the present AEC separation method

The AEC method presented in this study allows the pre-purification of PSI and PSII core and FCP complexes of the centric diatom T. pseudonana. It was also tested for the separation of the pigment proteins of the well-characterized centric diatom C. meneghiniana. These analyses yielded a comparable fractionation of the solubilized thylakoid membranes (see Additional file 2B). The isolated pigment protein complexes can serve as starting material for the final purification of the respective complexes using a separate protein purification method such as size exclusion chromatography or sucrose density gradient centrifugation. The partial purification, i.e. the isolation of PSI and PSII core complex fractions which contain FCP complexes, makes it possible to differentiate between FCP complexes which are more closely associated with the PSI and PSII core complexes and FCP complexes which build-up the peripheral antenna complexes, providing that the occurrence of FCPs in the PSI and PSII fractions does not represent a co-elution of FCP-A complexes and PS core complexes.

Heterogeneity of FCPs

Fraction 5 of the present AEC separation contained the peripheral FCP complexes which were not associated with the PSI and PSII core complexes. The peripheral FCP complexes were dominated by the presence of the 21 kDa protein band and the 18 kDa FCP band was only detected in low concentration. According to the analysis by mass spectrometry the 21 kDa band was exclusively composed of both the Lhcf8 and Lhcf9 proteins. Interestingly, Lhcf8 and Lhcf9 were also found in the 18 kDa band but, based on the data of analysis 1 of the present study, not in all fractions of the AEC. Additional Lhc proteins were not detected in the peripheral FCP. According to the AEC separation presented by Gundermann et al. [16] Fraction 5 corresponds to the FCPb complexes of C. meneghiniana. Gundermann et al. [16] detected Fcp5/Lhcf3 as the most prominent Lhc protein in the FCPb complexes. Lhcf3 was accompanied by low concentrations of Lhcf1 and Lhcf4/Lhcf6. The latter, however, was only found in the FCPb2 complex. Taking into account that in T. pseudonana the similar Lhcf3, Lhcf8 and Lhcf9 genes code for an identical protein, the data of the present study on the protein composition of the peripheral FCP complexes are in agreement with the data of Gundermann et al. [16] concerning the FCPb. In addition to the Lhcf proteins, Gundermann et al. [16] observed the presence of the Lhcx1 and Lhcx6_1 proteins in the FCPb which was purified from high light grown cultures. These proteins were not detected in the present study as components of the peripheral FCP. The occurrence of the Lhcf8 protein in the 21 kDa FCP band is in line with the data published by Nagao et al. [27] who detected the Lhcf8 protein in the 21 kDa bands of the oligomeric and trimeric FCP of T. pseudonana. The Lhcf9 protein, which was detected as an additional component of the 21 kDa band in the present study, was not observed by Nagao et al. [27]. However, this protein is likely identical to the Lhcf8 gene product. The oligomeric FCP of T. pseudonana, which was purified by Nagao et al. [27] by clear-native PAGE, was characterized by the single presence of the 21 kDa FCP band. It is thus comparable to the peripheral FCP complexes isolated in the present study which were also dominated by the 21 kDa band. The findings of the present study are also in line with recent observations by Calvaruso et al. [6] that the peripheral FCPb complex of T. pseudonana consists of the Lhcf8/Lhcf9 proteins. Like the 21 kDa band of Fraction 5 the 21 kDa bands of the other AEC fractions contained only Lhcf8 and Lhcf9. Like the 21 kDa band the 18 kDa band showed a comparable protein composition in the different fractions with the exception of Lhcf8 and Lhcf9 which, according to analysis 1, were only found in the 18 kDa band of Fractions 2 and 5. Lhcf1, Lhcf2, Lhcf5, and Lhcf6 on the other hand, were found in all fractions and thus seem to represent the main Lhcf proteins of the 18 kDa band. While in the first MS analysis performed in the present study Lhcf4 was only detected in the 18 kDa band of Fraction 2, the second MS analysis indicated the presence of the Lhcf4 protein in the 18 kDa band of Fractions 2 to 4. The presence of Lhcf5 in the 18 kDa FCP band is in line with the data of Nagao et al. [27] who observed two FCP bands in the 18 kDa region of trimeric FCP complexes of T. pseudonana. According to their mass spectrometric analysis the major 18 kDa band contained Lhcf5 and additionally Lhcf1 and Lhcf4. In the present study both Lhcf1 and Lhcf4 were also detected in the 18 kDa band of Fractions 2 to 4. The minor 18 kDa band in the study of Nagao et al. [27] was characterized by the additional presence of Lhcf6, Lhcf7, and Lhcf11. Lhcf6 represented a component of Fractions 2 to 4 of the present AEC separation, whereas Lhcf7 was only detected in the second analysis by mass spectrometry and only occurred in Fraction 2. Lhcf11, however, was not observed in the respective fractions of the present study. The absence of Lhcf11 is most probably explained by the fact that in the present protein separations by SDS-PAGE a minor 18 kDa FCP band was not resolved. The presence of Lhcf4 in the 18 kDA band of Fraction 2, which seems to be enriched in PSII, is in line with the recent isolation of a PSII-FCP supercomplex of C. gracilis [35]. Based on the structural data it was proposed that the FCP-E monomer, which is rather tightly associated with the PSII core complex and mediates the interaction of one of the FCP-A tetramers with the core, represents an Lhcf4-like subunit. Interestingly, the FCP-D monomer, which is also involved in the interaction of the peripheral FCP-A with the PSII core complex, seems to be an Lhca-related LHC protein.

Taking into account that a co-elution of the PSII and PSI core complexes and the FCPa complexes might have taken place in the AEC separation of the present study a comparison to the protein composition of the different FCPa complexes published by Gundermann et al. [16] seems valuable. In the present study Lhcf1, Lhcf2, Lhcf5, and Lhcf6, with an additional presence of Lhcf8 and Lhcf9, seemed to represent the main proteins of AEC fractions 2 to 4, which would correspond to the different FCPa complexes. Gundermann et al. [16] observed that the FCPa complexes of C. meneghiniana were dominated by the Lhcf1, Lhcf4/Lhcf6 and Lhcf3 proteins. According to their analysis by mass spectrometry, FCPa1, FCPa3 and FCPa4 contain Lhcf1 as the main Lhcf protein whereas FCPa2 is characterized by a high concentration of Lhcf4/Lhcf6.

Additional FCP proteins that were detected by Gundermann et al. [16] as constituents of the FCPa complexes were the Lhcx1 and Lhcx6_1 protein. In the present study four different Lhcx proteins were found, namely Lhcx1, Lhcx2, Lhcx5 and Lhcx6_1. The Lhcx1, Lhcx2 and Lhcx5 proteins were observed in Fraction 2 of the AEC separation, which according to its protein composition, represents a fraction enriched in PSII core complexes. Lhcx6_1 was a constituent of Fraction 1 which, due to its high concentration of DD, Dt and Fx, has to be regarded as a mixed protein and free pigment fraction.

Fractions 3 and 4 did not contain Lhcx proteins but, in addition to the Lhcf proteins, were characterized by the presence of two Lhcr proteins in each fraction. While Fraction 3 contained Lhcr3 and Lhcr14, Lhcr1 and Lhcr3 were found in Fraction 4. In addition, Lhcr3 was detected in Fraction 2. The presence of Lhcr proteins in the AEC fractions containing PSI core complex proteins is in line with data from the literature which describe the Lhcr proteins as PSI-specific antenna proteins [13, 18]. While in the present study Lhcr1, Lhcr3 and Lhcr14 were detected, Grouneva et al. [13] observed Lhcr1, Lhcr3, Lhcr4, Lhcr7, Lhcr10, Lhcr11 and Lhcr14 in their analysis of the thylakoid proteome of T. pseudonana. The analysis of PSI-FCPI complexes of T. pseudonana isolated by a combination of sucrose gradient centrifugation and AEC [18] showed the presence of Lhcr1, Lhcr3, Lhcr4, Lhcr10, Lhcr13 and Lhcr14 as PSI antenna proteins.

Assignment of other important proteins

Fraction 2 of the present AEC separation, which is enriched in PSII core complexes, contains another interesting protein, namely the DD de-epoxidase (DDE). DDE is the enzyme which catalyses the forward reaction of the xanthophyll cycle of diatoms, the de-epoxidation of DD to Dt (for a review on xanthophyll cycles and NPQ see [11]). Dt is one of the components responsible for the process of NPQ. Another important factor for NPQ is the presence of Lhcx proteins which also occur in the PSII-containing Fraction 2 of the AEC gradient. Dt and Lhcx proteins are thought to play a role in the quenching site Q2 which is located in the vicinity of the PSII core complex and, together with quenching site Q1, provides protection of PSII against damages caused by excessive excitation energy.


Materials and Methods

Strains

An myc epitope was introduced at the NH2 terminus of mature Emp24p. The myc-tagged emp24-E178A mutant was constructed by substituting an appropriate fragment of EMP24 with the sequenced mutant version obtained by PCR techniques. RH4443 (MATα, ura3, leu2, his4, bar1, emp24:: KanMx) was obtained by replacing the entire EMP24 coding sequence of RH1959 (MATα, ura3, leu2, his4, bar1) with a KanMx cassette. EMP24 alleles were cloned into a YCplac111 (CEN/ARS) plasmid. The S. cerevisiae strain RH696-2B (MATα, sec18-20 gap1Δ::LEU2 ura3 ade2 leu2 lys2Δ201 pPL269) was obtained by crossing PLY129 (pPL269) (MATα gap1Δ::LEU2 ura3 ade2 leu2 lys2Δ201 pPL269) (Kuehn et al. 1996), with RH478 (MATα sec18-20 leu2 his4). Cytosols were prepared from RH732 (MATα his4 leu2 ura3 lys2 pep4::URA3 bar1) and RH2043 (MATα sec18-20 his4 leu2 ura3 pep4::URA3 bar1).

Protein Techniques

Protein levels were determined by extraction of log phase cultures and Western blotting (Sütterlin et al. 1997) using antibodies raised against the Emp24p cytosolic tail or the myc epitope. Analysis of Gas1p transport in vivo was by a pulse-chase protocol (Sütterlin et al. 1997). Gas1p maturation was analyzed using a 4-min pulse and subsequent chase at 30°C, followed by immunoprecipitation, SDS-PAGE, and fluorography. Immature (105 kD) and mature (125 kD) forms of Gas1p were quantified using a densitometer. The percentage of mature Gas1p was used as an indication of transport to the Golgi apparatus. Determination of partitioning of Gas1p between aqueous and detergent phases of Triton X-114 was performed as described (Nuoffer et al. 1993), except that the media fractions were not analyzed. After separation into detergent and aqueous phases, Gas1p was denatured, immunoprecipitated, resolved by SDS-PAGE, visualized, and quantified using a PhosphorImager.

In Vitro ER-budding Assay

The permeabilized, cell-based, ER cargo packaging assay was performed as described (Kuehn et al. 1996) with a few modifications. Strains RH1959, RH4438, and RH696-2B were transformed with pCNYG1 to overexpress Gas1p (approximately fivefold) (Nuoffer et al. 1991). Overexpression did not affect Gas1p transport kinetics or dependence on EMP24. 10 mM 2-mercaptoethanesulfonic acid replaced 10 mM dithiothreitol. Budding reactions contained 30 μl of membranes (from 12 × 10 7 cells), 300 μg crude cytosol, 3 μg Sar1p, 1× ATP mix, and 0.2 mM GTP in a 150-μl volume. After 2 h at 25°C, a portion of the sample was removed for analysis (total), the remaining aliquot was sedimented (14,000 rpm, 2 min, 4°C), and 125 μl of the supernatant collected and subjected to flotation on a Nycodenz ® step gradient (Barlowe et al. 1994). 100 μl from the top was discarded and the next 800 μl transferred to a new tube. 400 μl was diluted threefold with B88 and centrifuged (100,000 g, 1 h, 4°C). The membrane pellet was dissolved in 1% SDS in TEPI buffer (Sütterlin et al. 1997) for 10 min at 55°C, subjected to immunoprecipitation, and analyzed by SDS-PAGE with subsequent exposure and quantitation using a PhosphorImager.

Vesicle Immunoisolation

Vesicles were produced in a budding reaction using sec18 membranes and cytosol. The sec18 membranes were prepared as above, except that the last 5 min of depletion and the pulse-labeling were at 32°C. The sec18 cytosol was preincubated (32°C, 10 min) before use. Vesicles were immunoisolated with or without Emp24p anti-tail antibody and processed according to Kuehn et al. 1996.

Cross-Linking

Nycodenz-purified vesicles produced in a budding reaction were adjusted to 2.5 M urea in B88 and incubated with 1 mM dithiobis(succinimidylpropionate) (DSP) or various amounts of disuccinimidyl glutarate (DSG Pierce) (20°C, 20 min). The cross-linking reaction was quenched by addition of glycine (50 mM final, 5 min, 20°C). Vesicles were sedimented at 100,000 g (1 h, 4°C), dissolved with 1% SDS in TEPI (5 min, 95°C for Gas1p and glycosylated pro α factor [gpαF], or 55°C for Gap1p), and immunoprecipitated with Emp24p anti-tail antibody or Erv25 antibody (Belden and Barlowe 1996) and protein A–Sepharose. Precipitated material was eluted from the Sepharose beads by incubation with 1% SDS in TEPI (5 min, 95 or 55°C) and reimmunoprecipitated with anti-Gas1p or anti-Gap1p antibody.


The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Keywords : bacteriophage, T4, NMR, cryo-EM, Mrh, Cef, Y04L and Gp57B

Citation: Zhang K, Li X, Wang Z, Li G, Ma B, Chen H, Li N, Yang H, Wang Y and Liu B (2021) Systemic Expression, Purification, and Initial Structural Characterization of Bacteriophage T4 Proteins Without Known Structure Homologs. Front. Microbiol. 12:674415. doi: 10.3389/fmicb.2021.674415

Received: 01 March 2021 Accepted: 22 March 2021
Published: 13 April 2021.

Shuai Le, Army Medical University, China

Jianfeng Yu, Shenzhen University, China
Nan Hou, Chinese Academy of Medical Sciences, China

Copyright © 2021 Zhang, Li, Wang, Li, Ma, Chen, Li, Yang, Wang and Liu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.



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