Regulation of CLC-1 chloride channel biosynthesis by FKBP8 and Hsp90β.

Mutations in human CLC-1 chloride channel are associated with the skeletal muscle disorder myotonia congenita. The disease-causing mutant A531V manifests enhanced proteasomal degradation of CLC-1. We recently found that CLC-1 degradation is mediated by cullin 4 ubiquitin ligase complex. It is currently unclear how quality control and protein degradation systems coordinate with each other to process the biosynthesis of CLC-1. Herein we aim to ascertain the molecular nature of the protein quality control system for CLC-1. We identified three CLC-1-interacting proteins that are well-known heat shock protein 90 (Hsp90)-associated co-chaperones: FK506-binding protein 8 (FKBP8), activator of Hsp90 ATPase homolog 1 (Aha1), and Hsp70/Hsp90 organizing protein (HOP). These co-chaperones promote both the protein level and the functional expression of CLC-1 wild-type and A531V mutant. CLC-1 biosynthesis is also facilitated by the molecular chaperones Hsc70 and Hsp90β. The protein stability of CLC-1 is notably increased by FKBP8 and the Hsp90β inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) that substantially suppresses cullin 4 expression. We further confirmed that cullin 4 may interact with Hsp90β and FKBP8. Our data are consistent with the idea that FKBP8 and Hsp90β play an essential role in the late phase of CLC-1 quality control by dynamically coordinating protein folding and degradation

Cbl negatively regulates nlrp3 inflammasome activation through glut1-dependent glycolysis inhibition

Activation of the nod-like receptor 3 (NLRP3) inflammasomes is crucial for immune defense, but improper and excessive activation causes inflammatory diseases. We previously reported that Cbl plays a pivotal role in suppressing NLRP3 inflammasome activation by inhibiting Pyk2-mediated apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization. Here, we showed that Cbl dampened NLRP3 inflammasome activation by inhibiting glycolysis, as demonstrated with Cbl knockout cells and treatment with the Cbl inhibitor hydrocotarnine. We revealed that the inhibition of Cbl promoted caspase-1 cleavage and interleukin (IL)-1β secretion through a glycolysis-dependent mechanism. Inhibiting Cbl increased cellular glucose uptake, glycolytic capacity, and mitochondrial oxidative phosphorylation capacity. Upon NLRP3 inflammasome activation, inhibiting Cbl increased glycolysis-dependent activation of mitochondrial respiration and increased the production of reactive oxygen species, which contributes to NLRP3 inflammasome activation and IL-1β secretion. Mechanistically, inhibiting Cbl increased surface expression of glucose transporter 1 (GLUT1) protein through post-transcriptional regulation, which increased cellular glucose uptake and consequently raised glycolytic capacity, and in turn enhanced NLRP3 inflammasome activation. Together, our findings provide new insights into the role of Cbl in NLRP3 inflammasome regulation through GLUT1 downregulation. We also show that a novel Cbl inhibitor, hydrocortanine, increased NLRP3 inflammasome activity via its effect on glycolysis

Kinetics of cytokine expression in cirrhotic rats

AbstractBackgroundCytokines are involved in liver injury and cirrhosis and systemic and hepatic cytokine levels may help predict cirrhosis evolution. However, the relevant survey has not been performed.MethodsMale Sprague-Dawley rats (240–270g) received either common bile duct ligation (BDL, animal model of cholestatic liver injury) or sham operation (control). Five rats were sacrificed and liver and serum were collected from each in weeks 1, 2, 4, 6, 8 and 10 after surgery. Hepatic expression of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) were analyzed by immunohistochemial staining. The corresponding serum levels were measured by ELISA.ResultsCompared to the corresponding sham groups, hepatic expression of these cytokines in BDL rats was significantly and progressively enhanced during cirrhosis development. However, serum IFN-γ levels of BDL rats did not change significantly. Serum TNF-α of BDL rats increased gradually and reached a peak in week 6. Serum TGF-β level was elevated up to week 8, whereas IL-10 level decreased progressively until week 6.ConclusionCirrhosis development in BDL rats is associated with progressively enhanced expression of hepatic pro-inflammatory and anti-inflammatory cytokines, which is not in accord with the corresponding serum concentration. The circulating cytokine concentration may not totally reflect the hepatic expression level throughout the development of cirrhosis

High Prevalence of Mutations in Quinolone-resistance-determining Regions and mtrR Loci in Polyclonal Neisseria gonorrhoeae Isolates at a Tertiary Hospital in Southern Taiwan

Background/PurposeThe emergence of multidrug-resistant Neisseria gonorrhoeae is a great challenge in controlling gonorrhea. This study was conducted to survey the prevalence of molecular mechanisms of antimicrobial resistance among 45 clinical isolates of N. gonorrhoeae collected at a university hospital in Southern Taiwan during 1999-2004.MethodsMutations in mtrR loci and quinolone-resistance-determining regions (QRDRs) were examined by gene sequencing. Polymerase chain reactions with specific primers were performed to detect ermA, ermB, ermC, and ermF. Serogroups and serovars were determined by commercial kits.ResultsThe percentage of multidrug resistance, that is, resistance to penicillin, tetracycline, erythromycin, and ciprofloxacin, among the 45 isolates was 40%. Ceftriaxone and spectinomycin were active against all isolates in vitro. The frequency of mutations in the QRDR and mtrR promoter was 82.2% and 93.3%, respectively. Eighty-two percent of the isolates carried mutations both in the QRDR and mtrR loci. Of nine mutation profiles with QRDR mutations (n =37), gyrA-Ser91Phe/gyrA-Asp95Gly/parC-Ser87Arg was the most common type (56.8%). Acquired genes for rRNA methylase were detected in 11 isolates (10 ermB and 1 ermA). Twenty-seven serovars were identified and all belonged to serogroup B, which suggested that multiple clones of N. gonorrhoeae were circulating in the community in the Tainan area.ConclusionThe high prevalence of multidrug resistance caused by varied resistance mechanisms in N. gonorrhoeae limits the drug choice. Ongoing surveillance of antimicrobial resistance and discovery of new effective antibiotic therapy are warranted in endemic areas