RESUMO
The von Hippel-Lindau (VHL) tumour suppressor gene encodes a substrate-specifying component of an E3 ubiquitin ligase that targets hypoxia-inducible factor (HIF) alpha subunits for degradation under normoxia. The VHL protein is composed of an N-terminal HIFalpha-binding beta domain and a C-terminal alpha domain, which is necessary and sufficient for the formation of the E3 multiprotein enzyme. A large number of disease-causing mutations in either the alpha or beta domain renders HIFalpha stable irrespective of oxygen tension, leading to the upregulation of numerous HIF-target genes, such as GLUT1 and VEGF. Here, we show that VHL forms a self-associated complex in vivo, but not in vitro, and demonstrate that coexpression of two different VHL missense mutants -- one in the alpha domain and the other in the beta domain -- restores HIF-mediated gene expression profile. These findings indicate that VHL homotypic complexes can function in vivo in a complementary fashion to target HIFalpha for ubiquitin-mediated proteolysis, and potentially explain why VHL-associated tumours with a missense mutation-carrying VHL allele is almost invariably accompanied by a second VHL allele harbouring a gross truncation or deletion.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Complexos Ubiquitina-Proteína Ligase/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Proteína Supressora de Tumor Von Hippel-Lindau/fisiologia , Substituição de Aminoácidos , Neoplasias Ósseas/patologia , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Dimerização , Elonguina , Humanos , Neoplasias Renais/patologia , Mutação de Sentido Incorreto , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Osteossarcoma/patologia , Oxigênio/metabolismo , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína/genética , Relação Estrutura-Atividade , Fatores de Transcrição/metabolismo , Transfecção , Complexos Ubiquitina-Proteína Ligase/química , Regulação para Cima , Proteína Supressora de Tumor Von Hippel-Lindau/química , Proteína Supressora de Tumor Von Hippel-Lindau/genéticaRESUMO
Na+/H+ exchanger (NHE) activity is exquisitely dependent on the intra- and extracellular concentrations of Na+ and H+. In addition, Cl- ions have been suggested to modulate NHE activity, but little is known about the underlying mechanism, and the Cl- sensitivity of the individual isoforms has not been established. To explore their Cl- sensitivity, types 1, 2, and 3 Na+/H+ exchangers (NHE1, NHE2, and NHE3) were heterologously expressed in antiport-deficient cells. Bilateral replacement of Cl- with nitrate or thiocyanate inhibited the activity of all isoforms. Cl- depletion did not affect cell volume or the cellular ATP content, which could have indirectly altered NHE activity. The number of plasmalemmal exchangers was unaffected by Cl- removal, implying that inhibition was due to a decrease in the intrinsic activity of individual exchangers. Analysis of truncated mutants of NHE1 revealed that the anion sensitivity resides, at least in part, in the COOH-terminal domain of the exchanger. Moreover, readdition of Cl- into the extracellular medium failed to restore normal transport, suggesting that intracellular Cl- is critical for activity. Thus interaction of intracellular Cl- with the COOH terminus of NHE1 or with an associated protein is essential for optimal activity.