Upregulation of Unc-51-like kinase 1 by nitric oxide stabilizes SIRT1, independent of autophagy.

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

Achievement of high strength-ductility combination in austenitic and ferritic duplex stainless steel by heterogeneous deformation

In this work, heterostructured duplex stainless steel consisting of fine-grained austenite and bimodal-grained ferrite was prepared by heavy cold rolling and 2-min annealing at 1050 °C. The tailored steel achieved an excellent combination of high strength with a yield stress of ∼710 MPa and high ductility with a tensile elongation of ∼43%. The results revealed that the plastic incompatibilities between the constituent phases arising from the microstructural heterogeneity contributed to the significant partitioning of plastic strain, and the respectable strength–ductility synergy was attributed to hetero-deformation induced strengthening and strain hardening

ULK1 regulates SIRT1 protein expression via 26S proteasomes.

<p>(A) HUVECs were transfected with control or <i>ULK1</i> SiRNA for 48 h, then treated with epoxomicin (0.1 µM) for 4 h; (B) HUVECs were transfected with control or <i>ULK1</i> siRNA for 48 h, SIRT1 mRNA levels were determined by RT-PCR; (C) HUVECs were transfected with control or <i>ULK1</i> or β-TrCP1 siRNA for 48 h. The western blots are representative of three independent experiments. *represents <i>p</i><0.05 vs control (<i>n</i> = 3); # represents <i>p</i><0.05 vs <i>si-ULK1</i> alone (<i>n</i> = 3). Si, siRNA.</p

NO stabilizes and upregulates ULK1 protein expression.

<p>(A) HUVECs were transfected with GFP or eNOS adenovirus for 48 h; (B) HUVECs were treated with A23187 (1 µM) for the indicated time; (C) HUVECs were treated with NONOate (50 µM) for the indicated time; (D) HUVECs were treated with CHX (5 µM) for the indicated time, followed by incubation of NONOate (50 µM) for 4 h; (E) HUVECs were treated with CHX (5 µM) for the indicated time, followed by incubation of A23187 (1 µM) for 4 h. The western blots are representative of three independent experiments. *represents <i>p</i><0.05 vs control (<i>n</i> = 3); NS, not significant. GFP, green fluorescent protein; eNOS, endothelial nitric oxide synthase; Ad, Adenovirus; CHX, cycloheximide.</p

ULK1 modulates 26S proteasome functionality.

<p>GFPu-1 cells were transfected with (A) control or <i>ULK1</i> plasmid for 48 h, and (B) the accumulated GFP fluorescence in <i>ULK1</i> plasmid-expressing cells was captured with a fluorescent microscope; (C) HEK293 cells were transfected with control or <i>ULK1</i> plasmid for 48 h and chymotrypsin-like activity was measured; (D) The <i>Ulk1</i>^−/− and <i>WT</i> MEF were transfected with Ub^G76V-GFP plasmid for 48 h; (E) Chymotrypsin-like activity was measured in the <i>Ulk1</i>^−/− and <i>WT</i> MEF; (F) HUVECs were transfected with control or <i>ULK1</i> SiRNA for 48 h and chymotrypsin-like activity was measured. The western blots are representative of three independent experiments. *represents <i>p</i><0.05 vs control (<i>n</i> = 3). Si, siRNA.</p

ULK1 mediates the NO-induced SIRT1 upregulation.

<p>(A) MEF from the <i>Ulk1</i>^−/−, <i>Ulk2</i>^−/−, <i>Ulk1/2</i>^−/−, and <i>WT</i> mice were transfected with GFP or eNOS Adenovirus for 48 h; (B) MEF from the <i>Ulk1</i>^−/−, <i>Ulk2</i>^−/−, <i>Ulk1/2</i>^−/−, and <i>WT</i> mice were treated with NONOate (50 µM) or A23187 (1 µM) for 4 h; (C) HUVECs were transfected with control or <i>ULK1</i> SiRNA for 48 h, then treated with A23187 (1 µM) for 4 h; (D) HUVECs were transfected with control or <i>ULK1</i> SiRNA for 48 h, then treated with NONOate (50 µM) for 4 h. The western blots shown are representative of three independent experiments. *represents <i>p</i><0.05 vs control (<i>n</i> = 3); #represents <i>p</i><0.05 vs A23187 or NONOate alone (<i>n</i> = 3); NS, not significant. GFP, green fluorescent protein; eNOS, endothelial nitric oxide synthase; Ad, Adenovirus; Si-SiRNA.</p

ULK1 regulates SIRT1 protein expression which is independent of autophagy.

<p>(A) HEK293 cells were transfected with <i>pcDNA</i> or <i>ULK1</i> plasmid for 48 h; (B) HEK293 cells were transfected with <i>pcDNA</i> or <i>ULK1</i> plasmid for 48 h. SIRT1 mRNA levels were determined by RT-PCR; (C) HUVECs were treated with rapamycin (2 µM) for the indicated time; (D) U20S cells which expressed GFP-LC3 reporter were treated with rapamycin (2 µM) or NONOate (50 µM) for 12 h; GFP-LC3 imaging was captured using fluorescent microscope according to instructions of the manufacturer, EMD Millipore; (E) MEF from the <i>Ulk1</i>^−/−, <i>Ulk2</i>^−/−, <i>Ulk1/2</i>^−/−, and <i>WT</i> mice were treated with bafilomycin A1 (10 nM) for 4 h; (F) HUVECs were treated with NONOate (50 µM) for the indicated time; (G) HUVECs were transfected with GFP or eNOS adenovirus for 48 h; (H) HUVECs were treated with A23187 (1 µM) for the indicated time. The western blots are representative of three independent experiments. *represent <i>p</i><0.05 vs control (<i>n</i> = 3). NS, not significant. GFP, green fluorescent protein; eNOS, endothelial nitric oxide synthase; Ad, adenovirus; Baf A1, Bafilomycin A1.</p