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1.
Cancer Sci ; 110(10): 3275-3287, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31368616

RESUMO

p97/VCP is an endoplasmic reticulum (ER)-associated protein that belongs to the AAA (ATPases associated with diverse cellular activities) ATPase family. It has a variety of cellular functions including ER-associated protein degradation, autophagy, and aggresome formation. Recent studies have shown emerging roles of p97/VCP and its potential as a therapeutic target in several cancer subtypes including multiple myeloma (MM). We conducted a cell-based compound screen to exploit novel small compounds that have cytotoxic activity in myeloma cells. Among approximately 2000 compounds, OSSL_325096 showed relatively strong antiproliferative activity in MM cell lines (IC50 , 100-500 nmol/L). OSSL_325096 induced apoptosis in myeloma cell lines, including a bortezomib-resistant cell line and primary myeloma cells purified from patients. Accumulation of poly-ubiquitinated proteins, PERK, CHOP, and IREα, was observed in MM cell lines treated with OSSL_325096, suggesting that it induces ER stress in MM cells. OSSL_325096 has a similar chemical structure to DBeQ, a known p97/VCP inhibitor. Knockdown of the gene encoding p97/VCP induced apoptosis in myeloma cells, accompanied by accumulation of poly-ubiquitinated protein. IC50 of OSSL_325096 to myeloma cell lines were found to be lower (0.1-0.8 µmol/L) than those of DBeQ (2-5 µmol/L). In silico protein-drug-binding simulation suggested possible binding of OSSL_325096 to the ATP binding site in the D2 domain of p97/VCP. In cell-free ATPase assays, OSSL_325096 showed dose-dependent inhibition of p97/VCP ATPase activity. Finally, OSSL_325096 inhibited the growth of subcutaneous myeloma cell tumors in vivo. The present data suggest that OSSL_325096 exerts anti-myeloma activity, at least in part through p97/VCP inhibition.


Assuntos
Adenosina Trifosfatases/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Inibidores Enzimáticos/administração & dosagem , Mieloma Múltiplo/tratamento farmacológico , Proteínas Nucleares/metabolismo , Bibliotecas de Moléculas Pequenas/administração & dosagem , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/química , Animais , Sítios de Ligação , Bortezomib/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático , Endorribonucleases/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Camundongos , Modelos Moleculares , Mieloma Múltiplo/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/química , Proteínas Serina-Treonina Quinases/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Fator de Transcrição CHOP/metabolismo , Ubiquitinação , Ensaios Antitumorais Modelo de Xenoenxerto , eIF-2 Quinase/metabolismo
2.
Nucleic Acids Res ; 47(10): 5260-5275, 2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-30997503

RESUMO

Eukaryotic translation initiation requires unwinding of secondary structures in the 5'-untranslated region of mRNA. The DEAD-box helicase eIF4A is thought to unwind structural elements in the 5'-UTR in conjunction with eIF4G and eIF4B. Both factors jointly stimulate eIF4A activities by modulation of eIF4A conformational cycling between open and closed states. Here we examine how RNA substrates modulate eIF4A activities. The RNAs fall into two classes: Short RNAs only partially stimulate the eIF4A ATPase activity, and closing is rate-limiting for the conformational cycle. By contrast, longer RNAs maximally stimulate ATP hydrolysis and promote closing of eIF4A. Strikingly, the rate constants of unwinding do not correlate with the length of a single-stranded region preceding a duplex, but reach a maximum for RNA with a single-stranded region of six nucleotides. We propose a model in which RNA substrates affect eIF4A activities by modulating the kinetic partitioning of eIF4A between futile, unproductive, and productive cycles.


Assuntos
Regiões 5' não Traduzidas , Adenosina Trifosfatases/química , Fator de Iniciação 4F em Eucariotos/química , RNA Helicases/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Clonagem Molecular , Fator de Iniciação 4F em Eucariotos/genética , Hidrólise , Cinética , Nucleotídeos/genética , Domínios Proteicos , RNA/genética , RNA Helicases/genética , Proteínas de Saccharomyces cerevisiae/genética
3.
Nucleic Acids Res ; 47(7): 3699-3710, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30993346

RESUMO

DEAD-box helicases are involved in all steps of RNA metabolism. They are ATP-dependent RNA binding proteins and RNA-dependent ATPases. They can displace short duplexes, but they lack processivity. Their mechanism and functioning are not clearly understood; classical or bulk biochemical assays are not sufficient to answer these questions. Single-molecule techniques provide useful tools, but they are limited in cases where the proteins are nonprocessive and give weak signals. We present here a new, magnetic-tweezers-based, single-molecule assay that is simple and that can sensitively measure the displacement time of a small, hybridized, RNA oligonucleotide. Tens of molecules can be analyzed at the same time. Comparing the displacement times with and without a helicase gives insights into the enzymatic activity of the protein. We used this assay to study yeast Ded1, which is orthologous to human DDX3. Although Ded1 acts on a variety of substrates, we find that Ded1 requires an RNA substrate for its ATP-dependent unwinding activity and that ATP hydrolysis is needed to see this activity. Further, we find that only intramolecular single-stranded RNA extensions enhance this activity. We propose a model where ATP-bound Ded1 stabilizes partially unwound duplexes and where multiple binding events may be needed to see displacement.


Assuntos
RNA Helicases DEAD-box/química , RNA/química , Proteínas de Saccharomyces cerevisiae/química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/química , Trifosfato de Adenosina/genética , Sequência de Aminoácidos/genética , RNA Helicases DEAD-box/genética , Humanos , Fenômenos Mecânicos , RNA/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
4.
Plant Mol Biol ; 100(1-2): 163-179, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30937701

RESUMO

KEY MESSAGE: We demonstrate that the C-terminus of OsCDC48 is essential for maintaining its full ATPase activity and OsCDC48/48E interaction is required to modulate cellular processes and plant survival in rice. Cell division cycle 48 (CDC48) belongs to the superfamily protein of ATPases associated with diverse cellular activities (AAA). We previously isolated a rice CDC48 mutant (psd128) displaying premature senescence and death phenotype. Here, we showed that OsCDC48 (Os03g0151800) interacted with OsCDC48E (Os10g0442600), a homologue of OsCDC48, to control plant survival in rice. OsCDC48E knockout plants exhibited similar behavior to psd128 with premature senescence and plant death. Removal of the C-terminus of OsCDC48 caused altered expression of cell cycle-related genes, changed the percentage of cells in G1 and G2/M phases, and abolished the interaction between OsCDC48 itself and between OsCDC48 and OsCDC48E, respectively. Furthermore, the truncated OsCDC48-PSD128 protein lacking the C-terminal 27 amino acid residues showed a decreased level of ATPase activity. Overexpression of OsCDC48-psd128 resulted in differential expression of AAA-ATPase associated genes leading to increased total ATPase activity, accumulation of reactive oxygen species and decreased plant tiller numbers while overexpression of OsCDC48 also resulted in differential expression of AAA-ATPase associated genes leading to increased total ATPase activity, but increased plant tiller numbers and grain yield, indicating its potential utilization for yield improvement. Our results demonstrated that the C-terminal region of OsCDC48 was essential for maintaining the full ATPase activity and OsCDC48/48E complex might function in form of heteromultimers to modulate cellular processes and plant survival in rice.


Assuntos
Oryza/fisiologia , Proteínas de Plantas/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Sequência de Bases , Ciclo Celular/genética , Núcleo Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Mutação/genética , Oryza/genética , Oryza/crescimento & desenvolvimento , Fenótipo , Desenvolvimento Vegetal , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Ligação Proteica , Transporte Proteico , Deleção de Sequência
5.
Oncol Rep ; 41(6): 3179-3188, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30942458

RESUMO

Multidrug resistance (MDR) is a major reason for the failure of acute myeloid leukemia (AML) therapy. Agents that reverse MDR and sensitize AML cells to chemotherapy are of great clinical significance. The present study developed Adriamycin (Adr)­resistant cell lines, namely K562/Adr200 and K562/Adr500, which exhibited MDR. The upregulation of ATP­binding cassette subfamily B member 1 (ABCB1) was confirmed as the mechanism of resistance by reverse transcription­quantitative polymerase chain reaction and western blot analyses. Subsequently, the role of the mammalian target of rapamycin (mTOR) kinase inhibitor, WYE­354, in sensitizing the K562/Adr200 and K562/Adr500 cell lines to Adr was evaluated. At sub­cytotoxic concentrations, WYE­354 increased Adr cytotoxicity in the K562/Adr200 and K562/Adr500 cells. WYE­354 restored Adr sensitivity in the resistant cells by inhibiting ABCB1­mediated substrate efflux, thereby leading to an accumulation of Adr, an increase in Adr­mediated G2/M cell cycle arrest and the induction of apoptosis. Furthermore, WYE­354 stimulated the ATPase activity of ABCB1, which was consistent with in silico predictions using a human ABCB1 mouse homology model, indicating that WYE­354 is a potent substrate of ABCB1. WYE­354 did not regulate the expression of ABCB1 at the concentrations used in the present study. These findings indicate that WYE­354 may be a competitive inhibitor of ABCB1­mediated efflux and a potential candidate in combination with standard chemotherapy for overcoming MDR. Further clinical investigations are warranted to validate this combination in vivo.


Assuntos
Doxorrubicina/administração & dosagem , Leucemia Mieloide Aguda/tratamento farmacológico , Purinas/administração & dosagem , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/química , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Adenosina Trifosfatases/química , Animais , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Células K562 , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos , Especificidade por Substrato , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética
7.
Nat Commun ; 10(1): 1273, 2019 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30894538

RESUMO

Hsp90 is a dimeric molecular chaperone that is essential for the folding and activation of hundreds of client proteins. Co-chaperone proteins regulate the ATP-driven Hsp90 client activation cycle. Aha-type co-chaperones are the most potent stimulators of the Hsp90 ATPase activity but the relationship between ATPase regulation and in vivo activity is poorly understood. We report here that the most strongly conserved region of Aha-type co-chaperones, the N terminal NxNNWHW motif, modulates the apparent affinity of Hsp90 for nucleotide substrates. The ability of yeast Aha-type co-chaperones to act in vivo is ablated when the N terminal NxNNWHW motif is removed. This work suggests that nucleotide exchange during the Hsp90 functional cycle may be more important than rate of catalysis.


Assuntos
Adenosina Trifosfatases/química , Chaperoninas/química , Proteínas de Choque Térmico HSP90/química , Chaperonas Moleculares/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Sítios de Ligação , Chaperoninas/genética , Chaperoninas/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Cinética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
8.
Nature ; 567(7748): 409-413, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30867599

RESUMO

Chromatin remodellers include diverse enzymes with distinct biological functions, but nucleosome-sliding activity appears to be a common theme1,2. Among the remodelling enzymes, Snf2 serves as the prototype to study the action of this protein family. Snf2 and related enzymes share two conserved RecA-like lobes3, which by themselves are able to couple ATP hydrolysis to chromatin remodelling. The mechanism by which these enzymes couple ATP hydrolysis to translocate the nucleosome along the DNA remains unclear2,4-8. Here we report the structures of Saccharomyces cerevisiae Snf2 bound to the nucleosome in the presence of ADP and ADP-BeFx. Snf2 in the ADP-bound state adopts an open conformation similar to that in the apo state, and induces a one-base-pair DNA bulge at superhelix location 2 (SHL2), with the tracking strand showing greater distortion than the guide strand. The DNA distortion propagates to the proximal end, leading to staggered translocation of the two strands. The binding of ADP-BeFx triggers a closed conformation of the enzyme, resetting the nucleosome to a relaxed state. Snf2 shows altered interactions with the DNA in different nucleotide states, providing the structural basis for DNA translocation. Together, our findings suggest a fundamental mechanism for the DNA translocation that underlies chromatin remodelling.


Assuntos
Adenosina Trifosfatases/metabolismo , Montagem e Desmontagem da Cromatina , Cromatina/genética , Cromatina/metabolismo , DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae , Fatores de Transcrição/metabolismo , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/química , Apoproteínas/química , Apoproteínas/metabolismo , Transporte Biológico , Cromatina/química , DNA/química , DNA/genética , Transferência Ressonante de Energia de Fluorescência , Modelos Moleculares , Nucleossomos/química , Nucleossomos/metabolismo , Nucleotídeos/química , Nucleotídeos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição/química
9.
Nucleic Acids Res ; 47(8): 4349-4362, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-30828714

RESUMO

DEAH-box adenosine triphosphatases (ATPases) play a crucial role in the spliceosome-mediated excision of pre-mRNA introns. Recent spliceosomal cryo-EM structures suggest that these proteins utilize translocation to apply forces on ssRNAs rather than direct RNA duplex unwinding to ensure global rearrangements. By solving the crystal structure of Prp22 in different adenosine nucleotide-free states, we identified two missing conformational snapshots of genuine DEAH-box ATPases that help to unravel the molecular mechanism of translocation for this protein family. The intrinsic mobility of the RecA2 domain in the absence of adenosine di- or triphosphate (ADP/ATP) and RNA enables DEAH-box ATPases to adopt different open conformations of the helicase core. The presence of RNA suppresses this mobility and stabilizes one defined open conformation when no adenosine nucleotide is bound. A comparison of this novel conformation with the ATP-bound state of Prp43 reveals that these ATPases cycle between closed and open conformations of the helicase core, which accommodate either a four- or five-nucleotide stack in the RNA-binding tunnel, respectively. The continuous repetition of these states enables these proteins to translocate in 3'-5' direction along an ssRNA with a step-size of one RNA nucleotide per hydrolyzed ATP. This ATP-driven motor function is maintained by a serine in the conserved motif V that senses the catalytic state and accordingly positions the RecA2 domain.


Assuntos
Adenosina Trifosfatases/química , Trifosfato de Adenosina/química , Chaetomium/química , RNA Helicases DEAD-box/química , Proteínas Fúngicas/química , Fatores de Processamento de RNA/química , RNA Fúngico/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Chaetomium/enzimologia , Chaetomium/genética , Clonagem Molecular , Cristalografia por Raios X , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Termodinâmica
10.
Nucleic Acids Res ; 47(9): 4814-4830, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-30916349

RESUMO

Posttranscriptional RNA modifications occur in all domains of life. Modifications of anticodon bases are of particular importance for ribosomal decoding and proteome homeostasis. The Elongator complex modifies uridines in the wobble position and is highly conserved in eukaryotes. Despite recent insights into Elongator's architecture, the structure and function of its regulatory factor Kti12 have remained elusive. Here, we present the crystal structure of Kti12's nucleotide hydrolase domain trapped in a transition state of ATP hydrolysis. The structure reveals striking similarities to an O-phosphoseryl-tRNA kinase involved in the selenocysteine pathway. Both proteins employ similar mechanisms of tRNA binding and show tRNASec-dependent ATPase activity. In addition, we demonstrate that Kti12 binds directly to Elongator and that ATP hydrolysis is crucial for Elongator to maintain proper tRNA anticodon modification levels in vivo. In summary, our data reveal a hitherto uncharacterized link between two translational control pathways that regulate selenocysteine incorporation and affect ribosomal tRNA selection via specific tRNA modifications.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Adenosina Trifosfatases/genética , Processamento Pós-Transcricional do RNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Adenosina Trifosfatases/química , Anticódon/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Chaetomium/química , Chaetomium/enzimologia , Cristalografia por Raios X , Conformação Proteica , RNA de Transferência/genética , Ribossomos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Uridina/genética
11.
Proc Natl Acad Sci U S A ; 116(13): 6111-6119, 2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30850548

RESUMO

Microrchidia 3 (MORC3) is a human protein linked to autoimmune disorders, Down syndrome, and cancer. It is a member of a newly identified family of human ATPases with an uncharacterized mechanism of action. Here, we elucidate the molecular basis for inhibition and activation of MORC3. The crystal structure of the MORC3 region encompassing the ATPase and CW domains in complex with a nonhydrolyzable ATP analog demonstrates that the two domains are directly coupled. The extensive ATPase:CW interface stabilizes the protein fold but inhibits the catalytic activity of MORC3. Enzymatic, NMR, mutational, and biochemical analyses show that in the autoinhibited, off state, the CW domain sterically impedes binding of the ATPase domain to DNA, which in turn is required for the catalytic activity. MORC3 autoinhibition is released by disrupting the intramolecular ATPase:CW coupling through the competitive interaction of CW with histone H3 tail or by mutating the interfacial residues. Binding of CW to H3 leads to a marked rearrangement in the ATPase-CW cassette, which frees the DNA-binding site in active MORC3 (on state). We show that ATP-induced dimerization of the ATPase domain is strictly required for the catalytic activity and that the dimeric form of ATPase-CW might cooperatively bind to dsDNA. Together, our findings uncovered a mechanism underlying the fine-tuned regulation of the catalytic domain of MORC3 by the epigenetic reader, CW.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/isolamento & purificação , Catálise , Domínio Catalítico , Cristalografia por Raios X , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/isolamento & purificação , Ativação Enzimática , Polarização de Fluorescência , Histonas/metabolismo , Humanos , Espectroscopia de Ressonância Magnética
12.
Int J Biol Macromol ; 131: 67-73, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30857964

RESUMO

KaiC, the core protein of the cyanobacterial clock, assembles into a hexamer upon ATP-binding. The hexameric KaiC from a cyanobacterium Synechococcus elongatus PCC 7942 (Se-KaiC) is a multifunctional enzyme with autokinase, autophosphatase and ATPase and these activities show a circadian rhythm in the presence of two other clock proteins, KaiA and KaiB both in vivo and in vitro. While an interplay among three enzymatic activities has been pointed out through studies on Se-KaiC as the basis of circadian rhythmicity in cyanobacteria, little is known about the structure and functions of KaiC from other cyanobacterial species. In this study, we established a protocol to prepare KaiC from Gloeocapsa sp. PCC 7428 (Gl-KaiC) belonging to a distinct genus from Synechococcus and characterized its oligomeric structure and function. The results demonstrate that Gl-KaiC shares the basic properties with Se-KaiC. The present protocol offers practical means for further analysis of structure and function of Gl-KaiC, which would provide insights into diversity and evolution of the clock systems in cyanobacteria.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Relógios Circadianos , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/química , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/metabolismo , Synechococcus/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Relógios Circadianos/genética , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/genética , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/isolamento & purificação , Clonagem Molecular , Expressão Gênica , Peso Molecular , Fosforilação , Synechococcus/fisiologia
13.
RNA ; 25(6): 685-701, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30910870

RESUMO

Eukaryotic ribosome biogenesis is a highly orchestrated process involving numerous assembly factors including ATP-dependent RNA helicases. The DEAH helicase DHX37 (Dhr1 in yeast) is activated by the ribosome biogenesis factor UTP14A to facilitate maturation of the small ribosomal subunit. We report the crystal structure of DHX37 in complex with single-stranded RNA, revealing a canonical DEAH ATPase/helicase architecture complemented by a structurally unique carboxy-terminal domain (CTD). Structural comparisons of the nucleotide-free DHX37-RNA complex with DEAH helicases bound to RNA and ATP analogs reveal conformational changes resulting in a register shift in the bound RNA, suggesting a mechanism for ATP-dependent 3'-5' RNA translocation. We further show that a conserved sequence motif in UTP14A interacts with and activates DHX37 by stimulating its ATPase activity and enhancing RNA binding. In turn, the CTD of DHX37 is required, but not sufficient, for interaction with UTP14A in vitro and is essential for ribosome biogenesis in vivo. Together, these results shed light on the mechanism of DHX37 and the function of UTP14A in controlling its recruitment and activity during ribosome biogenesis.


Assuntos
Adenosina Trifosfatases/química , Trifosfato de Adenosina/análogos & derivados , RNA Helicases DEAD-box/química , Biogênese de Organelas , RNA Helicases/química , RNA/química , Ribonucleoproteínas Nucleolares Pequenas/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cinética , Camundongos , Modelos Moleculares , Ligação Proteica , Biossíntese de Proteínas , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/genética , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Especificidade por Substrato
14.
Nucleic Acids Res ; 47(7): 3795-3810, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30788511

RESUMO

Upon triggering by their inducer, signal transduction ATPases with numerous domains (STANDs), initially in monomeric resting forms, multimerize into large hubs that activate target macromolecules. This process requires conversion of the STAND conserved core (the NOD) from a closed form encasing an ADP molecule to an ATP-bound open form prone to multimerize. In the absence of inducer, autoinhibitory interactions maintain the NOD closed. In particular, in resting STAND proteins with an LRR- or WD40-type sensor domain, the latter establishes interactions with the NOD that are disrupted in the multimerization-competent forms. Here, we solved the first crystal structure of a STAND with a tetratricopeptide repeat sensor domain, PH0952 from Pyrococcus horikoshii, revealing analogous NOD-sensor contacts. We use this structural information to experimentally demonstrate that similar interactions also exist in a PH0952 homolog, the MalT STAND archetype, and actually contribute to the MalT autoinhibition in vitro and in vivo. We propose that STAND activation occurs by stepwise release of autoinhibitory contacts coupled to the unmasking of inducer-binding determinants. The MalT example suggests that STAND weak autoinhibitory interactions could assist the binding of inhibitory proteins by placing in register inhibitor recognition elements born by two domains.


Assuntos
Adenosina Trifosfatases/química , Conformação Proteica , Domínios Proteicos/genética , Repetições de Tetratricopeptídeos/genética , Adenosina Trifosfatases/genética , Cristalografia por Raios X , Escherichia coli/genética , Humanos , Modelos Moleculares , Estrutura Terciária de Proteína/genética , Transdução de Sinais/genética , Repetições WD40/genética
15.
J Biol Chem ; 294(15): 5970-5979, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30760526

RESUMO

The P-type ATPase protein family includes, in addition to ion pumps such as Ca2+-ATPase and Na+,K+-ATPase, also phospholipid flippases that transfer phospholipids between membrane leaflets. P-type ATPase ion pumps translocate their substrates occluded between helices in the center of the transmembrane part of the protein. The large size of the lipid substrate has stimulated speculation that flippases use a different transport mechanism. Information on the functional importance of the most centrally located helices M5 and M6 in the transmembrane domain of flippases has, however, been sparse. Using mutagenesis, we examined the entire M5-M6 region of the mammalian flippase ATP8A2 to elucidate its possible function in the lipid transport mechanism. This mutational screen yielded an informative map assigning important roles in the interaction with the lipid substrate to only a few M5-M6 residues. The M6 asparagine Asn-905 stood out as being essential for the lipid substrate-induced dephosphorylation. The mutants N905A/D/E/H/L/Q/R all displayed very low activities and a dramatic insensitivity to the lipid substrate. Strikingly, Asn-905 aligns with key ion-binding residues of P-type ATPase ion pumps, and N905D was recently identified as one of the mutations causing the neurological disorder cerebellar ataxia, mental retardation, and disequilibrium (CAMRQ) syndrome. Moreover, the effects of substitutions to the adjacent residue Val-906 (i.e. V906A/E/F/L/Q/S) suggest that the lipid substrate approaches Val-906 during the translocation. These results favor a flippase mechanism with strong resemblance to the ion pumps, despite a location of the translocation pathway in the periphery of the transmembrane part of the flippase protein.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Substituição de Aminoácidos , Animais , Asparagina/química , Asparagina/genética , Asparagina/metabolismo , Bovinos , Células HEK293 , Humanos , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Proteínas de Transferência de Fosfolipídeos/química , Proteínas de Transferência de Fosfolipídeos/genética , Fosforilação
16.
J Biol Chem ; 294(13): 5050-5059, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30723158

RESUMO

TraB is an FtsK-like DNA translocase responsible for conjugative plasmid transfer in mycelial Streptomyces Unlike other conjugative systems, which depend on a type IV secretion system, Streptomyces requires only TraB protein to transfer the plasmid as dsDNA. The γ-domain of this protein specifically binds to repeated 8-bp motifs on the plasmid sequence, following a mechanism that is reminiscent of the FtsK/SpoIIIE chromosome segregation system. In this work, we purified and characterized the enzymatic activity of TraB, revealing that it is a DNA-dependent ATPase that is highly stimulated by dsDNA substrates. Interestingly, we found that unlike the SpoIIIE protein, the γ-domain of TraB does not confer sequence-specific ATPase stimulation. We also found that TraB binds G-quadruplex DNA structures with higher affinity than TraB-recognition sequences (TRSs). An EM-based structural analysis revealed that TraB tends to assemble as large complexes comprising four TraB hexamers, which might be a prerequisite for DNA translocation across cell membranes. In summary, our findings shed light on the molecular mechanism used by the DNA-translocating motor TraB, which may be shared by other membrane-associated machineries involved in DNA binding and translocation.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Streptomyces/metabolismo , Adenosina Trifosfatases/química , Proteínas de Bactérias/química , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Quadruplex G , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Streptomyces/química
17.
Int J Biol Macromol ; 129: 792-798, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30771393

RESUMO

GroEL is the most commonly used chaperonin protein for both in-vitro refolding of aggregating proteins as well as in-vivo solubilization of over-expressed aggregation-prone proteins of therapeutic and biotechnological applications. But sometimes the stress conditions like heat and a load of over-expressed/unfolded/misfolded proteins lead to a decrease in structural stability and functional efficiency of GroEL, which results in less recovery of substrate protein through the chaperone-mediated refolding process. So, to amend it, we have been able to optimize physicochemical conditions utilizing a cumulation of (NH4)2SO4/MgCl2 in the buffer. Interestingly, we found a consequential enhancement in the aggregation prevention efficiency, refolding of the denatured substrate and ATPase activity of GroEL protein. The reason for the increased refolding and aggregation prevention efficiency might be the exposure of hydrophobic sites and enhanced ATP hydrolysis rate in presence of buffer containing (NH4)2SO4/MgCl2. The present study withal shows that GroEL under optimized conditions exhibits consequential amelioration in thermal aggregation at high temperature. Hence the optimized buffer conditions are utilizable for the folding of substrate proteins under a broad temperature range.


Assuntos
Chaperonina 60/química , Sais/química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Chaperonina 60/metabolismo , Hidrólise , Cinética , Agregados Proteicos , Ligação Proteica , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Redobramento de Proteína , Estabilidade Proteica , Proteínas Recombinantes , Análise Espectral , Relação Estrutura-Atividade , Temperatura Ambiente
18.
J Biol Chem ; 294(10): 3577-3587, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30602566

RESUMO

During posttranslational translocation in Escherichia coli, polypeptide substrates are driven across the membrane through the SecYEG protein-conducting channel using the ATPase SecA, which binds to SecYEG and couples nucleotide hydrolysis to polypeptide movement. Recent studies suggest that SecA is a highly dynamic enzyme, able to repeatedly bind and dissociate from SecYEG during substrate translocation, but other studies indicate that these dynamics, here referred to as "SecA processivity," are not a requirement for transport. We employ a SecA mutant (PrlD23) that associates more tightly to membranes than WT SecA, in addition to a SecA-SecYEG cross-linked complex, to demonstrate that SecA-SecYEG binding and dissociation events are important for efficient transport of the periplasmic protein proPhoA. Strikingly however, we find that transport of the precursor of the outer membrane protein proOmpA does not depend on SecA processivity. By exchanging signal sequence and protein domains of similar size between PhoA and OmpA, we find that SecA processivity is not influenced by the sequence of the protein substrate. In contrast, using an extended proOmpA variant and a truncated derivative of proPhoA, we show that SecA processivity is affected by substrate length. These findings underscore the importance of the dynamic nature of SecA-SecYEG interactions as a function of the preprotein substrate, features that have not yet been reported using other biophysical or in vivo methods.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Escherichia coli/citologia , Escherichia coli/metabolismo , Canais de Translocação SEC/metabolismo , Adenosina Trifosfatases/química , Proteínas de Bactérias/química , Domínios Proteicos , Estabilidade Proteica , Transporte Proteico , Canais de Translocação SEC/química
19.
J Biol Chem ; 294(9): 3100-3116, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30617180

RESUMO

Eukaryotic cell homeostasis requires transfer of cellular components among organelles and relies on membrane fusion catalyzed by SNARE proteins. Inactive SNARE bundles are reactivated by hexameric N-ethylmaleimide-sensitive factor, vesicle-fusing ATPase (Sec18/NSF)-driven disassembly that enables a new round of membrane fusion. We previously found that phosphatidic acid (PA) binds Sec18 and thereby sequesters it from SNAREs and that PA dephosphorylation dissociates Sec18 from the membrane, allowing it to engage SNARE complexes. We now report that PA also induces conformational changes in Sec18 protomers and that hexameric Sec18 cannot bind PA membranes. Molecular dynamics (MD) analyses revealed that the D1 and D2 domains of Sec18 contain PA-binding sites and that the residues needed for PA binding are masked in hexameric Sec18. Importantly, these simulations also disclosed that a major conformational change occurs in the linker region between the D1 and D2 domains, which is distinct from the conformational changes that occur in hexameric Sec18 during SNARE priming. Together, these findings indicate that PA regulates Sec18 function by altering its architecture and stabilizing membrane-bound Sec18 protomers.


Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Ácidos Fosfatídicos/farmacologia , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas SNARE/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/metabolismo , Trifosfato de Adenosina/metabolismo , Simulação de Dinâmica Molecular , Proteínas Sensíveis a N-Etilmaleimida/metabolismo , Ácidos Fosfatídicos/metabolismo , Fosforilação , Domínios Proteicos , Multimerização Proteica , Estrutura Secundária de Proteína/efeitos dos fármacos , Proteínas SNARE/química , Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato
20.
J Basic Microbiol ; 59(3): 302-313, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30614541

RESUMO

Δlon mutant of Escherichia coli becomes hypersensitive to DNA damaging agents and over-produce capsule due to stabilization of the Lon substrates, namely, SulA and RcsA, respectively. These phenotypes were earlier found to be suppressed in Δlon ssrA::cat/pUC4 K and Δlon faa (DnaJ, G232D) strains, called as "Alp" strains. We observed that a plasmid carrying an E. coli chromosomal fragment harboring few genes, a heat shock gene htpY and a portion of dnaK capable of encoding truncated N-terminal ATPase domain (244 aa) could suppress lon mutant phenotypes. Deletion of htpY did not affect the efficiency of suppression. Clones expressing DnaK' (244 aa) peptide alone could suppress both Δlon phenotypes in copy number dependent manner. Inactivation of clpQ did not affect the MMSR phenotype of Δlon strain carrying dnaK' clones indicating that ClpYQ protease does not degrade SulA. We hypothesize that the high levels of defective DnaK'-DnaJ chaperone complex formed in these strains might lead to aggregation of SulA and RcsA and, thereby the suppression of Δlon phenotypes. Systematic deletion analysis of dnaK' revealed that, ∼220 aa N-terminal DnaK peptide is required for suppression of cps-lac over-expression and ∼169 aa peptide is enough for the suppression of MMSS phenotype of Δlon mutant.


Assuntos
Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Proteínas de Choque Térmico HSP70/metabolismo , Protease La/genética , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/genética , Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/química , Expressão Gênica , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/genética , Metanossulfonato de Metila/farmacologia , Viabilidade Microbiana/efeitos dos fármacos , Peptídeos/química , Peptídeos/metabolismo , Fenótipo , Plasmídeos/genética , Plasmídeos/metabolismo , Protease La/deficiência , Deleção de Sequência
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