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1.
Mol Cell ; 41(6): 672-81, 2011 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-21419342

RESUMO

Heat shock protein 90 (Hsp90) is an essential molecular chaperone whose activity is regulated not only by cochaperones but also by distinct posttranslational modifications. We report here that casein kinase 2 phosphorylates a conserved threonine residue (T22) in α helix-1 of the yeast Hsp90 N-domain both in vitro and in vivo. This α helix participates in a hydrophobic interaction with the catalytic loop in Hsp90's middle domain, helping to stabilize the chaperone's ATPase-competent state. Phosphomimetic mutation of this residue alters Hsp90 ATPase activity and chaperone function and impacts interaction with the cochaperones Aha1 and Cdc37. Overexpression of Aha1 stimulates the ATPase activity, restores cochaperone interactions, and compensates for the functional defects of these Hsp90 mutants.


Assuntos
Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Treonina/metabolismo , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Chaperoninas/química , Chaperoninas/genética , Chaperoninas/metabolismo , Proteínas Fúngicas/genética , Proteínas de Choque Térmico HSP90/genética , Humanos , Chaperonas Moleculares/genética , Fosforilação , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
2.
Proc Natl Acad Sci U S A ; 113(32): 9009-14, 2016 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-27466404

RESUMO

The serine/threonine phosphatase protein phosphatase 5 (PP5) regulates hormone- and stress-induced cellular signaling by association with the molecular chaperone heat shock protein 90 (Hsp90). PP5-mediated dephosphorylation of the cochaperone Cdc37 is essential for activation of Hsp90-dependent kinases. However, the details of this mechanism remain unknown. We determined the crystal structure of a Cdc37 phosphomimetic peptide bound to the catalytic domain of PP5. The structure reveals PP5 utilization of conserved elements of phosphoprotein phosphatase (PPP) structure to bind substrate and provides a template for many PPP-substrate interactions. Our data show that, despite a highly conserved structure, elements of substrate specificity are determined within the phosphatase catalytic domain itself. Structure-based mutations in vivo reveal that PP5-mediated dephosphorylation is required for kinase and steroid hormone receptor release from the chaperone complex. Finally, our data show that hyper- or hypoactivity of PP5 mutants increases Hsp90 binding to its inhibitor, suggesting a mechanism to enhance the efficacy of Hsp90 inhibitors by regulation of PP5 activity in tumors.


Assuntos
Proteínas Nucleares/química , Fosfoproteínas Fosfatases/química , Domínio Catalítico , Proteínas de Ciclo Celular/química , Chaperoninas/química , Cristalização , Proteínas de Choque Térmico HSP90/fisiologia , Proteínas Nucleares/fisiologia , Fosfoproteínas Fosfatases/fisiologia , Fosforilação , Especificidade por Substrato
3.
Plant Cell ; 26(4): 1746-1763, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24748042

RESUMO

Plant BZR1-BAM transcription factors contain a ß-amylase (BAM)-like domain, characteristic of proteins involved in starch breakdown. The enzyme-derived domains appear to be noncatalytic, but they determine the function of the two Arabidopsis thaliana BZR1-BAM isoforms (BAM7 and BAM8) during transcriptional initiation. Removal or swapping of the BAM domains demonstrates that the BAM7 BAM domain restricts DNA binding and transcriptional activation, while the BAM8 BAM domain allows both activities. Furthermore, we demonstrate that BAM7 and BAM8 interact on the protein level and cooperate during transcriptional regulation. Site-directed mutagenesis of residues in the BAM domain of BAM8 shows that its function as a transcriptional activator is independent of catalysis but requires an intact substrate binding site, suggesting it may bind a ligand. Microarray experiments with plants overexpressing truncated versions lacking the BAM domain indicate that the pseudo-enzymatic domain increases selectivity for the preferred cis-regulatory element BBRE (BZR1-BAM Responsive Element). Side specificity toward the G-box may allow crosstalk to other signaling networks. This work highlights the importance of the enzyme-derived domain of BZR1-BAMs, supporting their potential role as metabolic sensors.

4.
Mol Cell ; 31(6): 886-95, 2008 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-18922470

RESUMO

Activation of protein kinase clients by the Hsp90 system is mediated by the cochaperone protein Cdc37. Cdc37 requires phosphorylation at Ser13, but little is known about the regulation of this essential posttranslational modification. We show that Ser13 of uncomplexed Cdc37 is phosphorylated in vivo, as well as in binary complex with a kinase (C-K), or in ternary complex with Hsp90 and kinase (H-C-K). Whereas pSer13-Cdc37 in the H-C-K complex is resistant to nonspecific phosphatases, it is efficiently dephosphorylated by the chaperone-targeted protein phosphatase 5 (PP5/Ppt1), which does not affect isolated Cdc37. We show that Cdc37 and PP5/Ppt1 associate in Hsp90 complexes in yeast and in human tumor cells, and that PP5/Ppt1 regulates phosphorylation of Ser13-Cdc37 in vivo, directly affecting activation of protein kinase clients by Hsp90-Cdc37. These data reveal a cyclic regulatory mechanism for Cdc37, in which its constitutive phosphorylation is reversed by targeted dephosphorylation in Hsp90 complexes.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Chaperoninas/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas Quinases/metabolismo , Especificidade de Anticorpos , Quinase 4 Dependente de Ciclina/metabolismo , Ativação Enzimática , Células HCT116 , Humanos , Modelos Biológicos , Mutação/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Fosfosserina/metabolismo , Ligação Proteica , Proteína Fosfatase 1/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Saccharomyces cerevisiae , Especificidade por Substrato
5.
Plant Cell ; 23(4): 1391-403, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21487098

RESUMO

Plants contain ß-amylase-like proteins (BAMs; enzymes usually associated with starch breakdown) present in the nucleus rather than targeted to the chloroplast. They possess BRASSINAZOLE RESISTANT1 (BZR1)-type DNA binding domains--also found in transcription factors mediating brassinosteroid (BR) responses. The two Arabidopsis thaliana BZR1-BAM proteins (BAM7 and BAM8) bind a cis-regulatory element that both contains a G box and resembles a BR-responsive element. In protoplast transactivation assays, these BZR1-BAMs activate gene expression. Structural modeling suggests that the BAM domain's glucan binding cleft is intact, but the recombinant proteins are at least 1000 times less active than chloroplastic ß-amylases. Deregulation of BZR1-BAMs (the bam7bam8 double mutant and BAM8-overexpressing plants) causes altered leaf growth and development. Of the genes upregulated in plants overexpressing BAM8 and downregulated in bam7bam8 plants, many carry the cis-regulatory element in their promoters. Many genes that respond to BRs are inversely regulated by BZR1-BAMs. We propose a role for BZR1-BAMs in controlling plant growth and development through crosstalk with BR signaling. Furthermore, we speculate that BZR1-BAMs may transmit metabolic signals by binding a ligand in their BAM domain, although diurnal changes in the concentration of maltose, a candidate ligand produced by chloroplastic ß-amylases, do not influence their transcription factor function.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/crescimento & desenvolvimento , Brotos de Planta/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo , beta-Amilase/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/química , Sequência de Bases , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Glucanos/metabolismo , Hidrólise , Modelos Biológicos , Dados de Sequência Molecular , Proteínas Mutantes/isolamento & purificação , Proteínas Mutantes/metabolismo , Mutação/genética , Proteínas Nucleares/metabolismo , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Brotos de Planta/enzimologia , Ligação Proteica , Estrutura Terciária de Proteína , Elementos de Resposta/genética , Transativadores/metabolismo , Fatores de Transcrição/química , beta-Amilase/química
6.
Nat Struct Mol Biol ; 30(9): 1346-1356, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37653239

RESUMO

Telomeres replicated by leading-strand synthesis lack the 3' overhang required for telomere protection. Surprisingly, resection of these blunt telomeres is initiated by the telomere-specific 5' exonuclease Apollo rather than the Mre11-Rad50-Nbs1 (MRN) complex, the nuclease that acts at DNA breaks. Without Apollo, leading-end telomeres undergo fusion, which, as demonstrated here, is mediated by alternative end joining. Here, we show that DNA-PK and TRF2 coordinate the repression of MRN at blunt mouse telomeres. DNA-PK represses an MRN-dependent long-range resection, while the endonuclease activity of MRN-CtIP, which could cleave DNA-PK off of blunt telomere ends, is inhibited in vitro and in vivo by the iDDR of TRF2. AlphaFold-Multimer predicts a conserved association of the iDDR with Rad50, potentially interfering with CtIP binding and MRN endonuclease activation. We propose that repression of MRN-mediated resection is a conserved aspect of telomere maintenance and represents an ancient feature of DNA-PK and the iDDR.


Assuntos
Quebras de DNA , Proteína Quinase Ativada por DNA , Animais , Camundongos , Endonucleases , Telômero , DNA
7.
Nature ; 440(7087): 1013-7, 2006 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-16625188

RESUMO

Hsp90 (heat shock protein of 90 kDa) is a ubiquitous molecular chaperone responsible for the assembly and regulation of many eukaryotic signalling systems and is an emerging target for rational chemotherapy of many cancers. Although the structures of isolated domains of Hsp90 have been determined, the arrangement and ATP-dependent dynamics of these in the full Hsp90 dimer have been elusive and contentious. Here we present the crystal structure of full-length yeast Hsp90 in complex with an ATP analogue and the co-chaperone p23/Sba1. The structure reveals the complex architecture of the 'closed' state of the Hsp90 chaperone, the extensive interactions between domains and between protein chains, the detailed conformational changes in the amino-terminal domain that accompany ATP binding, and the structural basis for stabilization of the closed state by p23/Sba1. Contrary to expectations, the closed Hsp90 would not enclose its client proteins but provides a bipartite binding surface whose formation and disruption are coupled to the chaperone ATPase cycle.


Assuntos
Proteínas de Choque Térmico HSP90/química , Chaperonas Moleculares/química , Nucleotídeos/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Ativação Enzimática , Proteínas de Choque Térmico HSP90/metabolismo , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Nucleotídeos/metabolismo , Conformação Proteica , Proteínas de Saccharomyces cerevisiae/metabolismo
8.
Biochem J ; 413(2): 261-8, 2008 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-18412542

RESUMO

Tah1 [TPR (tetratricopeptide repeat)-containing protein associated with Hsp (heat-shock protein) 90] has been identified as a TPR-domain protein. TPR-domain proteins are involved in protein-protein interactions and a number have been characterized that interact either with Hsp70 or Hsp90, but a few can bind both chaperones. Independent studies suggest that Tah1 interacts with Hsp90, but whether it can also interact with Hsp70/Ssa1 has not been investigated. Amino-acid-sequence alignments suggest that Tah1 is most similar to the TPR2b domain of Hop (Hsp-organizing protein) which when mutated reduces binding to both Hsp90 and Hsp70. Our alignments suggest that there are three TPR-domain motifs in Tah1, which is consistent with the architecture of the TPR2b domain. In the present study we find that Tah1 is specific for Hsp90, and is able to bind tightly the yeast Hsp90, and the human Hsp90alpha and Hsp90beta proteins, but not the yeast Hsp70 Ssa1 isoform. Tah1 acheives ligand discrimination by favourably binding the methionine residue in the conserved MEEVD motif (Hsp90) and positively discriminating against the first valine residue in the VEEVD motif (Ssa1). In the present study we also show that Tah1 can affect the ATPase activity of Hsp90, in common with some other TPR-domain proteins.


Assuntos
Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/fisiologia , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Motivos de Aminoácidos , Calorimetria , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/química , Humanos , Cinética , Ligantes , Chaperonas Moleculares/metabolismo , Mutação , Ligação Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Ultracentrifugação
9.
Artigo em Inglês | MEDLINE | ID: mdl-31428588

RESUMO

Invasive, motile life cycle stages (zoites) of apicomplexan parasites possess a cortical membrane skeleton composed of intermediate filaments with roles in zoite morphogenesis, tensile strength and motility. Its building blocks include a family of proteins called alveolins that are characterized by conserved "alveolin" domains composed of tandem repeat sequences. A subset of alveolins possess additional conserved domains that are structurally unrelated and the roles of which remain unclear. In this structure-function analysis we investigated the functional contributions of the "alveolin" vs. "non-alveolin" domains of IMC1h, a protein expressed in the ookinete and sporozoite life cycle stages of malaria parasites and essential for parasite transmission. Using allelic replacement in Plasmodium berghei, we show that the alveolin domain is responsible for targeting IMC1h to the membrane skeleton and, consequently, its deletion from the protein results in loss of function manifested by abnormally-shaped ookinetes and sporozoites with reduced tensile strength, motility and infectivity. Conversely, IMC1h lacking its non-alveolin conserved domain is correctly targeted and can facilitate tensile strength but not motility. Our findings support the concept that the alveolin module contains the properties for filament formation, and show for the first time that tensile strength makes an important contribution to zoite infectivity. The data furthermore provide new insight into the underlying molecular mechanisms of motility, indicating that tensile strength is mechanistically uncoupled from locomotion, and pointing to a role of the non-alveolin domain in the motility-enhancing properties of IMC1h possibly by engaging with the locomotion apparatus.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Metaloendopeptidases/metabolismo , Plasmodium berghei/citologia , Plasmodium berghei/fisiologia , Proteínas de Protozoários/metabolismo , Animais , Sequência Conservada , Proteínas do Citoesqueleto/genética , Modelos Animais de Doenças , Locomoção , Malária/parasitologia , Malária/patologia , Metaloendopeptidases/genética , Camundongos , Plasmodium berghei/genética , Plasmodium berghei/patogenicidade , Domínios Proteicos , Transporte Proteico , Proteínas de Protozoários/genética , Deleção de Sequência
10.
Cell Rep ; 24(3): 744-754, 2018 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-30021170

RESUMO

The centromere binding factor 3 (CBF3) complex binds the third centromere DNA element in organisms with point centromeres, such as S. cerevisiae. It is an essential complex for assembly of the kinetochore in these organisms, as it facilitates genetic centromere specification and allows association of all other kinetochore components. We determined high-resolution structures of the core complex of CBF3 alone and in association with a monomeric construct of Ndc10, using cryoelectron microscopy (cryo-EM). We identify the DNA-binding site of the complex and present a model in which CBF3 induces a tight bend in centromeric DNA, thus facilitating assembly of the centromeric nucleosome.


Assuntos
Centrômero/metabolismo , Microscopia Crioeletrônica , DNA Fúngico/química , Proteínas de Ligação a DNA/ultraestrutura , Cinetocoros/ultraestrutura , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Sequência de Bases , Sítios de Ligação , Proteínas de Ligação a DNA/química , Cinetocoros/química , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química
11.
Sci Rep ; 7: 41626, 2017 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-28139700

RESUMO

The essential cochaperone Sgt1 recruits Hsp90 chaperone activity to a range of cellular factors including SCF E3 ubiquitin ligases and the kinetochore in eukaryotes. In these pathways Sgt1 interacts with Skp1, a small protein that heterodimerizes with proteins containing the F-box motif. We have determined the crystal structure of the interacting domains of Saccharomyces cerevisiae Sgt1 and Skp1 at 2.8 Å resolution and validated the interface in the context of the full-length proteins in solution. The BTB/POZ domain of Skp1 associates with Sgt1 via the concave surface of its TPR domain using residues that are conserved in humans. Dimerization of yeast Sgt1 occurs via an insertion that is absent from monomeric human Sgt1. We identify point mutations that disrupt dimerization and Skp1 binding in vitro and find that the interaction with Skp1 is an essential function of Sgt1 in yeast. Our data provide a structural rationale for understanding the phenotypes of temperature-sensitive Sgt1 mutants and for linking Skp1-associated proteins to Hsp90-dependent pathways.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas F-Box/química , Proteínas de Choque Térmico HSP90/química , Cinetocoros/química , Modelos Moleculares , Conformação Proteica , Proteínas Ligases SKP Culina F-Box/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Sequência Conservada , Proteínas F-Box/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Cinetocoros/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes , Proteínas Ligases SKP Culina F-Box/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
12.
Cell Rep ; 21(7): 1883-1895, 2017 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-29141220

RESUMO

The serine/threonine protein phosphatase 5 (PP5) regulates multiple cellular signaling networks. A number of cellular factors, including heat shock protein 90 (Hsp90), promote the activation of PP5. However, it is unclear whether post-translational modifications also influence PP5 phosphatase activity. Here, we show an "on/off switch" mechanism for PP5 regulation. The casein kinase 1δ (CK1δ) phosphorylates T362 in the catalytic domain of PP5, which activates and enhances phosphatase activity independent of Hsp90. Overexpression of the phosphomimetic T362E-PP5 mutant hyper-dephosphorylates substrates such as the co-chaperone Cdc37 and glucocorticoid receptor in cells. Our proteomic approach revealed that the tumor suppressor von Hippel-Lindau protein (VHL) interacts with and ubiquitinates K185/K199-PP5 for proteasomal degradation in a hypoxia- and prolyl-hydroxylation-independent manner. Finally, VHL-deficient clear cell renal cell carcinoma (ccRCC) cell lines and patient tumors exhibit elevated PP5 levels. Downregulation of PP5 causes ccRCC cells to undergo apoptosis, suggesting a prosurvival role for PP5 in kidney cancer.


Assuntos
Apoptose , Carcinoma de Células Renais/metabolismo , Glicoproteínas/metabolismo , Neoplasias Renais/metabolismo , Ubiquitinação , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Glicoproteínas/genética , Humanos , Neoplasias Renais/patologia , Fosforilação , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo
13.
Nat Commun ; 8: 14279, 2017 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-28176794

RESUMO

By moving essential body fluids and molecules, motile cilia and flagella govern respiratory mucociliary clearance, laterality determination and the transport of gametes and cerebrospinal fluid. Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder frequently caused by non-assembly of dynein arm motors into cilia and flagella axonemes. Before their import into cilia and flagella, multi-subunit axonemal dynein arms are thought to be stabilized and pre-assembled in the cytoplasm through a DNAAF2-DNAAF4-HSP90 complex akin to the HSP90 co-chaperone R2TP complex. Here, we demonstrate that large genomic deletions as well as point mutations involving PIH1D3 are responsible for an X-linked form of PCD causing disruption of early axonemal dynein assembly. We propose that PIH1D3, a protein that emerges as a new player of the cytoplasmic pre-assembly pathway, is part of a complementary conserved R2TP-like HSP90 co-chaperone complex, the loss of which affects assembly of a subset of inner arm dyneins.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Dineínas do Axonema/metabolismo , Genes Ligados ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Síndrome de Kartagener/genética , Proteínas dos Microtúbulos/genética , Chaperonas Moleculares/genética , Adolescente , Adulto , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Axonema/patologia , Criança , Pré-Escolar , Cílios/patologia , Cílios/ultraestrutura , Citoplasma/patologia , Modelos Animais de Doenças , Feminino , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Células HEK293 , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Recém-Nascido , Peptídeos e Proteínas de Sinalização Intracelular , Síndrome de Kartagener/patologia , Masculino , Microscopia Eletrônica de Transmissão , Linhagem , Filogenia , Mutação Puntual , Dobramento de Proteína , Alinhamento de Sequência , Deleção de Sequência , Motilidade dos Espermatozoides/genética , Sequenciamento do Exoma , Peixe-Zebra
14.
Structure ; 22(6): 799-800, 2014 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-24918336

RESUMO

Phosphatidylinositol-3 kinase-like kinases (PIKKs) are dependent on Hsp90 for their activation via the R2TP complex and Tel2. In this issue of Structure, Pal and colleagues present the molecular mechanism by which PIKKs are recruited to Hsp90.


Assuntos
Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a Telômeros/química , Proteínas de Ligação a Telômeros/metabolismo
16.
Methods Mol Biol ; 1008: 63-99, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23729249

RESUMO

Experimental approaches to detect, measure, and quantify protein-ligand binding, along with their theoretical bases, are described. A range of methods for detection of protein-ligand interactions is summarized. Specific protocols are provided for a nonequilibrium procedure pull-down assay, for an equilibrium direct binding method and its modification into a competition-based measurement and for steady-state measurements based on the effects of ligands on enzyme catalysis.


Assuntos
Ligantes , Simulação de Dinâmica Molecular , Proteínas/química , Autorradiografia , Sítios de Ligação , Ligação Competitiva , Catálise , Transferência Ressonante de Energia de Fluorescência , Humanos , Cinética , Medições Luminescentes , Ligação Proteica , Termodinâmica
17.
FEBS J ; 276(1): 199-209, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19032597

RESUMO

The conformationally coupled mechanism by which ATP is utilized by yeast Hsp90 is now well characterized. In contrast, ATP utilization by human Hsp90s is less well studied, and appears to operate differently. To resolve these conflicting models, we have conducted a side-by-side biochemical analysis in a series of mutant yeast and human Hsp90s that have been both mechanistically and structurally characterized with regard to the crystal structure of the yeast Hsp90 protein. We show that each monomer of the human Hsp90 dimer is mutually dependent on the other for ATPase activity. Fluorescence studies confirmed that the N-terminal domains of Hsp90beta come into close association with each other. Mutations that directly affect the conformational dynamics of the ATP-lid segment had marked effects, with T31I (yeast T22I) and A116N (yeast A107N) stimulating, and T110I (yeast T101I) inhibiting, human and yeast ATPase activity to similar extents, showing that ATP-dependent lid closure is a key rate-determining step in both systems. Mutation of residues implicated in N-terminal dimerization of yeast Hsp90 (L15R and L18R in yeast, L24R and L27R in humans) significantly reduced the ATPase activity of yeast and human Hsp90s, showing that ATP-dependent association of the N-terminal domains in the Hsp90 dimer is also essential in both systems. Furthermore, cross-linking studies of the hyper-active yeast A107N and human A116N ATP-lid mutants showed enhanced dimerization, suggesting that N-terminal association is a direct consequence of ATP binding and lid closure in both systems.


Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Citoplasma/metabolismo , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/genética , Calorimetria , Dimerização , Humanos , Cinética , Modelos Moleculares , Mutagênese , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo
18.
Development ; 135(6): 1147-56, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18256191

RESUMO

The mechanisms that regulate sarcomere assembly during myofibril formation are poorly understood. In this study, we characterise the zebrafish sloth(u45) mutant, in which the initial steps in sarcomere assembly take place, but thick filaments are absent and filamentous I-Z-I brushes fail to align or adopt correct spacing. The mutation only affects skeletal muscle and mutant embryos show no other obvious phenotypes. Surprisingly, we find that the phenotype is due to mutation in one copy of a tandemly duplicated hsp90a gene. The mutation disrupts the chaperoning function of Hsp90a through interference with ATPase activity. Despite being located only 2 kb from hsp90a, hsp90a2 has no obvious role in sarcomere assembly. Loss of Hsp90a function leads to the downregulation of genes encoding sarcomeric proteins and upregulation of hsp90a and several other genes encoding proteins that may act with Hsp90a during sarcomere assembly. Our studies reveal a surprisingly specific developmental role for a single Hsp90 gene in a regulatory pathway controlling late steps in sarcomere assembly.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/deficiência , Adenosina Trifosfatases/genética , Animais , Sequência de Bases , Sítios de Ligação , Primers do DNA/genética , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/deficiência , Proteínas de Choque Térmico HSP90/genética , Resposta ao Choque Térmico , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Mutação , Miofibrilas/metabolismo , Fenótipo , Sarcômeros/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética
19.
Plant Cell ; 20(4): 1040-58, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18390594

RESUMO

This work investigated the roles of beta-amylases in the breakdown of leaf starch. Of the nine beta-amylase (BAM)-like proteins encoded in the Arabidopsis thaliana genome, at least four (BAM1, -2, -3, and -4) are chloroplastic. When expressed as recombinant proteins in Escherichia coli, BAM1, BAM2, and BAM3 had measurable beta-amylase activity but BAM4 did not. BAM4 has multiple amino acid substitutions relative to characterized beta-amylases, including one of the two catalytic residues. Modeling predicts major differences between the glucan binding site of BAM4 and those of active beta-amylases. Thus, BAM4 probably lost its catalytic capacity during evolution. Total beta-amylase activity was reduced in leaves of bam1 and bam3 mutants but not in bam2 and bam4 mutants. The bam3 mutant had elevated starch levels and lower nighttime maltose levels than the wild type, whereas bam1 did not. However, the bam1 bam3 double mutant had a more severe phenotype than bam3, suggesting functional overlap between the two proteins. Surprisingly, bam4 mutants had elevated starch levels. Introduction of the bam4 mutation into the bam3 and bam1 bam3 backgrounds further elevated the starch levels in both cases. These data suggest that BAM4 facilitates or regulates starch breakdown and operates independently of BAM1 and BAM3. Together, our findings are consistent with the proposal that beta-amylase is a major enzyme of starch breakdown in leaves, but they reveal unexpected complexity in terms of the specialization of protein function.


Assuntos
Arabidopsis/enzimologia , Cloroplastos/enzimologia , Amido/metabolismo , beta-Amilase/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Catálise , Primers do DNA , Escherichia coli/genética , Microscopia de Fluorescência , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Homologia de Sequência de Aminoácidos , beta-Amilase/química , beta-Amilase/genética
20.
Mol Cell ; 23(5): 697-707, 2006 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-16949366

RESUMO

Activation of many protein kinases depends on their interaction with the Hsp90 molecular chaperone system. Recruitment of protein kinase clients to the Hsp90 chaperone system is mediated by the cochaperone adaptor protein Cdc37, which acts as a scaffold, simultaneously binding protein kinases and Hsp90. We have now expressed and purified an Hsp90-Cdc37-Cdk4 complex, defined its stoichiometry, and determined its 3D structure by single-particle electron microscopy. Comparison with the crystal structure of Hsp90 allows us to identify the locations of Cdc37 and Cdk4 in the complex and suggests a mechanism by which conformational changes in the kinase are coupled to the Hsp90 ATPase cycle.


Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/ultraestrutura , Chaperoninas/química , Chaperoninas/ultraestrutura , Quinase 4 Dependente de Ciclina/química , Quinase 4 Dependente de Ciclina/ultraestrutura , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/ultraestrutura , Proteínas de Ciclo Celular/isolamento & purificação , Chaperoninas/isolamento & purificação , Quinase 4 Dependente de Ciclina/isolamento & purificação , Proteínas de Choque Térmico HSP90/isolamento & purificação , Humanos , Microscopia Eletrônica , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/isolamento & purificação , Complexos Multiproteicos/ultraestrutura , Ligação Proteica
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