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N‐Glycosylated substrates that undergo ER‐associated degradation (ERAD) are often deglycosylated by the cytosolic peptide:N‐glycanase (Ngly1) during their proteasomal degradation in the cytosol. Consequently, the presence of non‐ or deglycosylated forms of such proteins after treatment with proteasome inhibitors is widely used as evidence for cytosolic deglycosylation by Ngly1. However, in this study, the accumulation of nonglycosylated RTA∆m, a model ERAD substrate, was still observed in mouse cells lacking cytosolic de‐N‐glycosylating enzymes, when treated with proteasome inhibitors. It was found that RTA∆m is normally partially N‐glycosylated, while the nonglycosylated form is rapidly degraded by proteasomes in the cytosol. Our results suggest that the occurrence of 'nonglycosylated' ERAD substrates upon treatment with proteasome inhibitors is not necessarily a clue for cytosolic deglycosylation. [ABSTRACT FROM AUTHOR]

Abstract: PUB domains are identified in several proteins functioning in the ubiquitin (Ub)–proteasome system and considered as p97-binding modules. To address the further functional roles of these domains, we herein characterized the interactions of the PUB domain of peptide:N-glycanase (PNGase) with Ub and Ub-like domain (UBL) of the proteasome shuttle factor HR23. NMR data indicated that PNGase-PUB exerts an acceptor preferentially for HR23–UBL, electrostatically interacting with the UBL surface employed for binding to other Ub/UBL motifs. Our findings imply that PNGase-PUB serves not only as p97-binding module but also as a possible activator of HR23 in endoplasmic reticulum-associated degradation mechanisms. Structured summary of protein interactions: PNGase binds to HR23A by affinity chromatography technology (View interaction) PNGase and HR23A bind by nuclear magnetic resonance (View interaction) PNGase and HR23B bind by nuclear magnetic resonance (View interaction) [Copyright &y& Elsevier]

Abstract: Peptide:N-glycanase (PNGase) is a deglycosylating enzyme that catalyzes the hydrolysis of the β-aspartylglycosylamine bond of aspargine-linked glycopeptides and glycoproteins. Earlier studies from our laboratory indicated that PNGase catalyzed de-N-glycosylation was limited to glycopeptide substrates, but recent reports have demonstrated that it also acts upon full-length misfolded glycoproteins. In this study, we utilized two glycoprotein substrates, yeast carboxypeptidase and chicken egg albumin (ovalbumin), to study the deglycosylation activity of yeast PNGase and its mutants. Our results provide further evidence that PNGase acts upon full-length glycoprotein substrates and clearly establish that PNGase acts only on misfolded or denatured glycoproteins. [Copyright &y& Elsevier]