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1.
Proc Natl Acad Sci U S A ; 118(33)2021 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-34385319

RESUMO

The protein kinase Akt is one of the primary effectors of growth factor signaling in the cell. Akt responds specifically to the lipid second messengers phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3] and phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2] via its PH domain, leading to phosphorylation of its activation loop and the hydrophobic motif of its kinase domain, which are critical for activity. We have now determined the crystal structure of Akt1, revealing an autoinhibitory interface between the PH and kinase domains that is often mutated in cancer and overgrowth disorders. This interface persists even after stoichiometric phosphorylation, thereby restricting maximum Akt activity to PI(3,4,5)P3- or PI(3,4)P2-containing membranes. Our work helps to resolve the roles of lipids and phosphorylation in the activation of Akt and has wide implications for the spatiotemporal control of Akt and potentially lipid-activated kinase signaling in general.

2.
Mol Cell ; 81(12): 2520-2532.e16, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-33930333

RESUMO

The tRNA ligase complex (tRNA-LC) splices precursor tRNAs (pre-tRNA), and Xbp1-mRNA during the unfolded protein response (UPR). In aerobic conditions, a cysteine residue bound to two metal ions in its ancient, catalytic subunit RTCB could make the tRNA-LC susceptible to oxidative inactivation. Here, we confirm this hypothesis and reveal a co-evolutionary association between the tRNA-LC and PYROXD1, a conserved and essential oxidoreductase. We reveal that PYROXD1 preserves the activity of the mammalian tRNA-LC in pre-tRNA splicing and UPR. PYROXD1 binds the tRNA-LC in the presence of NAD(P)H and converts RTCB-bound NAD(P)H into NAD(P)+, a typical oxidative co-enzyme. However, NAD(P)+ here acts as an antioxidant and protects the tRNA-LC from oxidative inactivation, which is dependent on copper ions. Genetic variants of PYROXD1 that cause human myopathies only partially support tRNA-LC activity. Thus, we establish the tRNA-LC as an oxidation-sensitive metalloenzyme, safeguarded by the flavoprotein PYROXD1 through an unexpected redox mechanism.


Assuntos
Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , RNA Ligase (ATP)/metabolismo , RNA de Transferência/metabolismo , Animais , Antioxidantes/fisiologia , Domínio Catalítico , Feminino , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NAD/metabolismo , NADP/metabolismo , Oxirredução , Oxirredutases/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/fisiologia , RNA Ligase (ATP)/química , RNA Ligase (ATP)/genética , Splicing de RNA/genética , Splicing de RNA/fisiologia , Resposta a Proteínas não Dobradas/fisiologia , Proteína 1 de Ligação a X-Box/metabolismo
3.
Biochemistry ; 60(17): 1347-1355, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33876640

RESUMO

Protein phosphorylation is an abundant post-translational modification (PTM) and an essential modulator of protein functionality in living cells. Intrinsically disordered proteins (IDPs) are particular targets of PTM protein kinases due to their involvement in fundamental protein interaction networks. Despite their dynamic nature, IDPs are far from having random-coil conformations but exhibit significant structural heterogeneity. Changes in the molecular environment, most prominently in the form of PTM via phosphorylation, can modulate these structural features. Therefore, how phosphorylation events can alter conformational ensembles of IDPs and their interactions with binding partners is of great interest. Here we study the effects of hyperphosphorylation on the IDP osteopontin (OPN), an extracellular target of the Fam20C kinase. We report a full characterization of the phosphorylation sites of OPN using a combined nuclear magnetic resonance/mass spectrometry approach and provide evidence for an increase in the local flexibility of highly phosphorylated regions and the ensuing overall structural elongation. Our study emphasizes the simultaneous importance of electrostatic and hydrophobic interactions in the formation of compact substates in IDPs and their relevance for molecular recognition events.


Assuntos
Osteopontina/química , Osteopontina/metabolismo , Humanos , Simulação de Dinâmica Molecular , Fosforilação , Conformação Proteica , Dobramento de Proteína
4.
Cell Cycle ; 19(14): 1777-1785, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32594847

RESUMO

Meiosis is the process by which haploid gametes are produced from diploid precursor cells. We used stable isotope labeling by amino acids in cell culture (SILAC) to characterize the meiotic proteome in the fission yeast Schizosaccharomyces pombe. We compared relative levels of proteins extracted from cells harvested around meiosis I with those of meiosis II, and proteins from premeiotic S phase with the interval between meiotic divisions, when S phase is absent. Our proteome datasets revealed peptides corresponding to short open reading frames (sORFs) that have been previously identified by ribosome profiling as new translated regions. We verified expression of selected sORFs by Western blotting and analyzed the phenotype of deletion mutants. Our data provide a resource for studying meiosis that may help understand differences between meiosis I and meiosis II and how S phase is suppressed between the two meiotic divisions.


Assuntos
Meiose , Fases de Leitura Aberta/genética , Proteômica , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Deleção de Genes , Marcação por Isótopo , Meiose/genética , Fenótipo , Proteoma/metabolismo , Reprodutibilidade dos Testes , Ribossomos/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo
5.
Cell Rep ; 30(9): 3171-3182.e6, 2020 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-32130916

RESUMO

Protein phosphatase 2A (PP2A) is an important regulator of signal transduction pathways and a tumor suppressor. Phosphorylation of the PP2A catalytic subunit (PP2AC) at tyrosine 307 has been claimed to inactivate PP2A and was examined in more than 180 studies using commercial antibodies, but this modification was never identified using mass spectrometry. Here we show that the most cited pTyr307 monoclonal antibodies, E155 and F-8, are not specific for phosphorylated Tyr307 but instead are hampered by PP2AC methylation at leucine 309 or phosphorylation at threonine 304. Other pTyr307 antibodies are sensitive to PP2AC methylation as well, and some cross-react with pTyr residues in general, including phosphorylated hemagglutinin tags. We identify pTyr307 using targeted mass spectrometry after transient overexpression of PP2AC and Src kinase. Yet under such conditions, none of the tested antibodies show exclusive pTyr307 specificity. Thus, data generated using these antibodies need to be revisited, and the mechanism of PP2A inactivation needs to be redefined.


Assuntos
Anticorpos/metabolismo , Especificidade de Anticorpos/imunologia , Leucina/metabolismo , Fosfotirosina/metabolismo , Proteína Fosfatase 2/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos/efeitos dos fármacos , Reações Cruzadas/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Células HEK293 , Humanos , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Peptídeos/química , Peptídeos/metabolismo , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Vanadatos/farmacologia , Quinases da Família src/metabolismo
6.
RNA Biol ; 17(8): 1104-1115, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32138588

RESUMO

During particular stress conditions, transfer RNAs (tRNAs) become substrates of stress-induced endonucleases, resulting in the production of distinct tRNA-derived small RNAs (tsRNAs). These small RNAs have been implicated in a wide range of biological processes, but how isoacceptor and even isodecoder-specific tsRNAs act at the molecular level is still poorly understood. Importantly, stress-induced tRNA cleavage affects only a few tRNAs of a given isoacceptor or isodecoder, raising the question as to how such limited molecule numbers could exert measurable biological impact. While the molecular function of individual tsRNAs is likely mediated through association with other molecules, addressing the interactome of specific tsRNAs has only been attempted by using synthetic RNA sequences. Since tRNAs carry post-transcriptional modifications, tsRNAs are likely modified but the extent of their modifications remains largely unknown. Here, we developed a biochemical framework for the production and purification of specific tsRNAs using human cells. Preparative scale purification of tsRNAs from biological sources should facilitate experimentally addressing as to how exactly these small RNAs mediate the multitude of reported molecular functions.


Assuntos
Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/isolamento & purificação , RNA de Transferência/genética , Morte Celular , Linhagem Celular , Fracionamento Químico , Expressão Ectópica do Gene , Dosagem de Genes , Regulação da Expressão Gênica , Humanos , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , RNA de Transferência/química , Estresse Fisiológico/genética
7.
Elife ; 82019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30767890

RESUMO

Many marine animals, ranging from corals to fishes, synchronise reproduction to lunar cycles. In the annelid Platynereis dumerilii, this timing is orchestrated by an endogenous monthly (circalunar) clock entrained by moonlight. Whereas daily (circadian) clocks cause extensive transcriptomic and proteomic changes, the quality and quantity of regulations by circalunar clocks have remained largely elusive. By establishing a combined transcriptomic and proteomic profiling approach, we provide first systematic insight into the molecular changes in Platynereis heads between circalunar phases, and across sexual differentiation and maturation. Whereas maturation elicits large transcriptomic and proteomic changes, the circalunar clock exhibits only minor transcriptomic, but strong proteomic regulation. Our study provides a versatile extraction technique and comprehensive resources. It corroborates that circadian and circalunar clock effects are likely distinct and identifies key molecular brain signatures for reproduction, sex and circalunar clock phase. Examples include prepro-whitnin/proctolin and ependymin-related proteins as circalunar clock targets.


Assuntos
Anelídeos/genética , Encéfalo/metabolismo , Relógios Circadianos/genética , Proteoma/metabolismo , Proteômica , Maturidade Sexual/genética , Transcriptoma/genética , Animais , Feminino , Masculino , Filogenia , Transdução de Sinais/genética
8.
Nucleic Acids Res ; 47(1): 3-14, 2019 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-30462291

RESUMO

RNA modifications are present in all classes of RNAs. They control the fate of mRNAs by affecting their processing, translation, or stability. Inosine is a particularly widespread modification in metazoan mRNA arising from deamination of adenosine catalyzed by the RNA-targeting adenosine deaminases ADAR1 or ADAR2. Inosine is commonly thought to be interpreted as guanosine by cellular machines and during translation. Here, we systematically test ribosomal decoding using mass spectrometry. We show that while inosine is primarily interpreted as guanosine it can also be decoded as adenosine, and rarely even as uracil. Decoding of inosine as adenosine and uracil is context-dependent. In addition, mass spectrometry analysis indicates that inosine causes ribosome stalling especially when multiple inosines are present in the codon. Indeed, ribosome profiling data from human tissues confirm inosine-dependent ribosome stalling in vivo. To our knowledge this is the first study where decoding of inosine is tested in a comprehensive and unbiased way. Thus, our study shows novel, unanticipated functions for inosines in mRNAs, further expanding coding potential and affecting translational efficiency.


Assuntos
Código Genético , Inosina/genética , Biossíntese de Proteínas , Edição de RNA , RNA Mensageiro/genética , Adenosina/genética , Adenosina/metabolismo , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sistema Livre de Células/química , Sistema Livre de Células/metabolismo , Clonagem Molecular , Desaminação , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Guanosina/genética , Guanosina/metabolismo , Humanos , Inosina/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reticulócitos/química , Reticulócitos/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Uracila/metabolismo
9.
Sci Signal ; 10(469)2017 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-28270554

RESUMO

The budding yeast Saccharomyces cerevisiae reacts to increased external osmolarity by modifying many cellular processes. Adaptive signaling relies primarily on the high-osmolarity glycerol (HOG) pathway, which is closely related to the mammalian p38 mitogen-activated protein kinase (MAPK) pathway in core architecture. To identify target proteins of the MAPK Hog1, we designed a mass spectrometry-based high-throughput experiment to measure the impact of Hog1 activation or inhibition on the Scerevisiae phosphoproteome. In addition, we analyzed how deletion of RCK2, which encodes a known effector protein kinase target of Hog1, modulated osmotic stress-induced phosphorylation. Our results not only provide an overview of the diversity of cellular functions that are directly and indirectly affected by the activity of the HOG pathway but also enabled an assessment of the Hog1-independent events that occur under osmotic stress conditions. We extended the number of putative Hog1 direct targets by analyzing the modulation of motifs consisting of serine or threonine followed by a proline (S/T-P motif) and subsequently validated these with an in vivo interaction assay. Rck2 appears to act as a central hub for many Hog1-mediated secondary phosphorylation events. This study clarifies many of the direct and indirect effects of HOG signaling and its stress-adaptive functions.


Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Pressão Osmótica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Espectrometria de Massas/métodos , Proteínas Quinases Ativadas por Mitógeno/genética , Mutação , Concentração Osmolar , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais/genética
10.
Sci Rep ; 6: 27383, 2016 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-27272015

RESUMO

The activity of several cytosolic proteins critically depends on the concentration of calcium ions. One important intracellular calcium-sensing protein is α-actinin-1, the major actin crosslinking protein in focal adhesions and stress fibers. The actin crosslinking activity of α-actinin-1 has been proposed to be negatively regulated by calcium, but the underlying molecular mechanisms are poorly understood. To address this, we determined the first high-resolution NMR structure of its functional calmodulin-like domain (CaMD) in calcium-bound and calcium-free form. These structures reveal that in the absence of calcium, CaMD displays a conformationally flexible ensemble that undergoes a structural change upon calcium binding, leading to limited rotation of the N- and C-terminal lobes around the connecting linker and consequent stabilization of the calcium-loaded structure. Mutagenesis experiments, coupled with mass-spectrometry and isothermal calorimetry data designed to validate the calcium binding stoichiometry and binding site, showed that human non-muscle α-actinin-1 binds a single calcium ion within the N-terminal lobe. Finally, based on our structural data and analogy with other α-actinins, we provide a structural model of regulation of the actin crosslinking activity of α-actinin-1 where calcium induced structural stabilisation causes fastening of the juxtaposed actin binding domain, leading to impaired capacity to crosslink actin.


Assuntos
Actinina/metabolismo , Cálcio/metabolismo , Calmodulina/metabolismo , Actinina/química , Sequência de Aminoácidos , Calmodulina/química , Humanos , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica , Homologia de Sequência de Aminoácidos
11.
Mol Biol Cell ; 27(2): 397-409, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26582391

RESUMO

Nutrient-sensitive phosphorylation of the S6 protein of the 40S subunit of the eukaryote ribosome is highly conserved. However, despite four decades of research, the functional consequences of this modification remain unknown. Revisiting this enigma in Saccharomyces cerevisiae, we found that the regulation of Rps6 phosphorylation on Ser-232 and Ser-233 is mediated by both TOR complex 1 (TORC1) and TORC2. TORC1 regulates phosphorylation of both sites via the poorly characterized AGC-family kinase Ypk3 and the PP1 phosphatase Glc7, whereas TORC2 regulates phosphorylation of only the N-terminal phosphosite via Ypk1. Cells expressing a nonphosphorylatable variant of Rps6 display a reduced growth rate and a 40S biogenesis defect, but these phenotypes are not observed in cells in which Rps6 kinase activity is compromised. Furthermore, using polysome profiling and ribosome profiling, we failed to uncover a role of Rps6 phosphorylation in either global translation or translation of individual mRNAs. Taking the results together, this work depicts the signaling cascades orchestrating Rps6 phosphorylation in budding yeast, challenges the notion that Rps6 phosphorylation plays a role in translation, and demonstrates that observations made with Rps6 knock-ins must be interpreted cautiously.


Assuntos
Complexos Multiproteicos/metabolismo , Proteína S6 Ribossômica/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Fosforilação/fisiologia , Polirribossomos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína S6 Ribossômica/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/metabolismo
12.
Nucleic Acids Res ; 44(4): 1703-17, 2016 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-26682798

RESUMO

The formation of RNA-DNA hybrids, referred to as R-loops, can promote genome instability and cancer development. Yet the mechanisms by which R-loops compromise genome instability are poorly understood. Here, we establish roles for the evolutionarily conserved Nrl1 protein in pre-mRNA splicing regulation, R-loop suppression and in maintaining genome stability. nrl1Δ mutants exhibit endogenous DNA damage, are sensitive to exogenous DNA damage, and have defects in homologous recombination (HR) repair. Concomitantly, nrl1Δ cells display significant changes in gene expression, similar to those induced by DNA damage in wild-type cells. Further, we find that nrl1Δ cells accumulate high levels of R-loops, which co-localize with HR repair factors and require Rad51 and Rad52 for their formation. Together, our findings support a model in which R-loop accumulation and subsequent DNA damage sequesters HR factors, thereby compromising HR repair at endogenously or exogenously induced DNA damage sites, leading to genome instability.


Assuntos
Processamento Alternativo/genética , Instabilidade Genômica/genética , Recombinação Homóloga/genética , Precursores de RNA/genética , Proteínas de Schizosaccharomyces pombe/genética , DNA/química , DNA/genética , Reparo do DNA/genética , RNA/química , RNA/genética , Rad51 Recombinase/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Schizosaccharomyces/genética , Spliceossomos/genética , Spliceossomos/metabolismo
13.
Elife ; 42015 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-26263501

RESUMO

Metabolic adjustment to changing environmental conditions, particularly balancing of growth and defense responses, is crucial for all organisms to survive. The evolutionary conserved AMPK/Snf1/SnRK1 kinases are well-known metabolic master regulators in the low-energy response in animals, yeast and plants. They act at two different levels: by modulating the activity of key metabolic enzymes, and by massive transcriptional reprogramming. While the first part is well established, the latter function is only partially understood in animals and not at all in plants. Here we identified the Arabidopsis transcription factor bZIP63 as key regulator of the starvation response and direct target of the SnRK1 kinase. Phosphorylation of bZIP63 by SnRK1 changed its dimerization preference, thereby affecting target gene expression and ultimately primary metabolism. A bzip63 knock-out mutant exhibited starvation-related phenotypes, which could be functionally complemented by wild type bZIP63, but not by a version harboring point mutations in the identified SnRK1 target sites.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Regulação da Expressão Gênica de Plantas , Multimerização Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Adaptação Fisiológica , Arabidopsis/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/deficiência , Técnicas de Inativação de Genes , Teste de Complementação Genética , Fosforilação , Processamento de Proteína Pós-Traducional
14.
Mol Biol Cell ; 26(17): 3013-29, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26133384

RESUMO

Trypanosoma brucei is the causative agent of African sleeping sickness, a devastating disease endemic to sub-Saharan Africa with few effective treatment options. The parasite is highly polarized, including a single flagellum that is nucleated at the posterior of the cell and adhered along the cell surface. These features are essential and must be transmitted to the daughter cells during division. Recently we identified the T. brucei homologue of polo-like kinase (TbPLK) as an essential morphogenic regulator. In the present work, we conduct proteomic screens to identify potential TbPLK binding partners and substrates to better understand the molecular mechanisms of kinase function. These screens identify a cohort of proteins, most of which are completely uncharacterized, which localize to key cytoskeletal organelles involved in establishing cell morphology, including the flagella connector, flagellum attachment zone, and bilobe structure. Depletion of these proteins causes substantial changes in cell division, including mispositioning of the kinetoplast, loss of flagellar connection, and prevention of cytokinesis. The proteins identified in these screens provide the foundation for establishing the molecular networks through which TbPLK directs cell morphogenesis in T. brucei.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/metabolismo , Animais , Divisão Celular/fisiologia , Células Cultivadas , Citocinese , Flagelos/metabolismo , Morfogênese , Fosforilação , Ligação Proteica , Proteômica/métodos , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/genética
15.
Cell Cycle ; 14(15): 2439-50, 2015 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-26083678

RESUMO

DNA double-strand break repair by the error-free pathway of homologous recombination (HR) requires the concerted action of several factors. Among these, EXO1 and DNA2/BLM are responsible for the extensive resection of DNA ends to produce 3'-overhangs, which are essential intermediates for downstream steps of HR. Here we show that EXO1 is a SUMO target and that sumoylation affects EXO1 ubiquitylation and protein stability. We identify an UBC9-PIAS1/PIAS4-dependent mechanism controlling human EXO1 sumoylation in vivo and demonstrate conservation of this mechanism in yeast by the Ubc9-Siz1/Siz2 using an in vitro reconstituted system. Furthermore, we show physical interaction between EXO1 and the de-sumoylating enzyme SENP6 both in vitro and in vivo, promoting EXO1 stability. Finally, we identify the major sites of sumoylation in EXO1 and show that ectopic expression of a sumoylation-deficient form of EXO1 rescues the DNA damage-induced chromosomal aberrations observed upon wt-EXO1 expression. Thus, our study identifies a novel layer of regulation of EXO1, making the pathways that regulate its function an ideal target for therapeutic intervention.


Assuntos
Quebras de DNA de Cadeia Dupla , Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA/genética , Exodesoxirribonucleases/metabolismo , Sumoilação/fisiologia , Antineoplásicos Fitogênicos/farmacologia , Camptotecina/farmacologia , Linhagem Celular , Cisteína Endopeptidases/metabolismo , Enzimas Reparadoras do DNA/genética , Exodesoxirribonucleases/genética , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Proteínas de Ligação a Poli-ADP-Ribose , Proteínas Inibidoras de STAT Ativados/genética , Proteínas Inibidoras de STAT Ativados/metabolismo , Estabilidade Proteica , Proteína SUMO-1/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
16.
PLoS Genet ; 11(5): e1005225, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25993311

RESUMO

Proper meiotic chromosome segregation, essential for sexual reproduction, requires timely formation and removal of sister chromatid cohesion and crossing-over between homologs. Early in meiosis cohesins hold sisters together and also promote formation of DNA double-strand breaks, obligate precursors to crossovers. Later, cohesin cleavage allows chromosome segregation. We show that in fission yeast redundant casein kinase 1 homologs, Hhp1 and Hhp2, previously shown to regulate segregation via phosphorylation of the Rec8 cohesin subunit, are also required for high-level meiotic DNA breakage and recombination. Unexpectedly, these kinases also mediate phosphorylation of a different meiosis-specific cohesin subunit Rec11. This phosphorylation in turn leads to loading of linear element proteins Rec10 and Rec27, related to synaptonemal complex proteins of other species, and thereby promotes DNA breakage and recombination. Our results provide novel insights into the regulation of chromosomal features required for crossing-over and successful reproduction. The mammalian functional homolog of Rec11 (STAG3) is also phosphorylated during meiosis and appears to be required for fertility, indicating wide conservation of the meiotic events reported here.


Assuntos
Caseína Quinase I/metabolismo , Segregação de Cromossomos , Proteínas Quinases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Caseína Quinase I/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Fúngico/genética , Recombinação Homóloga , Meiose , Mutação , Fosforilação , Regiões Promotoras Genéticas , Proteínas Quinases/genética , Proteínas de Schizosaccharomyces pombe/genética , Complexo Sinaptonêmico/metabolismo
17.
Environ Microbiol ; 17(4): 1397-413, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25212454

RESUMO

Chlamydiae are a highly successful group of obligate intracellular bacteria infecting a variety of eukaryotic hosts. Outer membrane proteins involved in attachment to and uptake into host cells, and cross-linking of these proteins via disulfide bonds are key features of the biphasic chlamydial developmental cycle. In this study, we used a consensus approach to predict outer membrane proteins in the genomes of members of three chlamydial families. By analysing outer membrane protein fractions of purified chlamydiae with highly sensitive mass spectrometry, we show that the protein composition differs strongly between these organisms. Large numbers of major outer membrane protein-like proteins are present at high abundance in the outer membrane of Simkania negevensis and Waddlia chondrophila, whereas yet uncharacterized putative porins dominate in Parachlamydia acanthamoebae. Simkania represents the first case of a chlamydia completely lacking stabilizing cysteine-rich proteins in its outer membrane. In agreement with this, and in contrast to Parachlamydia and Waddlia, the cellular integrity of Simkania is not impaired by conditions that reduce disulfide bonds of these proteins. The observed differences in the protein composition of the outer membrane among members of divergent chlamydial families suggest different stabilities of these organisms in the environment, probably due to adaption to different niches or transmission routes.


Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Chlamydia/genética , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Chlamydia/química , Chlamydia/classificação , Chlamydia/metabolismo , Sequência Conservada , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência
18.
Cell Rep ; 9(1): 143-152, 2014 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-25263559

RESUMO

DNA repair scaffolds mediate specific DNA and protein interactions in order to assist repair enzymes in recognizing and removing damaged sequences. Many scaffold proteins are dedicated to repairing a particular type of lesion. Here, we show that the budding yeast Saw1 scaffold is more versatile. It helps cells cope with base lesions and protein-DNA adducts through its known function of recruiting the Rad1-Rad10 nuclease to DNA. In addition, it promotes UV survival via a mechanism mediated by its sumoylation. Saw1 sumoylation favors its interaction with another nuclease Slx1-Slx4, and this SUMO-mediated role is genetically separable from two main UV lesion repair processes. These effects of Saw1 and its sumoylation suggest that Saw1 is a multifunctional scaffold that can facilitate diverse types of DNA repair through its modification and nuclease interactions.


Assuntos
Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Endonucleases/genética , Saccharomyces cerevisiae/citologia , Sumoilação , Análise de Sobrevida
19.
Mol Cell Biol ; 34(4): 711-24, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24324010

RESUMO

Downregulation of specific transcripts is one of the mechanisms utilized by eukaryotic checkpoint systems to prevent cell cycle progression. Here we identified and explored such a mechanism in the yeast Saccharomyces cerevisiae. It involves the Mec1-Rad53 kinase cascade, which attenuates G(2)/M-specific gene transcription upon genotoxic stress. This inhibition is achieved via multiple Rad53-dependent inhibitory phosphorylations on the transcriptional activator Ndd1 that prevent its chromatin recruitment via interactions with the forkhead factor Fkh2. Relevant modification sites on Ndd1 were identified by mass spectrometry, and corresponding alanine substitutions were able to suppress a methyl methanesulfonate-induced block in Ndd1 chromatin recruitment. Whereas effective suppression by these Ndd1 mutants is achieved for DNA damage, this is not the case under replication stress conditions, suggesting that additional mechanisms must operate under such conditions. We propose that budding yeast cells prevent the normal transcription of G(2)/M-specific genes upon genotoxic stress to precisely coordinate the timing of mitotic and postmitotic events with respect to S phase.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Divisão Celular/genética , Dano ao DNA/genética , Fase G2/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional/genética , Animais , Proteínas de Ciclo Celular/genética , Divisão Celular/fisiologia , Cromatina/metabolismo , Dano ao DNA/fisiologia , Replicação do DNA/genética , Replicação do DNA/fisiologia , Fase G2/fisiologia , Regulação Fúngica da Expressão Gênica/genética , Regulação Fúngica da Expressão Gênica/fisiologia , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética
20.
Nucleic Acids Res ; 41(10): 5341-53, 2013 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-23571759

RESUMO

Non-homologous end-joining (NHEJ) repairs DNA double-strand breaks by tethering and ligating the two DNA ends. The mechanisms regulating NHEJ efficiency and interplay between its components are not fully understood. Here, we identify and characterize the SUMOylation of budding yeast Lif1 protein, which is required for the ligation step in NHEJ. We show that Lif1 SUMOylation occurs throughout the cell cycle and requires the Siz SUMO ligases. Single-strand DNA, but not double-strand DNA or the Lif1 binding partner Nej1, is inhibitory to Lif1 SUMOylation. We identify lysine 301 as the major conjugation site and demonstrate that its replacement with arginine completely abolishes Lif1 SUMOylation in vivo and in vitro. The lif1-K301R mutant cells exhibit increased levels of NHEJ repair compared with wild-type cells throughout the cell cycle. This is likely due to the inhibitory effect of Lif1 SUMOylation on both its self-association and newly observed single-strand DNA binding activity. Taken together, these findings suggest that SUMOylation of Lif1 represents a new regulatory mechanism that downregulates NHEJ in a cell cycle phase-independent manner.


Assuntos
Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sumoilação , DNA/metabolismo , DNA Ligase Dependente de ATP , DNA Ligases/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Lisina/metabolismo , Mutação , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligases/genética
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