Ubiquitin-dependent down-regulation of the neurokinin-1 receptor

Transient stimulation with substance P (SP) induces endocytosis and recycling of the neurokinin-1 receptor (NK(1)R). The effects of sustained stimulation by high concentrations of SP on NK(1)R trafficking and Ca(2+) signaling, as may occur during chronic inflammation and pain, are unknown. Chronic exposure to SP (100 nm, 3 h) completely desensitized Ca(2+) signaling by wild-type NK(1)R (NK(1)Rwt). Resensitization occurred after 16 h, and cycloheximide prevented resensitization, implicating new receptor synthesis. Lysine ubiquitination of G-protein-coupled receptors is a signal for their trafficking and degradation. Lysine-deficient mutant receptors (NK(1)RDelta5K/R, C-terminal tail lysines; and NK(1)RDelta10K/R, all intracellular lysines) were expressed at the plasma membrane and were functional because they responded to SP by endocytosis and by mobilization of Ca(2+) ions. SP desensitized NK(1)Rwt, NK(1)RDelta5K/R, and NK(1)RDelta10K/R. However, NK(1)RDelta5K/R and NK(1)RDelta10K/R resensitized 4-8-fold faster than NK(1)Rwt by cycloheximide-independent mechanisms. NK(1)RDelta325 (a naturally occurring truncated variant) showed incomplete desensitization, followed by a marked sensitization of signaling. Upon labeling receptors in living cells using antibodies to extracellular epitopes, we observed that SP induced endocytosis of NK(1)Rwt, NK(1)RDelta5K/R, and NK(1)RDelta10K/R. After 4 h in SP-free medium, NK(1)RDelta5K/R and NK(1)RDelta10K/R recycled to the plasma membrane, whereas NK(1)Rwt remained internalized. SP induced ubiquitination of NK(1)Rwt and NK(1)RDelta5K/R as determined by immunoprecipitation under nondenaturing and denaturing conditions and detected with antibodies for mono- and polyubiquitin. NK(1)RDelta10K/R was not ubiquitinated. Whereas SP induced degradation of NK(1)Rwt, NK(1)RDelta5K/R and NK(1)RDelta10K/R showed approximately 50% diminished degradation. Thus, chronic stimulation with SP induces ubiquitination of the NK(1)R, which mediates its degradation and down-regulation.

A new tick Kunitz type inhibitor, Amblyomin-X, induces tumor cell death by modulating genes related to the cell cycle and targeting the ubiquitin-proteasome system

The aim of this study was to evaluate the anti-tumor activity of Amblyomin-X, a serine protease Kunitz-type inhibitor. Amblyomin-X induced tumor mass regression and decreased number of metastatic events in a B16F10 murine melanoma model. Alterations on expression of several genes related to cell cycle were observed when two tumor cell lines were treated with Amblyomin-X. PSMB2, which encodes a proteasome subunit, was differentially expressed, in agreement to inhibition of proteasomal activity in both cell lines. In conclusion, our results indicate that Amblyomin-X selectively acts on tumor cells by inducing apoptotic cell death, possibly by targeting the ubiquitin-proteasome system. (C) 2010 Elsevier Ltd. All rights reserved.

Genetic albation of the c-Cbl ubiquitin ligase domain results in increased energy expenditure and improved insulin action

Casitas b-lineage lymphoma (c-Cbl) is a multiadaptor protein with E3-ubiquitin ligase activity residing within its RING finger domain. We have previously reported that c-Cbl–deficient mice exhibit elevated energy expenditure, reduced adiposity, and improved insulin action. In this study, we examined mice expressing c-Cbl protein with a loss-of-function mutation within the RING finger domain (c-Cbl^A/– mice). Compared with control animals, c-Cbl^A/– mice display a phenotype that includes reduced adiposity, despite greater food intake; reduced circulating insulin, leptin, and triglyceride levels; and improved glucose tolerance. c-Cbl^A/– mice also display elevated oxygen consumption (13%) and are protected against high-fat diet–induced obesity and insulin resistance. Unlike c-Cbl^A/– mice, mice expressing a mutant c-Cbl with the phosphatidylinositol (PI) 3-kinase binding domain ablated (c-Cbl^F/F mice) exhibited an insulin sensitivity, body composition, and energy expenditure similar to that of wild-type animals. These results indicate that c-Cbl ubiquitin ligase activity, but not c-Cbl–dependent activation of PI 3-kinase, plays a key role in the regulation of whole-body energy metabolism.

A Pseudomonas aeruginosa toxin that hijacks the host ubiquitin proteolytic system

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen chronically infecting the lungs of patients with chronic obstructive pulmonary disease (COPD), pneumonia, cystic fibrosis (CF), and bronchiectasis. Cif (PA2934), a bacterial toxin secreted in outer membrane vesicles (OMV) by P. aeruginosa, reduces CFTR-mediated chloride secretion by human airway epithelial cells, a key driving force for mucociliary clearance. The aim of this study was to investigate the mechanism whereby Cif reduces CFTR-mediated chloride secretion. Cif redirected endocytosed CFTR from recycling endosomes to lysosomes by stabilizing an inhibitory effect of G3BP1 on the deubiquitinating enzyme (DUB), USP10, thereby reducing USP10-mediated deubiquitination of CFTR and increasing the degradation of CFTR in lysosomes. This is the first example of a bacterial toxin that regulates the activity of a host DUB. These data suggest that the ability of P. aeruginosa to chronically infect the lungs of patients with COPD, pneumonia, CF, and bronchiectasis is due in part to the secretion of OMV containing Cif, which inhibits CFTR-mediated chloride secretion and thereby reduces the mucociliary clearance of pathogens.

Decreased rate of protein synthesis, caspase-3 activity, and ubiquitin-proteasome proteolysis in soleus muscles from growing rats fed a low-protein, high-carbohydrate diet

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Exercise training prevents oxidative stress and ubiquitin-proteasome system overactivity and reverse skeletal muscle atrophy in heart failure

Background: Heart failure (HF) is known to lead to skeletal muscle atrophy and dysfunction. However, intracellular mechanisms underlying HF-induced myopathy are not fully understood. We hypothesized that HF would increase oxidative stress and ubiquitin-proteasome system (UPS) activation in skeletal muscle of sympathetic hyperactivity mouse model. We also tested the hypothesis that aerobic exercise training (AET) would reestablish UPS activation in mice and human HF. Methods/Principal Findings: Time-course evaluation of plantaris muscle cross-sectional area, lipid hydroperoxidation, protein carbonylation and chymotrypsin-like proteasome activity was performed in a mouse model of sympathetic hyperactivity-induced HF. At the 7th month of age, HF mice displayed skeletal muscle atrophy, increased oxidative stress and UPS overactivation. Moderate-intensity AET restored lipid hydroperoxides and carbonylated protein levels paralleled by reduced E3 ligases mRNA levels, and reestablished chymotrypsin-like proteasome activity and plantaris trophicity. In human HF (patients randomized to sedentary or moderate-intensity AET protocol), skeletal muscle chymotrypsin-like proteasome activity was also increased and AET restored it to healthy control subjects' levels. Conclusions: Collectively, our data provide evidence that AET effectively counteracts redox imbalance and UPS overactivation, preventing skeletal myopathy and exercise intolerance in sympathetic hyperactivity-induced HF in mice. Of particular interest, AET attenuates skeletal muscle proteasome activity paralleled by improved aerobic capacity in HF patients, which is not achieved by drug treatment itself. Altogether these findings strengthen the clinical relevance of AET in the treatment of HF.