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1.
Int J Mol Sci ; 22(5)2021 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-33806352

RESUMO

Lipids play essential roles in numerous cellular processes, including membrane remodeling, signal transduction, the modulation of hormone activity, and steroidogenesis. We chose steroidogenic MA-10 mouse tumor Leydig cells to investigate subcellular lipid localization during steroidogenesis. Electron microscopy showed that cAMP stimulation increased associations between the plasma membrane (PM) and the endoplasmic reticulum (ER) and between the ER and mitochondria. cAMP stimulation also increased the movement of cholesterol from the PM compared to untreated cells, which was partially inhibited when ATPase family AAA-domain containing protein 3 A (ATAD3A), which functions in ER and mitochondria interactions, was knocked down. Mitochondria, ER, cytoplasm, PM, PM-associated membranes (PAMs), and mitochondria-associated membranes (MAMs) were isolated from control and hormone-stimulated cells. Lipidomic analyses revealed that each isolated compartment had a unique lipid composition, and the induction of steroidogenesis caused the significant remodeling of its lipidome. cAMP-induced changes in lipid composition included an increase in phosphatidylserine and cardiolipin levels in PAM and PM compartments, respectively; an increase in phosphatidylinositol in the ER, mitochondria, and MAMs; and a reorganization of phosphatidic acid, cholesterol ester, ceramide, and phosphatidylethanolamine. Abundant lipids, such as phosphatidylcholine, were not affected by hormone treatment. Our data suggested that PM-ER-mitochondria tethering may be involved in lipid trafficking between organelles and indicated that hormone-induced acute steroid production involves extensive organelle remodeling.


Assuntos
Tumor de Células de Leydig/metabolismo , Lipídeos de Membrana/metabolismo , Esteroides/biossíntese , Neoplasias Testiculares/metabolismo , ATPases Associadas a Diversas Atividades Celulares/antagonistas & inibidores , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Animais , Bucladesina/farmacologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Colesterol/metabolismo , AMP Cíclico/farmacologia , Retículo Endoplasmático/metabolismo , Técnicas de Silenciamento de Genes , Tumor de Células de Leydig/ultraestrutura , Lipidômica , Masculino , Camundongos , Microscopia Eletrônica de Transmissão , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Biológicos , Neoplasias Testiculares/ultraestrutura
2.
Life Sci ; 276: 119322, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-33711386

RESUMO

ATAD2 is a promising oncoprotein with tumor-promoting functions in many cancers. It is a valid cancer drug-target and a potential cancer-biomarker for multiple malignancies. As a cancer/testis antigen (CTA), ATAD2 could also be a probable candidate for immunotherapy. It is a unique CTA that belongs to both AAA+ ATPase and bromodomain family proteins. Since 2007, several research groups have been reported on the pleiotropic oncogenic functions of ATAD2 in diverse signaling pathways, including Rb/E2F-cMyc pathway, steroid hormone signaling pathway, p53 and p38-MAPK-mediated apoptotic pathway, AKT pathway, hedgehog signaling pathway, HIF1α signaling pathway, and Epithelial to Mesenchymal Transition (EMT) pathway in various cancers. In all these pathways, ATAD2 participates in chromatin dynamics, DNA replication, and gene transcription, demonstrating its role as an epigenetic reader and transcription factor or coactivator to promote tumorigenesis. However, despite the progress, an overall mechanism of ATAD2-mediated oncogenesis in diverse origin is elusive. In this review, we summarize the accumulated evidence to envision the overall ATAD2 signaling networks during carcinogenesis and highlight the area where missing links await further research. Besides, the structure-function aspect of ATAD2 is also discussed. Since the efforts have already been initiated to explore targeted drug molecules and RNA-based therapeutic alternatives against ATAD2, their potency and prospects have been elucidated. Together, we believe this is a well-rounded review on ATAD2, facilitating a new drift in ATAD2 research, essential for its clinical implication as a biomarker and/or cancer drug-target.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Biomarcadores Tumorais/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Terapia de Alvo Molecular , Neoplasias/patologia , ATPases Associadas a Diversas Atividades Celulares/genética , Biomarcadores Tumorais/genética , Proteínas de Ligação a DNA/genética , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo
3.
Mol Cell Biol ; 41(4)2021 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-33526454

RESUMO

In eukaryotes, genomic DNA is packaged into nucleosomes, which are the basal components coordinating both the structures and functions of chromatin. In this study, we screened a collection of mutations for histone H3/H4 mutants in Saccharomyces cerevisiae that affect the DNA damage sensitivity of DNA damage tolerance (DDT)-deficient cells. We identified a class of histone H3/H4 mutations that suppress methyl methanesulfonate (MMS) sensitivity of DDT-deficient cells (referred to here as the histone SDD mutations), which likely cluster on a specific H3-H4 interface of the nucleosomes. The histone SDD mutations did not suppress the MMS sensitivity of DDT-deficient cells in the absence of Rad51, indicating that homologous recombination (HR) is responsible for DNA damage resistance. Furthermore, the histone SDD mutants showed reduced levels of PCNA ubiquitination after exposure to MMS or UV irradiation, consistent with decreased MMS-induced mutagenesis relative to that of wild-type cells. We also found that histone SDD mutants lacking the INO80 chromatin remodeler impair HR-dependent recovery from MMS-induced replication arrest, resulting in defective S-phase progression and increased Rad52 foci. Taken together, our data provide novel insights into nucleosome functions, which link INO80-dependent chromatin remodeling to the regulation of DDT and HR during the recovery from replication blockage.


Assuntos
Cromatina/metabolismo , Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Histonas/metabolismo , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Montagem e Desmontagem da Cromatina/fisiologia , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Recombinação Homóloga/fisiologia , Humanos , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
4.
Nat Genet ; 53(3): 279-287, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33558757

RESUMO

Chromatin accessibility is a hallmark of regulatory regions, entails transcription factor (TF) binding and requires nucleosomal reorganization. However, it remains unclear how dynamic this process is. In the present study, we use small-molecule inhibition of the catalytic subunit of the mouse SWI/SNF remodeler complex to show that accessibility and reduced nucleosome presence at TF-binding sites rely on persistent activity of nucleosome remodelers. Within minutes of remodeler inhibition, accessibility and TF binding decrease. Although this is irrespective of TF function, we show that the activating TF OCT4 (POU5F1) exhibits a faster response than the repressive TF REST. Accessibility, nucleosome depletion and gene expression are rapidly restored on inhibitor removal, suggesting that accessible chromatin is regenerated continuously and in a largely cell-autonomous fashion. We postulate that TF binding to chromatin and remodeler-mediated nucleosomal removal do not represent a stable situation, but instead accessible chromatin reflects an average of a dynamic process under continued renewal.


Assuntos
Cromatina/metabolismo , Complexos Multiproteicos/metabolismo , Fatores de Transcrição/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Sítios de Ligação , Linhagem Celular/efeitos dos fármacos , Cromatina/genética , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/fisiologia , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , DNA Helicases/antagonistas & inibidores , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Histonas/genética , Histonas/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Complexos Multiproteicos/efeitos dos fármacos , Complexos Multiproteicos/genética , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Receptores Estrogênicos/genética , Receptores Estrogênicos/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética
5.
Mol Cell ; 81(4): 830-844.e13, 2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33453168

RESUMO

The MYC oncoprotein globally affects the function of RNA polymerase II (RNAPII). The ability of MYC to promote transcription elongation depends on its ubiquitylation. Here, we show that MYC and PAF1c (polymerase II-associated factor 1 complex) interact directly and mutually enhance each other's association with active promoters. PAF1c is rapidly transferred from MYC onto RNAPII. This transfer is driven by the HUWE1 ubiquitin ligase and is required for MYC-dependent transcription elongation. MYC and HUWE1 promote histone H2B ubiquitylation, which alters chromatin structure both for transcription elongation and double-strand break repair. Consistently, MYC suppresses double-strand break accumulation in active genes in a strictly PAF1c-dependent manner. Depletion of PAF1c causes transcription-dependent accumulation of double-strand breaks, despite widespread repair-associated DNA synthesis. Our data show that the transfer of PAF1c from MYC onto RNAPII efficiently couples transcription elongation with double-strand break repair to maintain the genomic integrity of MYC-driven tumor cells.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/metabolismo , Elongação da Transcrição Genética , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Linhagem Celular Tumoral , Histonas/genética , Histonas/metabolismo , Humanos , Proteínas Proto-Oncogênicas c-myc/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
6.
Int J Mol Sci ; 22(2)2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33467681

RESUMO

Increasing evidence indicates that cancer metastasis is regulated by specific genetic pathways independent of those controlling tumorigenesis and cancer growth. WASF3, a Wiskott-Aldrich syndrome protein family member, appears to play a major role not only in the regulation of actin cytoskeleton dynamics but also in cancer cell invasion/metastasis. Recent studies have highlighted that WASF3 is a master regulator and acts as a pivotal scaffolding protein, bringing the various components of metastatic signaling complexes together both spatially and temporally. Herein, targeting WASF3 at the levels of transcription, protein stability, and phosphorylation holds great promise for metastasis suppression, regardless of the diverse genetic backgrounds associated with tumor development. This review focuses on the critical and distinct contributions of WASF3 in the regulation of signal pathways promoting cancer cell invasion and metastasis.


Assuntos
Regulação Neoplásica da Expressão Gênica , Metástase Neoplásica , Neoplasias/metabolismo , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Animais , Movimento Celular , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Janus Quinase 2/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , MicroRNAs/metabolismo , Proteínas Mitocondriais/metabolismo , Invasividade Neoplásica/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais
7.
Gene ; 764: 145100, 2021 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-32877748

RESUMO

Adipocyte differentiation is an essential part of adipose tissue development, and is closely related to obesity and obesity-related diseases. In this study, we found that the expression of PPARγ, RUVBL2 and Adiponectin were concurrently obviously increased in the 5th-7th day of 3T3-L1 cell differentiation. PPARγ overexpression or the PPARγ activator facilitated Adiponectin trafficking and secretion and upregulated RUVBL2 expression as well as AS160 phosphorylation during adipogenic differentiation of 3T3-L1 cells. Consistently RUVBL2 overexpression also enhanced the polymerization and secretion of Adiponectin, in contrast, RUVBL2 knockdown reduced Adiponectin secretion. Further, PPARγ significantly enhanced RUVBL2 promoter activity and transcription. The progressive deletions and mutations of RUVBL2 promoter for PPARγ binding sites suggested that the PPARγ binding motif situated at -804/-781 bp is an essential component required for RUVBL2 promoter activity. Chromatin immunoprecipitation (ChIP) assays determined that PPARγ can directly interact with the RUVBL2 promoter DNA. Taken together, these data suggest that PPARγ promotes the expression, polymerization and secretion of Adiponectin by activating RUVBL2 transcriptionally, which accelerates 3T3-L1 cell differentiation.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/genética , Adipócitos/fisiologia , Adipogenia/genética , Adiponectina/metabolismo , DNA Helicases/genética , PPAR gama/metabolismo , Células 3T3-L1 , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Animais , Sítios de Ligação/genética , Diferenciação Celular/genética , Imunoprecipitação da Cromatina , Clonagem Molecular , DNA Helicases/metabolismo , Camundongos , Mutação , Regiões Promotoras Genéticas/genética , Multimerização Proteica/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ativação Transcricional , Regulação para Cima
8.
Nucleic Acids Res ; 49(2): 1094-1113, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33367824

RESUMO

The PAQosome is a large complex composed of the HSP90/R2TP chaperone and a prefoldin-like module. It promotes the biogenesis of cellular machineries but it is unclear how it discriminates closely related client proteins. Among the main PAQosome clients are C/D snoRNPs and in particular their core protein NOP58. Using NOP58 mutants and proteomic experiments, we identify different assembly intermediates and show that C12ORF45, which we rename NOPCHAP1, acts as a bridge between NOP58 and PAQosome. NOPCHAP1 makes direct physical interactions with the CC-NOP domain of NOP58 and domain II of RUVBL1/2 AAA+ ATPases. Interestingly, NOPCHAP1 interaction with RUVBL1/2 is disrupted upon ATP binding. Moreover, while it robustly binds both yeast and human NOP58, it makes little interactions with NOP56 and PRPF31, two other closely related CC-NOP proteins. Expression of NOP58, but not NOP56 or PRPF31, is decreased in NOPCHAP1 KO cells. We propose that NOPCHAP1 is a client-loading PAQosome cofactor that selects NOP58 to promote box C/D snoRNP assembly.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Transporte/metabolismo , DNA Helicases/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/biossíntese , Trifosfato de Adenosina/metabolismo , Proteínas do Olho/metabolismo , Técnicas de Inativação de Genes , Genes Reporter , Proteínas de Choque Térmico HSP90/metabolismo , Células HeLa , Humanos , Complexos Multiproteicos , Domínios Proteicos , Mapeamento de Interação de Proteínas , Proteômica/métodos , Proteínas Recombinantes de Fusão/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
9.
J Comput Aided Mol Des ; 34(12): 1237-1259, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33034007

RESUMO

Computational protein-ligand docking is well-known to be prone to inaccuracies in input receptor structures, and it is challenging to obtain good docking results with computationally predicted receptor structures (e.g. through homology modeling). Here we introduce a fragment-based docking method and test if it reduces requirements on the accuracy of an input receptor structures relative to non-fragment docking approaches. In this method, small rigid fragments are docked first using AutoDock Vina to generate a large number of favorably docked poses spanning the receptor binding pocket. Then a graph theory maximum clique algorithm is applied to find combined sets of docked poses of different fragment types onto which the complete ligand can be properly aligned. On the basis of these alignments, possible binding poses of complete ligand are determined. This docking method is first tested for bound docking on a series of Cytochrome P450 (CYP450) enzyme-substrate complexes, in which experimentally determined receptor structures are used. For all complexes tested, ligand poses of less than 1 Å root mean square deviations (RMSD) from the actual binding positions can be recovered. Then the method is tested for unbound docking with modeled receptor structures for a number of protein-ligand complexes from different families including the very recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protease. For all complexes, poses with RMSD less than 3 Å from actual binding positions can be recovered. Our results suggest that for docking with approximately modeled receptor structures, fragment-based methods can be more effective than common complete ligand docking approaches.


Assuntos
Betacoronavirus/enzimologia , Infecções por Coronavirus/tratamento farmacológico , Cisteína Endopeptidases/efeitos dos fármacos , Simulação de Acoplamento Molecular , Pandemias , Pneumonia Viral/tratamento farmacológico , Proteínas não Estruturais Virais/efeitos dos fármacos , ATPases Associadas a Diversas Atividades Celulares/química , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Ligantes , Modelos Químicos , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo
10.
Proc Natl Acad Sci U S A ; 117(38): 23571-23580, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32907938

RESUMO

DNA replication requires the sliding clamp, a ring-shaped protein complex that encircles DNA, where it acts as an essential cofactor for DNA polymerases and other proteins. The sliding clamp needs to be opened and installed onto DNA by a clamp loader ATPase of the AAA+ family. The human clamp loader replication factor C (RFC) and sliding clamp proliferating cell nuclear antigen (PCNA) are both essential and play critical roles in several diseases. Despite decades of study, no structure of human RFC has been resolved. Here, we report the structure of human RFC bound to PCNA by cryogenic electron microscopy to an overall resolution of ∼3.4 Å. The active sites of RFC are fully bound to adenosine 5'-triphosphate (ATP) analogs, which is expected to induce opening of the sliding clamp. However, we observe the complex in a conformation before PCNA opening, with the clamp loader ATPase modules forming an overtwisted spiral that is incapable of binding DNA or hydrolyzing ATP. The autoinhibited conformation observed here has many similarities to a previous yeast RFC:PCNA crystal structure, suggesting that eukaryotic clamp loaders adopt a similar autoinhibited state early on in clamp loading. Our results point to a "limited change/induced fit" mechanism in which the clamp first opens, followed by DNA binding, inducing opening of the loader to release autoinhibition. The proposed change from an overtwisted to an active conformation reveals an additional regulatory mechanism for AAA+ ATPases. Finally, our structural analysis of disease mutations leads to a mechanistic explanation for the role of RFC in human health.


Assuntos
ATPases Associadas a Diversas Atividades Celulares , Replicação do DNA/fisiologia , Antígeno Nuclear de Célula em Proliferação , Proteína de Replicação C , ATPases Associadas a Diversas Atividades Celulares/química , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Microscopia Crioeletrônica , Humanos , Antígeno Nuclear de Célula em Proliferação/química , Antígeno Nuclear de Célula em Proliferação/metabolismo , Antígeno Nuclear de Célula em Proliferação/ultraestrutura , Proteína de Replicação C/química , Proteína de Replicação C/metabolismo , Proteína de Replicação C/ultraestrutura
11.
PLoS One ; 15(9): e0237268, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32886660

RESUMO

Endosomal sorting complexes required for transport proteins (ESCRT) catalyze the fission of cellular membranes during budding of membrane away from the cytosol. Here we have used Total Internal Reflection Fluorescence (TIRF) microscopy to visualize the recruitment of ESCRTs specifically, ALIX, CHMP4b and VPS4 onto the budding HIV Gag virus-like particles (VLPs). We imaged the budding VLPs with 200 millisecond time resolution for 300 frames. Our data shows three phases for ESCRT dynamics: 1) recruitment in which subunits of ALIX, CHMP4b and VPS4 are recruited with constant proportions on the budding sites of HIV Gag virus like particles for nearly 10 seconds, followed by 2) disassembly of ALIX and CHMP4b while VPS4 signal remains constant for nearly 20 seconds followed by 3) disassembly of VPS4. We hypothesized that the disassembly observed in step 2 was catalyzed by VPS4 and powered by ATP hydrolysis. To test this hypothesis, we performed ATP depletion using (-) glucose medium, deoxyglucose and oligomycin. Imaging ATP depleted cells, we show that the disassembly of CHMP4b and ALIX observed in step 2 is ATP dependent. ATP depletion resulted in the recruitment of approximately 2-fold as many subunits of all ESCRTs. Resuming ATP production in cells, resulted in disassembly of the full ESCRT machinery which had been locked in place during ATP depletion. With some caveats, our experiments provide insight into the formation of the ESCRT machinery at the budding site of HIV during budding.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , HIV-1/fisiologia , ATPases Vacuolares Próton-Translocadoras/metabolismo , Liberação de Vírus , Células HEK293 , Infecções por HIV/metabolismo , Infecções por HIV/virologia , Células HeLa , Humanos , Ligação Proteica , Vírion/fisiologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo
12.
Nat Commun ; 11(1): 4534, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32913330

RESUMO

Collisions between the DNA replication machinery and co-transcriptional R-loops can impede DNA synthesis and are a major source of genomic instability in cancer cells. How cancer cells deal with R-loops to proliferate is poorly understood. Here we show that the ATP-dependent chromatin remodelling INO80 complex promotes resolution of R-loops to prevent replication-associated DNA damage in cancer cells. Depletion of INO80 in prostate cancer PC3 cells leads to increased R-loops. Overexpression of the RNA:DNA endonuclease RNAse H1 rescues the DNA synthesis defects and suppresses DNA damage caused by INO80 depletion. R-loops co-localize with and promote recruitment of INO80 to chromatin. Artificial tethering of INO80 to a LacO locus enabled turnover of R-loops in cis. Finally, counteracting R-loops by INO80 promotes proliferation and averts DNA damage-induced death in cancer cells. Our work suggests that INO80-dependent resolution of R-loops promotes DNA replication in the presence of transcription, thus enabling unlimited proliferation in cancers.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proliferação de Células/genética , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Neoplasias/genética , Estruturas R-Loop/genética , Apoptose/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Montagem e Desmontagem da Cromatina , Dano ao DNA , Instabilidade Genômica , Humanos , Neoplasias/patologia , Transcrição Genética
13.
Mol Cell ; 79(4): 615-628.e5, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32668200

RESUMO

Ribosome assembly is driven by numerous assembly factors, including the Rix1 complex and the AAA ATPase Rea1. These two assembly factors catalyze 60S maturation at two distinct states, triggering poorly understood large-scale structural transitions that we analyzed by cryo-electron microscopy. Two nuclear pre-60S intermediates were discovered that represent previously unknown states after Rea1-mediated removal of the Ytm1-Erb1 complex and reveal how the L1 stalk develops from a pre-mature nucleolar to a mature-like nucleoplasmic state. A later pre-60S intermediate shows how the central protuberance arises, assisted by the nearby Rix1-Rea1 machinery, which was solved in its pre-ribosomal context to molecular resolution. This revealed a Rix12-Ipi32 tetramer anchored to the pre-60S via Ipi1, strategically positioned to monitor this decisive remodeling. These results are consistent with a general underlying principle that temporarily stabilized immature RNA domains are successively remodeled by assembly factors, thereby ensuring failsafe assembly progression.


Assuntos
Subunidades Ribossômicas Maiores de Eucariotos/química , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Microscopia Crioeletrônica , Escherichia coli/genética , Modelos Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
14.
Proc Natl Acad Sci U S A ; 117(31): 18459-18469, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32694211

RESUMO

Mdn1 is an essential mechanoenzyme that uses the energy from ATP hydrolysis to physically reshape and remodel, and thus mature, the 60S subunit of the ribosome. This massive (>500 kDa) protein has an N-terminal AAA (ATPase associated with diverse cellular activities) ring, which, like dynein, has six ATPase sites. The AAA ring is followed by large (>2,000 aa) linking domains that include an ∼500-aa disordered (D/E-rich) region, and a C-terminal substrate-binding MIDAS domain. Recent models suggest that intramolecular docking of the MIDAS domain onto the AAA ring is required for Mdn1 to transmit force to its ribosomal substrates, but it is not currently understood what role the linking domains play, or why tethering the MIDAS domain to the AAA ring is required for protein function. Here, we use chemical probes, single-particle electron microscopy, and native mass spectrometry to study the AAA and MIDAS domains separately or in combination. We find that Mdn1 lacking the D/E-rich and MIDAS domains retains ATP and chemical probe binding activities. Free MIDAS domain can bind to the AAA ring of this construct in a stereo-specific bimolecular interaction, and, interestingly, this binding reduces ATPase activity. Whereas intramolecular MIDAS docking appears to require a treatment with a chemical inhibitor or preribosome binding, bimolecular MIDAS docking does not. Hence, tethering the MIDAS domain to the AAA ring serves to prevent, rather than promote, MIDAS docking in the absence of inducing signals.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/química , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , ATPases Associadas a Diversas Atividades Celulares/genética , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Sítios de Ligação , Domínios Proteicos , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
15.
Nucleic Acids Res ; 48(13): 7218-7238, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32542338

RESUMO

R-loops are formed when replicative forks collide with the transcriptional machinery and can cause genomic instability. However, it is unclear how R-loops are regulated at transcription-replication conflict (TRC) sites and how replisome proteins are regulated to prevent R-loop formation or mediate R-loop tolerance. Here, we report that ATAD5, a PCNA unloader, plays dual functions to reduce R-loops both under normal and replication stress conditions. ATAD5 interacts with RNA helicases such as DDX1, DDX5, DDX21 and DHX9 and increases the abundance of these helicases at replication forks to facilitate R-loop resolution. Depletion of ATAD5 or ATAD5-interacting RNA helicases consistently increases R-loops during the S phase and reduces the replication rate, both of which are enhanced by replication stress. In addition to R-loop resolution, ATAD5 prevents the generation of new R-loops behind the replication forks by unloading PCNA which, otherwise, accumulates and persists on DNA, causing a collision with the transcription machinery. Depletion of ATAD5 reduces transcription rates due to PCNA accumulation. Consistent with the role of ATAD5 and RNA helicases in maintaining genomic integrity by regulating R-loops, the corresponding genes were mutated or downregulated in several human tumors.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Ligação a DNA/metabolismo , Estruturas R-Loop , RNA Helicases DEAD-box/metabolismo , Células HEK293 , Células HeLa , Humanos , Antígeno Nuclear de Célula em Proliferação/metabolismo
16.
Nat Commun ; 11(1): 2663, 2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-32471988

RESUMO

Endosomal sorting complexes for transport-III (ESCRT-III) assemble in vivo onto membranes with negative Gaussian curvature. How membrane shape influences ESCRT-III polymerization and how ESCRT-III shapes membranes is yet unclear. Human core ESCRT-III proteins, CHMP4B, CHMP2A, CHMP2B and CHMP3 are used to address this issue in vitro by combining membrane nanotube pulling experiments, cryo-electron tomography and AFM. We show that CHMP4B filaments preferentially bind to flat membranes or to tubes with positive mean curvature. Both CHMP2B and CHMP2A/CHMP3 assemble on positively curved membrane tubes. Combinations of CHMP4B/CHMP2B and CHMP4B/CHMP2A/CHMP3 are recruited to the neck of pulled membrane tubes and reshape vesicles into helical "corkscrew-like" membrane tubes. Sub-tomogram averaging reveals that the ESCRT-III filaments assemble parallel and locally perpendicular to the tube axis, highlighting the mechanical stresses imposed by ESCRT-III. Our results underline the versatile membrane remodeling activity of ESCRT-III that may be a general feature required for cellular membrane remodeling processes.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Membranas Artificiais , Estresse Mecânico , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Fenômenos Bioquímicos , Microscopia Crioeletrônica , Humanos , Nanotubos , Polimerização , Ligação Proteica/fisiologia , Multimerização Proteica , ATPases Vacuolares Próton-Translocadoras/metabolismo
17.
J Med Chem ; 63(10): 5212-5241, 2020 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-32321240

RESUMO

Most bromodomain inhibitors mimic the interactions of the natural acetylated lysine (KAc) histone substrate through key interactions with conserved asparagine and tyrosine residues within the binding pocket. Herein we report the optimization of a series of phenyl sulfonamides that exhibit a novel mode of binding to non-bromodomain and extra terminal domain (non-BET) bromodomains through displacement of a normally conserved network of four water molecules. Starting from an initial hit molecule, we report its divergent optimization toward the ATPase family AAA domain containing 2 (ATAD2) and cat eye syndrome chromosome region, candidate 2 (CECR2) domains. This work concludes with the identification of (R)-55 (GSK232), a highly selective, cellularly penetrant CECR2 inhibitor with excellent physicochemical properties.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/antagonistas & inibidores , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Sulfonamidas/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Células HEK293 , Humanos , Ligação Proteica/fisiologia , Domínios Proteicos/efeitos dos fármacos , Domínios Proteicos/fisiologia , Estrutura Secundária de Proteína , Sulfonamidas/química , Sulfonamidas/farmacologia
18.
Nature ; 580(7804): 536-541, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32322060

RESUMO

Separation of eukaryotic sister chromatids during the cell cycle is timed by the spindle assembly checkpoint (SAC) and ultimately triggered when separase cleaves cohesion-mediating cohesin1-3. Silencing of the SAC during metaphase activates the ubiquitin ligase APC/C (anaphase-promoting complex, also known as the cyclosome) and results in the proteasomal destruction of the separase inhibitor securin1. In the absence of securin, mammalian chromosomes still segregate on schedule, but it is unclear how separase is regulated under these conditions4,5. Here we show that human shugoshin 2 (SGO2), an essential protector of meiotic cohesin with unknown functions in the soma6,7, is turned into a separase inhibitor upon association with SAC-activated MAD2. SGO2-MAD2 can functionally replace securin and sequesters most separase in securin-knockout cells. Acute loss of securin and SGO2, but not of either protein individually, resulted in separase deregulation associated with premature cohesin cleavage and cytotoxicity. Similar to securin8,9, SGO2 is a competitive inhibitor that uses a pseudo-substrate sequence to block the active site of separase. APC/C-dependent ubiquitylation and action of the AAA-ATPase TRIP13 in conjunction with the MAD2-specific adaptor p31comet liberate separase from SGO2-MAD2 in vitro. The latter mechanism facilitates a considerable degree of sister chromatid separation in securin-knockout cells that lack APC/C activity. Thus, our results identify an unexpected function of SGO2 in mitotically dividing cells and a mechanism of separase regulation that is independent of securin but still supervised by the SAC.


Assuntos
Pontos de Checagem do Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/metabolismo , Proteínas Mad2/metabolismo , Securina , Separase/antagonistas & inibidores , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas Cdc20/metabolismo , Linhagem Celular , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Humanos , Ligação Proteica , Securina/metabolismo , Separase/metabolismo
19.
Life Sci ; 253: 117680, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32305524

RESUMO

AIMS: To investigate the effect of lncRNA LCTS5 in the non-small cell lung cancer (NSCLC). MATERIALS AND METHODS: LncRNA profiling was used to identify the novel lncRNA LCTS5. Viability and migration assays were implemented to evaluate the in vitro effect of LCTS5. Transplantation study was designed to investigate the in vivo role. Short hairpin RNA (shRNA) and lentiviral vector were used to alter LCTS5 expression. KEY FINDINGS: We identified a novel lncRNA named LCTS5 whose abundance is dramatically decreased in NSCLC. Overexpressing LCTS5 effectively inhibits viability and migration. Meanwhile, LCTS5 overexpression retards xenograft tumor growth and proliferation. LCTS5 interacts with INO80 to reduce INO80 occupancy at enhancer regions of multiple lung cancer related genes without affecting INO80 decay. SIGNIFICANCE: The newly identified lncRNA LCTS5 impairs NSCLC progression and provides a compelling target for therapeutic intervention during NSCLC treatments.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Proteínas de Ligação a DNA/metabolismo , Neoplasias Pulmonares/patologia , RNA Longo não Codificante/genética , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Progressão da Doença , Humanos , Neoplasias Pulmonares/genética , Camundongos , RNA Interferente Pequeno/administração & dosagem , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Nucleic Acids Res ; 48(9): 4928-4939, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32297953

RESUMO

Replication factor C (RFC), a heteropentamer of RFC1-5, loads PCNA onto DNA during replication and repair. Once DNA synthesis has ceased, PCNA must be unloaded. Recent findings assign the uloader role primarily to an RFC-like (RLC) complex, in which the largest RFC subunit, RFC1, has been replaced with ATAD5 (ELG1 in Saccharomyces cerevisiae). ATAD5-RLC appears to be indispensable, given that Atad5 knock-out leads to embryonic lethality. In order to learn how the retention of PCNA on DNA might interfere with normal DNA metabolism, we studied the response of ATAD5-depleted cells to several genotoxic agents. We show that ATAD5 deficiency leads to hypersensitivity to methyl methanesulphonate (MMS), camptothecin (CPT) and mitomycin C (MMC), agents that hinder the progression of replication forks. We further show that ATAD5-depleted cells are sensitive to poly(ADP)ribose polymerase (PARP) inhibitors and that the processing of spontaneous oxidative DNA damage contributes towards this sensitivity. We posit that PCNA molecules trapped on DNA interfere with the correct metabolism of arrested replication forks, phenotype reminiscent of defective homologous recombination (HR). As Atad5 heterozygous mice are cancer-prone and as ATAD5 mutations have been identified in breast and endometrial cancers, our finding may open a path towards the therapy of these tumours.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/genética , Antineoplásicos/farmacologia , Dano ao DNA , Proteínas de Ligação a DNA/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Galinhas , Cromatina/enzimologia , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Instabilidade Genômica , Mutagênicos/toxicidade , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/metabolismo
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