Adaptor protein ClpS is an essential regulator of prokaryotic ATP-dependent protease ClpAP, which delivers certain protein substrates with specific amino acid sequences to ClpAP for degradation. However, ClpS also functions as the inhibitor of the ClpAP-mediated protein degradation for other proteins. Here, we constructed the clpS-overexpression Mycobacterium smegmatis strain, and showed for the first time that overexpression of ClpS increased the resistance of M. smegmatis to rifampicin that is one of most widely used antibiotic drugs in treatment of tuberculosis. Using quantitative proteomic technology, we systematically analyzed effects of ClpS overexpression on changes in M. smegmatis proteome, and proposed that the increased rifampicin resistance was caused by ClpS-regulated drug sedimentation and drug metabolism. Our results indicate that the changes in degradation related proteins enhanced drug resistance and quantitative proteomic analysis is an important tool for understanding molecular mechanisms responsible for bacteria drug resistance.
ATP-Dependent Proteases ; metabolism ; Drug Resistance, Bacterial ; Endopeptidase Clp ; metabolism ; Mycobacterium smegmatis ; drug effects ; metabolism ; Proteolysis ; Proteomics ; Rifampin ; pharmacology

BACKGROUND: Acute-on-chronic liver failure (ACLF) is a severe liver disease with complex pathogenesis. Clinical hypoglycemia is common in patients with ACLF and often predicts a worse prognosis. Accumulating evidence suggests that glucose metabolic disturbance, especially gluconeogenesis dysfunction, plays a critical role in the disease progression of ACLF. Lon protease-1 (LONP1) is a novel mediator of energy and glucose metabolism. However, whether gluconeogenesis is a potential mechanism through which LONP1 modulates ACLF remains unknown. METHODS: In this study, we collected liver tissues from ACLF patients, established an ACLF mouse model with carbon tetrachloride (CCl 4 ), lipopolysaccharide (LPS), and D-galactose (D-gal), and constructed an in vitro hypoxia and hyperammonemia-triggered hepatocyte injury model. LONP1 overexpression and knockdown adenovirus were used to assess the protective effect of LONP1 on liver injury and gluconeogenesis regulation. Liver histopathology, biochemical index, mitochondrial morphology, cell viability and apoptosis, and the expression and activity of key gluconeogenic enzymes were detected to explore the underlying protective mechanisms of LONP1 in ACLF. RESULTS: We found that LONP1 and the expressions of gluconeogenic enzymes were downregulated in clinical ACLF liver tissues. Furthermore, LONP1 overexpression remarkably attenuated liver injury, which was characterized by improved liver histopathological lesions and decreased serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in ACLF mice. Moreover, mitochondrial morphology was improved upon overexpression of LONP1. Meanwhile, the expression and activity of the key gluconeogenic enzymes were restored by LONP1 overexpression. Similarly, the hepatoprotective effect was also observed in the hepatocyte injury model, as evidenced by improved cell viability, reduced cell apoptosis, and improved gluconeogenesis level and activity, while LONP1 knockdown worsened liver injury and gluconeogenesis disorders. CONCLUSION We demonstrated that gluconeogenesis dysfunction exists in ACLF, and LONP1 could ameliorate liver injury and improve gluconeogenic dysfunction, which would provide a promising therapeutic target for patients with ACLF.
Animals ; Humans ; Mice ; Acute-On-Chronic Liver Failure/pathology* ; ATP-Dependent Proteases/metabolism* ; Gluconeogenesis ; Hepatocytes/pathology* ; Liver/metabolism* ; Mitochondrial Proteins/metabolism* ; Protease La/metabolism*