共查询到20条相似文献,搜索用时 0 毫秒
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Espeut J Gaussen A Bieling P Morin V Prieto S Fesquet D Surrey T Abrieu A 《Molecular cell》2008,29(5):637-643
During mitosis, chromosome alignment depends on the regulated dynamics of microtubules and on motor protein activities. At the kinetochore, the interplay between microtubule-binding proteins, motors, and kinases is poorly understood. Cenp-E is a kinetochore-associated kinesin involved in chromosome congression, but the mechanism by which this is achieved is unclear. Here, we present a study of the regulation of Cenp-E motility by using purified full-length (FL) Xenopus Cenp-E protein, which demonstrates that FL Cenp-E is a genuine plus-end-directed motor. Furthermore, we find that the Cenp-E tail completely blocks the motility of Cenp-E in vitro. This is achieved through direct interaction between its motor and tail domains. Finally, we show that Cenp-E autoinhibition is reversed by MPS1- or CDK1-cyclin B-mediated phosphorylation of the Cenp-E tail. This suggests a model of dynamic control of Cenp-E motility, and hence chromosome congression, dependent upon phosphorylation at the kinetochore. 相似文献
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Itsuki Y Saeki M Nakahara H Egusa H Irie Y Terao Y Kawabata S Yatani H Kamisaki Y 《FEBS letters》2008,582(16):2365-2370
We have previously reported that Monad, a novel WD40 repeat protein, potentiates apoptosis induced by tumor necrosis factor-alpha(TNF-alpha) and cycloheximide (CHX). By affinity purification and mass spectrometry, we identified RNA polymerase II-associated protein 3 (RPAP3) as a binding protein of Monad. Overexpression of RPAP3 in HEK 293 potentiated caspase-3 activation and apoptosis induced by TNF-alpha and CHX. In addition, knockdown of RPAP3 by RNA interference resulted in a significant reduction of apoptosis induced by TNF-alpha and CHX in HEK293 and HeLa cells. These results raise the possibility that RPAP3, together with Monad, may function as a novel modulator of apoptosis pathway. 相似文献
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Protein phosphatase 5 (PP5) exhibits low basal activity due to the autoinhibitory properties of its N-terminal and C-terminal domains but can be activated approximately 40-fold in vitro by polyunsaturated fatty acids. To identify residues involved in regulating PP5 activity, we performed scanning mutagenesis of its N-terminal tetratricopeptide repeat (TPR) domain and deletion mutagenesis of its C-terminal domain. Mutating residues in a groove of the TPR domain that binds to heat shock protein 90 had no effect on basal phosphatase activity. Mutation of Glu-76, however, whose side chain projects away from this groove, resulted in a 10-fold elevation of basal activity without affecting arachidonic acid-stimulated activity. Thus, the interface of the TPR domain involved in PP5 autoinhibition appears to be different from that involved in heat shock protein 90 binding. We also observed a 10-fold elevation of basal phosphatase activity upon removing the C-terminal 13 amino acids of PP5, with a concomitant 50% decrease in arachidonic acid-stimulated activity. These two effects were accounted for by two distinct amino acid deletions: deleting the four C-terminal residues (496-499) of PP5 had no effect on its activity, but removing Gln-495 elevated basal activity 10-fold. Removal of a further three amino acids had no additional effect, but deleting Asn-491 resulted in a 50% reduction in arachidonic acid-stimulated activity. Thus, Glu-76 in the TPR domain and Gln-495 at the C-terminus were implicated in maintaining the low basal activity of PP5. While the TPR domain alone has been thought to mediate fatty acid activation of PP5, our data suggest that Asn-491, near its C-terminus, may also be involved in this process. 相似文献
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Exner V Alexandre C Rosenfeldt G Alfarano P Nater M Caflisch A Gruissem W Batschauer A Hennig L 《Plant physiology》2010,154(4):1633-1645
Plants use different classes of photoreceptors to collect information about their light environment. Cryptochromes are blue light photoreceptors that control deetiolation, entrain the circadian clock, and are involved in flowering time control. Here, we describe the cry1-L407F allele of Arabidopsis (Arabidopsis thaliana), which encodes a hypersensitive cryptochrome1 (cry1) protein. Plants carrying the cry1-L407F point mutation have elevated expression of CONSTANS and FLOWERING LOCUS T under short-day conditions, leading to very early flowering. These results demonstrate that not only the well-studied cry2, with an unequivocal role in flowering promotion, but also cry1 can function as an activator of the floral transition. The cry1-L407F mutants are also hypersensitive toward blue, red, and far-red light in hypocotyl growth inhibition. In addition, cry1-L407F seeds are hypersensitive to germination-inducing red light pulses, but the far-red reversibility of this response is not compromised. This demonstrates that the cry1-L407F photoreceptor can increase the sensitivity of phytochrome signaling cascades. Molecular dynamics simulation of wild-type and mutant cry1 proteins indicated that the L407F mutation considerably reduces the structural flexibility of two solvent-exposed regions of the protein, suggesting that the hypersensitivity might result from a reduced entropic penalty of binding events during downstream signal transduction. Other nonmutually exclusive potential reasons for the cry1-L407F gain of function are the location of phenylalanine-407 close to three conserved tryptophans, which could change cry1's photochemical properties, and stabilization of ATP binding, which could extend the lifetime of the signaling state of cry1. 相似文献
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A gain-of-function mutation in STAT6 总被引:4,自引:0,他引:4
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Ramsey AJ Russell LC Whitt SR Chinkers M 《The Journal of biological chemistry》2000,275(23):17857-17862
The sequential binding of different tetratricopeptide repeat (TPR) proteins to heat shock protein 90 (hsp90) is essential to its chaperone function in vivo. We have previously shown that three basic residues in the TPR domain of PP5 are required for binding to the acidic C-terminal domain of hsp90. We have now tested which acidic residues in this C-terminal domain are required for binding to three different TPR proteins as follows: PP5, FKBP52, and Hop. Mutation of Glu-729, Glu-730, and Asp-732 at the C terminus of hsp90 interfered with binding of all three TPR proteins. Mutation of Glu-720, Asp-722, Asp-723, and Asp-724 inhibited binding of FKBP52 and PP5 but not of Hop. Mutation of Glu-651 and Asp-653 did not affect binding of FKBP52 or PP5 but inhibited both Hop binding and hsp90 chaperone activity. We also found that a conserved Lys residue required for PP5 binding to hsp90 was critical for the binding of FKBP52 but not for the binding of Hop to hsp90. These results suggest distinct but overlapping binding sites on hsp90 for different TPR proteins and indicate that the binding site for Hop, which is associated with hsp90 in intermediate stages of protein folding, overlaps with a site of chaperone activity. 相似文献
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Effect of mutation of the tetratricopeptide repeat and asparatate-proline 2 domains of Sti1 on Hsp90 signaling and interaction in Saccharomyces cerevisiae
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Through simultaneous interactions with Hsp70 and Hsp90 via separate tetratricopeptide repeat (TPR) domains, the cochaperone protein Hop/Sti1 has been proposed to play a critical role in the transfer of client proteins from Hsp70 to Hsp90. However, no prior mutational analysis demonstrating a critical in vivo role for the TPR domains of Sti1 has been reported. We used site-directed mutagenesis of the TPR domains combined with a genetic screen to isolate mutations that disrupt Sti1 function. A single amino acid alteration in TPR2A disrupted Hsp90 interaction in vivo but did not significantly affect function. However, deletion of a conserved residue in TPR2A or mutations in the carboxy-terminal DP2 domain completely disrupted Sti1 function. Surprisingly, mutations in TPR1, previously shown to interact with Hsp70, were not sufficient to disrupt in vivo functions unless combined with mutations in TPR2B, suggesting that TPR1 and TPR2B have redundant or overlapping in vivo functions. We further examined the genetic and physical interaction of Sti1 with a mutant form of Hsp90, providing insight into the importance of the TPR2A domain of Sti1 in regulating Hsp90 function. 相似文献
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Ward BK Allan RK Mok D Temple SE Taylor P Dornan J Mark PJ Shaw DJ Kumar P Walkinshaw MD Ratajczak T 《The Journal of biological chemistry》2002,277(43):40799-40809
Cyclophilin 40 (CyP40) is a tetratricopeptide repeat (TPR)-containing immunophilin and a modulator of steroid receptor function through its binding to heat shock protein 90 (Hsp90). Critical to this binding are the carboxyl-terminal MEEVD motif of Hsp90 and the TPR domain of CyP40. Two different models of the CyP40-MEEVD peptide interaction were used as the basis for a comprehensive mutational analysis of the Hsp90-interacting domain of CyP40. Using a carboxyl-terminal CyP40 construct as template, 24 amino acids from the TPR and flanking acidic and basic domains were individually mutated by site-directed mutagenesis, and the mutants were coexpressed in yeast with a carboxyl-terminal Hsp90beta construct and qualitatively assessed for binding using a beta-galactosidase filter assay. For quantitative assessment, mutants were expressed as glutathione S-transferase fusion proteins and assayed for binding to carboxyl-terminal Hsp90beta using conventional pulldown and enzyme-linked immunosorbent assay microtiter plate assays. Collectively, the models predict that the following TPR residues help define a binding groove for the MEEVD peptide: Lys-227, Asn-231, Phe-234, Ser-274, Asn-278, Lys-308, and Arg-312. Mutational analysis identified five of these residues (Lys-227, Asn-231, Asn-278, Lys-308, and Arg-312) as essential for Hsp90 binding. The other two residues (Phe-234 and Ser-274) and another three TPR domain residues not definitively associated with the binding groove (Leu-284, Lys-285, and Asp-329) are required for efficient Hsp90 binding. These data confirm the critical importance of the MEEVD binding groove in CyP40 for Hsp90 recognition and reveal that additional charged and hydrophobic residues within the CyP40 TPR domain are required for Hsp90 binding. 相似文献
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A mutation in the second nucleotide binding fold of the cystic fibrosis gene. 总被引:25,自引:13,他引:12
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The discovery last year of the deletion of a phenylalanine residue at amino acid position 508 of the cystic fibrosis (CF) gene has meant that approximately 70% of mutant chromosomes associated with CF can be accounted for. We report the finding of a substitution at nucleotide position 4041 of the CF gene, resulting in a change from asparagine to lysine at amino acid position 1303. We believe that this is a disease-causing mutation, as it involves a nonconservative amino acid change and has only been found on CF chromosomes with a consistent haplotype background. The mutation was detected using direct sequencing of PCR-amplified genomic DNA and was confirmed by dot hybridization to both normal and mutant allele-specific oligonucleotides. The mutation was detected on three chromosomes from four individuals but not on any normal chromosome. Its presence in the heterozygous state is not correlated with the clinical status of the individual patients. 相似文献
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Human Sgt1 binds HSP90 through the CHORD-Sgt1 domain and not the tetratricopeptide repeat domain 总被引:2,自引:0,他引:2
Lee YT Jacob J Michowski W Nowotny M Kuznicki J Chazin WJ 《The Journal of biological chemistry》2004,279(16):16511-16517
Sgt1 has been identified as a subunit of both core kinetochore and SCF (Skp1-Cul1-F-box) ubiquitin ligase complexes and is also implicated in plant disease resistance. Sgt1 has two putative HSP90 binding domains, a tetratricopeptide repeat and a p23-like CHORD and Sgt1 (CS) domain. Using NMR spectroscopy, we show that only the CS domain of human Sgt1 physically interacts with HSP90. The tetratricopeptide repeat domain does not bind to either HSP90 or HSP70. Determination of the three-dimensional structure showed that the Sgt1-CS domain shares the same beta-sandwich fold as p23 but lacks the last highly conserved beta-strand in p23. Analysis of the structures of Sgt1-CS and p23 revealed a similar charge distribution on one of two opposing surfaces that suggests that it is the binding region for HSP90 in Sgt1. Although ATP is absolutely required for p23 binding to HSP90, Sgt1 binds to HSP90 also in the absence of the non-hydrolyzable analog ATPgammaS. Our findings suggest the CS domain is a binding module for HSP90 distinct from p23-like domains, which implies that Sgt1 and related proteins function in recruiting heat shock protein activities to multiprotein assemblies. 相似文献
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Constitutive gain-of-function mutants in a nucleotide binding site-leucine rich repeat protein encoded at the Rx locus of potato 总被引:1,自引:0,他引:1
Bendahmane A Farnham G Moffett P Baulcombe DC 《The Plant journal : for cell and molecular biology》2002,32(2):195-204
Rx in potato encodes a protein with a nucleotide binding site (NBS) and leucine-rich repeats (LRR) that confers resistance against Potato virus X. The NBS and LRR domains in Rx are present in many disease resistance proteins in plants and in regulators of apoptosis in animals. To investigate structure-function relationships of NBS-LRR proteins we exploited the potential of Rx to mediate a cell death response. With wild-type Rx cell death is elicited only in the presence of the viral coat protein. However, following random mutagenesis of Rx, we identified mutants in which cell death is activated in the absence of viral coat protein. Out of 2500 Rx clones tested there were seven constitutive gain-of-function mutants carrying eight independent mutations. The mutations encoded changes in the LRR or in conserved RNBS-D and MHD motifs of the NBS. Based on these findings we propose that there are inhibitory domains in the NBS and LRR. The constitutive gain-of-function phenotypes would be due to deletion or modification of these inhibitory domains. However activation of Rx is not simply release of negative regulation by the LRR and adjacent sequence because deleted forms of Rx that lack constitutive gain of function mutations are not active unless the protein is overexpressed. 相似文献
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Takabayashi S Iwashita S Hirashima T Katoh H 《Experimental biology and medicine (Maywood, N.J.)》2007,232(5):695-699
We carried out molecular analyses of the novel flaky skin mutation, Ttc7(fsn-Jic )(a synonym for fsn(Jic)), which we found in a previous study. It was revealed that this mutation involved a genomic in-frame deletion including exons 9 and 10 of the Ttc7 gene, and that the genomic deletion in Ttc7 (fsn-Jic )may disrupt the tetratricopeptide repeat-2B domain of the TTC7 protein. Based on a comparison of three Ttc7 mutations, including Ttc7(fsn-J )(a synonym for fsn) and Ttc7(fsn-hea )(a synonym for hea), it was suggested that either exon 9 or exon 10 or both may play a more important role than the other exons of the Ttc7 gene. Ttc7 gene expression analyses using Northern blotting revealed that Ttc7 mRNA is expressed in 11 tissues, except muscle. In conclusion, we confirmed that the Ttc7 (fsn-Jic )mutation, as well as the Ttc7(fsn-J )and Ttc7 (fsn-hea )mutations, is responsible for abnormal phenotypes observed in various tissues of mice with the flaky skin mutation. 相似文献