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1.
Cancer Metab ; 7: 2, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30886710

RESUMO

Background: Tumour cells rely on glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) to survive. Thus, mitochondrial OXPHOS has become an increasingly attractive area for therapeutic exploitation in cancer. However, mitochondria are required for intracellular oxygenation and normal physiological processes, and it remains unclear which mitochondrial molecular mechanisms might provide therapeutic benefit. Previously, we discovered that coiled-coil-helix-coiled-coil-helix domain-containing protein 4 (CHCHD4) is critical for regulating intracellular oxygenation and required for the cellular response to hypoxia (low oxygenation) in tumour cells through molecular mechanisms that we do not yet fully understand. Overexpression of CHCHD4 in human cancers correlates with increased tumour progression and poor patient survival. Results: Here, we show that elevated CHCHD4 expression provides a proliferative and metabolic advantage to tumour cells in normoxia and hypoxia. Using stable isotope labelling with amino acids in cell culture (SILAC) and analysis of the whole mitochondrial proteome, we show that CHCHD4 dynamically affects the expression of a broad range of mitochondrial respiratory chain subunits from complex I-V, including multiple subunits of complex I (CI) required for complex assembly that are essential for cell survival. We found that loss of CHCHD4 protects tumour cells from respiratory chain inhibition at CI, while elevated CHCHD4 expression in tumour cells leads to significantly increased sensitivity to CI inhibition, in part through the production of mitochondrial reactive oxygen species (ROS). Conclusions: Our study highlights an important role for CHCHD4 in regulating tumour cell metabolism and reveals that CHCHD4 confers metabolic vulnerabilities to tumour cells through its control of the mitochondrial respiratory chain and CI biology.

2.
Kidney Int ; 92(4): 900-908, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28506759

RESUMO

Complement C1q is part of the C1 macromolecular complex that mediates the classical complement activation pathway: a major arm of innate immune defense. C1q is composed of A, B, and C chains that require post-translational prolyl 4-hydroxylation of their N-terminal collagen-like domain to enable the formation of the functional triple helical multimers. The prolyl 4-hydroxylase(s) that hydroxylate C1q have not previously been identified. Recognized prolyl 4-hydroxylases include collagen prolyl-4-hydroxylases (CP4H) and the more recently described prolyl hydroxylase domain (PHD) enzymes that act as oxygen sensors regulating hypoxia-inducible factor (HIF). We show that several small-molecule prolyl hydroxylase inhibitors that activate HIF also potently suppress C1q secretion by human macrophages. However, reducing oxygenation to a level that activates HIF does not compromise C1q hydroxylation. In vitro studies showed that a C1q A chain peptide is not a substrate for PHD2 but is a substrate for CP4H1. Circulating levels of C1q did not differ between wild-type mice or mice with genetic deficits in PHD enzymes, but were reduced by prolyl hydroxylase inhibitors. Thus, C1q is hydroxylated by CP4H, but not the structurally related PHD hydroxylases. Hence, reduction of C1q levels may be an important off-target side effect of small molecule PHD inhibitors developed as treatments for renal anemia.


Assuntos
Complemento C1q/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Hipóxia/metabolismo , Macrófagos/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Inibidores de Prolil-Hidrolase/farmacologia , Anemia/tratamento farmacológico , Anemia/etiologia , Animais , Linhagem Celular , Complemento C1q/análise , Via Clássica do Complemento , Feminino , Humanos , Hidroxilação , Nefropatias/sangue , Nefropatias/tratamento farmacológico , Nefropatias/patologia , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Inibidores de Prolil-Hidrolase/uso terapêutico , Processamento de Proteína Pós-Traducional
3.
PLoS Genet ; 13(3): e1006620, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28267784

RESUMO

Tubulointerstitial kidney disease is an important cause of progressive renal failure whose aetiology is incompletely understood. We analysed a large pedigree with maternally inherited tubulointerstitial kidney disease and identified a homoplasmic substitution in the control region of the mitochondrial genome (m.547A>T). While mutations in mtDNA coding sequence are a well recognised cause of disease affecting multiple organs, mutations in the control region have never been shown to cause disease. Strikingly, our patients did not have classical features of mitochondrial disease. Patient fibroblasts showed reduced levels of mitochondrial tRNAPhe, tRNALeu1 and reduced mitochondrial protein translation and respiration. Mitochondrial transfer demonstrated mitochondrial transmission of the defect and in vitro assays showed reduced activity of the heavy strand promoter. We also identified further kindreds with the same phenotype carrying a homoplasmic mutation in mitochondrial tRNAPhe (m.616T>C). Thus mutations in mitochondrial DNA can cause maternally inherited renal disease, likely mediated through reduced function of mitochondrial tRNAPhe.


Assuntos
DNA Mitocondrial/genética , Nefropatias/genética , Túbulos Renais/patologia , Mutação , Acetilglucosaminidase/urina , Biópsia , Feminino , Fibroblastos/metabolismo , Ligação Genética , Humanos , Leucina/química , Masculino , Mitocôndrias/metabolismo , Consumo de Oxigênio , Linhagem , Fenótipo , Fenilalanina/química , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Músculo Quadríceps/patologia , RNA de Transferência/genética
4.
J Biol Chem ; 285(48): 37641-9, 2010 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-20870717

RESUMO

Bone morphogenetic proteins (BMPs) are critically involved in early development and cell differentiation. In humans, dysfunction of the bone morphogenetic protein type II receptor (BMPR-II) is associated with pulmonary arterial hypertension (PAH) and neoplasia. The ability of Kaposi sarcoma-associated herpesvirus (KSHV), the etiologic agent of Kaposi sarcoma and primary effusion lymphoma, to down-regulate cell surface receptor expression is well documented. Here we show that KSHV infection reduces cell surface BMPR-II. We propose that this occurs through the expression of the viral lytic gene, K5, a ubiquitin E3 ligase. Ectopic expression of K5 leads to BMPR-II ubiquitination and lysosomal degradation with a consequent decrease in BMP signaling. The down-regulation by K5 is dependent on both its RING domain and a membrane-proximal lysine in the cytoplasmic domain of BMPR-II. We demonstrate that expression of BMPR-II protein is constitutively regulated by lysosomal degradation in vascular cells and provide preliminary evidence for the involvement of the mammalian E3 ligase, Itch, in the constitutive degradation of BMPR-II. Disruption of BMP signaling may therefore play a role in the pathobiology of diseases caused by KSHV infection, as well as KSHV-associated tumorigenesis and vascular disease.


Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Lisossomos/metabolismo , Sarcoma de Kaposi/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/química , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Células Cultivadas , Células Endoteliais/metabolismo , Células Endoteliais/virologia , Células HeLa , Herpesvirus Humano 8/enzimologia , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/fisiologia , Humanos , Lisossomos/química , Lisossomos/genética , Estrutura Terciária de Proteína , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Sarcoma de Kaposi/genética , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteínas Virais/genética , Proteínas Virais/metabolismo
5.
J Immunol ; 184(9): 5186-92, 2010 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-20357254

RESUMO

ORFK3 (K3) and ORFK5 (K5) are Kaposi's sarcoma-associated herpesvirus-encoded E3 ubiquitin ligases that differentially reduce surface expression of various proteins in infected cells. In this study, we describe their effects on human dermal microvascular endothelial cells (ECs), a natural target of Kaposi's sarcoma-associated herpesvirus infection. TNF-treated human dermal microvascular ECs transduced to express K5 show reduced capacity to capture effector memory (EM) CD4+ T cells under conditions of venular shear stress. K5 but not K3 transduction significantly reduces ICAM-1 expression and the inhibition of T cell capture was phenocopied by small interfering RNA knockdown of ICAM-1 and by anti-ICAM-1 Ab blocking. Cotransduction with an ICAM-1 truncated construct not subject to K5 ubiquitylation restored EM CD4+ T cell capture. K3 transductants effectively capture EM CD4+ T cells, but fail to support their transendothelial migration (TEM) in response to TCR engagement by superantigen presented by the ECs, leaving intact chemokine-dependent TEM. K3 but not K5 transduction significantly reduces PECAM-1 expression, and the effect on TCR-induced TEM is phenocopied by small interfering RNA knockdown of PECAM-1 and by anti-PECAM-1 Ab blocking. TCR-dependent TEM was restored in K3 transductants cotransduced to express a mutant of PECAM-1 not subject to K3-induced ubiquitylation. EM CD4+ T cells lack any known PECAM-1 counter receptor, but heterophilic engagement of PECAM-1 can involve glycosaminoglycans. In addition, TCR-induced TEM, but not chemokine-induced TEM, appears to involve a heparan- or chondroitin-like molecule on T cells. These results both identify specific roles of K5 and K3 in immune evasion and further differentiate the processes of inflammatory chemokine- versus TCR-dependent recruitment of human EM CD4+ T cells.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Inibição de Migração Celular/imunologia , Quimiotaxia de Leucócito/imunologia , Endotélio Vascular/imunologia , Proteínas Imediatamente Precoces/fisiologia , Memória Imunológica , Receptores de Antígenos de Linfócitos T/fisiologia , Proteínas Virais/fisiologia , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Regulação para Baixo/imunologia , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/biossíntese , Molécula 1 de Adesão Intercelular/metabolismo , Camundongos , Células NIH 3T3 , Molécula-1 de Adesão Celular Endotelial a Plaquetas/biossíntese , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Receptores de Antígenos de Linfócitos T/antagonistas & inibidores , Ubiquitina/fisiologia
6.
FEBS Lett ; 581(1): 45-51, 2007 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-17174307

RESUMO

The membrane associated RING-CH (MARCH) family of genes encode a novel group of RING-type ubiquitin E3 ligases. Overexpression of one of these family members, MARCH-IX, leads to a downregulation of cell surface major histocompatibility complex (MHC) class I and CD4. Here, we identify MARCH-IX as the first ubiquitin E3 ligase to control expression of the critical cell adhesion molecule ICAM-1. MARCH-IX expression causes ubiquitination and downregulation of ICAM-1 and a short alternative transcript of MARCH-IX lacking the RING-CH domain, termed MARCH-IX RINGless, is shown to act as a positive regulator of MARCH-IX activity.


Assuntos
Molécula 1 de Adesão Intercelular/metabolismo , Proteínas de Membrana/metabolismo , Ubiquitina-Proteína Ligases/biossíntese , Ubiquitina/metabolismo , Processamento Alternativo/fisiologia , Antígenos CD4/metabolismo , Regulação para Baixo/fisiologia , Ativação Enzimática/fisiologia , Células HeLa , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/genética , Proteínas de Membrana/genética , Estrutura Terciária de Proteína/fisiologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
7.
Immunol Rev ; 207: 112-25, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16181331

RESUMO

The mK3, K3, and K5 gene products from the gamma2 group of gamma-herpesviruses are the founding members of a family of membrane-associated ubiquitin E3 ligases. As part of the viral immunoevasion strategy, expression of these proteins results in a decrease in cell-surface major histocompatibility complex class I molecules and other immunoreceptors including intercellular adhesion molecule-1, CD86, and CD1d. These viral gene products all possess a characteristic cytosolic N-terminal RING-CH domain, responsible for ubiquitination of the target protein, and two membrane-spanning segments required for substrate specificity. For the majority of substrates, ubiquitination at the cell surface leads to rapid internalization and endolysosomal degradation, while mK3 ubiquitinates class I molecules associated with the peptide-loading complex resulting in proteasome-mediated degradation. Related viral genes with similar functions have been found in poxviruses, suggesting appropriation of these genes from the eukaryotic host. Ten membrane-associated RING-CH (MARCH) human genes with a similar organization have now been identified, and their overexpression leads to ubiquitination and downregulation of a variety of cell-surface immunoreceptors. While all the MARCH proteins are predicted to act as ubiquitin E3 ligases, their physiological role and substrates remain to be defined.


Assuntos
Modelos Imunológicos , Receptores de Superfície Celular/imunologia , Ubiquitina-Proteína Ligases/imunologia , Proteínas Virais/imunologia , Viroses/imunologia , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Proteínas de Transporte/metabolismo , Regulação para Baixo , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Homologia de Sequência de Aminoácidos , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo
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