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)

Npl4 is a 67 kDa protein forming a stable heterodimer with Ufd1, which in turn binds the ubiquitous p97/VCP ATPase. According to a widely accepted model, VCP(Ufd1-Npl4) promotes the retrotranslocation of emerging ER proteins, their ubiquitination by associated ligases, and handling to the 26S proteasome for degradation in a process known as ERAD (ER-associated degradation). Using a series of Npl4 deletion mutants we have revealed that the binding of Ufd1 to Npl4 is mediated by two regions: a conserved stretch of amino acids from 113 to 255 within the zf-Npl4 domain and by the Npl4 homology domain between amino acids 263 and 344. Within the first region, we have identified two discrete subdomains: one involved in Ufd1 binding and one regulating VCP binding. Expression of any one of the mutants failed to induce any changes in the morphology of the ER or Golgi compartments. Moreover, we have observed that overexpression of all the analyzed mutants induced mild ER stress, as evidenced by increased Grp74/BiP expression without associated XBP1 splicing or induction of apoptosis. Surprisingly, we have not observed any accumulation of the typical ERAD substrate alphaTCR. This favors the model where the Ufd1-Npl4 dimer forms a regulatory gate at the exit from the retrotranslocone, rather than actively promoting retrotranslocation like the p97VCP ATPase.
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PMID:Analysis of Npl4 deletion mutants in mammalian cells unravels new Ufd1-interacting motifs and suggests a regulatory role of Npl4 in ERAD. 1858 29

In eukaryotic cells, the endoplasmic reticulum-associated degradation (ERAD) pathway is essential for the disposal of misfolded proteins. Recently, we demonstrated the existence of a higher order complex consisting of the ER bound E3 ligase gp78, p97, PNGase, and HR23B in mammals. This complex may serve to facilitate the routing of misfolded glycoproteins out of the ER to the cytosol where they are degraded by the proteasome. In this complex, p97 functions as an organizer to mediate the interactions with gp78 and the deglycosylating enzyme PNGase. A novel protein-binding motif of mouse p97 was identified that consists of its last 10 amino acid residues; this motif is sufficient to mediate the interaction of p97 with PNGase and Ufd3. Phosphorylation of p97's highly conserved penultimate tyrosine residue, completely blocks binding of both PNGase and Ufd3 to mp97. We have found that c-Src kinase directly and selectively phosphorylated the penultimate tyrosine of p97 in vitro, and that overexpression of c-Src significantly increased the phosphorylation level of p97 in cells and caused accumulation of the ERAD substrate TCRalpha-GFP, as well as ubiquitin-conjugated substrates. These results suggest a role for p97 phosphorylation in the degradation of misfolded glycoproteins.
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PMID:Tyrosine phosphorylation of ATPase p97 regulates its activity during ERAD. 1870 91

Mutations in p97/VCP cause the multisystem disease inclusion body myopathy, Paget disease of the bone and frontotemporal dementia (IBMPFD). p97/VCP is a member of the AAA+ (ATPase associated with a variety of activities) protein family and has been implicated in multiple cellular processes. One pathologic feature in IBMPFD is ubiquitinated inclusions, suggesting that mutations in p97/VCP may affect protein degradation. The present study shows that IBMPFD mutant expression increases ubiquitinated proteins and susceptibility to proteasome inhibition. Co-expression of an aggregate prone protein such as expanded polyglutamine in IBMPFD mutant cells results in an increase in aggregated protein that localizes to small inclusions instead of a single perinuclear aggresome. These small inclusions fail to co-localize with autophagic machinery. IBMPFD mutants avidly bind to these small inclusions and may not allow them to traffic to an aggresome. This is rescued by HDAC6, a p97/VCP-binding protein that facilitates the autophagic degradation of protein aggregates. Expression of HDAC6 improves aggresome formation and protects IBMPFD mutant cells from polyglutamine-induced cell death. Our study emphasizes the importance of protein aggregate trafficking to inclusion bodies in degenerative diseases and the therapeutic benefit of inclusion body formation.
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PMID:Impaired protein aggregate handling and clearance underlie the pathogenesis of p97/VCP-associated disease. 1871 68

NCLs (neuronal ceroid lipofuscinoses), a group of inherited neurodegenerative lysosomal storage diseases that predominantly affect children, are the result of autosomal recessive mutations within one of the nine cln genes. The wild-type cln gene products are composed of membrane and soluble proteins that localize to the lysosome or the ER (endoplasmic reticulum). However, the destiny of the Cln variants has not been fully characterized. To explore a possible link between ER quality control and processing of Cln mutants, we investigated the fate of two NCL-related Cln6 mutants found in patient samples (Cln6(G123D) and Cln6(M241T)) in neuronal-derived human cells. The point mutations are predicted to be in the putative transmembrane domains and most probably generate misfolded membrane proteins that are subjected to ER quality control. Consistent with this paradigm, both mutants underwent rapid proteasome-mediated degradation and complexed with components of the ER extraction apparatus, Derlin-1 and p97. In addition, knockdown of SEL1L [sel-1 suppressor of lin-12-like (Caenorhabditis elegans)], a member of an E3 ubiquitin ligase complex involved in ER protein extraction, rescued significant amounts of Cln6(G123D) and Cln6(M241T) polypeptides. The results implicate ER quality control in the instability of the Cln variants that probably contributes to the development of NCL.
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PMID:Cln6 mutants associated with neuronal ceroid lipofuscinosis are degraded in a proteasome-dependent manner. 1881 91

Inclusion body myopathy (IBM) associated with Paget disease of the bone (PDB) and frontotemporal dementia (FTD) (now called IBMPFD), is a progressive autosomal dominant disorder that was recently identified as being caused by mutations in the VCP (p97 or CDC48) gene which plays a key role in the ubiquitin-proteasome dependent degradation of cytosolic proteins and in the retro translocation of misfolded proteins from the endoplasmic reticulum into the cytoplasm. Approximately 90% of the affected persons in the study have myopathy or muscle weakness particularly of the shoulder and hip girdles, which can lead to loss of walking ability and even death by complications of respiratory and cardiac failure. About half of affected study participants have Paget disease of bone characterized by abnormal rates of bone growth that can result in bone pain, enlargement and fractures. Findings of premature FTD affecting behavior and personality are seen in a third of affected individuals. Within 20 IBMPFD families whose data was analyzed for this study, ten missense mutations have been identified, the majority of which are located in the N-terminal ubiquitin binding domain. Inclusions seen in the muscle, brain and heart in VCP disease contain ubiquitin, beta amyloid and TDP-43, also seen in other neurodegenerative disorders thus implicating common pathways in their pathogenesis.
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PMID:VCP disease associated with myopathy, Paget disease of bone and frontotemporal dementia: review of a unique disorder. 1884 50

In mammalian cells, abnormal proteins that escape proteasome-dependent degradation form small aggregates that can be transported into a centrosome-associated structure, called an aggresome. Here we demonstrate that in yeast a single aggregate formed by the huntingtin exon 1 with an expanded polyglutamine domain (103QP) represents a bona fide aggresome that colocalizes with the spindle pole body (the yeast centrosome) in a microtubule-dependent fashion. Since a polypeptide lacking the proline-rich region (P-region) of huntingtin (103Q) cannot form aggresomes, this domain serves as an aggresome-targeting signal. Coexpression of 103Q with 25QP, a soluble polypeptide that also carries the P-region, led to the recruitment of 103Q to the aggresome via formation of hetero-oligomers, indicating the aggresome targeting in trans. To identify additional factors involved in aggresome formation and targeting, we purified 103QP aggresomes and 103Q aggregates and identified the associated proteins using mass spectrometry. Among the aggresome-associated proteins we identified, Cdc48 (VCP/p97) and its cofactors, Ufd1 and Nlp4, were shown genetically to be essential for aggresome formation. The 14-3-3 protein, Bmh1, was also found to be critical for aggresome targeting. Its interaction with the huntingtin fragment and its role in aggresome formation required the huntingtin N-terminal N17 domain, adjacent to the polyQ domain. Accordingly, the huntingtin N17 domain, along with the P-region, plays a role in aggresome targeting. We also present direct genetic evidence for the protective role of aggresomes by demonstrating genetically that aggresome targeting of polyglutamine polypeptides relieves their toxicity.
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PMID:Abnormal proteins can form aggresome in yeast: aggresome-targeting signals and components of the machinery. 1885 35

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.
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PMID:UBXD1 binds p97 through two independent binding sites. 1917 49

Recent studies, mainly in yeast, have identified various cofactors that associate with the 26S proteasome and appear to influence its function. To identify these proteins in different cells and physiological states, we developed a method to gently and rapidly isolate 26S proteasomes and associated proteins without the need for genetic modifications of the proteasome. This method is based on the affinity of this complex for the ubiquitin-like (UBL) domain of hHR23B and elution with a competing polypeptide containing a ubiquitin-interacting motif. Associated with 26S proteasomes from rat muscle were a variety of known proteasome-interacting proteins, activators, and ubiquitin conjugates. In addition, we identified over 40 proteins not previously known to associate with the 26S proteasome, some of which were tightly associated with the proteasome in a substoichiometric fashion, e.g., the deubiquitinating enzymes USP5/isopeptidase T and USP7/HAUSP and the ubiquitin ligases ARF-BP1/HUWE1 and p600/UBR4. By altering buffer conditions, we also purified by this approach complexes of the ATPase p97/VCP associated with its adaptor proteins Ufd1-Npl4, p47, SAKS1, and FAF1, all of which contain ubiquitin-binding motifs. These complexes were isolated with ubiquitin conjugates bound and were not previously known to bind to the UBL domain of hHR23B. These various UBL-interacting proteins, dubbed the UBL interactome, represent a network of proteins that function together in ubiquitin-dependent proteolysis, and the UBL method offers many advantages for studies of the diversity, functions, and regulation of 26S proteasomes and p97 complexes under different conditions.
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PMID:Isolation of mammalian 26S proteasomes and p97/VCP complexes using the ubiquitin-like domain from HHR23B reveals novel proteasome-associated proteins. 1918 4

Clearance of misfolded proteins by endoplasmic reticulum (ER)-associated degradation (ERAD) requires concerted activity of chaperones, adaptor proteins, ubiquitin ligases, and proteasomes. RNF5 is a ubiquitin ligase anchored to the ER membrane implicated in ERAD via ubiquitination of misfolded proteins. Among RNF5-associated proteins is JNK-associated membrane protein (JAMP), a 7-transmembrane protein located within the ER membrane that facilitates degradation of misfolded proteins through recruitment of proteasomes and ERAD regulatory components. Here we demonstrate that RNF5 associates with JAMP in the ER membrane. This association results in Ubc13-dependent RNF5-mediated noncanonical ubiquitination of JAMP. This ubiquitination does not alter JAMP stability but rather inhibits its association with Rpt5 and p97. Consequently, clearance of misfolded proteins, such as CFTRDelta508 and T cell receptor alpha, is less efficient, resulting in their greater accumulation. Significantly, the RNF5 effect on JAMP is seen prior to and after ER stress response, thereby highlighting a novel mechanism to limit ERAD and proteasome assembly at the ER, to the actual ER stress response.
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PMID:Regulation of endoplasmic reticulum-associated degradation by RNF5-dependent ubiquitination of JNK-associated membrane protein (JAMP). 1926 66

Misfolded proteins of the secretory pathway are recognized in the endoplasmic reticulum (ER), retrotranslocated into the cytoplasm, and degraded by the ubiquitin-proteasome system. Right after retrotranslocation and polyubiquitination, they are extracted from the cytosolic side of the ER membrane through a complex consisting of the AAA ATPase Cdc48 (p97 in mammals), Ufd1, and Npl4. This complex delivers misfolded proteins to the proteasome for final degradation. Extraction, delivery, and processing of ERAD (ER-associated degradation) substrates to the proteasome requires additional cofactors of Cdc48. Here we characterize the UBX domain containing protein Ubx4 (Cui1) as a crucial factor for the degradation of polyubiquitinated proteins via ERAD. Ubx4 modulates the Cdc48-Ufd1-Npl4 complex to guarantee its correct function. Mutant variants of Ubx4 lead to defective degradation of misfolded proteins and accumulation of polyubiquitinated proteins bound to Cdc48. We show the requirement of the UBX domain of Ubx4 for its function in ERAD. The observation that Ubx2 and Ubx4 are not found together in one complex with Cdc48 suggests several distinct steps in modulating the activity and localization of Cdc48 in ERAD.
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PMID:Ubx4 modulates cdc48 activity and influences degradation of misfolded proteins of the endoplasmic reticulum. 1935 48


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