INVESTIGADORES
PODEROSO Juan Jose
congresos y reuniones científicas
Título:
Neuronal nitric oxide synthase trafficking into mitochondria involves the processing of the PDZ domain.
Autor/es:
CARRERAS MC, FRANCO MC, MOLINAS MF, GONZALEZ AS, SERRA MP, FINOCCHIETTO PV, CONVERSO DP, PODEROSO JJ
Lugar:
Bregenz, Austria
Reunión:
Congreso; 5th International Conference Biology, Chemistry and Therapeutic Applications of Nitric Oxide.; 2008
Institución organizadora:
Nitric Oxide Society
Resumen:
Mitochondrial nitric oxide synthase (mtNOS) has been described as a post-translational modified neuronal NOS-alpha (130 kDa). Modulation of mtNOS was observed in different situations; however, the mechanism of translocation into mitochondria is still unknown. We therefore studied a) rat liver and brain mitochondrial translocation of nNOS-alpha in experimental hypothyroidism in vivo; b) mitochondria from HEK 293 cells that do not express either NOS isoform challenged with nNOS newly synthesized by a reticulocyte lysate translation system (TTS), and c) nNOS-transfected HEK 293 cells. The processing of nNOS protein was assayed in Western blots by using antibodies (Abs) against different domains (aa1-100, 144-262, 1095-1289 and 1380-1429 or against the V5 tag). After 14 days of hypothyroidism, a pike of mitochondrial translocated nNOS expression and activity was detected in liver and brain. Apparent MW of translocated nNOS resulted reduced from 160 to 130 kDa. Since imported nNOS was not detected by antibodies against aa 1-100 but by all the other Abs, we confirmed that the 130 kDa mtNOS band looses the N-terminal PDZ domain during nNOS import. In the in vitro assay, HEK 293 mitochondria were incubated with nNOS newly synthesized by TTS and an aliquot was treated with proteinase K to eliminate the protein attached to the cytosolic side of mitochondrial outer membrane.160 kDa nNOS was inserted in mitochondrial membrane and PK treatment cut the amino and carboxi terminal domains, resulting in a 130 kDa fragment only detected by Abs against aa144-262 and aa 1095-1289. Similar results were obtained in nNOS-transfected HEK 293 cells. These data suggest that nNOS is internalized in vivo loosing the PDZ domain and passes through the mitochondrial outer membrane translocation channels as a hairpin loop leading the extremes in the cytosolic side and susceptible to endogenous proteases or to PK treatment.