Search

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: The chaperone-related p97 protein plays a central role in various cellular processes involving the ubiquitin–proteasome system. The diverse functions of p97 are controlled by a large number of cofactors that recruit specific substrates or influence their ubiquitylation state. Many cofactors bind through a UBX or PUB domain, two major p97 binding modules. However, the recently identified UBXD1 cofactor possesses both domains. To elucidate the molecular basis underlying the interaction between UBXD1 and p97, we analyzed the contribution of both domains to p97 binding biochemically and in living cells. The PUB domain mediated robust binding to the carboxy-terminus of p97, while the UBX domain did not contribute to p97 binding. Importantly, we identified an additional p97 binding site in UBXD1 that competed with the p47 cofactor for binding to the N domain of p97. This novel, bipartite binding mode suggests that UBXD1 could be an efficient regulator of p97 cofactor interactions. [Copyright &y& Elsevier]

Abstract: Peptide:N-glycanase (PNGase) releases N-glycans from glycoproteins/glycopeptides. Cytoplasmic PNGase is widely recognized as a component of machinery for ER-associated degradation (ERAD), i.e. proteasomal degradation of misfolded, newly synthesized (glyco)proteins that have been exported from the ER. The enzyme belongs to the “transglutaminase superfamily” that contains a putative catalytic triad of cysteine, histidine, and aspartic acid. The mammalian orthologues of PNGase contain the N-terminal PUB domain that serves as the protein–protein interaction domain. The C-terminus of PNGase was recently found to be a novel carbohydrate-binding domain. Taken together, these observations indicate that C-terminus of mammalian PNGase is important for recognition of the substrates while N-terminus of this enzyme is involved in assembly of a degradation complex. [Copyright &y& Elsevier]

During endoplasmic reticulum-associated degradation, the multi-functional AAA ATPase p97 is part of a protein degradation complex. p97 associates via its N-terminal domain with various cofactors to recruit ubiquitinated substrates. It also interacts with alternative substrate-processing cofactors, such as Ufd2, Ufd3, and peptide:N-glycanase (PNGase) in higher eukaryotes. These cofactors determine different fates of the substrates and they all bind outside of the N-terminal domain of p97. Here, we describe a cofactor-binding motif of p97 contained within the last 10 amino acid residues of the C terminus, which is both necessary and sufficient to mediate interactions of p97 with PNGase and Ufd3. The crystal structure of the N-terminal domain of PNGase in complex with this motif provides detailed insight into the interaction between p97 and its substrate-processing cofactors. Phosphorylation of p97's highly conserved penultimate tyrosine residue, which is the main phosphorylation site during T cell receptor stimulation, completely blocks binding of either PNGase or Ufd3 to p97. This observation suggests that phosphorylation of this residue modulates endoplasmic reticulum-associated protein degradation activity by discharging substrate-processing cofactors. [ABSTRACT FROM AUTHOR]

AAA+ ATPase p97/valosin-containing protein (VCP)/Cdc48 is a key player in various cellular stress responses in which it unfolds ubiquitinated proteins to facilitate their degradation by the proteasome. P97 works in different cellular processes using alternative sets of cofactors and is implicated in multiple degenerative diseases. Ubiquitin regulatory X domain protein 1 (UBXD1) has been linked to pathogenesis and is unique amongst p97 cofactors because it interacts with both termini of p97. Its N-domain binds to the N-domain and N/D1 interface of p97 and regulates its ATPase activity. The PUB (peptide:N-glycanase and UBA or UBX-containing proteins) domain binds the p97 C-terminus, but how it controls p97 function is still unknown. Here we present the NMR structure of UBXD1-PUB together with binding studies, mutational analysis, and a model of UBXD1-PUB in complex with the p97 C-terminus. While the binding pocket is conserved among PUB domains, UBXD1-PUB features a unique loop and turn regions suggesting a role in coordinating interaction with downstream regulators and substrate processing [ABSTRACT FROM AUTHOR]