Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 41
Filtrar
1.
Cell Rep ; 40(7): 111218, 2022 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-35977518

RESUMO

Metabolic dysfunction mutations can impair energy sensing and cause cancer. Loss of function of the mitochondrial tricarboxylic acid (TCA) cycle enzyme subunit succinate dehydrogenase B (SDHB) results in various forms of cancer typified by pheochromocytoma (PC). Here we delineate a signaling cascade where the loss of SDHB induces the Warburg effect, triggers dysregulation of [Ca2+]i, and aberrantly activates calpain and protein kinase Cdk5, through conversion of its cofactor from p35 to p25. Consequently, aberrant Cdk5 initiates a phospho-signaling cascade where GSK3 inhibition inactivates energy sensing by AMP kinase through dephosphorylation of the AMP kinase γ subunit, PRKAG2. Overexpression of p25-GFP in mouse adrenal chromaffin cells also elicits this phosphorylation signaling and causes PC. A potent Cdk5 inhibitor, MRT3-007, reverses this phospho-cascade, invoking a senescence-like phenotype. This therapeutic approach halted tumor progression in vivo. Thus, we reveal an important mechanistic feature of metabolic sensing and demonstrate that its dysregulation underlies tumor progression in PC and likely other cancers.


Assuntos
Adenilato Quinase , Carcinoma Neuroendócrino , Adenilato Quinase/metabolismo , Animais , Quinase 5 Dependente de Ciclina/metabolismo , Metabolismo Energético , Quinase 3 da Glicogênio Sintase/metabolismo , Camundongos , Fosforilação , Succinatos
2.
Proc Natl Acad Sci U S A ; 117(31): 18401-18411, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32690709

RESUMO

Disparities in cancer patient responses have prompted widespread searches to identify differences in sensitive vs. nonsensitive populations and form the basis of personalized medicine. This customized approach is dependent upon the development of pathway-specific therapeutics in conjunction with biomarkers that predict patient responses. Here, we show that Cdk5 drives growth in subgroups of patients with multiple types of neuroendocrine neoplasms. Phosphoproteomics and high throughput screening identified phosphorylation sites downstream of Cdk5. These phosphorylation events serve as biomarkers and effectively pinpoint Cdk5-driven tumors. Toward achieving targeted therapy, we demonstrate that mouse models of neuroendocrine cancer are responsive to selective Cdk5 inhibitors and biomimetic nanoparticles are effective vehicles for enhanced tumor targeting and reduction of drug toxicity. Finally, we show that biomarkers of Cdk5-dependent tumors effectively predict response to anti-Cdk5 therapy in patient-derived xenografts. Thus, a phosphoprotein-based diagnostic assay combined with Cdk5-targeted therapy is a rational treatment approach for neuroendocrine malignancies.


Assuntos
Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Tumores Neuroectodérmicos/tratamento farmacológico , Fosfoproteínas/metabolismo , Inibidores de Proteínas Quinases/administração & dosagem , Animais , Biomarcadores/análise , Biomarcadores/metabolismo , Quinase 5 Dependente de Ciclina/antagonistas & inibidores , Quinase 5 Dependente de Ciclina/genética , Quinase 5 Dependente de Ciclina/metabolismo , Xenoenxertos , Humanos , Camundongos , Neoplasias/genética , Tumores Neuroectodérmicos/genética , Tumores Neuroectodérmicos/metabolismo , Fosfoproteínas/análise , Fosfoproteínas/genética , Fosforilação
3.
Nat Cell Biol ; 21(2): 226-237, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30692625

RESUMO

Aberrant activation of AKT disturbs the proliferation, survival and metabolic homeostasis of various human cancers. Thus, it is critical to understand the upstream signalling pathways governing AKT activation. Here, we report that AKT undergoes SETDB1-mediated lysine methylation to promote its activation, which is antagonized by the Jumonji-family demethylase KDM4B. Notably, compared with wild-type mice, mice harbouring non-methylated mutant Akt1 not only exhibited reduced body size but were also less prone to carcinogen-induced skin tumours, in part due to reduced AKT activation. Mechanistically, the interaction of phosphatidylinositol (3,4,5)-trisphosphate with AKT facilitates its interaction with SETDB1 for subsequent AKT methylation, which in turn sustains AKT phosphorylation. Pathologically, genetic alterations, including SETDB1 amplification, aberrantly promote AKT methylation to facilitate its activation and oncogenic functions. Thus, AKT methylation is an important step, synergizing with PI3K signalling to control AKT activation. This suggests that targeting SETDB1 signalling could be a potential therapeutic strategy for combatting hyperactive AKT-driven cancers.


Assuntos
Carcinogênese/metabolismo , Proteínas Metiltransferases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Carcinogênese/genética , Linhagem Celular Tumoral , Feminino , Células HEK293 , Histona-Lisina N-Metiltransferase , Humanos , Metilação , Camundongos Nus , Camundongos Transgênicos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Plicamicina/farmacologia , Proteínas Metiltransferases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Células Sf9 , Spodoptera , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
4.
J Biol Chem ; 293(33): 12770-12780, 2018 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-29959229

RESUMO

Set7/9 (also known as Set7, Set9, Setd7, and Kmt7) is a lysine methyltransferase that catalyzes the methylation of multiple substrates, including histone H3 and non-histone proteins. Although not essential for normal development and physiology, Set7/9-mediated methylation events play important roles in regulating cellular pathways involved in various human diseases, making Set7/9 a promising therapeutic target. Multiple Set7/9 inhibitors have been developed, which exhibit varying degrees of potency and selectivity in vitro However, validation of these compounds in vivo has been hampered by the lack of a reliable cellular biomarker for Set7/9 activity. Here, we report the identification of Rpl29, a ribosomal protein abundantly expressed in all cell types, as a major substrate of Set7/9. We show that Rpl29 lysine 5 (Rpl29K5) is methylated exclusively by Set7/9 and can be demethylated by Lsd1 (also known as Kdm1a). Rpl29 is not a core component of the ribosome translational machinery and plays a regulatory role in translation efficiency. Our results indicate that Rpl29 methylation has no effect on global protein synthesis but affects Rpl29 subcellular localization. Using an Rpl29 methylation-specific antibody, we demonstrate that Rpl29K5 methylation is present ubiquitously and validate that (R)-PFI-2, a Set7/9 inhibitor, efficiently reduces Rpl29K5 methylation in cell lines. Thus, Rpl29 methylation can serve as a specific cellular biomarker for measuring Set7/9 activity.


Assuntos
Fatores de Coagulação Sanguínea/genética , Metilação de DNA , Regulação da Expressão Gênica , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Lisina/química , Proteínas Ribossômicas/fisiologia , Animais , Fatores de Coagulação Sanguínea/metabolismo , Células Cultivadas , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Histona-Lisina N-Metiltransferase/genética , Humanos , Masculino , Camundongos Knockout , Processamento de Proteína Pós-Traducional , Proteínas de Ligação a RNA , Transcrição Gênica
5.
J Proteome Res ; 16(4): 1506-1514, 2017 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-28171727

RESUMO

A comprehensive view of protein phosphorylation remains an unmet challenge in the field of cell biology. Mass spectrometry-based proteomics is one of the most promising approaches for identifying thousands of phosphorylation events in a single experiment, yet the full breadth of the phosphoproteome has yet to be elucidated. In this article, we examined the complementarity of two methods for phosphopeptide enrichment based on either titanium dioxide (TiO2) enrichment or phosphorylation motif-specific immunoaffinity precipitation (IAP) with four different antibodies. Each method identified nearly 2000 phosphoproteins. However, distinct populations of phosphopeptides were observed. Despite quantifying over 10 000 unique phosphorylation events using TiO2 and over 3900 with IAP, less than 5% of the sites were in common. Agreeing with published literature, the ratio of pS:pT:pY phosphorylation for the TiO2-enriched data set approximated 90:10:<1. In contrast, that ratio for the combined IAP data sets was 51:29:20. These differences not only suggest the complementarity between multiple enrichment methods but also emphasize their collective importance in obtaining a comprehensive view of the phosphoproteome.


Assuntos
Fosfopeptídeos/biossíntese , Fosfoproteínas/biossíntese , Proteômica , Linhagem Celular Tumoral , Humanos , Espectrometria de Massas , Fosfopeptídeos/genética , Fosfoproteínas/genética , Fosforilação/genética , Titânio/química
6.
Sci Signal ; 10(460)2017 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-28049764

RESUMO

The SCFß-TRCP E3 ubiquitin ligase complex plays pivotal roles in normal cellular physiology and in pathophysiological conditions. Identification of ß-transducin repeat-containing protein (ß-TRCP) substrates is therefore critical to understand SCFß-TRCP biology and function. We used a ß-TRCP-phosphodegron motif-specific antibody in a ß-TRCP substrate screen coupled with tandem mass spectrometry and identified multiple ß-TRCP substrates. One of these substrates was Lipin1, an enzyme and suppressor of the family of sterol regulatory element-binding protein (SREBP) transcription factors, which activate genes encoding lipogenic factors. We showed that SCFß-TRCP specifically interacted with and promoted the polyubiquitination of Lipin1 in a manner that required phosphorylation of Lipin1 by mechanistic target of rapamycin 1 (mTORC1) and casein kinase I (CKI). ß-TRCP depletion in HepG2 hepatocellular carcinoma cells resulted in increased Lipin1 protein abundance, suppression of SREBP-dependent gene expression, and attenuation of triglyceride synthesis. Moreover, ß-TRCP1 knockout mice showed increased Lipin1 protein abundance and were protected from hepatic steatosis induced by a high-fat diet. Together, these data reveal a critical physiological function of ß-TRCP in regulating hepatic lipid metabolic homeostasis in part through modulating Lipin1 stability.


Assuntos
Lipogênese , Fígado/metabolismo , Proteínas Nucleares/metabolismo , Fosfatidato Fosfatase/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Animais , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Immunoblotting , Camundongos , Camundongos Knockout , Células NIH 3T3 , Proteínas Nucleares/genética , Fosfatidato Fosfatase/genética , Fosforilação , Ligação Proteica , Proteólise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Ligases SKP Culina F-Box/genética , Especificidade por Substrato , Ubiquitinação
8.
Elife ; 42015 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-26575292

RESUMO

RBM15, an RNA binding protein, determines cell-fate specification of many tissues including blood. We demonstrate that RBM15 is methylated by protein arginine methyltransferase 1 (PRMT1) at residue R578, leading to its degradation via ubiquitylation by an E3 ligase (CNOT4). Overexpression of PRMT1 in acute megakaryocytic leukemia cell lines blocks megakaryocyte terminal differentiation by downregulation of RBM15 protein level. Restoring RBM15 protein level rescues megakaryocyte terminal differentiation blocked by PRMT1 overexpression. At the molecular level, RBM15 binds to pre-messenger RNA intronic regions of genes important for megakaryopoiesis such as GATA1, RUNX1, TAL1 and c-MPL. Furthermore, preferential binding of RBM15 to specific intronic regions recruits the splicing factor SF3B1 to the same sites for alternative splicing. Therefore, PRMT1 regulates alternative RNA splicing via reducing RBM15 protein concentration. Targeting PRMT1 may be a curative therapy to restore megakaryocyte differentiation for acute megakaryocytic leukemia.


Assuntos
Processamento de Proteína Pós-Traducional , Proteína-Arginina N-Metiltransferases/metabolismo , Splicing de RNA , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , Linhagem Celular , Humanos , Metilação , Proteólise , Ubiquitinação
9.
Nat Commun ; 6: 6758, 2015 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-25849564

RESUMO

The impact of protein arginine methylation on the regulation of immune functions is virtually unknown. Here, we apply a novel method­isomethionine methyl-SILAC­coupled with antibody-mediated arginine-methylated peptide enrichment to identify methylated peptides in human T cells by mass spectrometry. This approach allowed the identification of 2,502 arginine methylation sites from 1,257 tissue-specific and housekeeping proteins. We find that components of T cell antigen receptor signal machinery and several key transcription factors that regulate T cell fate determination are methylated on arginine. Moreover, we demonstrate changes in arginine methylation stoichiometry during cellular stimulation in a subset of proteins critical to T cell differentiation. Our data suggest that protein arginine methyltransferases exert key regulatory roles in T cell activation and differentiation, opening a new field of investigation in T cell biology.


Assuntos
Arginina/metabolismo , Diferenciação Celular , Ativação Linfocitária , Proteína-Arginina N-Metiltransferases/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Fatores de Transcrição/metabolismo , Humanos , Espectrometria de Massas , Metilação , Processamento de Proteína Pós-Traducional
10.
PLoS Comput Biol ; 11(4): e1004130, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25884760

RESUMO

Protein phosphorylation plays a central role in creating a highly dynamic network of interacting proteins that reads and responds to signals from growth factors in the cellular microenvironment. Cells of the neural crest employ multiple signaling mechanisms to control migration and differentiation during development. It is known that defects in these mechanisms cause neuroblastoma, but how multiple signaling pathways interact to govern cell behavior is unknown. In a phosphoproteomic study of neuroblastoma cell lines and cell fractions, including endosomes and detergent-resistant membranes, 1622 phosphorylated proteins were detected, including more than half of the receptor tyrosine kinases in the human genome. Data were analyzed using a combination of graph theory and pattern recognition techniques that resolve data structure into networks that incorporate statistical relationships and protein-protein interaction data. Clusters of proteins in these networks are indicative of functional signaling pathways. The analysis indicates that receptor tyrosine kinases are functionally compartmentalized into distinct collaborative groups distinguished by activation and intracellular localization of SRC-family kinases, especially FYN and LYN. Changes in intracellular localization of activated FYN and LYN were observed in response to stimulation of the receptor tyrosine kinases, ALK and KIT. The results suggest a mechanism to distinguish signaling responses to activation of different receptors, or combinations of receptors, that govern the behavior of the neural crest, which gives rise to neuroblastoma.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Endossomos/metabolismo , Neuroblastoma/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Quinases da Família src/metabolismo , Linhagem Celular Tumoral , Simulação por Computador , Humanos , Microdomínios da Membrana , Modelos Biológicos , Proteínas de Neoplasias/metabolismo , Fosfoproteínas/metabolismo
11.
Nat Commun ; 6: 6428, 2015 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-25737013

RESUMO

The human genome encodes a family of nine protein arginine methyltransferases (PRMT1-9), whose members can catalyse three distinct types of methylation on arginine residues. Here we identify two spliceosome-associated proteins-SAP145 and SAP49-as PRMT9-binding partners, linking PRMT9 to U2 snRNP maturation. We show that SAP145 is methylated by PRMT9 at arginine 508, which takes the form of monomethylated arginine (MMA) and symmetrically dimethylated arginine (SDMA). PRMT9 thus joins PRMT5 as the only mammalian enzymes capable of depositing the SDMA mark. Methylation of SAP145 on Arg 508 generates a binding site for the Tudor domain of the Survival of Motor Neuron (SMN) protein, and RNA-seq analysis reveals gross splicing changes when PRMT9 levels are attenuated. These results identify PRMT9 as a nonhistone methyltransferase that primes the U2 snRNP for interaction with SMN.


Assuntos
Arginina/análogos & derivados , Metilação de DNA/fisiologia , Proteínas F-Box/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Arginina/genética , Arginina/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Cromatografia por Troca Iônica , Cromatografia em Camada Fina , Metilação de DNA/genética , Primers do DNA/genética , Imunofluorescência , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunoprecipitação , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fatores de Processamento de RNA , Alinhamento de Sequência , Análise de Sequência de RNA , Proteína 1 de Sobrevivência do Neurônio Motor/genética
12.
Neuron ; 81(5): 1070-1083, 2014 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-24607229

RESUMO

Many psychiatric and neurological disorders are characterized by learning and memory deficits, for which cognitive enhancement is considered a valid treatment strategy. The N-methyl-D-aspartate receptor (NMDAR) is a prime target for the development of cognitive enhancers because of its fundamental role in learning and memory. In particular, the NMDAR subunit NR2B improves synaptic plasticity and memory when overexpressed in neurons. However, NR2B regulation is not well understood and no therapies potentiating NMDAR function have been developed. Here, we show that serine 1116 of NR2B is phosphorylated by cyclin-dependent kinase 5 (Cdk5). Cdk5-dependent NR2B phosphorylation is regulated by neuronal activity and controls the receptor's cell surface expression. Disrupting NR2B-Cdk5 interaction via a small interfering peptide (siP) increases NR2B surface levels, facilitates synaptic transmission, and improves memory formation in vivo. Our results reveal a regulatory mechanism critical to NR2B function that can be targeted for the development of cognitive enhancers.


Assuntos
Quinase 5 Dependente de Ciclina/metabolismo , Transtornos da Memória/metabolismo , Memória/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Quinase 5 Dependente de Ciclina/genética , Feminino , Hipocampo/citologia , Masculino , Transtornos da Memória/terapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Plasticidade Neuronal/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Técnicas de Cultura de Órgãos , Fosforilação/fisiologia , Ratos , Ratos Sprague-Dawley , Transmissão Sináptica/fisiologia
13.
Cancer Cell ; 25(1): 21-36, 2014 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-24434208

RESUMO

Coactivator-associated arginine methyltransferase 1 (CARM1), a coactivator for various cancer-relevant transcription factors, is overexpressed in breast cancer. To elucidate the functions of CARM1 in tumorigenesis, we knocked out CARM1 from several breast cancer cell lines using Zinc-Finger Nuclease technology, which resulted in drastic phenotypic and biochemical changes. The CARM1 KO cell lines enabled identification of CARM1 substrates, notably the SWI/SNF core subunit BAF155. Methylation of BAF155 at R1064 was found to be an independent prognostic biomarker for cancer recurrence and to regulate breast cancer cell migration and metastasis. Furthermore, CARM1-mediated BAF155 methylation affects gene expression by directing methylated BAF155 to unique chromatin regions (e.g., c-Myc pathway genes). Collectively, our studies uncover a mechanism by which BAF155 acquires tumorigenic functions via arginine methylation.


Assuntos
Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Proteína-Arginina N-Metiltransferases/metabolismo , Fatores de Transcrição/metabolismo , Animais , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina , Metilação de DNA , Progressão da Doença , Feminino , Humanos , Camundongos , Camundongos Knockout , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Análise Serial de Tecidos
14.
Mol Cell ; 53(4): 577-90, 2014 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-24462114

RESUMO

The three Akt isoforms are functionally distinct. Here we show that their phosphoproteomes also differ, suggesting that their functional differences are due to differences in target specificity. One of the top cellular functions differentially regulated by Akt isoforms is RNA processing. IWS1, an RNA processing regulator, is phosphorylated by Akt3 and Akt1 at Ser720/Thr721. The latter is required for the recruitment of SETD2 to the RNA Pol II complex. SETD2 trimethylates histone H3 at K36 during transcription, creating a docking site for MRG15 and PTB. H3K36me3-bound MRG15 and PTB regulate FGFR-2 splicing, which controls tumor growth and invasiveness downstream of IWS1 phosphorylation. Twenty-one of the twenty-four non-small-cell-lung carcinomas we analyzed express IWS1. More importantly, the stoichiometry of IWS1 phosphorylation in these tumors correlates with the FGFR-2 splicing pattern and with Akt phosphorylation and Akt3 expression. These data identify an Akt isoform-dependent regulatory mechanism for RNA processing and demonstrate its role in lung cancer.


Assuntos
Processamento Alternativo , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sequência de Aminoácidos , Animais , Regulação da Expressão Gênica , Células HeLa , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Humanos , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Transplante de Neoplasias , Fosfoproteínas/metabolismo , Fosforilação , Isoformas de Proteínas/metabolismo , Proteômica , RNA/metabolismo , Proteínas de Ligação a RNA , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de Tempo , Fatores de Transcrição
15.
Mol Cell ; 53(2): 317-29, 2014 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-24462205

RESUMO

The stability and activity of numerous signaling proteins in both normal and cancer cells depends on the dimeric molecular chaperone heat shock protein 90 (Hsp90). Hsp90's function is coupled to ATP binding and hydrolysis and requires a series of conformational changes that are regulated by cochaperones and numerous posttranslational modifications (PTMs). SUMOylation is one of the least-understood Hsp90 PTMs. Here, we show that asymmetric SUMOylation of a conserved lysine residue in the N domain of both yeast (K178) and human (K191) Hsp90 facilitates both recruitment of the adenosine triphosphatase (ATPase)-activating cochaperone Aha1 and, unexpectedly, the binding of Hsp90 inhibitors, suggesting that these drugs associate preferentially with Hsp90 proteins that are actively engaged in the chaperone cycle. Importantly, cellular transformation is accompanied by elevated steady-state N domain SUMOylation, and increased Hsp90 SUMOylation sensitizes yeast and mammalian cells to Hsp90 inhibitors, providing a mechanism to explain the sensitivity of cancer cells to these drugs.


Assuntos
Trifosfato de Adenosina/metabolismo , Chaperoninas/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/fisiologia , Humanos , Estrutura Terciária de Proteína , Sumoilação
16.
Mol Cell Proteomics ; 13(1): 372-87, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24129315

RESUMO

Protein methylation is a common posttranslational modification that mostly occurs on arginine and lysine residues. Arginine methylation has been reported to regulate RNA processing, gene transcription, DNA damage repair, protein translocation, and signal transduction. Lysine methylation is best known to regulate histone function and is involved in epigenetic regulation of gene transcription. To better study protein methylation, we have developed highly specific antibodies against monomethyl arginine; asymmetric dimethyl arginine; and monomethyl, dimethyl, and trimethyl lysine motifs. These antibodies were used to perform immunoaffinity purification of methyl peptides followed by LC-MS/MS analysis to identify and quantify arginine and lysine methylation sites in several model studies. Overall, we identified over 1000 arginine methylation sites in human cell line and mouse tissues, and ∼160 lysine methylation sites in human cell line HCT116. The number of methylation sites identified in this study exceeds those found in the literature to date. Detailed analysis of arginine-methylated proteins observed in mouse brain compared with those found in mouse embryo shows a tissue-specific distribution of arginine methylation, and extends the types of proteins that are known to be arginine methylated to include many new protein types. Many arginine-methylated proteins that we identified from the brain, including receptors, ion channels, transporters, and vesicle proteins, are involved in synaptic transmission, whereas the most abundant methylated proteins identified from mouse embryo are transcriptional regulators and RNA processing proteins.


Assuntos
Arginina/metabolismo , Encéfalo/metabolismo , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Motivos de Aminoácidos/genética , Animais , Arginina/genética , Cromatografia Líquida , Células HCT116 , Humanos , Lisina/genética , Metilação , Camundongos , Espectrometria de Massas em Tandem
17.
Cell Metab ; 18(6): 920-33, 2013 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-24315375

RESUMO

Reversible posttranslational modifications are emerging as critical regulators of mitochondrial proteins and metabolism. Here, we use a label-free quantitative proteomic approach to characterize the lysine succinylome in liver mitochondria and its regulation by the desuccinylase SIRT5. A total of 1,190 unique sites were identified as succinylated, and 386 sites across 140 proteins representing several metabolic pathways including ß-oxidation and ketogenesis were significantly hypersuccinylated in Sirt5(-/-) animals. Loss of SIRT5 leads to accumulation of medium- and long-chain acylcarnitines and decreased ß-hydroxybutyrate production in vivo. In addition, we demonstrate that SIRT5 regulates succinylation of the rate-limiting ketogenic enzyme 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) both in vivo and in vitro. Finally, mutation of hypersuccinylated residues K83 and K310 on HMGCS2 to glutamic acid strongly inhibits enzymatic activity. Taken together, these findings establish SIRT5 as a global regulator of lysine succinylation in mitochondria and present a mechanism for inhibition of ketogenesis through HMGCS2.


Assuntos
Lisina/análogos & derivados , Lisina/metabolismo , Mitocôndrias Hepáticas/enzimologia , Sirtuínas/metabolismo , Succinatos/metabolismo , Motivos de Aminoácidos , Animais , Carnitina/química , Carnitina/metabolismo , Linhagem Celular , Humanos , Hidroxibutiratos/química , Hidroxibutiratos/metabolismo , Hidroximetilglutaril-CoA Sintase/química , Hidroximetilglutaril-CoA Sintase/genética , Hidroximetilglutaril-CoA Sintase/metabolismo , Corpos Cetônicos/biossíntese , Lisina/análise , Lisina/química , Masculino , Redes e Vias Metabólicas , Camundongos , Camundongos Knockout , Mitocôndrias Hepáticas/metabolismo , Proteínas Mitocondriais/metabolismo , Mutação , Oxirredução , Sirtuínas/deficiência , Sirtuínas/genética , Succinatos/análise , Succinatos/química
18.
Cancer Cell ; 24(4): 499-511, 2013 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-24135281

RESUMO

Medullary thyroid carcinoma (MTC) is a neuroendocrine cancer that originates from calcitonin-secreting parafollicular cells, or C cells. We found that Cdk5 and its cofactors p35 and p25 are highly expressed in human MTC and that Cdk5 activity promotes MTC proliferation. A conditional MTC mouse model was generated and corroborated the role of aberrant Cdk5 activation in MTC. C cell-specific overexpression of p25 caused rapid C cell hyperplasia leading to lethal MTC, which was arrested by repressing p25 overexpression. A comparative phosphoproteomic screen between proliferating and arrested MTC identified the retinoblastoma protein (Rb) as a crucial Cdk5 downstream target. Prevention of Rb phosphorylation at Ser807/Ser811 attenuated MTC proliferation. These findings implicate Cdk5 signaling via Rb as critical to MTC tumorigenesis and progression.


Assuntos
Carcinoma Medular/metabolismo , Carcinoma Neuroendócrino/metabolismo , Quinase 5 Dependente de Ciclina/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias da Glândula Tireoide/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Progressão da Doença , Humanos , Camundongos , Camundongos Transgênicos , Fosforilação , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais , Fatores de Tempo , Transgenes
19.
Nat Methods ; 10(7): 676-82, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23749301

RESUMO

Cross-talk between different types of post-translational modifications on the same protein molecule adds specificity and combinatorial logic to signal processing, but it has not been characterized on a large-scale basis. We developed two methods to identify protein isoforms that are both phosphorylated and ubiquitylated in the yeast Saccharomyces cerevisiae, identifying 466 proteins with 2,100 phosphorylation sites co-occurring with 2,189 ubiquitylation sites. We applied these methods quantitatively to identify phosphorylation sites that regulate protein degradation via the ubiquitin-proteasome system. Our results demonstrate that distinct phosphorylation sites are often used in conjunction with ubiquitylation and that these sites are more highly conserved than the entire set of phosphorylation sites. Finally, we investigated how the phosphorylation machinery can be regulated by ubiquitylation. We found evidence for novel regulatory mechanisms of kinases and 14-3-3 scaffold proteins via proteasome-independent ubiquitylation.


Assuntos
Proteínas Fúngicas/metabolismo , Mapeamento de Interação de Proteínas/métodos , Saccharomyces cerevisiae/metabolismo , Proteínas Ubiquitinadas/metabolismo , Ubiquitinação/fisiologia , Sítios de Ligação , Fosforilação , Ligação Proteica
20.
Mol Syst Biol ; 9: 652, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23549480

RESUMO

Src homology 3 (SH3) domains bind peptides to mediate protein-protein interactions that assemble and regulate dynamic biological processes. We surveyed the repertoire of SH3 binding specificity using peptide phage display in a metazoan, the worm Caenorhabditis elegans, and discovered that it structurally mirrors that of the budding yeast Saccharomyces cerevisiae. We then mapped the worm SH3 interactome using stringent yeast two-hybrid and compared it with the equivalent map for yeast. We found that the worm SH3 interactome resembles the analogous yeast network because it is significantly enriched for proteins with roles in endocytosis. Nevertheless, orthologous SH3 domain-mediated interactions are highly rewired. Our results suggest a model of network evolution where general function of the SH3 domain network is conserved over its specific form.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Domínios de Homologia de src/genética , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Sequência Conservada , Endocitose/genética , Evolução Molecular , Dados de Sequência Molecular , Mapeamento de Interação de Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia Estrutural de Proteína , Técnicas do Sistema de Duplo-Híbrido
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...