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:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have identified a novel DNA helicase in humans that belongs to members of the superfamily I helicase and found that it contains a well conserved F-box motif at its N terminus. We have named the enzyme hFBH1 (human F-box DNA helicase 1). Recombinant hFBH1, containing glutathione S-transferase at the N terminus, was expressed in Sf9 cells and purified. In this report, we show that hFBH1 exhibited DNA-dependent ATPase and DNA unwinding activities that displace duplex DNA in the 3' to 5' direction. The hFBH1 enzyme interacted with human SKP1 and formed an SCF (SKP1/Cullin/F-box) complex together with human Cullin and ROC1. In addition, the SCF complex containing hFBH1 as an F-box protein displayed ubiquitin ligase activity. We demonstrate that hFBH1 is the first F-box protein that possesses intrinsic enzyme activity. The potential role of the F-box motif and the helicase activity of the enzyme are discussed with regard to regulation of DNA metabolism.
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PMID:The novel human DNA helicase hFBH1 is an F-box protein. 1195 8

The present report deals with the functional relationships among protein complexes which, when mutated, are responsible for four human syndromes displaying cancer proneness, and whose cells are deficient in DNA double-strand break (DSB) repair. In some of them, the cells are also unable to activate the proper checkpoint, while in the others an unduly override of the checkpoint-induced arrest occurs. As a consequence, all these patients display genome instability. In ataxia-telangiectasia, the mutated protein (ATM) is a kinase, which acts as a transducer of DNA damage signalling. The defective protein in the ataxia-telangiectasia-like disorder is a DNase (the Mre11 nuclease) that in vivo produces single-strand tails at both sides of DSBs. Mre11 is always present with the Rad50 ATPase in a protein machine: the nuclease complex. In mammals, this complex also contains nibrin, the protein mutated in the Nijmegen syndrome. Nibrin confers new abilities to the nuclease complex, and can also bind to BRCA1 (one of the two proteins mutated in familial breast cancer). BRCA1 has a central motif that binds with high affinity to cruciform DNA, a structure present in places where the DNA loops are anchored to the chromosomal axis or scaffold. The BRCA1 x cruciform DNA complex should be released to allow the nuclease complex to work in DNA recombinational repair of DSBs. BRCA1 also acts as a scaffold for the assembly of ATPases such as Rad51, responsible for the somatic homologous recombination. Loss of the BRCA1 gene prevents cell survival after exposure to cross-linkers. The BRCA1-RING domain is an E3-ubiquitin ligase. It can mono-ubiquitinate the FANCD2 protein, mutated in one of the Fanconi anemia complementation groups, to regulate it. Finally, during DNA replication, the nuclease complex and its activating ATM kinase are integrated in the BRCA1-associated surveillance complex (BASC) that contains, among others, enzymes required for mismatch excision repair. In short, the proteins missing in these syndromes have in common their BRCA1-mediated assembly into multimeric machines responsible for the surveillance of DNA replication, DSB recombinational repair, and the removal of DNA cross-links.
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PMID:Human syndromes with genomic instability and multiprotein machines that repair DNA double-strand breaks. 1250 2

Aldosterone increases within 30 min renal Na+reabsorption and K+secretion by a mechanism that is triggered at the level of gene transcription. Thus, gene products that are rapidly up- or down-regulated transmit this effect to the transport machinery within the distal nephron target cells. One such rapidly up-regulated gene product is a structural element of the transport machinery, namely the a subunit of ENaC. Its amount might in certain conditions play a rate limiting role for Na+transport. Cell-surface localization and function of ENaC and of the Na,K-ATPase are also tightly controlled by a complex regulatory network and aldosterone appears to acutely regulate the expression of elements of this network such as the small G-protein K-Ras (in A6 cells) and the kinase SGK1 (also in ENaC-expressing cells of the mammalian distal nephron). The kinase SGK1 is an early aldosterone-induced protein that relays signals from pathways that are transmitted via PDK1/2 and possibly PKA. Active SGK1 has been shown to increase ENaC and Na,K-ATPase cell-surface expression in Xenopus oocytes. This effect at the level of ENaC has been recently shown to be mediated by the ubiquitin ligase Nedd4-2 which is a direct target of SGK1. Once phosphorylated by SGK1, Nedd4-2 is prevented from interacting with ENaC and thus from decreasing ENaC cell-surface expression. This SGK1-Nedd4-2-ENaC pathway is the first direct link between aldosterone-induced transcriptional regulation and the function of the Na+transport machinery to be unravelled. The physiological importance of this pathway for mediating the aldosterone response in different target epithelia remains to be verified in vivo, in particular in view of the axial gradient of ENaC apical translocation observed along the aldosterone-sensitive distal nephron.
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PMID:SGK1: aldosterone-induced relay of Na+ transport regulation in distal kidney nephron cells. 1264 99

Membrane proteins destined for the vacuolar or lysosomal lumen are typically ubiquitinated, the ubiquitin serving as a targeting signal for the multivesicular body pathway. The RING-domain ubiquitin ligase Tul1 is an integral membrane protein that modifies the yeast vacuolar enzyme carboxypeptidase S (Cps1), the polyphosphatase Ppn1/Phm5 and other proteins containing exposed hydrophilic residues within their transmembrane domains (TMDs). Here we show that Bsd2 provides an alternative ubiquitination mechanism for Cps1, Phm5 and other proteins. Bsd2 is a three-TMD protein with a PPXY motif that binds the HECT domain ubiquitin ligase Rsp5. It can thus act as a specific adaptor linking Rsp5 to its substrates. Like Tul1, the Bsd2 system recognises polar TMDs. Bsd2 also controls the vacuolar targeting of a manganese transporter and a mutant plasma membrane ATPase, and together with the ER retrieval receptor Rer1, it protects cells from stress. We suggest that Bsd2 has a wide role in the quality control of membrane proteins. Bsd2 is the yeast homologue of human NEDD4 binding protein N4WBP5, which may therefore have similar functions.
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PMID:Bsd2 binds the ubiquitin ligase Rsp5 and mediates the ubiquitination of transmembrane proteins. 1498 31

Pma1-7 is a mutant plasma membrane ATPase that is impaired in targeting to the cell surface at 37 degrees C and is delivered instead to the endosomal/vacuolar pathway for degradation. We have proposed that Pma1-7 is a substrate for a Golgibased quality control mechanism. By contrast with wild-type Pma1, Pma1-7 is ubiquitinated. Ubiquitination and endosomal targeting of Pma1-7 is dependent on the Rsp5-Bul1-Bul2 ubiquitin ligase protein complex but not the transmembrane ubiquitin ligase Tul1. Analysis of Pma1-7 ubiquitination in mutants blocked in protein transport at various steps of the secretory pathway suggests that ubiquitination occurs after ER exit but before endosomal entry. In the absence of ubiquitination in rsp5-1 cells, Pma1-7 is delivered to the cell surface and remains stable. Nevertheless, Pma1-7 remains impaired in association with detergent-insoluble glycolipid-enriched complexes in rsp5-1 cells, suggesting that ubiquitination is not the cause of Pma1-7 exclusion from rafts. In vps1 cells in which protein transport into the endosomal pathway is blocked, Pma1-7 is routed to the cell surface. On arrival at the plasma membrane in vps1 cells, Pma1-7 remains stable and its ubiquitination disappears, suggesting deubiquitination activity at the cell surface. We suggest that Pma1-7 sorting and fate are regulated by ubiquitination.
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PMID:Ubiquitin-mediated targeting of a mutant plasma membrane ATPase, Pma1-7, to the endosomal/vacuolar system in yeast. 1502 Jul 11

In our previous study, we found that a human F-box DNA helicase, named hFBH1, interacted with SKP1 to form an SCF (SKP1-Cul1-F-box protein) complex together with CUL1 and ROC1 in an F-box-dependent manner. The complex immunoprecipitated from crude cell extracts catalyzed polyubiquitin formation in the presence of the ubiquitin-activating and ubiquitin-conjugating enzymes, E1 and E2, respectively. In this report, we characterized the enzymatic properties of the recombinant SCF(hFBH1) complex purified from insect cells expressing hFBH1, SKP1, CUL1 and ROC1. The SCF(hFBH1) complex was isolated as a single tight complex that retained DNA helicase, DNA-dependent ATPase and E3 ubiquitin ligase activities. The helicase and ATPase activities residing in the SCF(hFBH1) complex were indistinguishable from those of the hFBH1 protein alone. Moreover, the ubiquitin ligase activity of the SCF(hFBH1) complex was hardly affected by single-stranded or double-stranded DNA. The multiple activities present in this complex act independently of each other, suggesting that the SCF(hFBH1) complex can catalyze a ubiquitination reaction while acting as a DNA helicase or translocating along DNA. The potential roles of the SCF(hFBH1) complex in DNA metabolism based upon the enzymatic activities associated with this complex are discussed.
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PMID:SCFhFBH1 can act as helicase and E3 ubiquitin ligase. 1511 74

Endoplasmic reticulum-associated degradation (ERAD) is a protein quality control mechanism that eliminates unwanted proteins from the endoplasmic reticulum (ER) through a ubiquitin-dependent proteasomal degradation pathway. gp78 is a previously described ER membrane-anchored ubiquitin ligase (E3) involved in ubiquitination of ER proteins. AAA ATPase (ATPase associated with various cellular activities) p97/valosin-containing protein (VCP) subsequently dislodges the ubiquitinated proteins from the ER and chaperones them to the cytosol, where they undergo proteasomal degradation. We now report that gp78 physically interacts with p97/VCP and enhances p97/VCP-polyubiquitin association. The enhanced association correlates with decreases in ER stress-induced accumulation of polyubiquitinated proteins. This effect is abolished when the p97/VCP-interacting domain of gp78 is removed. Further, using ERAD substrate CD3delta, gp78 consistently enhances p97/VCP-CD3delta binding and facilitates CD3delta degradation. Moreover, inhibition of endogenous gp78 expression by RNA interference markedly increases the levels of total polyubiquitinated proteins, including CD3delta, and abrogates VCP-CD3delta interactions. The gp78 mutant with deletion of its p97/VCP-interacting domain fails to increase CD3delta degradation and leads to accumulation of polyubiquitinated CD3delta, suggesting a failure in delivering ubiquitinated CD3delta for degradation. These data suggest that gp78-p97/VCP interaction may represent one way of coupling ubiquitination with retrotranslocation and degradation of ERAD substrates.
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PMID:AAA ATPase p97/valosin-containing protein interacts with gp78, a ubiquitin ligase for endoplasmic reticulum-associated degradation. 1533 98

Dorfin, a RING-IBR type ubiquitin ligase (E3), can ubiquitylate mutant superoxide dismutase 1, the causative gene of familial amyotrophic lateral sclerosis (ALS). Dorfin is located in ubiquitylated inclusions (UBIs) in various neurodegenerative disorders, such as ALS and Parkinson's disease (PD). Here we report that Valosin-containing protein (VCP) directly binds to Dorfin and that VCP ATPase activity profoundly contributes to the E3 activity of Dorfin. High through-put analysis using mass spectrometry identified VCP as a candidate of Dorfin-associated protein. Glycerol gradient centrifugation analysis showed that endogenous Dorfin consisted of a 400-600-kDa complex and was co-immunoprecipitated with endogenous VCP. In vitro experiments showed that Dorfin interacted directly with VCP through its C-terminal region. These two proteins were colocalized in aggresomes in HEK293 cells and UBIs in the affected neurons of ALS and PD. VCP(K524A), a dominant negative form of VCP, reduced the E3 activity of Dorfin against mutant superoxide dismutase 1, whereas it had no effect on the autoubiquitylation of Parkin. Our results indicate that VCPs functionally regulate Dorfin through direct interaction and that their functional interplay may be related to the process of UBI formation in neurodegenerative disorders, such as ALS or PD.
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PMID:Physical and functional interaction between Dorfin and Valosin-containing protein that are colocalized in ubiquitylated inclusions in neurodegenerative disorders. 1545 87

Many enveloped viruses exploit the class E vacuolar protein-sorting (VPS) pathway to bud from cells, and use peptide motifs to recruit specific class E VPS factors. Homologous to E6AP COOH terminus (HECT) ubiquitin ligases have been implicated as cofactors for PPXY motif-dependent budding, but precisely which members of this family are responsible, and how they access the VPS pathway is unclear. Here, we show that PPXY-dependent viral budding is unusually sensitive to inhibitory fragments derived from specific HECT ubiquitin ligases, namely WWP1 and WWP2. We also show that WWP1, WWP2, or Itch ubiquitin ligase recruitment promotes PPXY-dependent virion release, and that this function requires that the HECT ubiquitin ligase domain be catalytically active. Finally, we show that several mammalian HECT ubiquitin ligases, including WWP1, WWP2, and Itch are recruited to class E compartments induced by dominant negative forms of the class E VPS ATPase, VPS4. These data indicate that specific HECT ubiquitin ligases can link PPXY motifs to the VPS pathway to induce viral budding.
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PMID:HECT ubiquitin ligases link viral and cellular PPXY motifs to the vacuolar protein-sorting pathway. 1562 82

Proteins destined for secretion in eukaryotic cells enter the endoplasmic reticulum (ER) in an unfolded state and are properly folded in this organelle and sent to their final destination. Misfolded or orphan proteins are retained in the ER by a quality control system, retrotranslocated into the cytosol and degraded. Soluble and membrane proteins were found to require a basic machinery for elimination. It is composed of (1) the E1 (ubiquitin activating), E2 (ubiquitin conjugating), and E3 (ubiquitin ligase) enzymes, which polyubiquitinate the substrate proteins during retrotranslocation; (2) the trimeric AAA-ATPase complex Cdc48-Ufd1-Npl4p, which liberates the polyubiquitinated proteins from the ER; and (3) the 26S proteasome, finally degrading the misfolded proteins. Additional components for degradation of soluble or membrane proteins may vary depending on the nature of malfolded proteins. It is therefore of utmost importance to gain insight into the different components of the ER protein quality control and degradation system required for the elimination of the substrate variety. Protein quality control of the ER and subsequent degradation are evolutionarily highly conserved from yeast to human. The yeast Saccharomyces cerevisiae is therefore an elegant model organism for a search of new components of the ER quality control and degradation machinery, because it is easily amenable to genetic and molecular biological experimentation. In this chapter, a genetic approach is presented, which leads to the isolation of mutants and to the identification of proteins involved in protein quality control and ER-associated degradation (ERAD). The method resides in ethylmethane sulfonate (EMS) mutagenesis of a yeast strain followed by screening for stabilization of soluble ERAD substrates, two mutated and consequently malfolded vacuolar enzymes, carboxypeptidase yscY (CPY*) and proteinase yscA (PrA*). Both malfolded proteins are retained in the ER lumen and become substrates of the ERAD machinery.
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PMID:Endoplasmic reticulum-associated protein quality control and degradation: screen for ERAD mutants after ethylmethane sulfonate mutagenesis. 1591 39


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