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1.
CHD1 is a novel DNA-binding protein that contains both a chromatin organization modifier (chromo) domain and a helicase/ATPase domain. We show here that CHD1 preferentially binds to relatively long A.T tracts in double-stranded DNA via minor-groove interactions. Several CHD1-binding sites were found in a well-characterized nuclear-matrix attachment region, which is located adjacent to the intronic enhancer of the kappa immunoglobulin gene. The DNA-binding activity of CHD1 was localized to a 229-amino-acid segment in the C-terminal portion of the protein, which contains sequence motifs that have previously been implicated in the minor-groove binding of other proteins. We also demonstrate that CHD1 is a constituent of bulk chromatin and that it can be extracted from nuclei with 0.6 M NaCl or with 2 mM EDTA after mild digestion with micrococcal nuclease. In contrast to another chromo-domain protein, HP1, CHD1 is not preferentially located in condensed centromeric heterochromatin, even though centromeric DNA is highly enriched in (A+T)-rich tracts. Most interestingly, CHD1 is released into the cytoplasm when cells enter mitosis and is reincorporated into chromatin during telophase-cytokinesis. These observations lend credence to the idea that CHD1, like other proteins with chromo or helicase/ATPase domains, plays an important role in the determination of chromatin architecture. 相似文献
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Kwok Ki Ho Heng ZhangBarbara L. Golden Joe Ogas 《Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms》2013,1829(2):199-210
PICKLE plays a critical role in repression of genes that regulate development identity in Arabidopsis thaliana. PICKLE codes for a putative ATP-dependent chromatin remodeler that exhibits sequence similarity to members of subfamily II of animal CHD remodelers, which includes remodelers such as CHD3/Mi-2 that also restrict expression of developmental regulators. Whereas animal CHD3 remodelers are a component of the Mi-2/NuRD complex that promotes histone deacetylation, PICKLE promotes trimethylation of histone H3 lysine 27 suggesting that it acts via a distinct epigenetic pathway. Here, we examine whether PICKLE is also a member of a multisubunit complex and characterize the biochemical properties of recombinant PICKLE protein. Phylogenetic analysis indicates that PICKLE-related proteins in plants share a common ancestor with members of subfamily II of animal CHD remodelers. Biochemical characterization of PICKLE in planta, however, reveals that PICKLE primarily exists as a monomer. Recombinant PICKLE protein is an ATPase that is stimulated by ssDNA and mononucleosomes and binds to both naked DNA and mononucleosomes. Furthermore, recombinant PICKLE exhibits ATP-dependent chromatin remodeling activity. These studies demonstrate that subfamily II CHD proteins in plants, such as PICKLE, retain ATP-dependent chromatin remodeling activity but act through a mechanism that does not involve the ubiquitous Mi-2/NuRD complex. 相似文献
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Chromodomain from heterochromatin protein 1 and polycomb protein is known to be a lysine-methylated histone H3 tail-binding module. Chromo-helicase/ATPase DNA-binding protein 1 (CHD1) is an ATP-dependent chromatin remodeling factor, containing two tandem chromodomains. In human CHD1, both chromodomains are essential for specific binding to a K4 methylated histone H3 (H3 MeK4) peptide and are found to bind cooperatively in the crystal structure. For the budding yeast homologue, Chd1, the second but not the first chromodomain was once reported to bind to an H3 MeK4 peptide. Here, we reveal that neither the second chromodomain nor a region containing tandem chromodomains from yeast Chd1 bind to any lysine-methylated or arginine-methylated histone peptides that we examined. In addition, we examined the structures of the chromodomains from Chd1 by NMR. Although the tertiary structure of the region containing tandem chromodomains could not be obtained, the secondary structure deduced from NMR is well conserved in the tertiary structures of the corresponding first and second chromodomains determined individually by NMR. Both chromodomains of Chd1 demonstrate a structure similar to that of the corresponding part of CHD1, consisting of a three-stranded beta-sheet followed by a C-terminal alpha-helix. However, an additional helix between the first and second beta-strands, which is found in both of the first chromodomains of Chd1 and CHD1, is positioned in an entirely different manner in Chd1 and CHD1. In human CHD1 this helix forms the peptide-binding site. The amino acid sequences of the chromodomains could be well aligned on the basis of these structures. The alignment showed that yeast Chd1 lacks several key functional residues, which are responsible for specific binding to a methylated lysine residue in other chromodomains. Chd1 is likely to have no binding affinity for any H3 MeK peptide, as found in other chromodomain proteins. 相似文献
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Judith W. Bergs Nina Neuendorff Godfried van der Heijden Evelyne Wassenaar Peter Rexin Hans-Peter Els?sser Roland Moll Willy M. Baarends Alexander Brehm 《PloS one》2014,9(5)
ATP-dependent nucleosome remodelers of the CHD family play important roles in chromatin regulation during development and differentiation. The ubiquitously expressed CHD3 and CHD4 proteins are essential for stem cell function and serve to orchestrate gene expression in different developmental settings. By contrast, the closely related CHD5 is predominantly expressed in neural tissue and its role is believed to be restricted to neural differentiation. Indeed, loss of CHD5 contributes to neuroblastoma. In this study, we first demonstrate that CHD5 is a nucleosome-stimulated ATPase. We then compare CHD3/4 and CHD5 expression in mouse brain and show that CHD5 expression is restricted to a subset of cortical and hippocampal neurons whereas CHD3/4 expression is more widespread. We also uncover high levels of CHD5 expression in testis. CHD5 is transiently expressed in differentiating germ cells. Expression is first detected in nuclei of post-meiotic round spermatids, reaches a maximum in stage VIII spermatids and then falls to undetectable levels in stage IX spermatids. Surprisingly, CHD3/4 and CHD5 show complementary expression patterns during spermatogenesis with CHD3/4 levels progressively decreasing as CHD5 expression increases. In spermatocytes, CHD3/4 localizes to the pseudoautosomal region, the X centromeric region and then spreads into the XY body chromatin. In postmeiotic cells, CHD5 colocalises with macroH2A1.2 in association with centromeres and part of the Y chromosome. The subnuclear localisations of CHD4 and CHD5 suggest specific roles in regulation of sex chromosome chromatin and pericentromeric chromatin structure prior to the histone-protamine switch. 相似文献
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Jessica C. Liu Catarina G. Ferreira Timur Yusufzai 《The Journal of biological chemistry》2015,290(1):25-34
Chromodomain helicase DNA-binding protein 2 (CHD2) is an ATPase and a member of the SNF2-like family of helicase-related enzymes. Although deletions of CHD2 have been linked to developmental defects in mice and epileptic disorders in humans, little is known about its biochemical and cellular activities. In this study, we investigate the ATP-dependent activity of CHD2 and show that CHD2 catalyzes the assembly of chromatin into periodic arrays. We also show that the N-terminal region of CHD2, which contains tandem chromodomains, serves an auto-inhibitory role in both the DNA-binding and ATPase activities of CHD2. While loss of the N-terminal region leads to enhanced chromatin-stimulated ATPase activity, the N-terminal region is required for ATP-dependent chromatin remodeling by CHD2. In contrast, the C-terminal region, which contains a putative DNA-binding domain, selectively senses double-stranded DNA of at least 40 base pairs in length and enhances the ATPase and chromatin remodeling activities of CHD2. Our study shows that the accessory domains of CHD2 play central roles in both regulating the ATPase domain and conferring selectivity to chromatin substrates. 相似文献
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DMRP, an ABC transporter encoded by the dMRP/CG6214 gene, is the Drosophila melanogaster orthologue of the “long” human multidrug resistance-associated proteins (MRP1/ABCC1, MRP2/ABCC2, MRP3/ABCC3, MRP6/ABCC6, and MRP7/ABCC10). In order to provide a detailed biochemical characterisation we expressed DMRP in Sf9 insect cell membranes. We demonstrated DMRP as a functional orthologue of its human counterparts capable of transporting several human MRP substrates like β-estradiol 17-β-d-glucuronide, leukotriene C4, calcein, fluo3 and carboxydichlorofluorescein. Unexpectedly, we found DMRP to exhibit an extremely high turnover rate for the substrate transport as compared to its human orthologues. Furthermore, DMRP showed remarkably high basal ATPase activity (68-75 nmol Pi/mg membrane protein/min), which could be further stimulated by probenecid and the glutathione conjugate of N-ethylmaleimide. Surprisingly, this high level basal ATPase activity was inhibited by the transported substrates. We discussed this phenomenon in the light of a potential endogenous substrate (or activator) present in the Sf9 membrane. 相似文献
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Archaeal replication machinery represents a core version of this in eukaryotes. The crenarchaeon Sulfolobus solfataricus has the potential to be a powerful model system to understand the central mechanism of eukaryotic DNA replication because it contains three active origins of replication and three eukaryote-like Orc1/Cdc6 proteins (SsoCdc6-1, SsoCdc6-2, and SsoCdc6-3). In this study, we investigate the DNA-binding activities of the N-terminal AAA+ ATPase domains of these Orc1/Cdc6 proteins, including their functional interactions with the other SsoCdc6 proteins, on duplex DNA substrates derived from the origins of S. solfataricus. We showed that the ATPase domain of SsoCdc6-2 retained to a great extent the origin DNA-binding activity, and likewise maintained its stimulating effect on SsoCdc6-3. Second, the ATPase domain of SsoCdc6-1, which also stimulated the DNA-binding ability of SsoCdc6-3, demonstrated a significantly improved DNA-binding activity at the forked substrate, but only showed a very weak ability towards the blunt DNA. Third, the ATPase domain of SsoCdc6-3, although having lost much of its DNA-binding activity from the origin, inhibited both SsoCdc6-1 and SsoCdc6-2. These imply that the N-terminal AAA+ ATPase domain of archaeal Orc1/Cdc6 protein could be differentially involved in origin recognition during DNA replication initiation even if lacking conventional C-terminal winged helix DNA-binding elements. Our findings further propose that conserved AAA+ ATPase domains of Orc1/Cdc6 proteins determine their defined and coordinated functions not only in the archaeon species but also in eukaryotes during the early events of DNA replication. 相似文献
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The DNA-binding domain of the Chd1 chromatin-remodelling enzyme contains SANT and SLIDE domains 总被引:1,自引:0,他引:1
The ATP-dependent chromatin-remodelling enzyme Chd1 is a 168-kDa protein consisting of a double chromodomain, Snf2-related ATPase domain, and a C-terminal DNA-binding domain. Here, we show the DNA-binding domain is required for Saccharomyces cerevisiae Chd1 to bind and remodel nucleosomes. The crystal structure of this domain reveals the presence of structural homology to SANT and SLIDE domains previously identified in ISWI remodelling enzymes. The presence of these domains in ISWI and Chd1 chromatin-remodelling enzymes may provide a means of efficiently harnessing the action of the Snf2-related ATPase domain for the purpose of nucleosome spacing and provide an explanation for partial redundancy between these proteins. Site directed mutagenesis was used to identify residues important for DNA binding and generate a model describing the interaction of this domain with DNA. Through inclusion of Chd1 sequences in homology searches SLIDE domains were identified in CHD6-9 proteins. Point mutations to conserved amino acids within the human CHD7 SLIDE domain have been identified in patients with CHARGE syndrome. 相似文献
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CHD7 is a member of the chromodomain helicase DNA binding domain (CHD) family of ATP-dependent chromatin remodelling enzymes. It is mutated in CHARGE syndrome, a multiple congenital anomaly condition. CHD7 is one of a subset of CHD proteins, unique to metazoans that contain the BRK domain, a protein module also found in the Brahma/BRG1 family of helicases. We describe here the NMR solution structure of the two BRK domains of CHD7. Each domain has a compact betabetaalphabeta fold. The second domain has a C-terminal extension consisting of two additional helices. The structure differs from those of other domains present in chromatin-associated proteins. 相似文献
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VPS4 proteins are AAA+ ATPases required to form multivesicular bodies, release viral particles, and complete cytokinesis. They act by disassembling ESCRT-III heteropolymers during or after their proposed function in membrane scission. Here we show that purified human VPS4A is essentially inactive but can be stimulated to hydrolyze ATP by ESCRT-III proteins in a reaction that requires both their previously defined MIT interacting motifs and ∼50 amino acids of the adjacent sequence. Importantly, C-terminal fragments of all ESCRT-III proteins tested, including CHMP2A, CHMP1B, CHMP3, CHMP4A, CHMP6, and CHMP5, activated VPS4A suggesting that it disassembles ESCRT-III heteropolymers by affecting each component protein. VPS4A is thought to act as a ring-shaped cylindrical oligomer like other AAA+ ATPases, but this has been difficult to directly demonstrate. We found that concentrating His6-VPS4A on liposomes containing Ni2+-nitrilotriacetic acid-tagged lipid increased ATP hydrolysis, confirming the importance of inter-subunit interactions for activity. We also found that mutating pore loops expected to line the center of a cylindrical oligomer changed the response of VPS4A to ESCRT-III proteins. Based on these data, we propose that ESCRT-III proteins facilitate assembly of functional but transient VPS4A oligomers and interact with sequences inside the pore of the assembled enzyme. Deleting the N-terminal MIT domain and adjacent linker from VPS4A increased both basal and liposome-enhanced ATPase activity, indicating that these elements play a role in autoinhibiting VPS4A until it encounters ESCRT-III proteins. These findings reveal new ways in which VPS4 activity is regulated and specifically directed to ESCRT-III polymers. 相似文献
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Nucleosome-remodelling factors containing the ATPase ISWI, such as ACF, render DNA in chromatin accessible by promoting the sliding of histone octamers. Although the ATP-dependent repositioning of mononucleosomes is readily observable in vitro, it is unclear to which extent nucleosomes can be moved in physiological chromatin, where neighbouring nucleosomes, linker histones and the folding of the nucleosomal array restrict mobility. We assembled arrays consisting of 12 nucleosomes or 12 chromatosomes (nucleosomes plus linker histone) from defined components and subjected them to remodelling by ACF or the ATPase CHD1. Both factors increased the access to DNA in nucleosome arrays. ACF, but not CHD1, catalysed profound movements of nucleosomes throughout the array, suggesting different remodelling mechanisms. Linker histones inhibited remodelling by CHD1. Surprisingly, ACF catalysed significant repositioning of entire chromatosomes in chromatin containing saturating levels of linker histone H1. H1 inhibited the ATP-dependent generation of DNA accessibility by only about 50%. This first demonstration of catalysed chromatosome movements suggests that the bulk of interphase euchromatin may be rendered dynamic by dedicated nucleosome-remodelling factors. 相似文献
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Protein supra-domains are defined as recurring arrangements of two or three domains present adjacent to each other along a polypeptide chain. Such combinations have novel functions beyond those of the individual partner domains that compose them, which can exist in isolation. Here, we describe a new type of large supra-domain (∼ 360 residues) in which one of the component partners (∼ 200 residues) appears to be incapable of existing in a context other than immediately adjacent to the C-terminus of the well-characterized Hsp90-like ATPase domain. We found that this supra-domain has a broad phylogenetic distribution, with examples in Archaea, Bacteria, and Eukarya. There is strong selective pressure for this arrangement to occur as part of repeated regions of unprecedented length. We identified multiple strategies of convergent evolution to attain such configurations. In humans, this supra-domain is present in triplicate at the N-terminus of the protein sacsin (4579 residues), mutated in the neurodegenerative disorder known as spastic ataxia of Charlevoix-Saguenay, and thus, we termed it “sacsin repeating region” (SRR). Biochemical characterization demonstrated that SRRs possess ATPase activity, which appears to be a requirement for sacsin function, as a disease-causing mutation leads to an alternate conformation completely incapable of hydrolyzing ATP. We also found evidence of a convergent evolutionary strategy to place SRRs in proteins containing C-terminal J domains, which we demonstrated here to be capable of stimulating the intrinsic ATPase activity of Hsp70. Our sequence and biochemical analyses indicate that SRRs necessitate nucleotide hydrolysis for their function, provided by the common Hsp90 ATPase domain, which, when coupled to the unique adjacent sequence, may give rise to a novel activity related to protein quality control. 相似文献
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Natalie K. Horvat Heather Armstrong Brian L. Lee Rebecca Mercier Annemarie Wolmarans Jacob Knowles Leo Spyracopoulos Paul LaPointe 《Journal of molecular biology》2014
Heat shock protein 90 (Hsp90) is a molecular chaperone that plays a central role in maintaining cellular homeostasis by facilitating activation of a large number of client proteins. ATP-dependent client activation by Hsp90 is tightly regulated by a host of co-chaperone proteins that control progression through the activation cycle. ATPase stimulation of Hsp90 by Aha1p requires a conserved RKxK motif that interacts with the catalytic loop of Hsp90. In this study, we explore the role of this RKxK motif in the biological and biochemical properties of Hch1p. We found that this motif is required for Hch1p-mediated ATPase stimulation in vitro, but mutations that block stimulation do not impair the action of Hch1p in vivo. This suggests that the biological function of Hch1p is not directly linked to ATPase stimulation. Moreover, a mutation in the catalytic loop of Hsp90 specifically impairs ATPase stimulation by Aha1p but not by Hch1p. Our work here suggests that both Hch1p and Aha1p regulate Hsp90 function through interaction with the catalytic loop but do so in different ways. 相似文献
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To examine the expressed gene profile during encystation of Acanthamoeba castellanii Castellani, we used differentially expressed gene (DGE) screening by RT-PCR with 20 sets of random primers. From this analysis, we found that approximately 16 genes showed upregulation during encystation. We chose 6 genes, which had relatively higher expression levels, for further investigation. Based on homology search in database, DEG2 showed 55% of similarity with xylose isomerase, DEG9 showed 37% of similarity with Na P-type ATPase, and DEG14 showed 77% of similarity with subtilisin-like serine proteinase. DEG3 and DEG26 were identified as hypothetical proteins and DEG25 exhibited no significant similarity to any known protein. Encystation of Acanthamoeba has been suggested to be a process to resist adverse environmental or nutritional conditions. Further characterization studies of these genes may provide us with more information on the encystation mechanism of Acanthamoeba. 相似文献
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Reactive oxygen species (ROS) have the potential to damage cellular components, such as protein, resulting in loss of function and structural alteration of proteins. The oxidative process affects a variety of side amino acid groups, some of which are converted to carbonyl compounds. We have previously shown that a prostaglandin D2 metabolite, 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2), is the potent inducer of intracellular oxidative stress on human neuroblastoma SH-SY5Y cells [Kondo, M., Oya-Ito, T., Kumagai, T., Osawa, T., and Uchida, K. (2001) Cyclopentenone prostaglandins as potential inducers of intracellular oxidative stress, J. Biol. Chem. 276, 12076-12083]. In the present study, to elucidate the molecular mechanism underlying the oxidative stress-mediated cell degeneration, we analyzed the protein carbonylation on SH-SY5Y cells when these cells were submitted to an endogenous inducer of ROS production. Upon exposure of SH-SY5Y cells to this endogenous electrophile, we observed significant accumulation of protein carbonyls within the cells. Proteomic analysis of oxidation-sensitive proteins showed that the major intracellular target of protein carbonylation was one of the regulatory subunits in 26 S proteasome, S6 ATPase. Accompanied by a dramatic increase in protein carbonyls within S6 ATPase, the electrophile-induced oxidative stress exerted a significant decrease in the S6 ATPase activities and a decreased ability of the 26 S proteasome to degrade substrates. Moreover, in vitro oxidation of 26 S proteasome with a metal-catalyzed oxidation system also confirmed that S6 ATPase represents the most oxidation-sensitive subunit in the proteasome. These and the observation that down-regulation of S6 ATPase by RNA interference resulted in the enhanced accumulation of ubiquitinated proteins suggest that S6 ATPase is a molecular target of ROS under conditions of electrophile-induced oxidative stress and that oxidative modification of this regulatory subunit of proteasome may be functionally associated with the altered recognition and degradation of proteasomal substrates in the cells. 相似文献