RESUMO
The ubiquitin-specific protease (USP) family of deubiquitinases (DUBs) controls cellular ubiquitin-dependent signaling events. This generates therapeutic potential, with active-site inhibitors in preclinical and clinical studies. Understanding of the USP active site is primarily guided by USP7 data, where the catalytic triad consists of cysteine, histidine, and a third residue (third critical residue), which polarizes the histidine through a hydrogen bond. A conserved aspartate (fourth critical residue) is directly adjacent to this third critical residue. Although both critical residues accommodate catalysis in USP2, these residues have not been comprehensively investigated in other USPs. Here, we quantitatively investigate their roles in five USPs. Although USP7 relies on the third critical residue for catalysis, this residue is dispensable in USP1, USP15, USP40, and USP48, where the fourth critical residue is vital instead. Furthermore, these residues vary in importance for nucleophilic attack. The diverging catalytic mechanisms of USP1 and USP7 are independent of substrate and retained in cells for USP1. This unexpected variety of catalytic mechanisms in this well-conserved protein family may generate opportunities for selective targeting of individual USPs.
Assuntos
Histidina , Proteases Específicas de Ubiquitina , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismo , Peptidase 7 Específica de Ubiquitina/genética , Peptidase 7 Específica de Ubiquitina/metabolismo , CatáliseRESUMO
The urokinase receptor (uPAR) is a glycosylphosphatidylinositol (GPI)-anchored protein that promotes tissue remodeling, tumor cell adhesion, migration and invasion. uPAR mediates degradation of the extracellular matrix through protease recruitment and enhances cell adhesion, migration and signaling through vitronectin binding and interactions with integrins. Full-length uPAR is released from the cell surface, but the mechanism and significance of uPAR shedding remain obscure. Here we identify transmembrane glycerophosphodiesterase GDE3 as a GPI-specific phospholipase C that cleaves and releases uPAR with consequent loss of function, whereas its homologue GDE2 fails to attack uPAR. GDE3 overexpression depletes uPAR from distinct basolateral membrane domains in breast cancer cells, resulting in a less transformed phenotype, it slows tumor growth in a xenograft model and correlates with prolonged survival in patients. Our results establish GDE3 as a negative regulator of the uPAR signaling network and, furthermore, highlight GPI-anchor hydrolysis as a cell-intrinsic mechanism to alter cell behavior.
Assuntos
Neoplasias da Mama/genética , Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica , Diester Fosfórico Hidrolases/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Adesão Celular , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Feminino , Técnicas de Inativação de Genes/métodos , Células HEK293 , Humanos , Hidrólise , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Camundongos Nus , Modelos Moleculares , Transplante de Neoplasias , Diester Fosfórico Hidrolases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Transdução de Sinais , Carga Tumoral , Vitronectina/genética , Vitronectina/metabolismoRESUMO
Phosphoinositides (PI) are important signaling molecules in the nucleus that influence gene expression. However, if and how nuclear PI directly affects the transcriptional machinery is not known. We report that the lipid kinase PIP4K2B regulates nuclear PI5P and the expression of myogenic genes during myoblast differentiation. A targeted screen for PI interactors identified the PHD finger of TAF3, a TATA box binding protein-associated factor with important roles in transcription regulation, pluripotency, and differentiation. We show that the PI interaction site is distinct from the known H3K4me3 binding region of TAF3 and that PI binding modulates association of TAF3 with H3K4me3 in vitro and with chromatin in vivo. Analysis of TAF3 mutants indicates that TAF3 transduces PIP4K2B-mediated alterations in PI into changes in specific gene transcription. Our study reveals TAF3 as a direct target of nuclear PI and further illustrates the importance of basal transcription components as signal transducers.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Homeodomínio/metabolismo , Fosfatidilinositóis/metabolismo , Transcrição Gênica , Sequência de Aminoácidos , Animais , Diferenciação Celular/genética , Linhagem Celular , Núcleo Celular/genética , Eletroforese em Gel de Poliacrilamida , Perfilação da Expressão Gênica , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Lisina/metabolismo , Metilação , Camundongos , Antígenos de Histocompatibilidade Menor , Dados de Sequência Molecular , Mutação , Mioblastos/citologia , Mioblastos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ligação Proteica , Interferência de RNA , Homologia de Sequência de Aminoácidos , Fatores Associados à Proteína de Ligação a TATA , Fator de Transcrição TFIID/genética , Fator de Transcrição TFIID/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
Phosphatidylinositol-5-phosphate (PtdIns5P)-4-kinases (PIP4Ks) are stress-regulated lipid kinases that phosphorylate PtdIns5P to generate PtdIns(4,5)P2. There are three isoforms of PIP4Ks: PIP4K2A, 2B and 2C, which localise to different subcellular compartments with the PIP4K2B isoform being localised predominantly in the nucleus. Suppression of PIP4K expression selectively prevents tumour cell growth in vitro and prevents tumour development in mice that have lost the tumour suppressor p53. p53 is lost or mutated in over 70% of all human tumours. These studies suggest that inhibition of PIP4K signalling constitutes a novel anti-cancer therapeutic target. In this review we will discuss the role of PIP4K in tumour suppression and speculate on how PIP4K modulates nuclear phosphoinositides (PPIns) and how this might impact on nuclear functions to regulate cell growth. This article is part of a Special Issue entitled Phosphoinositides.
