RESUMO
Tah1 and Pih1 are novel Hsp90 interactors. Tah1 acts as a cofactor of Hsp90 to stabilize Pih1. In yeast, Hsp90, Tah1, and Pih1 were found to form a complex that is required for ribosomal RNA processing through their effect on box C/D small nucleolar ribonucleoprotein assembly. Tah1 is a minimal tetratricopeptide repeat protein of 111 amino acid residues that binds to the C terminus of the Hsp90 molecular chaperone, whereas Pih1 consists of 344 residues of unknown fold. The NMR structure of Tah1 has been solved, and this structure shows the presence of two tetratricopeptide repeat motifs followed by a C helix and an unstructured region. The binding of Tah1 to Hsp90 is mediated by the EEVD C-terminal residues of Hsp90, which bind to a positively charged channel formed by Tah1. Five highly conserved residues, which form a two-carboxylate clamp that tightly interacts with the ultimate Asp-0 residue of the bound peptide, are also present in Tah1. Tah1 was found to bind to the C terminus of Pih1 through the C helix and the unstructured region. The C terminus of Pih1 destabilizes the protein in vitro and in vivo, whereas the binding of Tah1 to Pih1 allows for the formation of a stable complex. Based on our data, a model for an Hsp90-Tah1-Pih1 ternary complex is proposed.
Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Proteínas Nucleares/química , Oligopeptídeos/metabolismo , Ligação Proteica , Estabilidade Proteica , Estrutura Secundária de ProteínaRESUMO
Rvb1 and Rvb2 are highly conserved, essential AAA+ helicases found in a wide range of eukaryotes. The versatility of these helicases and their central role in the biology of the cell is evident from their involvement in a wide array of critical cellular complexes. Rvb1 and Rvb2 are components of the chromatin-remodeling complexes INO80, Swr-C, and BAF. They are also members of the histone acetyltransferase Tip60 complex, and the recently identified R2TP complex present in Saccharomyces cerevisiae and Homo sapiens; a complex that is involved in small nucleolar ribonucleoprotein (snoRNP) assembly. Furthermore, in humans, Rvb1 and Rvb2 have been identified in the URI prefoldin-like complex. In Drosophila, the Polycomb Repressive complex 1 contains Rvb2, but not Rvb1, and the Brahma complex contains Rvb1 and not Rvb2. Both of these complexes are involved in the regulation of growth and development genes in Drosophila. Rvbs are therefore crucial factors in various cellular processes. Their importance in chromatin remodeling, transcription regulation, DNA damage repair, telomerase assembly, mitotic spindle formation, and snoRNP biogenesis is discussed in this review.
Assuntos
Proteínas de Transporte/metabolismo , DNA Helicases/metabolismo , Animais , Proteínas de Transporte/genética , Montagem e Desmontagem da Cromatina , DNA Helicases/genética , Eucariotos/enzimologia , Humanos , Modelos Biológicos , Modelos Moleculares , Ribonucleoproteínas Nucleolares Pequenas/metabolismoRESUMO
RUVBL1 and RUVBL2 are highly conserved ATPases that belong to the AAA+ (ATPases Associated with various cellular Activities) superfamily and are involved in various complexes and cellular processes, several of which are closely linked to oncogenesis. The proteins were implicated in DNA damage signaling and repair, chromatin remodeling, telomerase activity, and in modulating the transcriptional activities of proto-oncogenes such as c-Myc and ß-catenin. Moreover, both proteins were found to be overexpressed in several different types of cancers such as breast, lung, kidney, bladder, and leukemia. Given their various roles and strong involvement in carcinogenesis, the RUVBL proteins are considered to be novel targets for the discovery and development of therapeutic cancer drugs. Here, we describe the identification of sorafenib as a novel inhibitor of the ATPase activity of human RUVBL2. Enzyme kinetics and surface plasmon resonance experiments revealed that sorafenib is a weak, mixed non-competitive inhibitor of the protein's ATPase activity. Size exclusion chromatography and small angle X-ray scattering data indicated that the interaction of sorafenib with RUVBL2 does not cause a significant effect on the solution conformation of the protein; however, the data suggested that the effect of sorafenib on RUVBL2 activity is mediated by the insertion domain in the protein. Sorafenib also inhibited the ATPase activity of the RUVBL1/2 complex. Hence, we propose that sorafenib could be further optimized to be a potent inhibitor of the RUVBL proteins.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/antagonistas & inibidores , Proteínas de Transporte/antagonistas & inibidores , DNA Helicases/antagonistas & inibidores , Sorafenibe/farmacologia , ATPases Associadas a Diversas Atividades Celulares/química , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , DNA Helicases/química , DNA Helicases/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Concentração Inibidora 50 , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Agregados Proteicos/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Sorafenibe/químicaRESUMO
The two NBDs (nucleotide-binding domains) of ABC (ATP-binding-cassette) proteins function in a complex to mediate ATPase activity and this activity has been linked to their regulated transport activity. A similar model has been proposed for CFTR (cystic fibrosis transmembrane conductance regulator), the chloride channel defective in cystic fibrosis, wherein ATP binding and hydrolysis regulate the channel gate. Recently, it was shown that the individual NBDs isolated from CFTR primarily mediate adenylate kinase activity, raising the possibility that this activity may also contribute to gating of the CFTR channel. However, this present study shows that whereas the isolated NBDs exhibit adenylate kinase activity, the full-length purified and reconstituted CFTR protein functions as an ATPase, arguing that the enzymatic activity of the NBDs is dependent on their molecular context and appropriate domain-domain assembly. As expected, the disease-causing mutant bearing a mutation in the ABC signature motif, CFTR-G551D, exhibited a markedly reduced ATPase activity. Furthermore, mutation of the putative catalytic base in CFTR caused a reduction in ATPase activity, with the CFTR-E1371Q mutant supporting a low level of residual activity. Neither of these mutants exhibited detectable adenylate kinase activity. Together, these findings support the concept that the molecular mechanism of action of CFTR is dependent on ATP binding and hydrolysis, and that the structure of prokaryotic ABC ATPases provide a useful template for understanding their mechanism of action.
Assuntos
Adenosina Trifosfatases/metabolismo , Adenilato Quinase/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/metabolismo , Adenilato Quinase/antagonistas & inibidores , Adenilato Quinase/genética , Animais , Linhagem Celular , Cloretos/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fosfatos de Dinucleosídeos/metabolismo , Humanos , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
Structural information is required to define the molecular basis for chloride conduction through CFTR (cystic fibrosis transmembrane conductance regulator). Towards this goal, we expressed MSD2, the second of the two MSDs (membrane-spanning domains) of CFTR, encompassing residues 857-1158 in Sf9 cells using the baculovirus system. In Sf9 plasma membranes, MSD2 migrates as expected for a dimer in non-dissociative PAGE, and confers the appearance of an anion permeation pathway suggesting that dimeric MSD2 mediates anion flux. To assess directly the function and quaternary structure of MSD2, we purified it from Sf9 cells by virtue of its polyhistidine tag and nickel affinity. Reconstitution of MSD2 into liposomes conferred a 4,4'-di-isothiocyanostilbene-2,2'-disulphonate-inhibitable, chloride-selective electrodiffusion pathway. Further, this activity is probably mediated directly by MSD2 as reaction of its single cysteine residue (Cys866) with the thiol modifying reagent, N(alpha)(3-maleimidylpropionyl)biocytin, inhibited chloride flux. Only MSD2 dimers were labelled by N(alpha)(3-maleimidylpropionyl)biocytin, supporting the idea that only dimeric MSD2 can mediate anion flux. As a further test of this hypothesis, we conducted a second purification procedure, wherein purified dimeric and monomeric MSD2 proteins were reconstituted separately. Only proteoliposomes containing stable MSD2 dimers mediated chloride electrodiffusion, providing direct evidence that dimeric MSD2 mediates chloride channel function. In summary, we have shown that the second membrane domain of CFTR can be purified and functionally reconstituted as a chloride channel, providing a tool for probing the structural basis of chloride conduction through CFTR.
Assuntos
Canais de Cloreto/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Animais , Ânions/metabolismo , Sítios de Ligação/genética , Linhagem Celular , Membrana Celular/metabolismo , Canais de Cloreto/química , Canais de Cloreto/isolamento & purificação , Cromatografia por Troca Iônica/métodos , Dicroísmo Circular , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Difusão , Dimerização , Eletroforese em Gel de Poliacrilamida , Eletrofisiologia , Lipossomos/química , Lipossomos/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fosfolipídeos/química , Fosfolipídeos/metabolismo , SpodopteraRESUMO
Lecithin-cholesterol acyltransferase deficiency is frequently associated with hypertriglyceridemia (HTG) in animal models and humans. We investigated the mechanism of HTG in the ldlr-/- x lcat-/- (double knockout (dko)) mice using the ldlr-/- x lcat+/+ (knock-out (ko)) littermates as control. Mean fasting triglyceride (TG) levels in the dko mice were elevated 1.75-fold compared with their controls (p < 0.002). Both the very low density lipoprotein and the low density lipoprotein/intermediate density lipoprotein fractions separated by fast protein liquid chromatography were TG-enriched in the dko mice. In vitro lipolysis assay revealed that the dko mouse very low density lipoprotein (d < 1.019 g/ml) fraction separated by ultracentrifugation was a more efficient substrate for lipolysis by exogenous bovine lipoprotein lipase. Post-heparin lipoprotein lipase activity was reduced by 61% in the dko mice. Hepatic TG production rate, determined after intravenous Triton WR1339 injection, was increased 8-fold in the dko mice. Hepatic mRNA levels of sterol regulatory element binding protein-1 (srebp-1) and its target genes acetyl-CoA carboxylase-1 (acc-1), fatty acid synthase (fas), and stearoyl-CoA desaturase-1 (scd-1) were significantly elevated in the dko mice compared with the ko control. The hepatic mRNA levels of LXRalpha (lxralpha) and its target genes including angiopoietin-like protein 3 (angptl-3) in the dko mice were unchanged. Fasting glucose and insulin levels were reduced by 31 and 42%, respectively in the dko mice, in conjunction with a 49% reduction in hepatic pepck-1 mRNA (p = 0.014). Both the HTG and the improved fasting glucose phenotype seen in the dko mice are at least in part attributable to an up-regulation of the hepatic srebp-1c gene.