title: Upregulation of Unc-51-like kinase 1 by nitric oxide stabilizes SIRT1, independent of autophagy.
abstract: SIRT1 is central to the lifespan and vascular health, but undergoes degradation that contributes to several medical conditions, including diabetes. How SIRT1 turnover is regulated remains unclear. However, emerging evidence suggests that endothelial nitric oxide synthase (eNOS) positively regulates SIRT1 protein expression. We recently identified NO as an endogenous inhibitor of 26S proteasome functionality with a cellular reporter system. Here we extended this finding to a novel pathway that regulates SIRT1 protein breakdown. In cycloheximide (CHX)-treated endothelial cells, NONOate, an NO donor, and A23187, an eNOS activator, significantly stabilized SIRT1 protein. Similarly, NO enhanced SIRT1 protein, but not mRNA expression, in CHX-free cells. NO also stabilized an autophagy-related protein unc-51 like kinase (ULK1), but did not restore SIRT1 protein levels in ULK1-siRNA-treated cells or in mouse embryonic fibroblasts (MEF) from Ulk1-/- mice. This suggests that ULK1 mediated the NO regulation of SIRT1. Furthermore, adenoviral overexpression of ULK1 increased SIRT1 protein expression, while ULK1 siRNA treatment decreased it. Rapamycin-induced autophagy did not mimic these effects, suggesting that the effects of ULK1 were autophagy-independent. Treatment with MG132, a proteasome inhibitor, or siRNA of β-TrCP1, an E3 ligase, prevented SIRT1 reduction induced by ULK1-siRNA. Mechanistically, ULK1 negatively regulated 26S proteasome functionality, which was at least partly mediated by O-linked-GlcNAc transferase (OGT), probably by increased O-GlcNAc modification of proteasomal subunit Rpt2. The NO-ULK1-SIRT1 axis was likely operative in the whole animal: both ULK1 and SIRT1 protein levels were significantly reduced in tissue homogenates in eNOS-knockout mice (lung) and in db/db mice where eNOS is downregulated (lung and heart). Taken together, the results show that NO stabilizes SIRT1 by regulating 26S proteasome functionality through ULK1 and OGT, but not autophagy, in endothelial cells.
authors.affiliation: Department of Emergency, Qilu Hospital, Shandong University, Jinan, China; Department of Medicine and Harold Hamm Oklahoma Diabetes Center, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America.
authors.forename: Junhui
authors.initials: J
authors.lastname: Xing
authors.affiliation: Department of Medicine and Harold Hamm Oklahoma Diabetes Center, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America.
authors.forename: Hongtao
authors.initials: H
authors.lastname: Liu
authors.affiliation: Department of Medicine and Harold Hamm Oklahoma Diabetes Center, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America.
authors.forename: Huabing
authors.initials: H
authors.lastname: Yang
authors.affiliation: Department of Physiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America.
authors.forename: Rui
authors.initials: R
authors.lastname: Chen
authors.affiliation: Department of Emergency, Qilu Hospital, Shandong University, Jinan, China.
authors.forename: Yuguo
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Medicine and Harold Hamm Oklahoma Diabetes Center, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America.
authors.forename: Jian
authors.initials: J
authors.lastname: Xu
mesh_terms: D000818:Animals; D001343:Autophagy; D000071189:Autophagy-Related Protein-1 Homolog; D002460:Cell Line; D057809:HEK293 Cells; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D059748:Proteolysis; D056564:Sirtuin 1 / Q000378:metabolism*; D015854:Up-Regulation
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; D000071189:Autophagy-Related Protein-1 Homolog; D017346:Protein Serine-Threonine Kinases; C484754:ULK1 protein, human; C112444:Ulk1 protein, mouse; D046988:Proteasome Endopeptidase Complex; C447939:SIRT1 protein, human; C470545:Sirt1 protein, mouse; D056564:Sirtuin 1