Nitric Oxide Destabilizes Pias3 and Regulates Sumoylation

Small ubiquitin-related protein modifiers (SUMO) modification is an important mechanism for posttranslational regulation of protein function. However, it is largely unknown how the sumoylation pathway is regulated. Here, we report that nitric oxide (NO) causes global hyposumoylation in mammalian cells. Both SUMO E2 conjugating enzyme Ubc9 and E3 ligase protein inhibitor of activated STAT3 (Pias3) were targets for S-nitrosation. S-nitrosation did not interfere with the SUMO conjugating activity of Ubc9, but promoted Pias3 degradation by facilitating its interaction with tripartite motif-containing 32 (Trim32), a ubiquitin E3 ligase. On the one hand, NO promoted Trim32-mediated Pias3 ubiquitination. On the other hand, NO enhanced the stimulatory effect of Pias3 on Trim32 autoubiquitination. The residue Cys459 of Pias3 was identified as a target site for S-nitrosation. Mutation of Cys459 abolished the stimulatory effect of NO on the Pias3-Trim32 interaction, indicating a requirement of S-nitrosation at Cys459 for positive regulation of the Pias3-Trim32 interplay. This study reveals a novel crosstalk between S-nitrosation, ubiquitination, and sumoylation, which may be crucial for NO-related physiological and pathological processes

Inhibition of Siah ubiquitin ligase function

Tumor hypoxia induces the upregulation of hypoxiainducible factor 1a (Hif-1a), which in turn induces the expression of genes including VEGF to recruit new blood vessel outgrowth, enabling tumor growth andmetasta sis. Interference with the Hif-1 pathway and neoangiogenesis is an attractive antitumor target. The hydroxylation of Hif-1a by prolyl-hydroxylase (PHD) proteins during normoxia serves as a recognition motif for its proteasomal degradation. However, under hypoxic conditions, hydroxylation is inhibitedan dfurt hermore, PHD proteins are themselves polyubiquitylated and degraded by Siah ubiquitin ligases. Our data demonstrate for the first time that inhibition of the interaction between Siah and PHD proteins using a fragment derived from a Drosophila protein (phyllopod) interferes with the PHD degradation. Furthermore, cells stably expressing the phyllopod fragment display reduced upregulation of Hif-1a protein levels and Hif-1-mediated gene expression under hypoxia. In a syngeneic mouse model of breast cancer, the phyllopod fragment reduced tumor growth and neoangiogenesis and prolonged survival of the mice. In addition, levels of Hif-1a andits target Glut-1 are reduced in tumors expressing the phyllopodfragment . These data show, in a proof-of principle study, that Siah protein, the most upstream component of the hypoxia pathway yet identified, is a viable drug target for antitumor therapies.A. Möller, C.M. House, C.S.F. Wong, D.B. Scanlon, M.C.P. Liu, Z. Ronai and D.D.L. Bowtel