Edited by Noboru MizushimaKeywords: Mitophagy UNC-51 like kinase Adenosine 5 0 -monophosphate (AMP)-activated protein kinase Hypoxia Autophagy Mitochondria a b s t r a c t UNC-51 like kinase (ULK1) translocates to dysfunctional mitochondria and is involved in mitophagy, but the mechanisms responsible for ULK1 activation and translocation remain unclear. Here, we found that hypoxia induces phosphorylation of ULK1 at Serine-555 by Adenosine 5 0 -monophosphate (AMP)-activated protein kinase (AMPK). Unlike wild-type ULK1, an ULK1 (S555A) mutant cannot translocate to mitochondria in response to hypoxia. Inhibition or knockdown of AMPK prevents ULK1 translocation and inhibits mitophagy. Finally, the phospho-mimic ULK1 (S555D) mutant, but not ULK1 (S555A), rescues mitophagy in AMPK-knockdown cells. Thus, we conclude that AMPK-dependent phosphorylation of ULK1 is critical for translocation of ULK1 to mitochondria and for mitophagy in response to hypoxic stress.

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Podocyte injury has a pivotal role in the pathogenesis of diabetic nephropathy (DN). MicroRNA-27a (miR-27a), peroxisome proliferator-activated receptor γ (PPARγ) and β-catenin pathways have been involved in the pathogenesis of DN. Herein, we asked whether miR-27a mediates podocyte injury through PPARγ/β-catenin signaling in DN. The functional relevance of miR-27a, PPARγ and β-catenin were investigated in cultured podocytes and glomeruli of diabetic rats and patients using in vitro and in vivo approaches. Podocyte injury was assessed by migration, invasion and apoptosis assay. Biological parameters were analyzed using enzyme-linked immunosorbent assay. We found that high glucose stimulated miR-27a expression, which, by negatively targeting PPARγ, activated β-catenin signaling as evidenced by upregulation of β-catenin target genes, snail1 and α-smooth muscle actin (α-SMA) and downregulation of podocyte-specific markers podocin and synaptopodin. These changes caused podocyte injury as demonstrated by increased podocyte mesenchymal transition, disrupted podocyte architectural integrity and increased podocyte apoptosis. Furthermore, we provide evidence that miR-27a contributed to unfavorable renal function and increased podocyte injury in diabetic rats. Notably, miR-27a exhibited clinical and biological relevance as it was linked to elevated serum creatinine, proteinuria and reduced creatinine clearance rate. In addition, miR-27a upregulation and activation of PPARγ/β-catenin signaling were verified in renal biopsy samples from DN patients. We propose a novel role of the miR-27a/PPARγ/β-catenin axis in fostering the progression toward more deteriorated podocyte injury in DN. Targeting miR-27a could be a potential therapeutic approach for DN.

Only a few cold-adapted halophilic proteases have been reported. Here, the gene mcp03 encoding a cold-adapted halophilic protease MCP-03 was cloned from deep-sea psychrotolerant bacterium Pseudoalteromonas sp. SM9913, which contains a 2,130-bp ORF encoding a novel subtilase precursor. The recombinant MCP-03, expressed in Escherichia coli BL21 and purified from fermented broth, is a multi-domain protein with a catalytic domain and two PPC domains. Compared to mesophilic subtilisin Carlsberg, MCP-03 had characteristics of a typical cold-adapted enzyme (e.g., higher activity at low temperatures, lower optimum temperature and higher thermolability). MCP-03 also exhibited good halophilic ability with maximal activity at 3 M NaCl/KCl and good stability in 3 M NaCl. Deletion mutagenesis showed that the C-terminal PPC domains were unnecessary for enzyme secretion but had an inhibitory effect on MCP-03 catalytic efficiency and were essential for keeping MCP-03 thermostable.

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In conclusion, this study indicates that cirrhotic patients have an increased risk of fracture. Preventive measures should be instituted as early as possible.

MicroRNA-27a (miR-27a) upregulation has been identified in diabetes, but the pathogenesis of miR-27a in renal tubulointerstitial fibrosis (TIF) in diabetic nephropathy (DN) has not been elucidated. Herein, we found that high glucose stimulated miR-27a expression, which directly inhibited PPARγ and promoted fibrosis in NRK-52E cells. The functional relevance of miR-27a-dependent PPARγ decrease was proven by inhibition or overexpression of miR-27a both in vitro and in streptozotocin-induced diabetic rats. MiR-27a, via repression of PPARγ, activates the TGF-β/Smad3 signaling and contributes to the expressional changes of connective tissue growth factor (CTGF), Fibronectin and Collagen I, key mediators of fibrosis. Furthermore, we provide evidences that plasma miR-27a upregulation contributed to unfavorable renal function and increased TIF in renal tissues of diabetic rats and DN patients. Notably, miR-27a exhibited clinical and biological relevance as it was linked to elevated serum creatinine, proteinuria, urinary N-acetyl-β-D-glucosaminidase (NAG), and reduced estimated glomerular filtration rate (eGFR). Thus, we propose a novel role of the miR-27a-PPARγ axis in fostering the progression toward more deteriorated renal TIF in DN. Monitoring plasma miR-27a level and its association with PPARγ can be used to reflect the severity of renal TIF. Targeting miR-27a could be evaluated as a potential therapeutic approach for DN.