Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.25.1 (proteasome)
28,817 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A cytoplasmic peptide:N-glycanase has been implicated in the proteasomal degradation of newly synthesized misfolded glycoproteins that are exported from the endoplasmic reticulum to the cytosol. Recently, the gene encoding this enzyme (Png1p) was identified in yeast and shown to bind to the 26S proteasome through its interaction with a component of the DNA repair system, Rad23p. Moreover, a mouse homologue of Png1p (mPng1p), which has an extended N-terminal domain, was found to bind not only to the Rad23 protein, but also to various proteins related to the ubiquitin/proteasome pathway. An extended N-terminus of mPng1p, which is not found in yeast, contains a potential site of protein-protein interaction called the PUB/PUG domain. The PUB/PUG domain is predicted to be helix-rich and is found in various proteins that may be involved in the ubiquitin/proteasome-related pathway. This review will discuss the consequence of the deglycosylation reaction by peptide:N-glycanase in cellular processes. In addition, the potential importance of the PUB/PUG domain for the formation of a putative "glycoprotein-degradation complex" will be discussed.
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PMID:Hypothesis: a glycoprotein-degradation complex formed by protein-protein interaction involves cytoplasmic peptide:N-glycanase. 1259 38

Successful maturation determines the intracellular fate of secretory and membrane proteins in the endoplasmic reticulum (ER). Failure of proteins to fold or assemble properly can lead to their retention in the ER and redirects them to the cytosol for degradation by the proteasome. Proteasome inhibitors can yield deglycosylated cytoplasmic intermediates that are the result of an N-glycanase activity, believed to act prior to destruction of these substrates by the proteasome. A gene encoding a yeast peptide:N-glycanase, PNG1, has been cloned, but this N-glycanase and its mammalian homolog were reported to be incapable of deglycosylating full-length glycoproteins. We show that both the yeast PNG1 enzyme and its mammalian homolog display N-glycanase activity towards intact glycoproteins. As substrates, cytosolic PNGase activity prefers proteins containing high-mannose over those bearing complex type oligosaccharides. Importantly, PNG1 discriminates between non-native and folded glycoproteins, consistent with a role for N-glycanase in cytoplasmic turnover of glycoproteins.
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PMID:A role for N-glycanase in the cytosolic turnover of glycoproteins. 1260 69

In species as diverse as yeast and mammals, peptide:N-glycanase (PNG1 in yeast; Ngly1 in mouse) is believed to play a key role in the degradation of misfolded glycoproteins by the proteasome. In this study, we report the genomic organization and mRNA distribution of the mouse Ngly1. Mouse Ngly1 spans 61kb and is composed of 12 exons, the organization of which is conserved throughout vertebrates. Comparison of the mouse and human genomic sequence identifies a conserved gene structure with significant sequence similarity extending into introns. A 2.6kb Ngly1 message was detected in all mouse tissues examined, with the highest abundance in the testis. In addition, a lower molecular weight transcript of 2.4kb was detected in the testis. From analysis of dbESTs the alternative transcript of Ngly1 is predicted to be present in the human placenta. Given the key role Ngly1 plays in glycoprotein degradation, we predict that Ngly1 may be a contributing factor in "disease" susceptibility. To begin to address this question, we used radiation hybrid mapping to localize mouse Ngly1 to chromosome 14 and the human orthologue to chromosome 3 with a strong link with known genes.
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PMID:Ngly1, a mouse gene encoding a deglycosylating enzyme implicated in proteasomal degradation: expression, genomic organization, and chromosomal mapping. 1271 18

Recently, the role of N-linked glycans in the process of ERAD (endoplasmic reticulum-associated degradation) of proteins has been widely recognized. In the present study, we attempted to delineate further the sequence of events leading from a fully glycosylated soluble protein to its deglycosylated form. Degradation intermediates of a truncated form of ribophorin I, namely RI(332), which contains a single N-linked oligosaccharide and is a substrate for the ERAD/ubiquitin-proteasome pathway, were characterized in HeLa cells under conditions blocking proteasomal degradation. The action of a deoxymannojirimycin- and kifunensine-sensitive alpha1,2-mannosidase was shown here to be required for both further glycan processing and progression of RI(332) in the ERAD pathway. In a first step, the Man(8) isomer B, generated by ER mannosidase I, appears to be the major oligomannoside structure associated with RI(332) intermediates. Some other trimmed N-glycan species, in particular Glc(1)Man(7)GlcNAc(2), were also found on the protein, indicating that several mannosidases might be implicated in the initial trimming of the oligomannoside. Secondly, another intermediate of degradation of RI(332) accumulated after proteasome inhibition. We demonstrated that this completely deglycosylated form arose from the action of an N-glycanase closely linked to the ER membrane. Indeed, the deglycosylated form of the protein remained membrane-associated, while being accessible from the cytoplasm to ubiquitinating enzymes and to added protease. Our results indicate that deglycosylation of a soluble ERAD substrate glycoprotein occurs in at least two distinct steps and is coupled with the retro-translocation of the protein preceding its proteasomal degradation.
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PMID:Processing of N-linked glycans during endoplasmic-reticulum-associated degradation of a short-lived variant of ribophorin I. 1295 21

Peptide:N-glycanase (PNGase) has been proposed to participate in the proteasome-dependent glycoprotein degradation pathway. The finding that yeast PNGase interacts with the 19S proteasome subunit through the protein Rad23 supports this hypothesis. In this report, we have used immunofluorescence, subcellular fractionation, coimmunoprecipitation, and in vitro GST pull-down techniques for detecting intracellular localization and interactions of PNGase, HR23B, and S4 by using human (h) and mouse (m) homologs. Immunofluorescence studies revealed that hPNGase, hHR23B, and hS4 are present in close proximity to the endoplasmic reticulum (ER) when calnexin was used as an ER marker in HeLa cells. Subcellular fractionation suggests not only cytoplasmic but also ER association of hPNGase in HeLa cells. Immunoprecipitation analysis revealed the interaction of h/mPNGase with the 19S proteasome subunit, hS4, through hHR23B. Using an in vitro GST pull-down assay, we also have shown that recombinant mPNGase requires its N terminus and middle domain for interaction with mHR23B. Finally, using misfolded yeast carboxypeptidase Y and chicken ovalbumin as glycoprotein substrates, we have established that mHR23B acts as a receptor for deglycosylated proteins. Based on this finding, we propose that after deglycosylation of misfolded glycoproteins by PNGase, the aglyco forms of these proteins are recognized by HR23B and targeted for degradation.
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PMID:A complex between peptide:N-glycanase and two proteasome-linked proteins suggests a mechanism for the degradation of misfolded glycoproteins. 1535 61

A widely expressed protein containing UBA (ubiquitin-associated) and UBX (ubiquitin-like) domains was identified as a substrate of SAPKs (stress-activated protein kinases). Termed SAKS1 (SAPK substrate-1), it was phosphorylated efficiently at Ser200 in vitro by SAPK3/p38gamma, SAPK4/p38delta and JNK (c-Jun N-terminal kinase), but weakly by SAPK2a/p38alpha, SAPK2b/p38beta2 or ERK (extracellular-signal-regulated kinase) 2. Ser200, situated immediately N-terminal to the UBX domain, became phosphorylated in HEK-293 (human embryonic kidney) cells in response to stressors. Phosphorylation was not prevented by SB 203580 (an inhibitor of SAPK2a/p38alpha and SAPK2b/p38beta2) and/or PD 184352 (which inhibits the activation of ERK1 and ERK2), and was similar in fibroblasts lacking both SAPK3/p38gamma and SAPK4/p38delta or JNK1 and JNK2. SAKS1 bound ubiquitin tetramers and VCP (valosin-containing protein) in vitro via the UBA and UBX domains respectively. The amount of VCP in cell extracts that bound to immobilized GST (glutathione S-transferase)-SAKS1 was enhanced by elevating the level of polyubiquitinated proteins, while SAKS1 and VCP in extracts were coimmunoprecipitated with an antibody raised against S5a, a component of the 19 S proteasomal subunit that binds polyubiquitinated proteins. PNGase (peptide N-glycanase) formed a 1:1 complex with VCP and, for this reason, also bound to immobilized GST-SAKS1. We suggest that SAKS1 may be an adaptor that directs VCP to polyubiquitinated proteins, and PNGase to misfolded glycoproteins, facilitating their destruction by the proteasome.
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PMID:A novel UBA and UBX domain protein that binds polyubiquitin and VCP and is a substrate for SAPKs. 1536 74

Peptide:N-glycanase (PNGase) is ostensibly the sole enzyme responsible for deglycosylation of unfolded N-linked glycoproteins dislocated from the ER to the cytosol. Here we show the pan-caspase inhibitor, Z-VAD-fmk, to be an active site-directed irreversible inhibitor of yeast and mammalian PNGase at concentrations below those used to inhibit caspases in vivo. Through chemical synthesis we determined that the P1 residue, electrophile position, and leaving group are important structural parameters for PNGase inhibition. We show that Z-VAD-fmk inhibits PNGase in living cells and that degradation of class I MHC heavy chains and TCRalpha, in an identical cellular setting, is markedly different. Remarkably, proteasome-mediated turnover of class I MHC heavy chains proceeds even when PNGase is completely inhibited, suggesting that the function of PNGase may be to facilitate more efficient proteasomal proteolysis of N-linked glycoproteins through glycan removal.
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PMID:Using a small molecule inhibitor of peptide: N-glycanase to probe its role in glycoprotein turnover. 1561 Aug 52

The lysosomal catabolism of glycoproteins is part of the normal turnover of cellular constituents and the cellular homeostasis of glycosylation. Glycoproteins are delivered to lysosomes for catabolism either by endocytosis from outside the cell or by autophagy within the cell. Once inside the lysosome, glycoproteins are broken down by a combination of proteases and glycosidases, with the characteristic properties of soluble lysosomal hydrolases. The proteases consist of a mixture of endopeptidases and exopeptidases, which act in concert to produce a mixture of amino acids and dipeptides, which are transported across the lysosomal membrane into the cytosol by a combination of diffusion and carrier-mediated transport. Although the glycans of all mature glycoproteins are probably degraded in lysosomes, the breakdown of N-linked glycans has been studied most intensively. The catabolic pathways for high-mannose, hybrid, and complex glycans have been established. They are bidirectional with concurrent sequential removal of monosaccharides from the nonreducing end by exoglycosidases and proteolysis and digestion of the carbohydrate-polypeptide linkage at the reducing end. The process is initiated by the removal of any core and peripheral fucose, which is a prerequisite for the action of the peptide N-glycanase aspartylglucosaminidase, which hydrolyzes the glycan-peptide bond. This enzyme also requires free alpha carboxyl and amino groups on the asparagine residue, implying extensive prior proteolysis. The catabolism of O-linked glycans has not been studied so intensively, but many lysosomal glycosidases appear to act on the same linkages whether they are in N- or O-linked glycans, glycosaminoglycans, or glycolipids. The monosaccharides liberated during the breakdown of N- and O-linked glycans are transported across the lysosomal membrane into the cytosol by a combination of diffusion and carrier-mediated transport. Defects in these pathways lead to lysosomal storage diseases. The structures of some of the oligosaccharides that accumulate in these diseases are not digestion intermediates in the lysosomal catabolic pathways but correspond to intermediates in the biosynthetic pathway for N-linked glycans, suggesting another route of delivery of glycans to the lysosome. Incorrectly folded or glycosylated proteins that are rejected by the quality control mechanism are broken down in the ER and cytoplasm and the end product of the cytosolic degradation of N-glycans is delivered to the lysosomes. This route is enhanced in cells actively secreting glycoproteins or producing increased amounts of aberrant glycoproteins. Thus interaction between the lysosome and proteasome is important for the regulation of the biosynthesis and distribution of N-linked glycoproteins. Another example of the extralysosomal function of lysosomal enzymes is the release of lysosomal proteases into the cytosol to initiate the lysosomal pathway of apoptosis.
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PMID:Lysosomal metabolism of glycoproteins. 1564 14

A cytoplasmic peptide: N-glycanase (PNGase) has been implicated in the proteasomal degradation of aberrant glycoproteins synthesized in the endoplasmic reticulum. The reaction is believed to be important for subsequent proteolysis by the proteasome since bulky N-glycan chains on misfolded glycoproteins may impair their efficient entry into the interior of the cylinder-shaped 20S proteasome, where its active site resides. This cytoplasmic enzyme was first detected in 1993 by a simple, sensitive assay method using 14C-labeled glycopeptide as a substrate. The deglycosylation reaction by PNGase brings about two major changes on substrate the peptide; one is removal of the N-glycan chain and the other is the introduction of a negative charge into the core peptide by converting the glycosylated asparagine residue(s) into an aspartic acid residue(s). The assay method we developed monitors these major changes in the core peptide, and the respective changes were detected by distinct analytical methods: i.e., paper chromatography and paper electrophoresis. This chapter will describe the simple, sensitive in vitro assay method for PNGase.
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PMID:A simple, sensitive in vitro assay for cytoplasmic deglycosylation by peptide: N-glycanase. 1580 8

In eukaryotes, misfolded proteins must be distinguished from correctly folded proteins during folding and transport processes by quality control systems. Yeast peptide:N-glycanase (yPNGase) specifically deglycosylates the denatured form of N-linked glycoproteins in the cytoplasm and assists proteasome-mediated glycoprotein degradation by forming a complex with 26S proteasome through DNA repair protein, yRad23. Here, we describe the crystal structures of a yPNGase and XPC-binding domain of yRad23 (yRad23XBD, residues 238-309) complex and of a yPNGase-yRad23XBD complex bound to a caspase inhibitor, Z-VAD-fmk. yPNGase is formed with three domains, a core domain containing a Cys-His-Asp triad, a Zn-binding domain, and a Rad23-binding domain. Both N- and C-terminal helices of yPNGase interact with yRad23 through extensive hydrophobic interactions. The active site of yPNGase is located in a deep cleft that is formed with residues conserved in all PNGase members, and three sugar molecules are bound to this cleft. Complex structures in conjunction with mutational analyses revealed that the walls of the cleft block access to the active site of yPNGase by native glycoprotein, whereas the cleft is sufficiently wide to accommodate denatured glycoprotein, thus explaining the specificity of PNGase for denatured substrates.
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PMID:Structure of a peptide:N-glycanase-Rad23 complex: insight into the deglycosylation for denatured glycoproteins. 1596 83


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