No description available.

No description available.

The ubiquitin proteasome pathway is the predominant biological mechanism of myofibrillar protein (MF) degradation.  To test the hypothesis that amino acid and insulin act synergistically to regulate proteolysis, two experimental models were employed;  an <i>in vivo </i>study on growing calves and an <i>in vitro</i> C2C12 myotubes culture. Calves growing at 0.3kg/day, were constantly infused with glucose at a low (LDG) or high (HDG) dose (to stimulate insulin) with or without essential amino acids (EAA).  Glucose infusions increased plasma insulin and IGF-1 concentrations in a dose dependent manner (P<0.05).  HDG was associated with decreased plasma urea nitrogen and 3-MH concentrations and 3-MH:creatinine output (an index of MF degradation) (P < 0.05).  Glucose infusions down regulated the expression of 14-kDa E2 ubiquitin conjugating enzyme and C2 20 S proteasome sub unit, however EAA did not alter the effect of raised plasma insulin on muscle ubiquitin proteasome pathway suggesting that under the conditions employed, EAA do not act synergistically with insulin to decrease myofibrillar protein degradation, <i>in vivo.</i> In the <i>in vitro</i> experiments, amino acid deprivation (0.2 X physiological concentration amino acid;  PC AA) of myotubes for 8 h was associated with increased (P < 0.05) proteolysis (measured from TCA soluble ³H-tyrosine release in the medium), compared to controls (1.0 X PC AA).  Addition of insulin inhibited this increase (P < 0.05).  Rapamycin significantly increased proteolysis in 1.0 X PC AA media suggesting amino acid might regulate proteolysis through mTOR signalling pathway.  Reduced amino acid supply also increased 14-kDa E2 and C2 mRNA expression compared to controls (P < 0.05).  Increasing leucine concentration in 0.2 X PC AA basal media showed a dose dependent decrease in protein degradation and expression of 14-kDa E2, in the presence of insulin.  In conclusion, the results suggested that decreased availability of amino acids was associated with increased total proteolysis and that anti-catabolic effect of amino acid in C2C12 muscle cell cultures, was additive to that of insulin.

No description available.

Ubiquitin proteasome pathway is an important cellular pathway that affects the fate of almost all intracellular proteins. Misregulation of this pathway has been found to be associated with a broad range of human diseases, such as cancer, neurodegenerative diseases, as well as viral infections. Ubiquitin recognition by the proteasome is of central importance to this pathway. So far, two proteasome subunits, Rpn10 and Rpn13, have been identified as ubiquitin receptors. An alternative pathway is mediated by shuttling factors. In yeast, three shuttling factors, known as UBL-UBA proteins, have been found. A UBL receptor activity of the proteasome has been attributed to Rpn1. However, yeast cell mutated all five proteasomal ubiquitin receptors is still viable. To identify the additional proteasomal ubiquitin receptor in cells, I first obtained and characterized a new Rpn13 mutant allele. This Rpn13 mutant completely abolished its ubiquitin binding activity, and functionally resembles a null allele. Rpn13 substrate pool has also been sought in this mutant cells. In the second part of this dissertation, I reported a novel ubiquitin binding site on proteasomal subunit Rpn1. With the help of NMR analysis, Rpn1's ubiquitin and UBL binding surfaces were resolved at high resolution and found to substantially overlap. A specific Rpn1 mutation that disrupts both ubiquitin and UBL binding while not compromising the folding of Rpn1 was obtained. This mutant allele shows a pleiotropic proteasomal defect in vivo. Moreover, I found that the dual ubiquitin/UBL binding activity is not unique in Rpn1, but a common feature in all three proteasomal ubiquitin receptors. In summary, the proteasome adopts a multilayer ubiquitin/UBL binding surface to ensure flexible substrate recognition.

No description available.

Class of 2007 Abstract / Objectives: To explore the effects of human immunodeficiency virus protease inhibitors (HPI) on insulin metabolism and protein degradation in HepG2 hepatocytes in vitro. Methods: To see if HIV-protease inhibitors affect insulin degradation in a dose-dependent manner, HepG2 cells were incubated with various concentrations of tipranavir, indinavir, or atazanavir. After 125I-insulin was added, its degradation was measured by precipitation with trichloroacetic acid (TCA). To see the effect of HPIs on protein degradation, HepG2 cells labeled overnight with 3H-leucine were incubated with 50 mM of an HPI, followed by another HPI incubation including concentrations of insulin ranging from 10-12 to 10-6 M. Cells were solubilized and proteins were precipitated using TCA. Degradation was quantified as percent TCA soluble, normalized, plotted, and then compared using student’s t-test or one- way ANOVA. Results: Cellular insulin degradation was inhibited only by tipranavir at the highest concentrations of 75 and 100 mM (12.06 ± 1.07%, p=0.047 and 9.35 ± 0.44%, p=0.024, respectively) when compared to the control (17.01 ± 1.37%; n=3). None of the concentrations of indinavir or atazanavir decreased insulin degradation significantly. From the protein degradation experiments, the log EC50 of the control (no HPI) insulin dose-response curve was not statistically different compared to those of the individual HPIs. Conclusions: Except for high concentrations of tipranavir, it appears that HPI does not inhibit the cellular degradation of insulin. HPIs do not appear to inhibit the role of insulin in the inhibition of protein degradation significantly.

DYT1 dystonia (DYT1) is a disabling inherited neurological disorder with juvenile onset. The genetic mutation in DYT1 leads to the deletion of a glutamic acid (E) residue in the protein torsinA. The function of torsinA and how the mutation leads to DYT1 is poorly understood. We hypothesize that how efficiently the disease-linked mutant protein is cleared may be critical for DYT1 pathogenesis. Therefore we explored mechanisms of torsinA catabolism, employing biochemical, cellular, and animal-based approaches. We asked if torsinA(wt) and torsinA(DE) are degraded preferentially through different catabolic mechanisms, specifically the ubiquitin proteasome pathway (UPP) and autophagy. We determined that torsinA(wt) is cleared by autophagy while torsinA(DE) is efficiently degraded by the UPP suggesting degradation processes can modulate torsinA(DE) levels. Proteins implicated in recognizing motifs on torsinA(DE) for targeting to the UPP represent candidate proteins that may modify DYT1 pathogenesis. We examined how removal of the hydrophobic domain and mutation of glycosylated asparagine residues on torsinA altered stability and catabolic mechanism. We found the glycosylation sites on torsinA are important for stability modulate its degradation through the UPP. F-box G-domain protein 1 (FBG1) has been implicated in degradation of glycosylated ER proteins. We hypothesized that FBG1 would promote torsinA degradation and demonstrated that FBG1 modulates levels of torsinA in a non-canonical manner through the UPP and autophagy. We examined if lack of FBG1 in a torsinA(DE) mouse model altered motor phenotypes. We saw no effect which suggests FBG1 does not alter DYT1 pathogenesis despite its promotion of torsinA(DE) degradation. In addition, we explored a potential mechanism for the previously described role of torsinA in modulating cytoplasmic protein aggregation. We hypothesized this endoplasmic reticulum (ER) resident protein would indirectly alter cytoplasmic protein aggregation through modulation of ER stress. We employed a poly-glutamine expanded repeat protein and pharmacological ER stressors to determine that torsinA does not alter poly-glutamine protein aggregation nor ER stress in a mammalian system. In summary, this thesis suggests proteins involved in the catabolism of torsinA(DE) may modify DYT1 pathogenesis and that torsinA and its DYT1-linked mutant are model proteins for investigating ER protein degradation by the UPP and autophagy.

The type II tumour suppressor ING3 has been shown to modulate transcription, cell cycle control, and apoptosis. To investigate the putative role of ING3 in melanoma development, we examined the expression of ING3 in 58 dysplastic nevi, 114 primary melanomas, and 50 metastatic melanomas with tissue microarray and immunohistochemistry. Overall ING3 was reduced in metastatic melanomas compared with dyslastic nevi and primary melanomas. Reduced nuclear ING3 staining also correlated with melanoma progression, increased cytoplasmic ING3 level, tumour location at sun-exposed sites, and a poorer disease-specific 5-year survival of patients with primary melanoma. Multivariate analysis revealed that nuclear ING3 staining can independently predict patient outcome in primary melanomas. In melanoma cells, ING3 expression was rapidly induced by UV irradiation. Using stable clones of melanoma cells overexpressing ING3, we showed that ING3 significantly promoted UV-induced apoptosis. Unlike its homologues ING1b and ING2, ING3-enhanced apoptosis upon UV irradiation was independent of functional p53. Furthermore, ING3 did not affect the expression of mitochondrial proteins but increased the cleavage of Bid and caspases. Moreover, ING3 upregulated Fas expression and ING3-mediated apoptosis was blocked by inhibiting caspase-8 or Fas activation. Knockdown of ING3 expression decreased UV-induced apoptosis remarkably, suggesting that ING3 plays a crucial role in cellular response to UV radiation. To explore how ING3 is deregulated in advanced melanomas, we examined ING3 expression in metastatic melanoma cells and found that ING3 was downregulated due to a rapid protein turnover in these cells. Further studies demonstrated that ING3 undergoes degradation via the ubiquitin-proteasome pathway. We also demonstrate that ING3 interacts with the SCF (Skp1/Cul1/Roc1/Skp2) E3 ligase complex. Knockdown of Cul1 or Skp2 significantly stabilized ING3 in melanoma cells. In addition, lysine residue 96 is essential for ING3 ubiquitination as its mutation to arginine completely abrogated ING3 turnover and enhanced ING3-stimulatd apoptosis upon UV irradiation. Taken together, ING3 is deregulated in melanomas as a result of both nucleus-to-cytoplasm shift and rapid degradation. The level of ING3 in the nucleus may be an important marker for human melanoma progression and prognosis. Restoration of ING3 expression significantly sensitizes melanoma cells to UV radiation through the activation of Fas/caspase-8 pathway.

Whey protein supplementation, containing large amount of leucine, has been a traditional intervention to maintain net protein balance in the past decades. It has been recognized that leucine alone is able to stimulate protein synthesis by activating mTOR and its related downstream pathway without affecting protein degradation, whereas its metabolite β-hydroxy-β-methylbutyrate (HMB) is known to attenuate protein degradation when provided chronically. However, the mechanism of HMB’s benefit remains unclear. To address how HMB regulates protein synthesis and degradation signaling pathways, we compared one dose of whey protein (187.5mg/kg), HMB (400mg/kg) or leucine (1.4g/kg) by oral gavage. Blood was collected at 0, 45 and 90 min for blood glucose and plasma insulin analysis. Red and white gastrocnemius muscle was taken separately 90 min after gavage. Blood glucose was reduced by leucine at 45 and 90 min post gavage. Plasma insulin was enhanced by leucine at 45 min and then decreased at 90 min post gavage, whereas HMB decreased plasma insulin through 90 min post gavege. Western blot analysis showed that HMB phosphorylated Akt in red gastronemius, and enhanced phosphorylation of mTOR in both types of muscles. Leucine phosphorylated mTOR, p70s6k and 4E-BP1 in both red and white gastronemius. Regarding protein degradation signals, phosphorylation of FOXO3A was enhanced by HMB, but not in the other treatment groups. Whey protein had no effect on those cellular signaling. Our results indicate that both HMB and leucine may stimulate protein synthesis through the mTOR pathway in red and white gastrocnemius muscles by different degrees with leucine more effective than HMB. HMB may have a greater effect than leucine on limiting protein degradation by phosphorylating Akt and FOXO3A in red and white gastrocnemius muscles. A combination of HMB and leucine, as a new interventional strategy, is predicted to maximize protein accretion by increasing protein synthesis as well as inhibiting protein degradation. / text