Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 29
Filtrar
1.
Nature ; 619(7969): 385-393, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-37407816

RESUMO

The basic helix-loop-helix (bHLH) family of transcription factors recognizes DNA motifs known as E-boxes (CANNTG) and includes 108 members1. Here we investigate how chromatinized E-boxes are engaged by two structurally diverse bHLH proteins: the proto-oncogene MYC-MAX and the circadian transcription factor CLOCK-BMAL1 (refs. 2,3). Both transcription factors bind to E-boxes preferentially near the nucleosomal entry-exit sites. Structural studies with engineered or native nucleosome sequences show that MYC-MAX or CLOCK-BMAL1 triggers the release of DNA from histones to gain access. Atop the H2A-H2B acidic patch4, the CLOCK-BMAL1 Per-Arnt-Sim (PAS) dimerization domains engage the histone octamer disc. Binding of tandem E-boxes5-7 at endogenous DNA sequences occurs through direct interactions between two CLOCK-BMAL1 protomers and histones and is important for circadian cycling. At internal E-boxes, the MYC-MAX leucine zipper can also interact with histones H2B and H3, and its binding is indirectly enhanced by OCT4 elsewhere on the nucleosome. The nucleosomal E-box position and the type of bHLH dimerization domain jointly determine the histone contact, the affinity and the degree of competition and cooperativity with other nucleosome-bound factors.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , DNA , Histonas , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , DNA/genética , DNA/metabolismo , Sequências Hélice-Alça-Hélice/genética , Histonas/química , Histonas/metabolismo , Nucleossomos/química , Nucleossomos/genética , Nucleossomos/metabolismo , Ligação Proteica , Proteínas CLOCK/química , Proteínas CLOCK/metabolismo , Proteínas Proto-Oncogênicas c-myc/química , Proteínas Proto-Oncogênicas c-myc/metabolismo , Regulação Alostérica , Zíper de Leucina , Fator 3 de Transcrição de Octâmero/metabolismo , Multimerização Proteica
2.
Mol Cell ; 83(14): 2478-2492.e8, 2023 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-37369201

RESUMO

The RNA-binding protein TRIM71/LIN-41 is a phylogenetically conserved developmental regulator that functions in mammalian stem cell reprogramming, brain development, and cancer. TRIM71 recognizes target mRNAs through hairpin motifs and silences them through molecular mechanisms that await identification. Here, we uncover that TRIM71 represses its targets through RNA-supported interaction with TNRC6/GW182, a core component of the miRNA-induced silencing complex (miRISC). We demonstrate that AGO2, TRIM71, and UPF1 each recruit TNRC6 to specific sets of transcripts to silence them. As cellular TNRC6 levels are limiting, competition occurs among the silencing pathways, such that the loss of AGO proteins or of AGO binding to TNRC6 enhances the activities of the other pathways. We conclude that a miRNA-like silencing activity is shared among different mRNA silencing pathways and that the use of TNRC6 as a central hub provides a means to integrate their activities.


Assuntos
Proteínas Argonautas , MicroRNAs , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ligação Proteica , Células-Tronco/metabolismo , Mamíferos/metabolismo
4.
Mol Cell ; 82(7): 1359-1371.e9, 2022 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-35216668

RESUMO

The chromatin-binding protein 53BP1 promotes DNA repair by orchestrating the recruitment of downstream effectors including PTIP, RIF1, and shieldin to DNA double-strand break sites. While we know how PTIP recognizes 53BP1, the molecular details of RIF1 recruitment to DNA-damage sites remains undefined. Here, we report that RIF1 is a phosphopeptide-binding protein that directly interacts with three phosphorylated 53BP1 epitopes. The RIF1-binding sites on 53BP1 share an essential LxL motif followed by two closely apposed phosphorylated residues. Simultaneous mutation of these sites on 53BP1 abrogates RIF1 accumulation into ionizing-radiation-induced foci, but surprisingly, only fully compromises 53BP1-dependent DNA repair when an alternative mode of shieldin recruitment to DNA-damage sites is also disabled. Intriguingly, this alternative mode of recruitment still depends on RIF1 but does not require its interaction with 53BP1. RIF1 therefore employs phosphopeptide recognition to promote DNA repair but also modifies shieldin action independently of 53BP1 binding.


Assuntos
Fosfopeptídeos , Proteínas de Ligação a Telômeros , Proteína BRCA1/genética , Proteínas de Transporte/metabolismo , DNA/metabolismo , Reparo do DNA por Junção de Extremidades , Reparo do DNA , Fosfopeptídeos/genética , Fosfopeptídeos/metabolismo , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
5.
Nat Commun ; 13(1): 1059, 2022 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-35217664

RESUMO

The coordinated action of multiple replicative helicase loading factors is needed for the licensing of replication origins prior to DNA replication. Binding of the Origin Recognition Complex (ORC) to DNA initiates the ATP-dependent recruitment of Cdc6, Cdt1 and Mcm2-7 loading, but the structural details for timely ATPase site regulation and for how loading can be impeded by inhibitory signals, such as cyclin-dependent kinase phosphorylation, are unknown. Using cryo-electron microscopy, we have determined several structures of S. cerevisiae ORC·DNA·Cdc6 intermediates at 2.5-2.7 Å resolution. These structures reveal distinct ring conformations of the initiator·co-loader assembly and inactive ATPase site configurations for ORC and Cdc6. The Orc6 N-terminal domain laterally engages the ORC·Cdc6 ring in a manner that is incompatible with productive Mcm2-7 docking, while deletion of this Orc6 region alleviates the CDK-mediated inhibition of Mcm7 recruitment. Our findings support a model in which Orc6 promotes the assembly of an autoinhibited ORC·DNA·Cdc6 intermediate to block origin licensing in response to CDK phosphorylation and to avert DNA re-replication.


Assuntos
Proteínas de Ciclo Celular , Complexo de Reconhecimento de Origem , Proteínas de Saccharomyces cerevisiae , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Microscopia Crioeletrônica , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , DNA/metabolismo , DNA Helicases/metabolismo , Replicação do DNA , Componente 7 do Complexo de Manutenção de Minicromossomo/genética , Complexo de Reconhecimento de Origem/metabolismo , Ligação Proteica , Origem de Replicação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
6.
Nat Struct Mol Biol ; 29(2): 85-96, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35102319

RESUMO

Transcriptionally silenced heterochromatin bearing methylation of histone H3 on lysine 9 (H3K9me) is critical for maintaining organismal viability and tissue integrity. Here we show that in addition to ensuring H3K9me, MET-2, the Caenorhabditis elegans homolog of the SETDB1 histone methyltransferase, has a noncatalytic function that contributes to gene repression. Subnuclear foci of MET-2 coincide with H3K9me deposition, yet these foci also form when MET-2 is catalytically deficient and H3K9me is compromised. Whereas met-2 deletion triggers a loss of silencing and increased histone acetylation, foci of catalytically deficient MET-2 maintain silencing of a subset of genes, blocking acetylation on H3K9 and H3K27. In normal development, this noncatalytic MET-2 activity helps to maintain fertility. Under heat stress MET-2 foci disperse, coinciding with increased acetylation and transcriptional derepression. Our study suggests that the noncatalytic, focus-forming function of this SETDB1-like protein and its intrinsically disordered cofactor LIN-61 is physiologically relevant.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Animais , Animais Geneticamente Modificados , Biocatálise , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas Cromossômicas não Histona/deficiência , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Inativação Gênica , Heterocromatina/genética , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase/deficiência , Histona-Lisina N-Metiltransferase/genética , Histonas/metabolismo , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Metilação , Modelos Biológicos , Mutação , Transcrição Gênica
7.
STAR Protoc ; 2(4): 100825, 2021 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-34568845

RESUMO

Here, we describe a fractionation protocol optimized to quantify changes in relative abundance of the chromatin-bound proteome (chromatome) by tandem mass tag multiplexing-based tandem mass spectrometry. It has been applied to yeast cells before and after exposure to DNA-damaging drugs to characterize changes in chromatin composition induced by the DNA damage response. We detail steps for stringent chromatin fractionation, sample preparation for mass spectrometry, and its evaluation. For complete details on the use and execution of this protocol, please refer to Challa et al. (2021).


Assuntos
Cromatina , Proteoma , Proteômica/métodos , Saccharomycetales , Sacarose/química , Cromatina/química , Cromatina/genética , Cromatina/isolamento & purificação , Proteoma/análise , Proteoma/química , Proteoma/genética , Saccharomycetales/química , Saccharomycetales/genética , Saccharomycetales/metabolismo , Espectrometria de Massas em Tandem/métodos
8.
EMBO J ; 40(21): e108439, 2021 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-34569643

RESUMO

Upon replication stress, budding yeast checkpoint kinase Mec1ATR triggers the downregulation of transcription, thereby reducing the level of RNA polymerase (RNAP) on chromatin to facilitate replication fork progression. Here, we identify a hydroxyurea-induced phosphorylation site on Mec1, Mec1-S1991, that contributes to the eviction of RNAPII and RNAPIII during replication stress. The expression of the non-phosphorylatable mec1-S1991A mutant reduces replication fork progression genome-wide and compromises survival on hydroxyurea. This defect can be suppressed by destabilizing chromatin-bound RNAPII through a TAP fusion to its Rpb3 subunit, suggesting that lethality in mec1-S1991A mutants arises from replication-transcription conflicts. Coincident with a failure to repress gene expression on hydroxyurea in mec1-S1991A cells, highly transcribed genes such as GAL1 remain bound at nuclear pores. Consistently, we find that nuclear pore proteins and factors controlling RNAPII and RNAPIII are phosphorylated in a Mec1-dependent manner on hydroxyurea. Moreover, we show that Mec1 kinase also contributes to reduced RNAPII occupancy on chromatin during an unperturbed S phase by promoting degradation of the Rpb1 subunit.


Assuntos
Replicação do DNA , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , RNA Polimerase III/genética , RNA Polimerase II/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Cromatina/química , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Galactoquinase/genética , Galactoquinase/metabolismo , Regulação Fúngica da Expressão Gênica , Hidroxiureia/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fosfoproteínas , Fosforilação , Proteínas Serina-Treonina Quinases/genética , RNA Polimerase II/metabolismo , RNA Polimerase III/metabolismo , Fase S/efeitos dos fármacos , Fase S/genética , Saccharomyces cerevisiae/genética , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Transcrição Gênica
9.
PLoS Biol ; 19(7): e3001344, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34297726

RESUMO

A major cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) spectrum disorder is the hexanucleotide G4C2 repeat expansion in the first intron of the C9orf72 gene. Many underlying mechanisms lead to manifestation of disease that include toxic gain-of-function by repeat G4C2 RNAs, dipeptide repeat proteins, and a reduction of the C9orf72 gene product. The C9orf72 protein interacts with SMCR8 and WDR41 to form a trimeric complex and regulates multiple cellular pathways including autophagy. Here, we report the structure of the C9orf72-SMCR8 complex at 3.8 Å resolution using single-particle cryo-electron microscopy (cryo-EM). The structure reveals 2 distinct dimerization interfaces between C9orf72 and SMCR8 that involves an extensive network of interactions. Homology between C9orf72-SMCR8 and Folliculin-Folliculin Interacting Protein 2 (FLCN-FNIP2), a GTPase activating protein (GAP) complex, enabled identification of a key residue within the active site of SMCR8. Further structural analysis suggested that a coiled-coil region within the uDenn domain of SMCR8 could act as an interaction platform for other coiled-coil proteins, and its deletion reduced the interaction of the C9orf72-SMCR8 complex with FIP200 upon starvation. In summary, this study contributes toward our understanding of the biological function of the C9orf72-SMCR8 complex.


Assuntos
Proteína C9orf72/metabolismo , Proteínas de Transporte/metabolismo , Esclerose Lateral Amiotrófica/genética , Animais , Proteína C9orf72/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Linhagem Celular , Demência Frontotemporal/genética , Humanos , Estrutura Molecular , Fases de Leitura Aberta , Ligação Proteica , Mapas de Interação de Proteínas , Spodoptera
10.
Mol Cell ; 81(4): 811-829.e6, 2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33529595

RESUMO

Eukaryotic cells package their genomes around histone octamers. In response to DNA damage, checkpoint activation in yeast induces core histone degradation resulting in 20%-40% reduction in nucleosome occupancy. To gain insight into this process, we developed a new approach to analyze the chromatin-associated proteome comprehensively before and after damage. This revealed extensive changes in protein composition after Zeocin-induced damage. First, core histones and the H1 homolog Hho1 were partially lost from chromatin along with replication, transcription, and chromatin remodeling machineries, while ubiquitin ligases and the proteasome were recruited. We found that the checkpoint- and INO80C-dependent recruitment of five ubiquitin-conjugating factors (Rad6, Bre1, Pep5, Ufd4, and Rsp5) contributes to core and linker histone depletion, reducing chromatin compaction and enhancing DNA locus mobility. Importantly, loss of Rad6/Bre1, Ufd4/TRIP12, and Pep5/VPS11 compromise DNA strand invasion kinetics during homology-driven repair. Thus we provide a comprehensive overview of a functionally relevant genome-wide chromatin response to DNA damage.


Assuntos
Montagem e Desmontagem da Cromatina , Reparo do DNA , DNA Fúngico/metabolismo , Histonas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , DNA Fúngico/genética , Histonas/genética , Complexo de Endopeptidases do Proteassoma/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligases/genética
11.
Nat Commun ; 9(1): 4048, 2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30279501

RESUMO

Regulation of transcription, replication, and cell division relies on differential protein binding to DNA and chromatin, yet it is unclear which regulatory components remain bound to compacted mitotic chromosomes. By utilizing the buoyant density of DNA-protein complexes after cross-linking, we here develop a mass spectrometry-based approach to quantify the chromatin-associated proteome at separate stages of the cell cycle. While epigenetic modifiers that promote transcription are lost from mitotic chromatin, repressive modifiers generally remain associated. Furthermore, while proteins involved in transcriptional elongation are evicted, most identified transcription factors are retained on mitotic chromatin to varying degrees, including core promoter binding proteins. This predicts conservation of the regulatory landscape on mitotic chromosomes, which we confirm by genome-wide measurements of chromatin accessibility. In summary, this work establishes an approach to study chromatin, provides a comprehensive catalog of chromatin changes during the cell cycle, and reveals the degree to which the genomic regulatory landscape is maintained through mitosis.


Assuntos
Ciclo Celular , Cromatina/metabolismo , Regulação da Expressão Gênica , Proteômica/métodos , Linhagem Celular Tumoral , Cromatina/química , Humanos , Espectrometria de Massas , Fatores de Transcrição/metabolismo
12.
Nat Commun ; 8: 15398, 2017 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-28530236

RESUMO

Thalidomide and its derivatives lenalidomide and pomalidomide (IMiDs) are effective treatments of haematologic malignancies. It was shown that IMiDs impart gain-of-function properties to the CUL4-RBX1-DDB1-CRBN (CRL4CRBN) ubiquitin ligase that enable binding, ubiquitination and degradation of key therapeutic targets such as IKZF1, IKZF3 and CSNK1A1. While these substrates have been implicated as efficacy targets in multiple myeloma (MM) and 5q deletion associated myelodysplastic syndrome (del(5q)-MDS), other targets likely exist. Using a pulse-chase SILAC mass spectrometry-based proteomics approach, we demonstrate that lenalidomide induces the ubiquitination and degradation of ZFP91. We establish ZFP91 as a bona fide IMiD-dependent CRL4CRBN substrate and further show that ZFP91 harbours a zinc finger (ZnF) motif, related to the IKZF1/3 ZnF, critical for IMiD-dependent CRBN binding. These findings demonstrate that single time point pulse-chase SILAC mass spectrometry-based proteomics (pSILAC MS) is a sensitive approach for target identification of small molecules inducing selective protein degradation.


Assuntos
Ubiquitina-Proteína Ligases/química , Proteínas Adaptadoras de Transdução de Sinal , Motivos de Aminoácidos , Animais , Antineoplásicos/farmacologia , Deleção de Genes , Células HCT116 , Células HEK293 , Humanos , Lenalidomida/farmacologia , Espectrometria de Massas , Camundongos , Mieloma Múltiplo/metabolismo , Síndromes Mielodisplásicas/metabolismo , Peptídeo Hidrolases/química , Proteômica , Especificidade por Substrato , Ubiquitina/química , Ubiquitinação , Dedos de Zinco
13.
Cell Signal ; 28(9): 1412-1421, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27289018

RESUMO

Current standard-of-care treatment for malignant cancers includes radiotherapy and adjuvant chemotherapy. Here, we report increased MAP kinase-interacting kinase (MNK)-regulated phosphorylation of translation initiation factor 4E (eIF4E) in glioma cells upon temozolomide (TMZ) treatment and in medullary thyroid carcinoma (MTC) cells in response to targeted radionuclide therapy. Depletion of MNK activity by using two MNK inhibitors, CGP57380 or cercosporamide, as well as by MNK1-specific knockdown sensitized glioblastoma (GBM) cells and GBM-derived spheres to TMZ. Furthermore, CGP57380 treatment enhanced response of MTC cells to (177)Lu-labeled gastrin analogue. In order to understand how MNK signaling pathways support glioma survival we analyzed putative MNK substrates by quantitative phosphoproteomics in normal condition and in the presence of TMZ. We identified MNK inhibitor-sensitive phosphorylation sites on eIF4G1, mutations of which either influenced eIF4E phosphorylation or glioma cell response to TMZ, pointing to altered regulation of translation initiation as a resistance mechanism. Pharmacological inhibition of overexpressed MNK1 by CGP57380 reduced eIF4E phosphorylation and induced association of inactive MNK1 with eIF4G1. Taken together, our data show an activation of MNK-mediated survival mechanisms in response to either glioma chemotherapy or MTC targeted radiation and suggest that inhibition of MNK activity represents an attractive sensitizing strategy for cancer treatments.


Assuntos
Antineoplásicos/uso terapêutico , Dacarbazina/análogos & derivados , Glioma/tratamento farmacológico , Glioma/radioterapia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Radioisótopos/uso terapêutico , Transdução de Sinais , Compostos de Anilina , Antineoplásicos/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/radioterapia , Linhagem Celular Tumoral , Dacarbazina/farmacologia , Dacarbazina/uso terapêutico , Fator de Iniciação 4E em Eucariotos/metabolismo , Fator de Iniciação Eucariótico 4G/metabolismo , Gastrinas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lutécio , Fosfoproteínas/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/metabolismo , Proteômica , Purinas , Transdução de Sinais/efeitos dos fármacos , Temozolomida
14.
Elife ; 4: e08231, 2015 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-26182403

RESUMO

Cytosolic and nuclear iron-sulfur (Fe-S) proteins are involved in many essential pathways including translation and DNA maintenance. Their maturation requires the cytosolic Fe-S protein assembly (CIA) machinery. To identify new CIA proteins we employed systematic protein interaction approaches and discovered the essential proteins Yae1 and Lto1 as binding partners of the CIA targeting complex. Depletion of Yae1 or Lto1 results in defective Fe-S maturation of the ribosome-associated ABC protein Rli1, but surprisingly no other tested targets. Yae1 and Lto1 facilitate Fe-S cluster assembly on Rli1 in a chain of binding events. Lto1 uses its conserved C-terminal tryptophan for binding the CIA targeting complex, the deca-GX3 motifs in both Yae1 and Lto1 facilitate their complex formation, and Yae1 recruits Rli1. Human YAE1D1 and the cancer-related ORAOV1 can replace their yeast counterparts demonstrating evolutionary conservation. Collectively, the Yae1-Lto1 complex functions as a target-specific adaptor that recruits apo-Rli1 to the generic CIA machinery.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Transporte/metabolismo , Ferro/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Enxofre/metabolismo , Proteínas de Transporte/genética , Teste de Complementação Genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Ligação Proteica , Mapeamento de Interação de Proteínas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
15.
Sci Signal ; 7(329): ra56, 2014 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-24917593

RESUMO

Memo is an evolutionarily conserved protein with a critical role in cell motility. We found that Memo was required for migration and invasion of breast cancer cells in vitro and spontaneous lung metastasis from breast cancer cell xenografts in vivo. Biochemical assays revealed that Memo is a copper-dependent redox enzyme that promoted a more oxidized intracellular milieu and stimulated the production of reactive oxygen species (ROS) in cellular structures involved in migration. Memo was also required for the sustained production of the ROS O2- by NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase 1 (NOX1) in breast cancer cells. Memo abundance was increased in >40% of the primary breast tumors tested, was correlated with clinical parameters of aggressive disease, and was an independent prognostic factor of early distant metastasis.


Assuntos
Neoplasias da Mama/metabolismo , Movimento Celular , Cobre/metabolismo , Proteínas de Neoplasias/metabolismo , Ferroproteínas não Heme/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Xenoenxertos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , NADP/genética , NADP/metabolismo , NADPH Oxidase 1 , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Metástase Neoplásica , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Ferroproteínas não Heme/genética , Superóxidos/metabolismo
16.
Endocrinology ; 154(3): 1310-20, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23407452

RESUMO

Follistatin-like 3 (FSTL3) is a glycoprotein that binds and inhibits the action of TGFß ligands such as activin. The roles played by FSTL3 and activin signaling in organ development and homeostasis are not fully understood. The authors show mice deficient in FSTL3 develop markedly enlarged testes that are also delayed in their age-related regression. These FSTL3 knockout mice exhibit increased Sertoli cell numbers, allowing for increased spermatogenesis but otherwise showing normal testicular function. The data show that FSTL3 deletion leads to increased AKT signaling and SIRT1 expression in the testis. This demonstrates a cross-talk between TGFß ligand and AKT signaling and leads to a potential mechanism for increased cellular survival and antiaging. The findings identify crucial roles for FSTL3 in limiting testis organ size and promoting age-related testicular regression.


Assuntos
Envelhecimento/fisiologia , Proteínas Relacionadas à Folistatina/fisiologia , Proteínas/fisiologia , Testículo/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Envelhecimento/patologia , Animais , Contagem de Células , Proteínas Relacionadas à Folistatina/deficiência , Proteínas Relacionadas à Folistatina/genética , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Tamanho do Órgão/genética , Tamanho do Órgão/fisiologia , Proteínas/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células de Sertoli/patologia , Transdução de Sinais , Sirtuína 1/metabolismo , Espermatogênese/genética , Espermatogênese/fisiologia , Testículo/patologia
17.
Mol Syst Biol ; 8: 571, 2012 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-22373819

RESUMO

Protein post-translational modifications (PTMs) represent important regulatory states that when combined have been hypothesized to act as molecular codes and to generate a functional diversity beyond genome and transcriptome. We systematically investigate the interplay of protein phosphorylation with other post-transcriptional regulatory mechanisms in the genome-reduced bacterium Mycoplasma pneumoniae. Systematic perturbations by deletion of its only two protein kinases and its unique protein phosphatase identified not only the protein-specific effect on the phosphorylation network, but also a modulation of proteome abundance and lysine acetylation patterns, mostly in the absence of transcriptional changes. Reciprocally, deletion of the two putative N-acetyltransferases affects protein phosphorylation, confirming cross-talk between the two PTMs. The measured M. pneumoniae phosphoproteome and lysine acetylome revealed that both PTMs are very common, that (as in Eukaryotes) they often co-occur within the same protein and that they are frequently observed at interaction interfaces and in multifunctional proteins. The results imply previously unreported hidden layers of post-transcriptional regulation intertwining phosphorylation with lysine acetylation and other mechanisms that define the functional state of a cell.


Assuntos
Acetilesterase/metabolismo , Tamanho do Genoma/genética , Lisina/metabolismo , Redes e Vias Metabólicas/genética , Pneumonia por Mycoplasma/genética , Proteínas Quinases/metabolismo , Acetilação , Domínio Catalítico/genética , Evolução Molecular , Redes Reguladoras de Genes/genética , Redes Reguladoras de Genes/fisiologia , Genoma Bacteriano/genética , Redes e Vias Metabólicas/fisiologia , Modelos Biológicos , Organismos Geneticamente Modificados , Fosforilação/fisiologia , Pneumonia por Mycoplasma/metabolismo , Processamento de Proteína Pós-Traducional/genética , Proteoma/genética , Proteoma/metabolismo
18.
Cell ; 146(4): 607-20, 2011 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-21854985

RESUMO

Previous experiments suggest a connection between the N-alpha-acetylation of proteins and sensitivity of cells to apoptotic signals. Here, we describe a biochemical assay to detect the acetylation status of proteins and demonstrate that protein N-alpha-acetylation is regulated by the availability of acetyl-CoA. Because the antiapoptotic protein Bcl-xL is known to influence mitochondrial metabolism, we reasoned that Bcl-xL may provide a link between protein N-alpha-acetylation and apoptosis. Indeed, Bcl-xL overexpression leads to a reduction in levels of acetyl-CoA and N-alpha-acetylated proteins in the cell. This effect is independent of Bax and Bak, the known binding partners of Bcl-xL. Increasing cellular levels of acetyl-CoA by addition of acetate or citrate restores protein N-alpha-acetylation in Bcl-xL-expressing cells and confers sensitivity to apoptotic stimuli. We propose that acetyl-CoA serves as a signaling molecule that couples apoptotic sensitivity to metabolism by regulating protein N-alpha-acetylation.


Assuntos
Sobrevivência Celular , Proteínas/metabolismo , Proteína bcl-X/metabolismo , Acetilação , Animais , Apoptose , Caspase 2/metabolismo , Linhagem Celular , Embrião de Mamíferos/citologia , Técnicas de Inativação de Genes , Células HeLa , Humanos , Células Jurkat , Camundongos , Processamento de Proteína Pós-Traducional
20.
Bioinformatics ; 27(8): 1128-34, 2011 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-21349864

RESUMO

MOTIVATION: Although many methods and statistical approaches have been developed for protein identification by mass spectrometry, the problem of accurate assessment of statistical significance of protein identifications remains an open question. The main issues are as follows: (i) statistical significance of inferring peptide from experimental mass spectra must be platform independent and spectrum specific and (ii) individual spectrum matches at the peptide level must be combined into a single statistical measure at the protein level. RESULTS: We present a method and software to assign statistical significance to protein identifications from search engines for mass spectrometric data. The approach is based on asymptotic theory of order statistics. The parameters of the asymptotic distributions of identification scores are estimated for each spectrum individually. The method relies on new unbiased estimators for parameters of extreme value distribution. The estimated parameters are used to assign a spectrum-specific P-value to each peptide-spectrum match. The protein-level confidence measure combines P-values of peptide-to-spectrum matches. CONCLUSION: We extensively tested the method using triplicate mouse and yeast high-throughput proteomic experiments. The proposed statistical approach improves the sensitivity of protein identifications without compromising specificity. While the method was primarily designed to work with Mascot, it is platform-independent and is applicable to any search engine which outputs a single score for a peptide-spectrum match. We demonstrate this by testing the method in conjunction with X!Tandem. AVAILABILITY: The software is available for download at ftp://genetics.bwh.harvard.edu/SSPV/. CONTACT: ssunyaev@rics.bwh.harvard.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
Espectrometria de Massas/métodos , Proteínas/química , Algoritmos , Animais , Interpretação Estatística de Dados , Bases de Dados de Proteínas , Camundongos , Peptídeos/química , Proteínas/análise , Proteômica , Proteínas de Saccharomyces cerevisiae/análise , Proteínas de Saccharomyces cerevisiae/química , Software
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA