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1.
Nat Med ; 25(11): 1739-1747, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31700183

RESUMO

Type 2 diabetes is characterized by insulin resistance and a gradual loss of pancreatic beta cell mass and function1,2. Currently, there are no therapies proven to prevent beta cell loss and some, namely insulin secretagogues, have been linked to accelerated beta cell failure, thereby limiting their use in type 2 diabetes3,4. The adipokine adipsin/complement factor D controls the alternative complement pathway and generation of complement component C3a, which acts to augment beta cell insulin secretion5. In contrast to other insulin secretagogues, we show that chronic replenishment of adipsin in diabetic db/db mice ameliorates hyperglycemia and increases insulin levels while preserving beta cells by blocking dedifferentiation and death. Mechanistically, we find that adipsin/C3a decreases the phosphatase Dusp26; forced expression of Dusp26 in beta cells decreases expression of core beta cell identity genes and sensitizes to cell death. In contrast, pharmacological inhibition of DUSP26 improves hyperglycemia in diabetic mice and protects human islet cells from cell death. Pertaining to human health, we show that higher concentrations of circulating adipsin are associated with a significantly lower risk of developing future diabetes among middle-aged adults after adjusting for body mass index (BMI). Collectively, these data suggest that adipsin/C3a and DUSP26-directed therapies may represent a novel approach to achieve beta cell health to treat and prevent type 2 diabetes.


Assuntos
Complemento C3a/genética , Fator D do Complemento/farmacologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Fosfatases de Especificidade Dupla/genética , Células Secretoras de Insulina/efeitos dos fármacos , Fosfatases da Proteína Quinase Ativada por Mitógeno/genética , Animais , Índice de Massa Corporal , Desdiferenciação Celular/efeitos dos fármacos , Fator D do Complemento/genética , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Glucose/metabolismo , Humanos , Hiperglicemia/tratamento farmacológico , Hiperglicemia/genética , Hiperglicemia/patologia , Insulina/genética , Resistência à Insulina/genética , Células Secretoras de Insulina/patologia , Camundongos , Camundongos Endogâmicos NOD
2.
Cancer Cell ; 36(4): 402-417.e13, 2019 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-31564638

RESUMO

Metastasis is the leading cause of cancer mortality. Chromatin remodeling provides the foundation for the cellular reprogramming necessary to drive metastasis. However, little is known about the nature of this remodeling and its regulation. Here, we show that metastasis-inducing pathways regulate histone chaperones to reduce canonical histone incorporation into chromatin, triggering deposition of H3.3 variant at the promoters of poor-prognosis genes and metastasis-inducing transcription factors. This specific incorporation of H3.3 into chromatin is both necessary and sufficient for the induction of aggressive traits that allow for metastasis formation. Together, our data clearly show incorporation of histone variant H3.3 into chromatin as a major regulator of cell fate during tumorigenesis, and histone chaperones as valuable therapeutic targets for invasive carcinomas.

3.
Nat Cell Biol ; 21(10): 1179-1190, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31548608

RESUMO

Cell fate transitions are accompanied by global transcriptional, epigenetic and topological changes driven by transcription factors, as is exemplified by reprogramming somatic cells to pluripotent stem cells through the expression of OCT4, KLF4, SOX2 and cMYC. How transcription factors orchestrate the complex molecular changes around their target gene loci remains incompletely understood. Here, using KLF4 as a paradigm, we provide a transcription-factor-centric view of chromatin reorganization and its association with three-dimensional enhancer rewiring and transcriptional changes during the reprogramming of mouse embryonic fibroblasts to pluripotent stem cells. Inducible depletion of KLF factors in PSCs caused a genome-wide decrease in enhancer connectivity, whereas disruption of individual KLF4 binding sites within pluripotent-stem-cell-specific enhancers was sufficient to impair enhancer-promoter contacts and reduce the expression of associated genes. Our study provides an integrative view of the complex activities of a lineage-specifying transcription factor and offers novel insights into the nature of the molecular events that follow transcription factor binding.


Assuntos
Reprogramação Celular/genética , Montagem e Desmontagem da Cromatina/genética , Elementos Facilitadores Genéticos , Fatores de Transcrição Kruppel-Like/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Animais , Células Cultivadas , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Células-Tronco Pluripotentes/metabolismo
4.
J Clin Invest ; 130: 3924-3940, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31260412

RESUMO

Despite recent therapeutic advances, prostate cancer remains a leading cause of cancer-related death. A subset of castration resistant prostate cancers become androgen receptor (AR) signaling-independent and develop neuroendocrine prostate cancer (NEPC) features through lineage plasticity. These NEPC tumors, associated with aggressive disease and poor prognosis, are driven, in part, by aberrant expression of N-Myc, through mechanisms that remain unclear. Integrative analysis of the N-Myc transcriptome, cistrome and interactome using in vivo, in vitro and ex vivo models (including patient-derived organoids) identified a lineage switch towards a neural identity associated with epigenetic reprogramming. N-Myc and known AR-co-factors (e.g., FOXA1 and HOXB13) overlapped, independently of AR, at genomic loci implicated in neural lineage specification. Moreover, histone marks specifically associated with lineage-defining genes were reprogrammed by N-Myc. We also demonstrated that the N-Myc-induced molecular program accurately classifies our cohort of patients with advanced prostate cancer. Finally, we revealed the potential for EZH2 inhibition to reverse the N-Myc-induced suppression of epithelial lineage genes. Altogether, our data provide insights on how N-Myc regulates lineage plasticity and epigenetic reprogramming associated with lineage-specification. The N-Myc signature we defined could also help predict the evolution of prostate cancer and thus better guide the choice of future therapeutic strategies.

5.
Proc Natl Acad Sci U S A ; 116(21): 10382-10391, 2019 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-31072927

RESUMO

During skeletal muscle regeneration, muscle stem cells (MuSCs) respond to multiple signaling inputs that converge onto mammalian target of rapamycin complex 1 (mTORC1) signaling pathways. mTOR function is essential for establishment of the differentiation-committed progenitors (early stage of differentiation, marked by the induction of myogenin expression), myotube fusion, and, ultimately, hypertrophy (later stage of differentiation). While a major mTORC1 substrate, p70S6K, is required for myotube fusion and hypertrophy, an mTORC1 effector for the induction of myogenin expression remains unclear. Here, we identified Per-Arnt-Sim domain kinase (PASK) as a downstream phosphorylation target of mTORC1 in MuSCs during differentiation. We have recently shown that the PASK phosphorylates Wdr5 to stimulate MuSC differentiation by epigenetically activating the myogenin promoter. We show that phosphorylation of PASK by mTORC1 is required for the activation of myogenin transcription, exit from self-renewal, and induction of the myogenesis program. Our studies reveal that mTORC1-PASK signaling is required for the rise of myogenin-positive committed myoblasts (early stage of myogenesis), whereas mTORC1-S6K signaling is required for myoblast fusion (later stage of myogenesis). Thus, our discoveries allow molecular dissection of mTOR functions during different stages of the myogenesis program driven by two different substrates.

6.
Proc Natl Acad Sci U S A ; 116(24): 11796-11805, 2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31142645

RESUMO

The current model of polarized plasma membrane protein sorting in epithelial cells has been largely generated on the basis of experiments characterizing the polarized distribution of a relatively small number of overexpressed model proteins under various experimental conditions. Thus, the possibility exists that alternative roles of various types of sorting machinery may have been underestimated or missed. Here, we utilize domain-selective surface biotinylation combined with stable isotope labeling with amino acids in cell culture (SILAC) and mass spectrometry to quantitatively define large populations of apical and basolateral surface proteins in Madin-Darby canine kidney (MDCK) cells. We identified 313 plasma membrane proteins, of which 38% were apical, 51% were basolateral, and 11% were nonpolar. Silencing of clathrin adaptor proteins (AP) AP-1A, AP-1B, or both caused redistribution of basolateral proteins as expected but also, of a large population of apical proteins. Consistent with their previously reported ability to compensate for one another, the strongest loss of polarity was observed when we silenced AP-1A and AP-1B simultaneously. We found stronger evidence of compensation in the apical pathway compared with the basolateral pathway. Surprisingly, we also found subgroups of proteins that were affected after silencing just one adaptor, indicating previously unrecognized independent roles for AP-1A and AP-1B. While AP-1B silencing mainly affected basolateral polarity, AP-1A silencing seemed to cause comparable loss of apical and basolateral polarity. Our results uncover previously overlooked roles of AP-1 in polarized distribution of apical and basolateral proteins and introduce surface proteomics as a method to examine mechanisms of polarization with a depth not possible until now.

8.
Cancer Cell ; 35(4): 603-617.e8, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30930119

RESUMO

Deletion of the gene encoding the chromatin remodeler CHD1 is among the most common alterations in prostate cancer (PCa); however, the tumor-suppressive functions of CHD1 and reasons for its tissue-specific loss remain undefined. We demonstrated that CHD1 occupied prostate-specific enhancers enriched for the androgen receptor (AR) and lineage-specific cofactors. Upon CHD1 loss, the AR cistrome was redistributed in patterns consistent with the oncogenic AR cistrome in PCa samples and drove tumor formation in the murine prostate. Notably, this cistrome shift was associated with a unique AR transcriptional signature enriched for pro-oncogenic pathways unique to this tumor subclass. Collectively, these data credential CHD1 as a tumor suppressor in the prostate that constrains AR binding/function to limit tumor progression.


Assuntos
Carcinogênese , DNA Helicases/deficiência , Proteínas de Ligação a DNA/deficiência , Elementos Facilitadores Genéticos , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Transcrição Genética , Proteínas Supressoras de Tumor/deficiência , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , PTEN Fosfo-Hidrolase/deficiência , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Ligação Proteica , Receptores Androgênicos/genética , Transdução de Sinais , Técnicas de Cultura de Tecidos , Proteínas Supressoras de Tumor/genética
9.
BMC Bioinformatics ; 20(1): 7, 2019 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-30611210

RESUMO

BACKGROUND: To further our understanding of immunopeptidomics, improved tools are needed to identify peptides presented by major histocompatibility complex class I (MHC-I). Many existing tools are limited by their reliance upon chemical affinity data, which is less biologically relevant than sampling by mass spectrometry, and other tools are limited by incomplete exploration of machine learning approaches. Herein, we assemble publicly available data describing human peptides discovered by sampling the MHC-I immunopeptidome with mass spectrometry and use this database to train random forest classifiers (ForestMHC) to predict presentation by MHC-I. RESULTS: As measured by precision in the top 1% of predictions, our method outperforms NetMHC and NetMHCpan on test sets, and it outperforms both these methods and MixMHCpred on new data from an ovarian carcinoma cell line. We also find that random forest scores correlate monotonically, but not linearly, with known chemical binding affinities, and an information-based analysis of classifier features shows the importance of anchor positions for our classification. The random-forest approach also outperforms a deep neural network and a convolutional neural network trained on identical data. Finally, we use our large database to confirm that gene expression partially determines peptide presentation. CONCLUSIONS: ForestMHC is a promising method to identify peptides bound by MHC-I. We have demonstrated the utility of random forest-based approaches in predicting peptide presentation by MHC-I, assembled the largest known database of MS binding data, and mined this database to show the effect of gene expression on peptide presentation. ForestMHC has potential applicability to basic immunology, rational vaccine design, and neoantigen binding prediction for cancer immunotherapy. This method is publicly available for applications and further validation.


Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Aprendizado de Máquina , Peptídeos/imunologia , Proteoma/metabolismo , Algoritmos , Linhagem Celular Tumoral , Bases de Dados de Proteínas , Regulação da Expressão Gênica , Humanos , Peptídeos/química , Reprodutibilidade dos Testes
10.
J Proteome Res ; 18(3): 1411-1417, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30576142

RESUMO

Mass-spectrometry-based phosphoproteomics has revolutionized phosphoprotein analysis and enhanced our understanding of diverse and fundamental cellular processes important for human health and disease. Because of their relative scarcity, phosphopeptides must be enriched before analysis. Many different enrichment methods and materials have been described, and many reports have made claims about the advantages of particular materials and methodological variations. We demonstrate an effective and highly reproducible single-step enrichment method using an off-the-shelf preparation of calcium titanate. Using two different cell lines and replicate analysis, we show that our method achieves a purity and depth of analysis comparable or superior to a widely used TiO2-based method at a reduced cost and effort. This method provides a new and immediately available tool for expanding the reach of phosphoproteomic inquiry.

11.
Cell Rep ; 21(13): 3807-3818, 2017 12 26.
Artigo em Inglês | MEDLINE | ID: mdl-29281829

RESUMO

Aneuploidy disrupts cellular homeostasis. However, the molecular mechanisms underlying the physiological responses and adaptation to aneuploidy are not well understood. Deciphering these mechanisms is important because aneuploidy is associated with diseases, including intellectual disability and cancer. Although tumors and mammalian aneuploid cells, including several cancer cell lines, show altered levels of sphingolipids, the role of sphingolipids in aneuploidy remains unknown. Here, we show that ceramides and long-chain bases, sphingolipid molecules that slow proliferation and promote survival, are increased by aneuploidy. Sphingolipid levels are tightly linked to serine synthesis, and inhibiting either serine or sphingolipid synthesis can specifically impair the fitness of aneuploid cells. Remarkably, the fitness of aneuploid cells improves or deteriorates upon genetically decreasing or increasing ceramides, respectively. Combined targeting of serine and sphingolipid synthesis could be exploited to specifically target cancer cells, the vast majority of which are aneuploid.


Assuntos
Aneuploidia , Saccharomyces cerevisiae/metabolismo , Serina/metabolismo , Esfingolipídeos/biossíntese , Proliferação de Células , Ceramidas/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Serina/biossíntese , Transcrição Genética , Regulação para Cima/genética
12.
Cell ; 171(7): 1545-1558.e18, 2017 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-29153836

RESUMO

mTORC1 is a signal integrator and master regulator of cellular anabolic processes linked to cell growth and survival. Here, we demonstrate that mTORC1 promotes lipid biogenesis via SRPK2, a key regulator of RNA-binding SR proteins. mTORC1-activated S6K1 phosphorylates SRPK2 at Ser494, which primes Ser497 phosphorylation by CK1. These phosphorylation events promote SRPK2 nuclear translocation and phosphorylation of SR proteins. Genome-wide transcriptome analysis reveals that lipid biosynthetic enzymes are among the downstream targets of mTORC1-SRPK2 signaling. Mechanistically, SRPK2 promotes SR protein binding to U1-70K to induce splicing of lipogenic pre-mRNAs. Inhibition of this signaling pathway leads to intron retention of lipogenic genes, which triggers nonsense-mediated mRNA decay. Genetic or pharmacological inhibition of SRPK2 blunts de novo lipid synthesis, thereby suppressing cell growth. These results thus reveal a novel role of mTORC1-SRPK2 signaling in post-transcriptional regulation of lipid metabolism and demonstrate that SRPK2 is a potential therapeutic target for mTORC1-driven metabolic disorders.


Assuntos
Regulação da Expressão Gênica , Lipogênese , Processamento Pós-Transcricional do RNA , Transdução de Sinais , Animais , Núcleo Celular/metabolismo , Colesterol/metabolismo , Ácidos Graxos/metabolismo , Feminino , Xenoenxertos , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Nus , Transplante de Neoplasias , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo
13.
Mol Cell ; 67(3): 512-527.e4, 2017 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-28757207

RESUMO

Aberrant signaling by the mammalian target of rapamycin (mTOR) contributes to the devastating features of cancer cells. Thus, mTOR is a critical therapeutic target and catalytic inhibitors are being investigated as anti-cancer drugs. Although mTOR inhibitors initially block cell proliferation, cell viability and migration in some cancer cells are quickly restored. Despite sustained inhibition of mTORC1/2 signaling, Akt, a kinase regulating cell survival and migration, regains phosphorylation at its regulatory sites. Mechanistically, mTORC1/2 inhibition promotes reorganization of integrin/focal adhesion kinase-mediated adhesomes, induction of IGFR/IR-dependent PI3K activation, and Akt phosphorylation via an integrin/FAK/IGFR-dependent process. This resistance mechanism contributes to xenograft tumor cell growth, which is prevented with mTOR plus IGFR inhibitors, supporting this combination as a therapeutic approach for cancers.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/tratamento farmacológico , Movimento Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Quinase 1 de Adesão Focal/metabolismo , Melanoma/tratamento farmacológico , Complexos Multiproteicos/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Receptores de Somatomedina/antagonistas & inibidores , Neoplasias Cutâneas/tratamento farmacológico , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Quinase 1 de Adesão Focal/genética , Humanos , Integrina alfa2/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Melanoma/enzimologia , Melanoma/patologia , Camundongos Nus , Complexos Multiproteicos/metabolismo , Invasividade Neoplásica , Fosfatidilinositol 3-Quinase/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Receptores de Somatomedina/genética , Receptores de Somatomedina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias Cutâneas/enzimologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Serina-Treonina Quinases TOR/metabolismo , Fatores de Tempo , Transfecção , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Mol Biol Cell ; 28(20): 2589-2599, 2017 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-28794263

RESUMO

In budding yeast, cell cycle progression and ribosome biogenesis are dependent on plasma membrane growth, which ensures that events of cell growth are coordinated with each other and with the cell cycle. However, the signals that link the cell cycle and ribosome biogenesis to membrane growth are poorly understood. Here we used proteome-wide mass spectrometry to systematically discover signals associated with membrane growth. The results suggest that membrane trafficking events required for membrane growth generate sphingolipid-dependent signals. A conserved signaling network appears to play an essential role in signaling by responding to delivery of sphingolipids to the plasma membrane. In addition, sphingolipid-dependent signals control phosphorylation of protein kinase C (Pkc1), which plays an essential role in the pathways that link the cell cycle and ribosome biogenesis to membrane growth. Together these discoveries provide new clues as to how growth--dependent signals control cell growth and the cell cycle.


Assuntos
Esfingolipídeos/metabolismo , Esfingolipídeos/fisiologia , Ciclo Celular/fisiologia , Crescimento Celular , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Fosforilação , Proteína Quinase C/metabolismo , Transporte Proteico , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismo , Transdução de Sinais/genética
15.
J Biol Chem ; 292(12): 4925-4941, 2017 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-28100785

RESUMO

Protein phosphatase 2A (PP2A) plays important roles in controlling mitosis in all eukaryotic cells. The form of PP2A that controls mitosis is associated with a conserved regulatory subunit that is called B55 in vertebrates and Cdc55 in budding yeast. The activity of this form of PP2A can be inhibited by binding of conserved Igo/ENSA proteins. Although the mechanisms that activate Igo/ENSA to bind and inhibit PP2A are well understood, little is known about how Igo/Ensa are inactivated. Here, we have analyzed regulation of Igo/ENSA in the context of a checkpoint pathway that links mitotic entry to membrane growth in budding yeast. Protein kinase C (Pkc1) relays signals in the pathway by activating PP2ACdc55 We discovered that constitutively active Pkc1 can drive cells through a mitotic checkpoint arrest, which suggests that Pkc1-dependent activation of PP2ACdc55 plays a critical role in checkpoint signaling. We therefore used mass spectrometry to determine how Pkc1 modifies the PP2ACdc55 complex. This revealed that Pkc1 induces changes in the phosphorylation of multiple subunits of the complex, as well as dissociation of Igo/ENSA. Pkc1 directly phosphorylates Cdc55 and Igo/ENSA, and phosphorylation site mapping and mutagenesis indicate that phosphorylation of Cdc55 contributes to Igo/ENSA dissociation. Association of Igo2 with PP2ACdc55 is regulated during the cell cycle, yet mutation of Pkc1-dependent phosphorylation sites on Cdc55 and Igo2 did not cause defects in mitotic progression. Together, the data suggest that Pkc1 controls PP2ACdc55 by multiple overlapping mechanisms.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteína Quinase C/metabolismo , Proteína Fosfatase 2/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Proteínas de Ciclo Celular/análise , Modelos Moleculares , Fosforilação , Ligação Proteica , Proteína Quinase C/análise , Proteína Fosfatase 2/análise , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/análise , Alinhamento de Sequência
16.
J Proteome Res ; 16(3): 1121-1132, 2017 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-28102081

RESUMO

Labeling peptides with isobaric tags is a popular strategy in quantitative bottom-up proteomics. In this study, we labeled six breast tumor cell lysates (1.34 mg proteins per channel) using 10-plex tandem mass tag reagents and analyzed the samples on a Q Exactive HF Quadrupole-Orbitrap mass spectrometer. We identified a total of 8,706 proteins and 28,186 phosphopeptides, including 7,394 proteins and 23,739 phosphosites common to all channels. The majority of technical replicates correlated with a R2 ≥ 0.98, indicating minimum variability was introduced after labeling. Unsupervised hierarchical clustering of phosphopeptide data sets successfully classified the breast tumor samples into Her2 (epidermal growth factor receptor 2) positive and Her2 negative groups, whereas mRNA abundance did not. The tyrosine phosphorylation levels of receptor tyrosine kinases, phosphoinositide-3-kinase, protein kinase C delta, and Src homology 2, among others, were significantly higher in the Her2 positive than the Her2 negative group. Despite ratio compression in MS2-based experiments, we demonstrated the ratios calculated using an MS2 method are highly correlated (R2 > 0.65) with ratios obtained using MS3-based quantitation (using a Thermo Orbitrap Fusion mass spectrometer) with reduced ratio suppression. Given the deep coverage of global and phosphoproteomes, our data show that MS2-based quantitation using TMT can be successfully used for large-scale multiplexed quantitative proteomics.


Assuntos
Neoplasias da Mama/patologia , Proteômica/métodos , Coloração e Rotulagem , Linhagem Celular Tumoral , Análise por Conglomerados , Feminino , Humanos , Espectrometria de Massas/métodos , Fosfopeptídeos/análise , Fosforilação , Receptor ErbB-2/análise , Tirosina/metabolismo
17.
Cell Metab ; 24(1): 104-17, 2016 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-27411012

RESUMO

Naive T cell stimulation activates anabolic metabolism to fuel the transition from quiescence to growth and proliferation. Here we show that naive CD4(+) T cell activation induces a unique program of mitochondrial biogenesis and remodeling. Using mass spectrometry, we quantified protein dynamics during T cell activation. We identified substantial remodeling of the mitochondrial proteome over the first 24 hr of T cell activation to generate mitochondria with a distinct metabolic signature, with one-carbon metabolism as the most induced pathway. Salvage pathways and mitochondrial one-carbon metabolism, fed by serine, contribute to purine and thymidine synthesis to enable T cell proliferation and survival. Genetic inhibition of the mitochondrial serine catabolic enzyme SHMT2 impaired T cell survival in culture and antigen-specific T cell abundance in vivo. Thus, during T cell activation, mitochondrial proteome remodeling generates specialized mitochondria with enhanced one-carbon metabolism that is critical for T cell activation and survival.


Assuntos
Carbono/metabolismo , Ativação Linfocitária/imunologia , Biogênese de Organelas , Proteoma/metabolismo , Linfócitos T/metabolismo , Animais , Linfócitos T CD4-Positivos/metabolismo , Sobrevivência Celular , Metabolismo Energético , Epitopos , Redes e Vias Metabólicas , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Proteômica , Pirimidinas/biossíntese
18.
Proc Natl Acad Sci U S A ; 113(26): E3667-75, 2016 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-27298372

RESUMO

The DNA damage response (DDR) is regulated by a protein kinase signaling cascade that orchestrates DNA repair and other processes. Identifying the substrate effectors of these kinases is critical for understanding the underlying physiology and mechanism of the response. We have used quantitative mass spectrometry to profile DDR-dependent phosphorylation in budding yeast and genetically explored the dependency of these phosphorylation events on the DDR kinases MEC1, RAD53, CHK1, and DUN1. Based on these screens, a database containing many novel DDR-regulated phosphorylation events has been established. Phosphorylation of many of these proteins has been validated by quantitative peptide phospho-immunoprecipitation and examined for functional relevance to the DDR through large-scale analysis of sensitivity to DNA damage in yeast deletion strains. We reveal a link between DDR signaling and the metabolic pathways of inositol phosphate and phosphatidyl inositol synthesis, which are required for resistance to DNA damage. We also uncover links between the DDR and TOR signaling as well as translation regulation. Taken together, these data shed new light on the organization of DDR signaling in budding yeast.


Assuntos
Dano ao DNA , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Reparo do DNA , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
19.
Mol Cell ; 59(5): 867-81, 2015 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-26051181

RESUMO

Execution of the DNA damage response (DDR) relies upon a dynamic array of protein modifications. Using quantitative proteomics, we have globally profiled ubiquitination, acetylation, and phosphorylation in response to UV and ionizing radiation. To improve acetylation site profiling, we developed the strategy FACET-IP. Our datasets of 33,500 ubiquitination and 16,740 acetylation sites provide valuable insight into DDR remodeling of the proteome. We find that K6- and K33-linked polyubiquitination undergo bulk increases in response to DNA damage, raising the possibility that these linkages are largely dedicated to DDR function. We also show that Cullin-RING ligases mediate 10% of DNA damage-induced ubiquitination events and that EXO1 is an SCF-Cyclin F substrate in the response to UV radiation. Our extensive datasets uncover additional regulated sites on known DDR players such as PCNA and identify previously unknown DDR targets such as CENPs, underscoring the broad impact of the DDR on cellular physiology.


Assuntos
Dano ao DNA , Proteômica/métodos , Acetilação/efeitos da radiação , Proteínas Culina/metabolismo , Reparo do DNA , Enzimas Reparadoras do DNA/metabolismo , Bases de Dados de Proteínas , Exodesoxirribonucleases/metabolismo , Células HeLa , Humanos , Fosforilação/efeitos da radiação , Complexo de Endopeptidases do Proteassoma/metabolismo , Análise Serial de Proteínas/estatística & dados numéricos , Proteoma/metabolismo , Proteoma/efeitos da radiação , Proteômica/estatística & dados numéricos , Fuso Acromático/metabolismo , Ubiquitinação/efeitos da radiação
20.
J Cell Biol ; 209(3): 387-402, 2015 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-25963819

RESUMO

During each cell cycle, the mitotic spindle is efficiently assembled to achieve chromosome segregation and then rapidly disassembled as cells enter cytokinesis. Although much has been learned about assembly, how spindles disassemble at the end of mitosis remains unclear. Here we demonstrate that nucleocytoplasmic transport at the membrane domain surrounding the mitotic spindle midzone, here named the midzone membrane domain (MMD), is essential for spindle disassembly in Schizosaccharomyces pombe cells. We show that, during anaphase B, Imp1-mediated transport of the AAA-ATPase Cdc48 protein at the MMD allows this disassembly factor to localize at the spindle midzone, thereby promoting spindle midzone dissolution. Our findings illustrate how a separate membrane compartment supports spindle disassembly in the closed mitosis of fission yeast.


Assuntos
Anáfase/fisiologia , Núcleo Celular/metabolismo , Schizosaccharomyces/metabolismo , Fuso Acromático/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/genética , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Fuso Acromático/genética , Proteína com Valosina
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