RESUMEN
The androgen receptor (AR) N-terminal domain plays a critical role in androgen-responsive gene regulation. A novel AR N-terminal-interacting protein (ARNIP) was isolated using the yeast two-hybrid system and its interaction with amino acids 11-172 of the normal or corresponding region of the polyglutamine-expanded human AR confirmed by glutathione S-transferase pulldown assays. ARNIP cDNAs cloned from NSC-34 (mouse neuroblastoma/spinal cord) or PC-3 (human prostate adenocarcinoma) mRNA encoded highly homologous 30 kDa (261 amino acids) cysteine-rich proteins with a RING-H2 (C3H2C3 zinc finger) domain; this motif is highly conserved in predicted ARNIP-homologous proteins from several other species. Expression of the approximately 1.7 kb ARNIP mRNA was detected in various tissues by Northern blotting, but was highest in mouse testes, kidney and several neuronal cell lines. In addition, the human ARNIP protein was found to be encoded by nine exons spanning 32 kb on chromosome 4q21. In COS-1 cells, coexpression of ARNIP and AR did not affect AR ligand-binding kinetics, nor did ARNIP act as a coactivator or corepressor in transactivation assays. However, AR N-terminal:C-terminal interaction was reduced in the presence of ARNIP. Intriguingly, ARNIP, and in particular its RING-H2 domain, functioned as a ubiquitin-protein ligase in vitro in the presence of a specific ubiquitin-conjugating enzyme, Ubc4-1. Mutation of a single cysteine residue in the ARNIP RING-H2 domain (Cys145Ala) abolished this E3 ubiquitin ligase activity. Fluorescent protein tagging studies revealed that AR-ARNIP interaction was hormone-independent in COS-1 cells, and suggest that colocalization of both AR and ARNIP to the nucleus upon androgen addition may allow ARNIP to play a role in nuclear processes. Thus, identification of a novel AR-interacting protein with ubiquitin ligase activity will stimulate further investigation into the role of ubiquitination and the ubiquitin-proteasome system in AR-mediated cellular functions.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Ligasas/metabolismo , Receptores Androgénicos/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Andrógenos/metabolismo , Animales , Secuencia de Bases , Northern Blotting , Línea Celular , Clonación Molecular , Cartilla de ADN , ADN Complementario , Proteínas de Unión al ADN/genética , Exones , Regulación de la Expresión Génica , Humanos , Intrones , Ligasas/genética , Ratones , Datos de Secuencia Molecular , Mutagénesis , Sistemas de Lectura Abierta , Unión Proteica , Receptores Androgénicos/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Factores de Transcripción/genética , Técnicas del Sistema de Dos Híbridos , Ubiquitina-Proteína LigasasRESUMEN
Spinobulbar muscular atrophy (SBMA) is a neurodegenerative disease caused by the expansion of the polyglutamine (polyGln) tract in the human androgen receptor (hAR). One mechanism by which polyGln-expanded proteins are believed to cause neuronotoxicity is through aberrant interaction(s) with, and possible sequestration of, critical cellular protein(s). Our goal was to confirm and further characterize the interaction between hAR and cytochrome c oxidase subunit Vb (COXVb), a nuclear-encoded mitochondrial protein. We initially isolated COXVb as an AR-interacting protein in a yeast two-hybrid screen to identify candidate proteins that interacted with normal and polyGln-expanded AR. Using the mammalian two-hybrid system, we confirm that COXVb interacts with normal and mutant AR and demonstrated that the COXVb-normal AR interaction is stimulated by heat shock protein 70. In addition, blue fluorescent protein-tagged AR specifically co-localized with cytoplasmic aggregates formed by green fluorescent protein-labeled polyGln-expanded AR in androgen-treated cells. Mitochondrial dysfunction may precede neuropathological findings in polyGln-expanded disorders and may thus represent an early event in neuronotoxicity. Interaction of COXVb and hAR, with subsequent sequestration of COXVb, may provide a mechanism for putative mitochondrial dysfunction in SBMA.
Asunto(s)
Complejo IV de Transporte de Electrones/metabolismo , Trastornos Musculares Atróficos/genética , Trastornos Musculares Atróficos/metabolismo , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Animales , Células COS , Complejo IV de Transporte de Electrones/genética , Genes Reporteros , Proteínas Fluorescentes Verdes , Proteínas HSP70 de Choque Térmico/metabolismo , Hormonas/farmacología , Humanos , Indicadores y Reactivos/metabolismo , Proteínas Luminiscentes/genética , Mamíferos , Mitocondrias/metabolismo , Degeneración Nerviosa/genética , Degeneración Nerviosa/metabolismo , Péptidos/genética , Proteínas Recombinantes de Fusión/genética , Expansión de Repetición de Trinucleótido , Técnicas del Sistema de Dos HíbridosRESUMEN
Previous investigations into the relationship of CAG-repeat lengths in the androgen receptor (AR) gene to female breast cancer (BC) have yielded somewhat confusing results. Decreased AR transactivational activity lowers androgen:estrogen balance, and may thereby effect functional hyperestrogenicity. This may promote the pathogenesis of BC. To elucidate whether longer CAG repeats of the AR gene (AR), which correlate with lower transactivational activity of the AR, are associated with BC in women over 40, we examined the distribution of CAG-repeat lengths in BC tissue from this population. The BC tissue was histologically graded as: Grade 1, well differentiated (WD); Grade 2, moderately differentiated (MD); and Grade 3, poorly-differentiated (PD). Analysis showed significant differences as compared to controls when CAG lengths greater than 21 were examined, and that alleles with > or = 26 repeats were 2.4-fold more frequent in BC samples than in constitutional samples from a normal population. A significant shift to greater CAG-repeat lengths, appeared in WD and MD tumors only. Our results give some indication as to the progression of BC by suggesting that hypotransactive ARs with long polyglutamine (polyGln) tracts may have a role in the initiation and/or progression of BC. PD tumors tended to have shorter than normal CAG-repeat lengths. In this case it is hypothesized that the ARs have now become hypertransactive, possibly coinciding with the estrogen resistance that is associated with PD tumors. Whether this shift is of germline or somatic origin was not clear, though the appearance in 14% of the BC samples of a third CAG-repeat length indicates that it may be somatic.