RESUMEN
Poly(ADP-ribose) glycohydrolase (PARG) is a catabolic enzyme that cleaves ADP-ribose polymers formed by members of the PARP family of enzymes. Despite its discovery and subsequent partial purification in the 1970s and the cloning of its single gene in the late 1990s, little is known about the role of PARG in cell function. Because of its low abundance within cells and its extreme sensitivity to proteases, PARG has been difficult to study. The existence of several PARG isoforms with different subcellular localizations is still debated today after more than 30 years of intensive research. In this article, we want to summarize and discuss the current knowledge related to PARG, its different forms and subcellular distribution. We also examine the possible biological roles of PARG in modulating chromatin structure, transcription, DNA repair and apoptosis.
Asunto(s)
Apoptosis/fisiología , Cromatina/metabolismo , Reparación del ADN , Glicósido Hidrolasas/metabolismo , Poli Adenosina Difosfato Ribosa/metabolismo , Animales , Cromatina/genética , Glicósido Hidrolasas/genética , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Transcripción GenéticaRESUMEN
Sporadic clear cell renal carcinomas frequently harbor inactivating mutations in exon 2 of the von Hippel-Lindau (VHL) tumor suppressor gene. Here, we examine the effect of the loss of exon 2-encoded beta-domain function on VHL biochemical properties. Exon 2-encoded residues are required for VHL-mediated NEDD8 conjugation on cullin-2 and assembly with hypoxia-inducible factor alpha (HIFalpha) and fibronectin. These residues are not essential for VHL ability to assemble with elongin BC/cullin-2, to display E3 ubiquitin ligase activity in vitro and to confer energy-dependent nuclear import properties to a reporter protein. Localization studies in HIF-1alpha-null embryonic cells suggest that exon 2-encoded beta-domain mediates transcription-dependent nuclear/cytoplasmic shuttling of VHL independently of assembly with HIF-1alpha and oxygen concentration. Exon 3-encoded alpha-helical domain is required for VHL complex formation with BC/cullin-2 and E3 ubiquitin ligase activity, for binding to HIFalpha/fibronectin, but this domain is not essential for transcription-dependent nuclear/cytoplasmic trafficking. VHL(-/-) renal carcinoma cells expressing beta-domain mutants failed to produce an extracellular fibronectin matrix and to degrade HIFalpha, which accumulated exclusively in the nucleus of normoxic cells. These results demonstrate that exon 2-encoded residues are involved in two independent functions: substrate protein recognition and transcription-dependent nuclear/cytoplasmic trafficking. They also suggest that beta-domain mutations inactivate VHL function differently than alpha-domain mutations, potentially providing an explanation for the relationship between different mutations of the VHL gene and clinical outcome.
Asunto(s)
Proteínas Cullin , Genes Supresores de Tumor , Ligasas , Proteínas/genética , Eliminación de Secuencia , Factores de Transcripción , Proteínas Supresoras de Tumor , Ubiquitina-Proteína Ligasas , Adenoviridae , Carcinoma de Células Renales/genética , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Exones , Fibronectinas/metabolismo , Eliminación de Gen , Genes Reporteros , Células HeLa , Humanos , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Neoplasias Renales/genética , Proteína NEDD8 , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Conformación Proteica , Estructura Secundaria de Proteína , Transporte de Proteínas , Proteínas/química , Proteínas/metabolismo , Transfección , Células Tumorales Cultivadas , Ubiquitinas/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-LindauRESUMEN
Inactivating mutations of the von Hippel-Lindau (VHL) tumor suppressor gene cause the VHL cancer syndrome and sporadic renal clear cell carcinoma. VHL engages in a nucleocytoplasmic shuttle, which is required for its function. Here, we pursue our investigation to identify mechanisms by which VHL-green fluorescent protein (VHL-GFP) is exported from the nucleus. We show that nuclear export of VHL-GFP in living cells requires ongoing RNA polymerase II activity, and is mediated by mechanisms that are temperature-sensitive and energy-dependent. In vitro nuclear export of VHL-GFP is inhibited by nuclear pore-specific lectins, requires ATP hydrolysis and polyadenylated mRNAs, and occurs with kinetics that are similar to those of proteins containing a nuclear export signal. Biochemical fractionation has revealed that nuclear export of VHL-GFP occurs by way of a Ran-dependent pathway. Size exclusion column chromatography and deletion mutant analysis suggest that VHL-GFP does not require assembly with one of its associated proteins, cullin-2, to engage in nuclear export. These results demonstrate that nuclear export of VHL-GFP is Ran-mediated and ATP hydrolysis-dependent. They also suggest that sequences outside the elongin C binding box may function as a nuclear export domain, potentially providing a novel role for this region of VHL frequently mutated in renal cell carcinoma.