Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 96
Filtrar
Más filtros

Banco de datos
Tipo del documento
Intervalo de año de publicación
1.
Mol Cell ; 79(2): 332-341.e7, 2020 07 16.
Artículo en Inglés | MEDLINE | ID: mdl-32521225

RESUMEN

The Ddi1/DDI2 proteins are ubiquitin shuttling factors, implicated in a variety of cellular functions. In addition to ubiquitin-binding and ubiquitin-like domains, they contain a conserved region with similarity to retroviral proteases, but whether and how DDI2 functions as a protease has remained unknown. Here, we show that DDI2 knockout cells are sensitive to proteasome inhibition and accumulate high-molecular weight, ubiquitylated proteins that are poorly degraded by the proteasome. These proteins are targets for the protease activity of purified DDI2. No evidence for DDI2 acting as a de-ubiquitylating enzyme was uncovered, which could suggest that it cleaves the ubiquitylated protein itself. In support of this idea, cleavage of transcription factor NRF1 is known to require DDI2 activity in vivo. We show that DDI2 is indeed capable of cleaving NRF1 in vitro but only when NRF1 protein is highly poly-ubiquitylated. Together, these data suggest that DDI2 is a ubiquitin-directed endoprotease.


Asunto(s)
Proteasas de Ácido Aspártico/metabolismo , Factor Nuclear 1 de Respiración/metabolismo , Ubiquitina/metabolismo , Proteasas de Ácido Aspártico/genética , Sitios de Unión , Sistemas CRISPR-Cas , Línea Celular , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Biosíntesis de Proteínas , Proteolisis
2.
J Hepatol ; 80(3): 467-481, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-37972658

RESUMEN

BACKGROUND & AIMS: Metabolic dysfunction-associated steatohepatitis (MASH) is linked to insulin resistance and type 2 diabetes and marked by hepatic inflammation, microvascular dysfunction, and fibrosis, impairing liver function and aggravating metabolic derangements. The liver homeostatic interactions disrupted in MASH are still poorly understood. We aimed to elucidate the plasticity and changing interactions of non-parenchymal cells associated with advanced MASH. METHODS: We characterized a diet-induced mouse model of advanced MASH at single-cell resolution and validated findings by assaying chromatin accessibility, bioimaging murine and human livers, and via functional experiments in vivo and in vitro. RESULTS: The fibrogenic activation of hepatic stellate cells (HSCs) led to deterioration of a signaling module consisting of the bile acid receptor NR1H4/FXR and HSC-specific GS-protein-coupled receptors (GSPCRs) capable of preserving stellate cell quiescence. Accompanying HSC activation, we further observed the attenuation of HSC Gdf2 expression, and a MASH-associated expansion of a CD207-positive macrophage population likely derived from both incoming monocytes and Kupffer cells. CONCLUSION: We conclude that HSC-expressed NR1H4 and GSPCRs of the healthy liver integrate postprandial cues, which sustain HSC quiescence and, through paracrine signals, overall sinusoidal health. Hence HSC activation in MASH not only drives fibrogenesis but may desensitize the hepatic sinusoid to liver homeostatic signals. IMPACT AND IMPLICATIONS: Homeostatic interactions between hepatic cell types and their deterioration in metabolic dysfunction-associated steatohepatitis are poorly characterized. In our current single cell-resolved study of advanced murine metabolic dysfunction-associated steatohepatitis, we identified a quiescence-associated hepatic stellate cell-signaling module with potential to preserve normal sinusoid function. As expression levels of its constituents are conserved in the human liver, stimulation of the identified signaling module is a promising therapeutic strategy to restore sinusoid function in chronic liver disease.


Asunto(s)
Diabetes Mellitus Tipo 2 , Hígado Graso , Ratones , Humanos , Animales , Pericitos/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Hígado/patología , Transducción de Señal , Células Estrelladas Hepáticas/metabolismo , Hígado Graso/metabolismo , Cirrosis Hepática/patología , Factor 2 de Diferenciación de Crecimiento/metabolismo
3.
Genome Res ; 30(1): 127-137, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31831592

RESUMEN

Bone marrow-derived mesenchymal stem cells (MSCs) differentiate into osteoblasts upon stimulation by signals present in their niche. Because the global signaling cascades involved in the early phases of MSCs osteoblast (OB) differentiation are not well-defined, we used quantitative mass spectrometry to delineate changes in human MSCs proteome and phosphoproteome during the first 24 h of their OB lineage commitment. The temporal profiles of 6252 proteins and 15,059 phosphorylation sites suggested at least two distinct signaling waves: one peaking within 30 to 60 min after stimulation and a second upsurge after 24 h. In addition to providing a comprehensive view of the proteome and phosphoproteome dynamics during early MSCs differentiation, our analyses identified a key role of serine/threonine protein kinase D1 (PRKD1) in OB commitment. At the onset of OB differentiation, PRKD1 initiates activation of the pro-osteogenic transcription factor RUNX2 by triggering phosphorylation and nuclear exclusion of the histone deacetylase HDAC7.


Asunto(s)
Diferenciación Celular , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Osteoblastos/citología , Osteoblastos/metabolismo , Fosfoproteínas/metabolismo , Proteoma , Proteómica , Humanos , Filogenia , Proteómica/métodos
4.
Biochem J ; 479(23): 2419-2431, 2022 12 09.
Artículo en Inglés | MEDLINE | ID: mdl-36408944

RESUMEN

The E3 ligase HOIL-1 forms ester bonds in vitro between ubiquitin and serine/threonine residues in proteins. Here, we exploit UbiSite technology to identify serine and threonine residues undergoing HOIL-1 catalysed ubiquitylation in macrophages stimulated with R848, an activator of the TLR7/8 heterodimer. We identify Thr12, Thr14, Ser20 and Thr22 of ubiquitin as amino acid residues forming ester bonds with the C-terminal carboxylate of another ubiquitin molecule. This increases from 8 to 12 the number of ubiquitin linkage types that are formed in cells. We also identify Ser175 of IRAK4, Ser136, Thr163 and Ser168 of IRAK2 and Thr141 of MyD88 as further sites of HOIL-1-catalysed ubiquitylation together with lysine residues in these proteins that also undergo R848-dependent ubiquitylation. These findings establish that the ubiquitin chains attached to components of myddosomes are initiated by both ester and isopeptide bonds. Ester bond formation takes place within the proline, serine, threonine-rich (PST) domains of IRAK2 and IRAK4 and the intermediate domain of MyD88. The ubiquitin molecules attached to Lys162, Thr163 and Ser168 of IRAK2 are attached to different IRAK2 molecules.


Asunto(s)
Ésteres , Ubiquitina , Serina , Treonina
5.
J Proteome Res ; 20(4): 2042-2055, 2021 04 02.
Artículo en Inglés | MEDLINE | ID: mdl-33539096

RESUMEN

Small ubiquitin-like modifiers (SUMO) and ubiquitin are frequent post-translational modifications of proteins that play pivotal roles in all cellular processes. We previously reported mass spectrometry-based proteomics methods that enable profiling of lysines modified by endogenous SUMO or ubiquitin in an unbiased manner, without the need for genetic engineering. Here we investigated the applicability of precursor mass filtering enabled by MaxQuant.Live to our SUMO and ubiquitin proteomics workflows, which efficiently avoided sequencing of precursors too small to be modified but otherwise indistinguishable by mass-to-charge ratio. Using precursor mass filtering, we achieved a much higher selectivity of modified peptides, ultimately resulting in up to 30% more SUMO and ubiquitin sites identified from replicate samples. Real-time exclusion of unmodified peptides by MQL resulted in 90% SUMO-modified precursor selectivity from a 25% pure sample, demonstrating great applicability for digging deeper into ubiquitin-like modificomes. We adapted the precursor mass filtering strategy to the new Exploris 480 mass spectrometer, achieving comparable gains in SUMO precursor selectivity and identification rates. Collectively, precursor mass filtering via MQL significantly increased identification rates of SUMO- and ubiquitin-modified peptides from the exact same samples, without the requirement for prior knowledge or spectral libraries.


Asunto(s)
Ubiquitina , Ubiquitinas , Espectrometría de Masas , Péptidos , Procesamiento Proteico-Postraduccional , Proteómica , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Ubiquitinas/metabolismo
6.
Mol Cell ; 52(2): 206-20, 2013 Oct 24.
Artículo en Inglés | MEDLINE | ID: mdl-24055346

RESUMEN

Although the general relevance of chromatin modifications for genotoxic stress signaling, cell-cycle checkpoint activation, and DNA repair is well established, how these modifications reach initial thresholds in order to trigger robust responses remains largely unexplored. Here, we identify the chromatin-associated scaffold attachment factor SAFB1 as a component of the DNA damage response and show that SAFB1 cooperates with histone acetylation to allow for efficient γH2AX spreading and genotoxic stress signaling. SAFB1 undergoes a highly dynamic exchange at damaged chromatin in a poly(ADP-ribose)-polymerase 1- and poly(ADP-ribose)-dependent manner and is required for unperturbed cell-cycle checkpoint activation and guarding cells against replicative stress. Altogether, our data reveal that transient recruitment of an architectural chromatin component is required in order to overcome physiological barriers by making chromatin permissive for DNA damage signaling, whereas the ensuing exclusion of SAFB1 may help prevent excessive signaling.


Asunto(s)
Cromatina/genética , Daño del ADN , Proteínas de Unión a la Región de Fijación a la Matriz/genética , Proteínas Asociadas a Matriz Nuclear/genética , Receptores de Estrógenos/genética , Transducción de Señal/genética , Acetilación , Western Blotting , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Cromatina/metabolismo , Roturas del ADN de Doble Cadena/efectos de los fármacos , Roturas del ADN de Doble Cadena/efectos de la radiación , Reparación del ADN , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Histonas/metabolismo , Humanos , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Microscopía Fluorescente , Modelos Genéticos , Pruebas de Mutagenicidad , Proteínas Asociadas a Matriz Nuclear/metabolismo , Fosforilación , Poli Adenosina Difosfato Ribosa/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Interferencia de ARN , Receptores de Estrógenos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
7.
Mol Cell ; 51(6): 707-22, 2013 Sep 26.
Artículo en Inglés | MEDLINE | ID: mdl-24011590

RESUMEN

The stimulation of fibroblast growth factor receptors (FGFRs) with distinct FGF ligands generates specific cellular responses. However, the mechanisms underlying this paradigm have remained elusive. Here, we show that FGF-7 stimulation leads to FGFR2b degradation and, ultimately, cell proliferation, whereas FGF-10 promotes receptor recycling and cell migration. By combining mass-spectrometry-based quantitative proteomics with fluorescence microscopy and biochemical methods, we find that FGF-10 specifically induces the rapid phosphorylation of tyrosine (Y) 734 on FGFR2b, which leads to PI3K and SH3BP4 recruitment. This complex is crucial for FGFR2b recycling and responses, given that FGF-10 stimulation of either FGFR2b_Y734F mutant- or SH3BP4-depleted cells switches the receptor endocytic route to degradation, resulting in decreased breast cancer cell migration and the inhibition of epithelial branching in mouse lung explants. Altogether, these results identify an intriguing ligand-dependent mechanism for the control of receptor fate and cellular outputs that may explain the pathogenic role of deregulated FGFR2b, thus offering therapeutic opportunities.


Asunto(s)
Factor 10 de Crecimiento de Fibroblastos/metabolismo , Factor 7 de Crecimiento de Fibroblastos/metabolismo , Proteómica , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Animales , Movimiento Celular , Ligandos , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteolisis , Tirosina/metabolismo
8.
Mol Cell Proteomics ; 18(Suppl 1): S118-S131, 2019 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-30622161

RESUMEN

G-protein coupled receptors (GPCRs) belong to the seven transmembrane receptor superfamily that transduce signals via G proteins in response to external stimuli to initiate different intracellular signaling pathways which culminate in specific cellular responses. The expression of diverse GPCRs at the plasma membrane of human spermatozoa suggests their involvement in the regulation of sperm fertility. However, the signaling events downstream of many GPCRs in spermatozoa remain uncharacterized. Here, we selected the kappa-opioid receptor (KOR) as a study model and applied phosphoproteomic approach based on TMT labeling and LC-MS/MS analyses. Quantitative coverage of more than 5000 proteins with over 3500 phosphorylation sites revealed changes in the phosphorylation levels of sperm-specific proteins involved in the regulation of the sperm fertility in response to a specific agonist of KOR, U50488H. Further functional studies indicate that KOR could be involved in the regulation of sperm fertile capacity by modulation of calcium channels. Our findings suggest that human spermatozoa possess unique features in the molecular mechanisms downstream of GPCRs which could be key regulators of sperm fertility and improved knowledge of these specific processes may contribute to the development of useful biochemical tools for diagnosis and treatment of male infertility.


Asunto(s)
Fosfoproteínas/metabolismo , Proteómica , Receptores Opioides kappa/metabolismo , Espermatozoides/metabolismo , Reacción Acrosómica , Canales de Calcio/metabolismo , Humanos , Masculino , Fosforilación , Proteoma/metabolismo , Receptores Opioides kappa/agonistas
9.
Mol Cell Proteomics ; 16(8): 1433-1446, 2017 08.
Artículo en Inglés | MEDLINE | ID: mdl-28572092

RESUMEN

Cylindromatosis tumor suppressor protein (CYLD) is a deubiquitinase, best known as an essential negative regulator of the NFkB pathway. Previous studies have suggested an involvement of CYLD in epidermal growth factor (EGF)-dependent signal transduction as well, as it was found enriched within the tyrosine-phosphorylated complexes in cells stimulated with the growth factor. EGF receptor (EGFR) signaling participates in central cellular processes and its tight regulation, partly through ubiquitination cascades, is decisive for a balanced cellular homeostasis. Here, using a combination of mass spectrometry-based quantitative proteomic approaches with biochemical and immunofluorescence strategies, we demonstrate the involvement of CYLD in the regulation of the ubiquitination events triggered by EGF. Our data show that CYLD regulates the magnitude of ubiquitination of several major effectors of the EGFR pathway by assisting the recruitment of the ubiquitin ligase Cbl-b to the activated EGFR complex. Notably, CYLD facilitates the interaction of EGFR with Cbl-b through its Tyr15 phosphorylation in response to EGF, which leads to fine-tuning of the receptor's ubiquitination and subsequent degradation. This represents a previously uncharacterized strategy exerted by this deubiquitinase and tumors suppressor for the negative regulation of a tumorigenic signaling pathway.


Asunto(s)
Enzima Desubiquitinante CYLD/metabolismo , Receptores ErbB/metabolismo , Proteolisis , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Ubiquitinación , Cromatografía Liquida , Enzima Desubiquitinante CYLD/genética , Células HeLa , Humanos , Fosforilación , Proteómica , Espectrometría de Masas en Tándem , Tirosina/metabolismo
10.
J Proteome Res ; 17(1): 296-304, 2018 01 05.
Artículo en Inglés | MEDLINE | ID: mdl-29091453

RESUMEN

Modulation of protein activities by reversible post-translational modifications (PTMs) is a major molecular mechanism involved in the control of virtually all cellular processes. One of these PTMs is ubiquitination, which regulates key processes including protein degradation, cell cycle, DNA damage repair, and signal transduction. Because of its importance for numerous cellular functions, ubiquitination has become an intense topic of research in recent years, and proteomics tools have greatly facilitated the identification of many ubiquitination targets. Taking advantage of the StUbEx strategy for exchanging the endogenous ubiquitin with an epitope-tagged version, we created a modified system, StUbEx PLUS, which allows precise mapping of ubiquitination sites by mass spectrometry. Application of StUbEx PLUS to U2OS cells treated with proteasomal inhibitors resulted in the identification of 41 589 sites on 7762 proteins, which thereby revealed the ubiquitous nature of this PTM and demonstrated the utility of the approach for comprehensive ubiquitination studies at site-specific resolution.


Asunto(s)
Sitios de Unión , Péptidos/aislamiento & purificación , Ubiquitina/metabolismo , Ubiquitinación , Línea Celular , Humanos , Espectrometría de Masas , Péptidos/metabolismo , Procesamiento Proteico-Postraduccional
11.
Mol Cell ; 40(5): 810-22, 2010 Dec 10.
Artículo en Inglés | MEDLINE | ID: mdl-21145488

RESUMEN

The intimate relationship between mediators of the ubiquitin (Ub)-signaling system and human diseases has sparked profound interest in how Ub influences cell death and survival. While the consequence of Ub attachment is intensely studied, little is known with regards to the effects of other Ub-like proteins (UBLs), and deconjugating enzymes that remove the Ub or UBL adduct. Systematic in vivo RNAi analysis identified three NEDD8-specific isopeptidases that, when knocked down, suppress apoptosis. Consistent with the notion that attachment of NEDD8 prevents cell death, genetic ablation of deneddylase 1 (DEN1) suppresses apoptosis. Unexpectedly, we find that Drosophila and human inhibitor of apoptosis (IAP) proteins can function as E3 ligases of the NEDD8 conjugation pathway, targeting effector caspases for neddylation and inactivation. Finally, we demonstrate that DEN1 reverses this effect by removing the NEDD8 modification. Altogether, our findings indicate that IAPs not only modulate cellular processes via ubiquitylation but also through attachment of NEDD8, thereby extending the complexity of IAP-mediated signaling.


Asunto(s)
Proteínas Inhibidoras de la Apoptosis/metabolismo , Interferencia de ARN , Ubiquitina-Proteína Ligasas/genética , Ubiquitina/metabolismo , Animales , Drosophila/metabolismo , Endopeptidasas/metabolismo , Proteínas Inhibidoras de la Apoptosis/genética , Ubiquitina/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación
12.
Mol Cell Proteomics ; 15(6): 2076-92, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-27067055

RESUMEN

Anti-cancer immunotherapies commonly rely on the use of interleukin-2 (IL-2) to promote the expansion of T lymphocytes. IL-2- dependent proliferation is the culmination of a complex network of phosphorylation-driven signaling events that impact on gene transcription through mechanisms that are not clearly understood. To study the role of IL-2 in the regulation of nuclear protein function we have performed an unbiased mass spectrometry-based study of the nuclear phosphoproteome of resting and IL-2-treated CD4(+) T lymphocytes. We detected 8521distinct phosphosites including many that are not yet reported in curated phosphorylation databases. Although most phosphorylation sites remained unaffected upon IL-2 treatment, 391 sites corresponding to 288 gene products showed robust IL-2-dependent regulation. Importantly, we show that ATP-citrate lyase (ACLY) is a key phosphoprotein effector of IL-2-mediated T-cell responses. ACLY becomes phosphorylated on serine 455 in T lymphocytes upon IL-2-driven activation of AKT, and depletion or inactivation of ACLY compromises IL-2-promoted T-cell growth. Mechanistically, we demonstrate that ACLY is required for enhancing histone acetylation levels and inducing the expression of cell cycle regulating genes in response to IL-2. Thus, the metabolic enzyme ACLY emerges as a bridge between cytokine signaling and proliferation of T lymphocytes, and may be an attractive candidate target for the development of more efficient anti-cancer immunotherapies.


Asunto(s)
ATP Citrato (pro-S)-Liasa/aislamiento & purificación , Linfocitos T CD4-Positivos/citología , Interleucina-2/farmacología , Proteómica/métodos , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Redes Reguladoras de Genes/efectos de los fármacos , Humanos , Proteínas Nucleares/análisis , Proteínas Nucleares/efectos de los fármacos , Fosfoproteínas/análisis , Fosfoproteínas/efectos de los fármacos
13.
J Proteome Res ; 16(1): 106-121, 2017 01 06.
Artículo en Inglés | MEDLINE | ID: mdl-27463037

RESUMEN

It remains a paradox that IL-2 and IL-15 can differentially modulate the immune response using the same signaling receptors. We have previously dissected the phosphotyrosine-driven signaling cascades triggered by both cytokines in Kit225 T-cells, unveiling subtle differences that may contribute to their functional dichotomy. In this study, we aimed to decipher the receptor complex assembly in IL-2- and IL-15-activated T-lymphocytes that is highly orchestrated by site-specific phosphorylation events. Comparing the cytokine-induced interactome of the interleukin receptor beta and gamma subunits shared by the two cytokines, we defined the components of the early IL-2 and IL-15 receptor-associated complex discovering novel constituents. Additionally, phosphopeptide-directed analysis allowed us to detect several cytokine-dependent and -independent phosphorylation events within the activated receptor complex including novel phosphorylated sites located in the cytoplasmic region of IL-2 receptor ß subunit (IL-2Rß). We proved that the distinct phosphorylations induced by the cytokines serve for recruiting different types of effectors to the initial receptor/ligand complex. Overall, our study sheds new light into the initial molecular events triggered by IL-2 and IL-15 and constitutes a further step toward a better understanding of the early signaling aspects of the two closely related cytokines in T-lymphocytes.


Asunto(s)
Subunidad gamma Común de Receptores de Interleucina/inmunología , Interleucina-15/farmacología , Subunidad beta del Receptor de Interleucina-2/inmunología , Interleucina-2/farmacología , Janus Quinasa 1/inmunología , Janus Quinasa 3/inmunología , Linfocitos T/efectos de los fármacos , Secuencia de Aminoácidos , Línea Celular Tumoral , Regulación de la Expresión Génica , Humanos , Subunidad gamma Común de Receptores de Interleucina/genética , Interleucina-15/genética , Interleucina-15/inmunología , Interleucina-2/genética , Interleucina-2/inmunología , Subunidad beta del Receptor de Interleucina-2/genética , Janus Quinasa 1/genética , Janus Quinasa 3/genética , Activación de Linfocitos , Fosforilación , Fosfotirosina/genética , Fosfotirosina/inmunología , Unión Proteica , Mapeo de Interacción de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacología , Transducción de Señal , Linfocitos T/citología , Linfocitos T/inmunología
14.
Am J Physiol Endocrinol Metab ; 310(2): E116-28, 2016 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-26578713

RESUMEN

The tumor suppressor p53 (TRP53 in mice) is known for its involvement in carcinogenesis, but work during recent years has underscored the importance of p53 in the regulation of whole body metabolism. A general notion is that p53 is necessary for efficient oxidative metabolism. The importance of UCP1-dependent uncoupled respiration and increased oxidation of glucose and fatty acids in brown or brown-like adipocytes, termed brite or beige, in relation to energy balance and homeostasis has been highlighted recently. UCP1-dependent uncoupled respiration in classic interscapular brown adipose tissue is central to cold-induced thermogenesis, whereas brite/beige adipocytes are of special importance in relation to diet-induced thermogenesis, where the importance of UCP1 is only clearly manifested in mice kept at thermoneutrality. We challenged wild-type and TRP53-deficient mice by high-fat feeding under thermoneutral conditions. Interestingly, mice lacking TRP53 gained less weight compared with their wild-type counterparts. This was related to an increased expression of Ucp1 and other PPARGC1a and PPARGC1b target genes but not Ppargc1a or Ppargc1b in inguinal white adipose tissue of mice lacking TRP53. We show that TRP53, independently of its ability to bind DNA, inhibits the activity of PPARGC1a and PPARGC1b. Collectively, our data show that TRP53 has the ability to regulate the thermogenic capacity of adipocytes through modulation of PPARGC1 activity.


Asunto(s)
Tejido Adiposo Pardo/metabolismo , Canales Iónicos/metabolismo , Proteínas Mitocondriales/metabolismo , Termogénesis/genética , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Adipocitos/metabolismo , Tejido Adiposo Blanco/metabolismo , Animales , Células Cultivadas , Dieta Alta en Grasa , Femenino , Regulación de la Expresión Génica , Canales Iónicos/genética , Ratones , Ratones Noqueados , Proteínas Mitocondriales/genética , Factores de Transcripción/genética , Proteína p53 Supresora de Tumor/genética , Proteína Desacopladora 1 , Aumento de Peso/fisiología
15.
Mol Cell ; 32(4): 540-53, 2008 Nov 21.
Artículo en Inglés | MEDLINE | ID: mdl-19026784

RESUMEN

Ubiquitin-mediated inactivation of caspases has long been postulated to contribute to the regulation of apoptosis. However, detailed mechanisms and functional consequences of caspase ubiquitylation have not been demonstrated. Here we show that the Drosophila Inhibitor of Apoptosis 1, DIAP1, blocks effector caspases by targeting them for polyubiquitylation and nonproteasomal inactivation. We demonstrate that the conjugation of ubiquitin to drICE suppresses its catalytic potential in cleaving caspase substrates. Our data suggest that ubiquitin conjugation sterically interferes with substrate entry and reduces the caspase's proteolytic velocity. Disruption of drICE ubiquitylation, either by mutation of DIAP1's E3 activity or drICE's ubiquitin-acceptor lysines, abrogates DIAP1's ability to neutralize drICE and suppress apoptosis in vivo. We also show that DIAP1 rests in an "inactive" conformation that requires caspase-mediated cleavage to subsequently ubiquitylate caspases. Taken together, our findings demonstrate that effector caspases regulate their own inhibition through a negative feedback mechanism involving DIAP1 "activation" and nondegradative polyubiquitylation.


Asunto(s)
Inhibidores de Caspasas , Ubiquitinación , Animales , Apoptosis/genética , Apoptosis/fisiología , Caspasas/genética , Caspasas Efectoras/genética , Caspasas Efectoras/metabolismo , Células Cultivadas , Drosophila/citología , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/antagonistas & inhibidores , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/metabolismo , Cinética , Modelos Biológicos , Péptido Hidrolasas/metabolismo , Conformación Proteica , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo
16.
Proteomics ; 15(2-3): 520-31, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25142963

RESUMEN

Common γ-chain family of cytokines (IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, where IL stands for interleukin) are key regulators of the immune homeostasis that exhibit pleiotropic biological activities and even sometimes redundant roles as a result of the utilization of the same receptor subunit. However, they also exert distinct functions that make each of them to be indispensable. For instance, all family members can act as T-cell growth factors; however, we found that IL-15 but not IL-7 can replace IL-2 to promote and sustain the proliferation of Kit225T cells. In addition to the γ-chain, IL-2 and IL-15 share the ß-chain, which creates the paradox of how they can trigger diverse phenotypes despite signaling through the same receptors. To investigate this paradigm, we combined SILAC with enrichment of tyrosine-phosphorylated proteins and peptides followed by mass spectrometric analysis to quantitatively assess the signaling networks triggered downstream IL-2/IL-2R and IL-15/IL-15R. This study confirmed that the transduction pathways initiated by both cytokines are highly similar and revealed that the main signaling branches, JAK/STAT, RAS/MAPK and PI3K/AKT, were nearly equivalently activated in response to both ILs. Despite that, our study revealed that receptor internalization rates differ in IL-2- and IL-15-treated cells indicating a discrete modulation of cytokine signaling. All MS data have been deposited in the ProteomeXchange with identifier PXD001129 (http://proteomecentral.proteomexchange.org/dataset/PXD001129).


Asunto(s)
Interleucina-15/inmunología , Interleucina-2/inmunología , Transducción de Señal , Linfocitos T/inmunología , Línea Celular Tumoral , Proliferación Celular , Endocitosis , Humanos , Interleucina-7/inmunología , Fosforilación , Proteómica , Linfocitos T/citología
17.
J Proteome Res ; 14(8): 3348-61, 2015 Aug 07.
Artículo en Inglés | MEDLINE | ID: mdl-26074025

RESUMEN

Muscle stem cells, or satellite cells, play an important role in the maintenance and repair of muscle tissue and have the capacity to proliferate and differentiate in response to physiological or environmental changes. Although they have been extensively studied, the key regulatory steps and the complex temporal protein dynamics accompanying the differentiation of primary human muscle cells remain poorly understood. Here, we demonstrate the advantages of applying a MS-based quantitative approach, stable isotope labeling by amino acids in cell culture (SILAC), for studying human myogenesis in vitro and characterize the fine-tuned changes in protein expression underlying the dramatic phenotypic conversion of primary mononucleated human muscle cells during in vitro differentiation to form multinucleated myotubes. Using an exclusively optimized triple encoding SILAC procedure, we generated dynamic expression profiles during the course of myogenic differentiation and quantified 2240 proteins, 243 of which were regulated. These changes in protein expression occurred in sequential waves and underlined vast reprogramming in key processes governing cell fate decisions, i.e., cell cycle withdrawal, RNA metabolism, cell adhesion, proteolysis, and cytoskeletal organization. In silico transcription factor target analysis demonstrated that the observed dynamic changes in the proteome could be attributed to a cascade of transcriptional events involving key myogenic regulatory factors as well as additional regulators not yet known to act on muscle differentiation. In addition, we created of a dynamic map of the developing myofibril, providing valuable insights into the formation and maturation of the contractile apparatus in vitro. Finally, our SILAC-based quantitative approach offered the possibility to follow the expression profiles of several muscle disease-associated proteins simultaneously and therefore could be a valuable resource for future studies investigating pathogenesis of degenerative muscle disorders as well as assessing new therapeutic strategies.


Asunto(s)
Diferenciación Celular , Fibras Musculares Esqueléticas/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Células Satélite del Músculo Esquelético/metabolismo , Aminoácidos/metabolismo , Western Blotting , Células Cultivadas , Cromatografía Liquida , Análisis por Conglomerados , Humanos , Inmunohistoquímica , Recién Nacido , Marcaje Isotópico/métodos , Cinética , Fibras Musculares Esqueléticas/citología , Proteoma/clasificación , Células Satélite del Músculo Esquelético/citología , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en Tándem , Factores de Tiempo
18.
Semin Cell Dev Biol ; 23(8): 863-71, 2012 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-22677334

RESUMEN

Reversible protein phosphorylation is involved in the regulation of most, if not all, major cellular processes via dynamic signal transduction pathways. During the last decade quantitative phosphoproteomics have evolved from a highly specialized area to a powerful and versatile platform for analyzing protein phosphorylation at a system-wide scale and has become the intuitive strategy for comprehensive characterization of signaling networks. Contemporary phosphoproteomics use highly optimized procedures for sample preparation, mass spectrometry and data analysis algorithms to identify and quantify thousands of phosphorylations, thus providing extensive overviews of the cellular signaling networks. As a result of these developments quantitative phosphoproteomics have been applied to study processes as diverse as immunology, stem cell biology and DNA damage. Here we review the developments in phosphoproteomics technology that have facilitated the application of phosphoproteomics to signaling networks and introduce examples of recent system-wide applications of quantitative phosphoproteomics. Despite the great advances in phosphoproteomics technology there are still several outstanding issues and we provide here our outlook on the current limitations and challenges in the field.


Asunto(s)
Fosfoproteínas/análisis , Proteómica/métodos , Transducción de Señal , Animales , Humanos , Espectrometría de Masas , Fosforilación
19.
J Lipid Res ; 55(12): 2491-500, 2014 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-25312885

RESUMEN

Adipocyte differentiation is orchestrated by the ligand-activated nuclear receptor PPARγ. Endogenous ligands comprise oxidized derivatives of arachidonic acid and structurally similar PUFAs. Although expression of PPARγ peaks in mature adipocytes, ligands are produced primarily at the onset of differentiation. Concomitant with agonist production, murine fibroblasts undergo two rounds of mitosis referred to as mitotic clonal expansion. Here we show that mouse embryonic fibroblasts deficient in either of two cell cycle inhibitors, the transcription factor p53 or its target gene encoding the cyclin-dependent kinase inhibitor p21, exhibit increased adipogenic potential. The antiadipogenic effect of p53 relied on its transcriptional activity and p21 expression but was circumvented by administration of an exogenous PPARγ agonist suggesting a linkage between cell cycling and PPARγ ligand production. Indeed, cell cycle inhibitory compounds decreased PPARγ ligand production in differentiating 3T3-L1 preadipocytes. Furthermore, these inhibitors abolished the release of arachidonic acid induced by the hormonal cocktail initiating adipogenesis. Collectively, our results suggest that murine fibroblasts require clonal expansion for PPARγ ligand production at the onset of adipocyte differentiation.


Asunto(s)
Adipocitos Blancos/metabolismo , Adipogénesis , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Regulación del Desarrollo de la Expresión Génica , PPAR gamma/agonistas , Proteína p53 Supresora de Tumor/metabolismo , Células 3T3-L1 , Adipocitos Blancos/citología , Animales , Ácido Araquidónico/antagonistas & inhibidores , Ácido Araquidónico/metabolismo , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos/citología , Cinética , Ligandos , Ratones , Ratones Noqueados , Mitosis/efectos de los fármacos , PPAR gamma/genética , PPAR gamma/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/genética
20.
J Proteome Res ; 13(9): 4192-204, 2014 Sep 05.
Artículo en Inglés | MEDLINE | ID: mdl-25093938

RESUMEN

Post-translational modification of proteins with the small polypeptide ubiquitin plays a pivotal role in many cellular processes, altering protein lifespan, location, and function and regulating protein-protein interactions. Ubiquitination exerts its diverse functions through complex mechanisms by formation of different polymeric chains and subsequent recognition of the ubiquitin signal by specific protein interaction domains. Despite some recent advances in the analytical tools for the analysis of ubiquitination by mass spectrometry, there is still a need for additional strategies suitable for investigation of cellular ubiquitination at the proteome level. Here, we present a stable tagged ubiquitin exchange (StUbEx) cellular system in which endogenous ubiquitin is replaced with an epitope-tagged version, thereby allowing specific and efficient affinity purification of ubiquitinated proteins for global analyses of protein ubiquitination. Importantly, the overall level of ubiquitin in the cell remains virtually unchanged, thus avoiding ubiquitination artifacts associated with overexpression. The efficiency and reproducibility of the method were assessed through unbiased analysis of epidermal growth factor (EGF) signaling by quantitative mass spectrometry, covering over 3400 potential ubiquitinated proteins. The StUbEx system is applicable to virtually any cell line and can be readily adapted to any of the ubiquitin-like post-translational modifications.


Asunto(s)
Marcaje Isotópico/métodos , Proteómica/métodos , Ubiquitina/química , Ubiquitina/metabolismo , Cromatografía de Afinidad/métodos , Bases de Datos de Proteínas , Células HeLa , Histidina , Humanos , Oligopéptidos , Proteínas Recombinantes de Fusión , Reproducibilidad de los Resultados , Ubiquitinación
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA