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1.
Cell ; 156(4): 771-85, 2014 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-24529379

RESUMO

mTORC1 promotes cell growth in response to nutrients and growth factors. Insulin activates mTORC1 through the PI3K-Akt pathway, which inhibits the TSC1-TSC2-TBC1D7 complex (the TSC complex) to turn on Rheb, an essential activator of mTORC1. However, the mechanistic basis of how this pathway integrates with nutrient-sensing pathways is unknown. We demonstrate that insulin stimulates acute dissociation of the TSC complex from the lysosomal surface, where subpopulations of Rheb and mTORC1 reside. The TSC complex associates with the lysosome in a Rheb-dependent manner, and its dissociation in response to insulin requires Akt-mediated TSC2 phosphorylation. Loss of the PTEN tumor suppressor results in constitutive activation of mTORC1 through the Akt-dependent dissociation of the TSC complex from the lysosome. These findings provide a unifying mechanism by which independent pathways affecting the spatial recruitment of mTORC1 and the TSC complex to Rheb at the lysosomal surface serve to integrate diverse growth signals.


Assuntos
Insulina/metabolismo , Lisossomos/metabolismo , Complexos Multiproteicos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Aminoácidos/metabolismo , Animais , Linhagem Celular , GTP Fosfo-Hidrolases/metabolismo , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/metabolismo
2.
J Biol Chem ; 294(10): 3359-3366, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30647128

RESUMO

Bile acids are critical metabolites in the gastrointestinal tract and contribute to maintaining intestinal immune homeostasis through cross-talk with the gut microbiota. The conversion of bile acids by the gut microbiome is now recognized as a factor affecting both host metabolism and immune responses, but its physiological roles remain unclear. We conducted a screen for microbiome metabolites that would function as inflammasome activators and herein report the identification of 12-oxo-lithocholic acid (BAA485), a potential microbiome-derived bile acid metabolite. We demonstrate that the more potent analogue 11-oxo-12S-hydroxylithocholic acid methyl ester (BAA473) can induce secretion of interleukin-18 (IL-18) through activation of the inflammasome in both myeloid and intestinal epithelial cells. Using a genome-wide CRISPR screen with compound induced pyroptosis in THP-1 cells, we identified that inflammasome activation by BAA473 is pyrin-dependent (MEFV). To our knowledge, the bile acid analogues BAA485 and BAA473 are the first small molecule activators of the pyrin inflammasome. We surmise that pyrin inflammasome activation through microbiota-modified bile acid metabolites such as BAA473 and BAA485 plays a role in gut microbiota regulated intestinal immune response. The discovery of these two bioactive compounds may help to further unveil the importance of pyrin in gut homeostasis and autoimmune diseases.


Assuntos
Ácidos e Sais Biliares/imunologia , Células Epiteliais/imunologia , Microbioma Gastrointestinal/imunologia , Imunidade nas Mucosas , Inflamassomos/imunologia , Mucosa Intestinal/imunologia , Pirina/imunologia , Ácidos e Sais Biliares/química , Humanos , Células Mieloides/imunologia , Células THP-1
3.
Mol Cell ; 47(4): 535-46, 2012 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-22795129

RESUMO

The tuberous sclerosis complex (TSC) tumor suppressors form the TSC1-TSC2 complex, which limits cell growth in response to poor growth conditions. Through its GTPase-activating protein (GAP) activity toward Rheb, this complex inhibits the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1), a key promoter of cell growth. Here, we identify and biochemically characterize TBC1D7 as a stably associated and ubiquitous third core subunit of the TSC1-TSC2 complex. We demonstrate that the TSC1-TSC2-TBC1D7 (TSC-TBC) complex is the functional complex that senses specific cellular growth conditions and possesses Rheb-GAP activity. Sequencing analyses of samples from TSC patients suggest that TBC1D7 is unlikely to represent TSC3. TBC1D7 knockdown decreases the association of TSC1 and TSC2 leading to decreased Rheb-GAP activity, without effects on the localization of TSC2 to the lysosome. Like the other TSC-TBC components, TBC1D7 knockdown results in increased mTORC1 signaling, delayed induction of autophagy, and enhanced cell growth under poor growth conditions.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Sequência de Aminoácidos , Proteínas de Transporte/genética , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lisossomos/genética , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Dados de Sequência Molecular , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Complexos Multiproteicos , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Ligação Proteica , Proteínas/genética , Proteína Enriquecida em Homólogo de Ras do Encéfalo , Transdução de Sinais , Serina-Treonina Quinases TOR , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose Tuberosa , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/genética
4.
Mol Cell ; 39(2): 171-83, 2010 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-20670887

RESUMO

Aberrant activation of the mammalian target of rapamycin complex 1 (mTORC1) is a common molecular event in a variety of pathological settings, including genetic tumor syndromes, cancer, and obesity. However, the cell-intrinsic consequences of mTORC1 activation remain poorly defined. Through a combination of unbiased genomic, metabolomic, and bioinformatic approaches, we demonstrate that mTORC1 activation is sufficient to stimulate specific metabolic pathways, including glycolysis, the oxidative arm of the pentose phosphate pathway, and de novo lipid biosynthesis. This is achieved through the activation of a transcriptional program affecting metabolic gene targets of hypoxia-inducible factor (HIF1alpha) and sterol regulatory element-binding protein (SREBP1 and SREBP2). We find that SREBP1 and 2 promote proliferation downstream of mTORC1, and the activation of these transcription factors is mediated by S6K1. Therefore, in addition to promoting protein synthesis, mTORC1 activates specific bioenergetic and anabolic cellular processes that are likely to contribute to human physiology and disease.


Assuntos
Regulação da Expressão Gênica/fisiologia , Glicólise/fisiologia , Lipídeos/biossíntese , Via de Pentose Fosfato/fisiologia , Biossíntese de Proteínas/fisiologia , Fatores de Transcrição/metabolismo , Transcrição Gênica/fisiologia , Animais , Linhagem Celular Transformada , Proliferação de Células , Genômica/métodos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Lipídeos/genética , Alvo Mecanístico do Complexo 1 de Rapamicina , Metabolômica/métodos , Camundongos , Complexos Multiproteicos , Neoplasias/genética , Neoplasias/metabolismo , Obesidade/genética , Obesidade/metabolismo , Proteínas , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Serina-Treonina Quinases TOR , Fatores de Transcrição/genética
5.
J Biol Chem ; 288(28): 20443-52, 2013 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-23689509

RESUMO

The COP9 signalosome (CSN) is a conserved protein complex known to be involved in developmental processes of eukaryotic organisms. Genetic disruption of a CSN gene causes arrest during early embryonic development in mice. The Csn8 subunit is the smallest and the least conserved subunit, being absent from the CSN complex of several fungal species. Nevertheless, Csn8 is an integral component of the CSN complex in higher eukaryotes, where it is essential for life. By characterizing the mouse embryonic fibroblasts (MEFs) that express Csn8 at a low level, we found that Csn8 plays an important role in maintaining the proper duration of the G1 phase of the cell cycle. A decreased level of Csn8, either in Csn8 hypomorphic MEFs or following siRNA-mediated knockdown in HeLa cells, accelerated cell growth rate. Csn8 hypomorphic MEFs exhibited a shortened G1 duration and affected expression of G1 regulators. In contrast to Csn8, down-regulation of Csn5 impaired cell proliferation. Csn5 proteins were found both as a component of the CSN complex and outside of CSN (Csn5-f), and the amount of Csn5-f relative to CSN was increased in the Csn8 hypomorphic cells. We conclude that CSN harbors both positive and negative regulators of the cell cycle and therefore is poised to influence the fate of a cell at the crossroad of cell division, differentiation, and senescence.


Assuntos
Proteínas de Transporte/metabolismo , Proliferação de Células , Fibroblastos/metabolismo , Fase G1 , Animais , Western Blotting , Complexo do Signalossomo COP9 , Proteínas de Transporte/genética , Células Cultivadas , Embrião de Mamíferos/citologia , Feminino , Fibroblastos/citologia , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Camundongos , Camundongos Knockout , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Proteínas/genética , Proteínas/metabolismo , Interferência de RNA , Fatores de Tempo
7.
bioRxiv ; 2024 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-39386441

RESUMO

Mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) integrates diverse intracellular and extracellular growth signals to regulate cell and tissue growth. How the molecular mechanisms regulating mTORC1 signaling established through biochemical and cell biological studies function under physiological states in specific mammalian tissues are unknown. Here, we characterize a genetic mouse model lacking the 5 phosphorylation sites on the tuberous sclerosis complex 2 (TSC2) protein through which the growth factor-stimulated protein kinase AKT can active mTORC1 signaling in cell culture models. These phospho-mutant mice (TSC2-5A) are developmentally normal but exhibit reduced body weight and the weight of specific organs, such as brain and skeletal muscle, associated with cell intrinsic decreases in growth factor-stimulated mTORC1 signaling. The TSC2-5A mouse model demonstrates that TSC2 phosphorylation is a primary mechanism of mTORC1 activation in some, but not all, tissues and provides a genetic tool to facilitate studies on the physiological regulation of mTORC1.

8.
Circ Res ; 108(1): 40-50, 2011 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-21051661

RESUMO

RATIONALE: Ubiquitin-proteasome system (UPS) dysfunction has been implicated in cardiac pathogenesis. Understanding how cardiac UPS function is regulated will facilitate delineating the pathophysiological significance of UPS dysfunction and developing new therapeutic strategies. The COP9 (constitutive photomorphogenesis mutant 9) signalosome (CSN) may regulate the UPS, but this has not been tested in a critical vertebrate organ. Moreover, the role of CSN in a postmitotic organ and the impact of cardiomyocyte-restricted UPS dysfunction on the heart have not been reported. OBJECTIVE: We sought to determine the role of CSN-mediated deneddylation in UPS function and postnatal cardiac development and function. METHODS AND RESULTS: Cardiomyocyte-restricted Csn8 gene knockout (CR-Csn8KO) in mice was achieved using a Cre-LoxP system. CR-Csn8KO impaired CSN holocomplex formation and cullin deneddylation and resulted in decreases in F-box proteins. Probing with a surrogate misfolded protein revealed severe impairment of UPS function in CR-Csn8KO hearts. Consequently, CR-Csn8KO mice developed cardiac hypertrophy, which rapidly progressed to heart failure and premature death. Massive cardiomyocyte necrosis rather than apoptosis appears to be the primary cause of the heart failure. This is because (1) massive necrotic cell death and increased infiltration of leukocytes were observed before increased apoptosis; (2) increased apoptosis was not detectable until overt heart failure was observed; and (3) cardiac overexpression of Bcl2 failed to ameliorate CR-Csn8KO mouse premature death. CONCLUSIONS: Csn8/CSN plays an essential role in cullin deneddylation, UPS-mediated degradation of a subset of proteins, and the survival of cardiomyocytes and, therefore, is indispensable in postnatal development and function of the heart. Cardiomyocyte-restricted UPS malfunction can cause heart failure.


Assuntos
Cardiomiopatia Dilatada/metabolismo , Proteínas de Transporte/metabolismo , Proteínas Culina/metabolismo , Complexos Multiproteicos/metabolismo , Miócitos Cardíacos/metabolismo , Peptídeo Hidrolases/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Animais , Apoptose/genética , Complexo do Signalossomo COP9 , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/patologia , Proteínas de Transporte/genética , Sobrevivência Celular/genética , Proteínas Culina/genética , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Leucócitos/metabolismo , Leucócitos/patologia , Camundongos , Camundongos Knockout , Complexos Multiproteicos/genética , Miócitos Cardíacos/patologia , Necrose , Peptídeo Hidrolases/genética , Complexo de Endopeptidases do Proteassoma/genética , Ubiquitina/genética
9.
Mol Aspects Med ; 82: 101018, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34489092

RESUMO

Autophagy is a catabolic process that promotes cellular fitness by clearing aggregated protein species, pathogens and damaged organelles through lysosomal degradation. The autophagic process is particularly important in the nervous system where post-mitotic neurons rely heavily on protein and organelle quality control in order to maintain cellular health throughout the lifetime of the organism. Alterations of autophagy and lysosomal function are hallmarks of various neurodegenerative disorders. In this review, we conceptualize some of the mechanistic and genetic evidence pointing towards autophagy and lysosomal dysfunction as a causal driver of neurodegeneration. Furthermore, we discuss rate-limiting pathway nodes and potential approaches to restore pathway activity, from autophagy initiation, cargo sequestration to lysosomal capacity.


Assuntos
Lisossomos , Doenças Neurodegenerativas , Autofagia/genética , Humanos , Doenças Neurodegenerativas/genética , Neurônios
10.
Mol Cell Biol ; 27(13): 4708-19, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17452440

RESUMO

DET1 (de-etiolated 1) is an essential negative regulator of plant light responses, and it is a component of the Arabidopsis thaliana CDD complex containing DDB1 and COP10 ubiquitin E2 variant. Human DET1 has recently been isolated as one of the DDB1- and Cul4A-associated factors, along with an array of WD40-containing substrate receptors of the Cul4A-DDB1 ubiquitin ligase. However, DET1 differs from conventional substrate receptors of cullin E3 ligases in both biochemical behavior and activity. Here we report that mammalian DET1 forms stable DDD-E2 complexes, consisting of DDB1, DDA1 (DET1, DDB1 associated 1), and a member of the UBE2E group of canonical ubiquitin-conjugating enzymes. DDD-E2 complexes interact with multiple ubiquitin E3 ligases. We show that the E2 component cannot maintain the ubiquitin thioester linkage once bound to the DDD core, rendering mammalian DDD-E2 equivalent to the Arabidopsis CDD complex. While free UBE2E-3 is active and able to enhance UbcH5/Cul4A activity, the DDD core specifically inhibits Cul4A-dependent polyubiquitin chain assembly in vitro. Overexpression of DET1 inhibits UV-induced CDT1 degradation in cultured cells. These findings demonstrate that the conserved DET1 complex modulates Cul4A functions by a novel mechanism.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas Culina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Sequência de Aminoácidos , Proteínas de Transporte/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Dados de Sequência Molecular , Ligação Proteica/efeitos da radiação , Processamento de Proteína Pós-Traducional/efeitos da radiação , Subunidades Proteicas/metabolismo , Especificidade por Substrato/efeitos da radiação , Termodinâmica , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/química , Raios Ultravioleta
11.
PLoS One ; 15(8): e0235551, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833964

RESUMO

VPS34 is a key regulator of endomembrane dynamics and cargo trafficking, and is essential in cultured cell lines and in mice. To better characterize the role of VPS34 in cell growth, we performed unbiased cell line profiling studies with the selective VPS34 inhibitor PIK-III and identified RKO as a VPS34-dependent cellular model. Pooled CRISPR screen in the presence of PIK-III revealed endolysosomal genes as genetic suppressors. Dissecting VPS34-dependent alterations with transcriptional profiling, we found the induction of hypoxia response and cholesterol biosynthesis as key signatures. Mechanistically, acute VPS34 inhibition enhanced lysosomal degradation of transferrin and low-density lipoprotein receptors leading to impaired iron and cholesterol uptake. Excess soluble iron, but not cholesterol, was sufficient to partially rescue the effects of VPS34 inhibition on mitochondrial respiration and cell growth, indicating that iron limitation is the primary driver of VPS34-dependency in RKO cells. Loss of RAB7A, an endolysosomal marker and top suppressor in our genetic screen, blocked transferrin receptor degradation, restored iron homeostasis and reversed the growth defect as well as metabolic alterations due to VPS34 inhibition. Altogether, our findings suggest that impaired iron mobilization via the VPS34-RAB7A axis drive VPS34-dependence in certain cancer cells.


Assuntos
Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Ferro/metabolismo , Neoplasias/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Proliferação de Células , Colesterol/biossíntese , Colesterol/genética , Classe III de Fosfatidilinositol 3-Quinases/genética , Endossomos/metabolismo , Células HEK293 , Humanos , Lisossomos/metabolismo , Receptores de LDL/metabolismo , Transferrina/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7
12.
Nat Cell Biol ; 21(5): 662-663, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30783264

RESUMO

In the version of this Article originally published, the labels for Rictor and mTOR in the whole cell lysate (WCL) blots were swapped in Fig. 3b and the mTOR blot was placed upside down. Unprocessed blots of mTOR were also missing from Supplementary Fig. 9. The corrected Figs are shown below. In addition, control blots for the mTOR antibody (Cell Signalling Technology #2972) were also missing. These are now provided below, as Fig. 9, and show that the lower band is likely non-specific.

13.
BMC Biochem ; 9: 1, 2008 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-18173839

RESUMO

BACKGROUND: Cullin-RING ubiquitin E3 ligases (CRLs) are regulated by modification of an ubiquitin-like protein, Nedd8 (also known as Rub1) on the cullin subunit. Neddylation is shown to facilitate E3 complex assembly; while un-neddylated cullins are bound by CAND1 that prevents recruitment of the substrates. The level of Nedd8 modification is critically dependent on the COP9 signalosome (CSN), an eight-subunit protein complex containing Nedd8 isopeptidase activity. RESULTS: We report isolation of SAP130 (SF3b-3) as a CSN1 interacting protein. SAP130 is homologous to DDB1, and is a component of SF3b RNA splicing complex and STAGA/TFTC transcription complexes, but its specific function within these complexes is unknown. We show that SAP130 can interact with a variety of cullin proteins. It forms tertiary complexes with fully assembled CRL E3 complexes such as SCFSkp2, Elongin B/C -Cul2- VHL and Cul4-DDB complex by binding to both N-terminal and C-terminal domain of cullins. SAP130 preferentially associates with neddylated cullins in vivo. However knock-down of CAND1 abolished this preference and increased association of SAP130 with Cul2. Furthermore, we provide evidence that CSN regulates SAP130-Cul2 interaction and SAP130-associated polyubiquitinating activity. CONCLUSION: SAP130 is a cullin binding protein that is likely involved in the Nedd8 pathway. The association of SAP130 with various cullin member proteins such as Cul1, Cul2 and Cul4A is modulated by CAND1 and CSN. As an established component of transcription and RNA processing complexes, we hypothesis that SAP130 may link CRL mediated ubiquitination to gene expression.


Assuntos
Proteínas Culina/metabolismo , Complexos Multiproteicos/metabolismo , Peptídeo Hidrolases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Sítios de Ligação , Complexo do Signalossomo COP9 , Linhagem Celular , Proteínas Culina/química , Humanos , Camundongos , Poliubiquitina/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/isolamento & purificação , Coelhos , Especificidade por Substrato , Fatores de Transcrição/metabolismo , Ubiquitinas/metabolismo
14.
Curr Biol ; 12(8): 667-72, 2002 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-11967155

RESUMO

The COP9 signalosome (CSN) is a conserved protein complex with homologies to the lid subcomplex of the 26S proteasome. It promotes cleavage of the Nedd8 conjugate (deneddylation) from the cullin component of SCF ubiquitin ligases. We provide evidence that cullin neddylation and deneddylation is highly dynamic, that its equilibrium can be effectively modulated by CSN, and that neddylation allows Cul1 to form larger protein complexes. CSN2 integrates into the CSN complex via its C-terminal region and its N-terminal half region is necessary for direct interaction with Cul1. The polyclonal antibodies against CSN2 but not other CSN subunits cause accumulation of neddylated Cul1/Cul2 in HeLa cell extract, indicating that CSN2 is essential in cullin deneddylation. Further, CSN inhibits ubiquitination and degradation of the cyclin-dependent kinase inhibitor p27(kip1) in vitro. Microinjection of the CSN complex impeded the G1 cells from entering the S phase. Moreover, anti-CSN2 antibodies negate the CSN-dependent p27 stabilization and the G1/S blockage, suggesting that these functions require the deneddylation activity. We conclude that CSN inhibits SCF ubiquitin ligase activity in targeting p27 proteolysis and negatively regulates cell cycle at the G1 phase by promoting deneddylation of Cul1.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Culina , Fase G1 , Proteínas Nucleares , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Receptores dos Hormônios Tireóideos , Fase S , Fatores de Transcrição , Proteínas Supressoras de Tumor/metabolismo , Animais , Complexo do Signalossomo COP9 , Linhagem Celular , Cromatografia em Gel , Inibidor de Quinase Dependente de Ciclina p27 , Células HeLa , Humanos , Peso Molecular , Complexos Multiproteicos , Peptídeo Hidrolases , Ligação Proteica , Suínos , Transfecção
15.
Mol Cell Biol ; 23(19): 6790-7, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12972599

RESUMO

Csn2 (Trip15/Cops2/Alien) encodes the second subunit of the COP9 signalosome (CSN), an eight-subunit heteromeric complex homologous to the lid subcomplex of the 26S proteasome. CSN is a regulator of SCF (Skp1-cullin-F-box protein)ubiquitin ligases, mostly through the enzymatic activity that deconjugates the ubiquitin-like protein Nedd8 from the SCF Cul1 component. In addition, CSN associates with protein kinase activities targeting p53, c-Jun, and IkappaB for phosphorylation. Csn2 also interacts with and regulates a subset of nuclear hormone receptors and is considered a novel corepressor. We report that targeted disruption of Csn2 in mice caused arrest of embryo development at the peri-implantation stage. Csn2(-/-) blastocysts failed to outgrow in culture and exhibited a cell proliferation defect in inner cell mass, accompanied by a slight decrease in Oct4. In addition, lack of Csn2 disrupted the CSN complex and resulted in a drastic increase in cyclin E, supporting a role for CSN in cooperating with the SCF-ubiquitin-proteasome system to regulate protein turnover. Furthermore, Csn2(-/-) embryos contained elevated levels of p53 and p21, which may contribute to premature cell cycle arrest of the mutant.


Assuntos
Ciclina E/metabolismo , Proteínas/metabolismo , Transdução de Sinais , Fatores de Transcrição , Proteína Supressora de Tumor p53/metabolismo , Animais , Blastocisto/metabolismo , Complexo do Signalossomo COP9 , Divisão Celular/fisiologia , Cruzamentos Genéticos , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Genes Essenciais , Células HeLa , Heterozigoto , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexos Multiproteicos , Fator 3 de Transcrição de Octâmero , Peptídeo Hidrolases , Subunidades Proteicas , Proteínas/genética , Recombinação Genética
16.
JAMA Neurol ; 73(7): 836-845, 2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-27159400

RESUMO

IMPORTANCE: Focal cortical dysplasia (FCD), hemimegalencephaly, and megalencephaly constitute a spectrum of malformations of cortical development with shared neuropathologic features. These disorders are associated with significant childhood morbidity and mortality. OBJECTIVE: To identify the underlying molecular cause of FCD, hemimegalencephaly, and diffuse megalencephaly. DESIGN, SETTING, AND PARTICIPANTS: Patients with FCD, hemimegalencephaly, or megalencephaly (mean age, 11.7 years; range, 2-32 years) were recruited from Pediatric Hospital A. Meyer, the University of Hong Kong, and Seattle Children's Research Institute from June 2012 to June 2014. Whole-exome sequencing (WES) was performed on 8 children with FCD or hemimegalencephaly using standard-depth (50-60X) sequencing in peripheral samples (blood, saliva, or skin) from the affected child and their parents and deep (150-180X) sequencing in affected brain tissue. Targeted sequencing and WES were used to screen 93 children with molecularly unexplained diffuse or focal brain overgrowth. Histopathologic and functional assays of phosphatidylinositol 3-kinase-AKT (serine/threonine kinase)-mammalian target of rapamycin (mTOR) pathway activity in resected brain tissue and cultured neurons were performed to validate mutations. MAIN OUTCOMES AND MEASURES: Whole-exome sequencing and targeted sequencing identified variants associated with this spectrum of developmental brain disorders. RESULTS: Low-level mosaic mutations of MTOR were identified in brain tissue in 4 children with FCD type 2a with alternative allele fractions ranging from 0.012 to 0.086. Intermediate-level mosaic mutation of MTOR (p.Thr1977Ile) was also identified in 3 unrelated children with diffuse megalencephaly and pigmentary mosaicism in skin. Finally, a constitutional de novo mutation of MTOR (p.Glu1799Lys) was identified in 3 unrelated children with diffuse megalencephaly and intellectual disability. Molecular and functional analysis in 2 children with FCD2a from whom multiple affected brain tissue samples were available revealed a mutation gradient with an epicenter in the most epileptogenic area. When expressed in cultured neurons, all MTOR mutations identified here drive constitutive activation of mTOR complex 1 and enlarged neuronal size. CONCLUSIONS AND RELEVANCE: In this study, mutations of MTOR were associated with a spectrum of brain overgrowth phenotypes extending from FCD type 2a to diffuse megalencephaly, distinguished by different mutations and levels of mosaicism. These mutations may be sufficient to cause cellular hypertrophy in cultured neurons and may provide a demonstration of the pattern of mosaicism in brain and substantiate the link between mosaic mutations of MTOR and pigmentary mosaicism in skin.


Assuntos
Malformações do Desenvolvimento Cortical/genética , Megalencefalia/genética , Mosaicismo , Mutação/genética , Serina-Treonina Quinases TOR/genética , Adolescente , Adulto , Aminoácidos/farmacologia , Animais , Células Cultivadas , Córtex Cerebral/citologia , Criança , Pré-Escolar , Deficiências do Desenvolvimento/diagnóstico por imagem , Deficiências do Desenvolvimento/genética , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Estudos de Associação Genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/deficiência , Masculino , Malformações do Desenvolvimento Cortical/diagnóstico por imagem , Alvo Mecanístico do Complexo 1 de Rapamicina , Megalencefalia/diagnóstico por imagem , Complexos Multiproteicos/farmacologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Ratos , Estudos Retrospectivos , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/farmacologia , Adulto Jovem
17.
Methods Enzymol ; 398: 468-81, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16275351

RESUMO

COP9 signalosome (CSN) is an evolutionarily conserved multisubunit protein complex involved in diverse cellular and developmental processes in eukaryotes. CSN functions in the cell as proteases that deconjugate Nedd8/Rub1 from cullin family proteins and depolymerize ubiquitin chains. As such, CSN represents an important regulator of multiple cullin-based E3 ubiquitin ligases. CSN has also been shown to associate with protein kinase activities. This chapter describes purification of the CSN complex by classical chromatography from porcine spleen and by immunoaffinity purification procedures from cultured human cells and transgenic Arabidopsis plants expressing epitope-tagged CSN subunits. It also describes in vitro deneddylation assays using the HeLa cell extract or the Arabidopsis cell extract, which we have used to test and compare the activity of purified CSN complexes.


Assuntos
Proteínas de Arabidopsis/isolamento & purificação , Arabidopsis/enzimologia , Complexos Multiproteicos/isolamento & purificação , Peptídeo Hidrolases/isolamento & purificação , Baço/enzimologia , Suínos , Animais , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Complexo do Signalossomo COP9 , Linhagem Celular , Cromatografia de Afinidade , Cromatografia em Gel , Cromatografia por Troca Iônica , Células HeLa , Humanos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteína NEDD8 , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Polietilenoglicóis , Ubiquitinas/metabolismo
18.
Nat Cell Biol ; 16(11): 1069-79, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25327288

RESUMO

Cells rely on autophagy to clear misfolded proteins and damaged organelles to maintain cellular homeostasis. In this study we use the new autophagy inhibitor PIK-III to screen for autophagy substrates. PIK-III is a selective inhibitor of VPS34 that binds a unique hydrophobic pocket not present in related kinases such as PI(3)Kα. PIK-III acutely inhibits autophagy and de novo lipidation of LC3, and leads to the stabilization of autophagy substrates. By performing ubiquitin-affinity proteomics on PIK-III-treated cells we identified substrates including NCOA4, which accumulates in ATG7-deficient cells and co-localizes with autolysosomes. NCOA4 directly binds ferritin heavy chain-1 (FTH1) to target the iron-binding ferritin complex with a relative molecular mass of 450,000 to autolysosomes following starvation or iron depletion. Interestingly, Ncoa4(-/-) mice exhibit a profound accumulation of iron in splenic macrophages, which are critical for the reutilization of iron from engulfed red blood cells. Taken together, the results of this study provide a new mechanism for selective autophagy of ferritin and reveal a previously unappreciated role for autophagy and NCOA4 in the control of iron homeostasis in vivo.


Assuntos
Autofagia/fisiologia , Classe III de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Ferritinas/metabolismo , Homeostase/fisiologia , Ferro/metabolismo , Coativadores de Receptor Nuclear/metabolismo , Animais , Autofagia/efeitos dos fármacos , Células Cultivadas , Humanos , Lisossomos/metabolismo , Camundongos , Fagossomos/metabolismo , Ligação Proteica
19.
Nat Cell Biol ; 15(11): 1340-50, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24161930

RESUMO

The mechanistic target of rapamycin (mTOR) functions as a critical regulator of cellular growth and metabolism by forming multi-component, yet functionally distinct complexes mTORC1 and mTORC2. Although mTORC2 has been implicated in mTORC1 activation, little is known about how mTORC2 is regulated. Here we report that phosphorylation of Sin1 at Thr 86 and Thr 398 suppresses mTORC2 kinase activity by dissociating Sin1 from mTORC2. Importantly, Sin1 phosphorylation, triggered by S6K or Akt, in a cellular context-dependent manner, inhibits not only insulin- or IGF-1-mediated, but also PDGF- or EGF-induced Akt phosphorylation by mTORC2, demonstrating a negative regulation of mTORC2 independent of IRS-1 and Grb10. Finally, a cancer-patient-derived Sin1-R81T mutation impairs Sin1 phosphorylation, leading to hyper-activation of mTORC2 by bypassing this negative regulation. Together, our results reveal a Sin1-phosphorylation-dependent mTORC2 regulation, providing a potential molecular mechanism by which mutations in the mTORC1-S6K-Sin1 signalling axis might cause aberrant hyper-activation of the mTORC2-Akt pathway, which facilitates tumorigenesis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Carcinogênese , Complexos Multiproteicos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Humanos , Alvo Mecanístico do Complexo 2 de Rapamicina , Mutação , Fosforilação
20.
Sci Signal ; 5(217): ra24, 2012 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-22457330

RESUMO

The mammalian target of rapamycin (mTOR) complex 1 (mTORC1) is a nutrient-sensitive protein kinase that is aberrantly activated in many human cancers. Whether dysregulation of mTORC1 signaling in normal tissues increases the risk for cancer, however, is unknown. We focused on hepatocellular carcinoma, which has been linked to environmental factors that affect mTORC1 activity, including diet. Ablation of the gene encoding TSC1 (tuberous sclerosis complex 1), which as part of the TSC1-TSC2 complex is an upstream inhibitor of mTORC1, results in constitutively increased mTORC1 signaling, an effect on this pathway similar to that of obesity. We found that mice with liver-specific knockout of Tsc1 developed sporadic hepatocellular carcinoma with heterogeneous histological and biochemical features. The spontaneous development of hepatocellular carcinoma in this mouse model was preceded by a series of pathological changes that accompany the primary etiologies of this cancer in humans, including liver damage, inflammation, necrosis, and regeneration. Chronic mTORC1 signaling led to unresolved endoplasmic reticulum stress and defects in autophagy, factors that contributed to hepatocyte damage and hepatocellular carcinoma development. Therefore, we conclude that increased activation of mTORC1 can promote carcinogenesis and may thus represent a key molecular link between cancer risk and environmental factors, such as diet.


Assuntos
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas/metabolismo , Fatores de Transcrição/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Células Cultivadas , Progressão da Doença , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Feminino , Hepatócitos/metabolismo , Hepatócitos/patologia , Immunoblotting , Imuno-Histoquímica , Fígado/metabolismo , Fígado/patologia , Fígado/fisiopatologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexos Multiproteicos/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Sirolimo/farmacologia , Serina-Treonina Quinases TOR , Proteína 1 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
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