CONICET | Buscador de Institutos y Recursos Humanos

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 and b) HEK 293 cells transfection with two vectors: wild type nNOS and delta-pdz nNOS. 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), and internalization of the protein by proteinase K to eliminate the protein attached to the cytosolic side of mitochondrial outer membrane. 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. Wild type 160 kDa nNOS transfected to HEK 293 cells, that do not express either NOS isoform, was inserted in mitochondrial outer 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 with the 130 kDa deltapdz nNOS, detecting a 120 kDa band after PK treatment. All the proteins were functional. 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.

enviar mensaje