z-VAD-fmk inhibits peptide:N-glycanase and may result in ER stress


After insertion into the endoplasmic reticulum (ER), many proteins are glycosylated and participate in chaperone-assisted folding by the lectins calnexin and calreticulin. A failure to degrade misfolded proteins, such as occurs in protein folding diseases or caused by small molecules that interfere with normal protein maturation in the ER (e.g. DTT, thapsigargin, tunicamycin), results in ER stress. Connections between ER stress and caspase activation have been observed in mouse and human cells for both caspase-4 and caspase-12. The list has recently been expanded to include the initiator caspase-2, and effector caspases-3, and -7, which are activated by depletion of Ca2þ stores in the ER. The steps involved in the overall degradation pathway(s) include dislocation of the misfolded glycoprotein from the ER to the cytosol, followed by deglycosylation, ubiquitination, and proteasomal proteolysis. Deglycosylation occurs in the cytosol and is carried out by peptide:N-glycanase (PNGase). This enzyme cleaves the b-aspartyl-glucosamine bond between the first N-acetylglucosamine (GlcNAc) of the glycan and the amide side chain of asparagine, converting the asparagine to an aspartate residue. PNGase has been shown to discriminate between folded and unfolded glycoproteins and to deglycosylate only unfolded glycoproteins. PNGases have conserved residues essential for function. These include a putative cysteine–histidine–aspartate catalytic triad and four additional cysteines, each in a C-X-X-C motif (Figure 1a). We recently identified the general caspase inhibitor, z-VAD-fmk (Figure 1b), as a potent inhibitor of yeast and mammalian PNGase. Complete inactivation of PNGase is afforded at concentrations lower than those typically used for caspase blockade. We found that more selective tetrapeptide fmk inhibitors also blocked PNGase in vitro. To better understand the crossreactivity of z-VAD-fmk and related inhibitors with caspases and PNGase we sought to determine the site(s) of modification of PNGase by z-VAD-fmk. Fluoromethylketones react with cysteine proteases like caspases to yield a covalent thioether adduct. Yeast PNGase (YPng1) contains 14 cysteines, five of which are absolutely conserved. Mutation of any of the putative active site residues – Cys191, His218 and Asp235 – or the other four conserved cysteines – Cys129, Cys132, Cys165 and Cys168 – results in loss of enzymatic activity. Although mutation of the proposed active site cysteine to alanine (C191A) indeed renders PNGase inactive, it is not clear whether inactivity is a consequence of eliminating the active site nucleophile or distortion of the active enzyme’s conformation. Conformational distortion is suggested by an altered circular dichroism spectrum for the mutant. To determine the site(s) of adduct formation between zVAD-fmk and YPng1, we used mass spectrometry. Following incubation of z-VAD-fmk (Sigma) with either wild-type YPng1 (WT) or the C191A mutant, the proteins were denatured, reduced and alkylated with iodoacetamide to afford carboxamidomethyl (CAM) modification of cysteine thiols. MALDITOF analysis shows a mass increase of 392 daltons (Da) (Figure 1c, left panel), for WT, consistent with the addition of a single z-VAD-fmk molecule per YPng1. The difference between the calculated mass increase for a 1 : 1 adduct (433 Da) and the observed increase (392 Da, 0.09% error) is the result of measurement on the full-length protein (B44 000 Da). Small differences (o0.1% error) are common at higher mass ranges. Under identical conditions, no mass increase was observed for the C191A mutant. To map the site(s) of modification, z-VAD-fmk-treated WT and C191A were digested with trypsin, chymotrypsin or endo Glu-C peptidases and analyzed by MALDI-MS. Tryptic digests revealed peptides of m/z 3043.4 and 2015.9 Da, corresponding to unmodified peptides containing Asp235 and His218, respectively (Figure 1d, left and center panels). We observe no evidence for z-VAD-fmk modification of these peptides. This result shows that putative catalytic triad residues, His218, and Asp235, are not modified by z-VADfmk. Additionally, we observed peptides mapping conserved cysteine residues Cys129 and Cys132, each of which was found only as a CAM adduct (Figure 1d, right panel). We also observed seven additional nonconserved cysteine residues, each of which was modified only with CAM. We did not observe any tryptic peptides containing Cys63, Cys195, or the conserved cysteine residues Cys165, Cys168, and Cys191. Digestion with chymotrypsin yielded peptides mapping 12 of 14 cysteines, with only Cys165 and Cys168 remaining unaccounted. Comparison of WT and C191A chymotryptic digests revealed the detection of a peptide, of m/z 2188.9 Da, unique to the WT digest (Figure 1e). This peptide contains Cys191 and Cys195, and the observed mass indicates modification by one each of z-VAD-mk and CAM. Peptides corresponding to these same peptide sequences but lacking z-VAD-mk modification were not observed. As we observe CAM modification of the other 10 cysteine residues that were mapped in the chymotryptic digest, and find no evidence for their modification by z-VAD-fmk, we concluded that the site(s) of WT modification by z-VAD-mk occurs at one or more of the Cell Death and Differentiation (2006) 13, 163–165 & 2006 Nature Publishing Group All rights reserved 1350-9047/06 $30.00

DOI: 10.1038/sj.cdd.4401716


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@article{Misaghi2006zVADfmkIP, title={z-VAD-fmk inhibits peptide:N-glycanase and may result in ER stress}, author={Shahram Misaghi and Gregory Alan Korbel and Benedikt M. Kessler and Eric Spooner and Hidde L. Ploegh}, journal={Cell Death and Differentiation}, year={2006}, volume={13}, pages={163-165} }