Investigations concerning metallothionein (MT) have covered 4 main areas. Initial studies were carried out to develop an immunocytochemical technique for the detection of MT. An indirect peroxidase technique was used to localise MT within the livers and kidneys of rats injected with CuSO4. An increase in immunocytochemical staining was observed following Cu injection which was consistent with the increase in MT-I concentrations as detected by RIA. To establish whether degradation of MT in vitro is influenced by prior exposure of protein to oxygen free radicals. (Cu,Zn)-MT with Cu:Zn ratio 1:1, purified from pig liver following injection with diethylamine copper oxyquinoline sulphonate (Cujec), was found to be extensively degraded after incubation with a free radical generating system (xanthine/xanthine oxidase) and subsequently with trypsin. However proteins with Cu:Zn ratios of 2:1 or 5:1 were not greatly affected. This indicates that an oxidative step may be involved in the degradation pathway and/or aggregation of MT but the magnitude of the effect is ultimately determined by the ratio of metals present within MT. To establish whether the turnover rate of hepatic CuMT is increased in vivo in animals subjected to oxidant stress. Iron overload was used to initiate oxidant stress in rats prior to injection of Cu using a mixture of Cujec and CuSO4. It could not be concluded, however, if in vivo degradation of CuMT was influenced by the application of iron-induced oxidant stress. Subcellular localisation of MT by fractionation of liver and kidney homogenates using preformed Percoll gradients did, however, demonstrate that MT was not associated with the lysosomal fraction but within the nuclear fraction in correlation with previous studies. To identify specific chelators for selective removal of Cu from CuMT in vitro and to establish the effect of administration of such chelators on the turnover of CuMT in vivo. Ammonium terathiomolybdate [(NH4)2MoS4] was incubated with (Cu,Zn)-MT and Cd, resulting in the complete removal of Cu from protein and replacement with Cd. The effect of this chelating action for Cu was then studied in vivo by administration of (NH4)2MoS4 to rats following injection of Cu using a mixture of Cujec and CuSO4. The turnover and degradation of induced CuMT, however, could not be said to be increased by the addition of the Cu chelator, (NH4)2MoS4, conclusively.

This work examines skeletal muscle catabolism in cancer and its attenuation by Eicosapentaenoic Acid (EPA). In vivo studies in mice bearing a cachexia inducing murine colon adenocarcinoma - MAC16, demonstrated an elevation in the gastrocnemius muscle in the activity and expression of regulatory components of the ubiquitin-proteasome proteolytic pathway. This was accompanied by an accelerated loss of muscle tissue correlating with an increase in overall weight loss, all of which were attenuated by prior daily dosing with EPA. Recently a proteolysis inducing factor (PIF) has been isolated from the MAC16 tumour, and from the serum and urine of cachectic cancer patients. Previous studies have shown that PIF induces protein degradation in vitro, and that this is possibly mediated through 15-hydroxyeicosatetraenoic acid (15-HETE), a metabolite of the n-6 polyunsaturated fatty acid- arachidonate. Employing the murine myoblast cell line C2C12, it was shown that both PIF and 15-HETE increased protein degradation and expression of proteasome subunits, processes which were again attenuated by prior incubation in EPA. Similarly, in NMRI mice which had been fasted for 24hours, EPA and the lipoxygenase inhibitor CV-6504 (but not structurally related fatty acids) inhibited skeletal muscle proteolysis and expression of various proteasome subunits, showing that firstly, EPA may be anti-cachexic partly through its ability to influence 15-HETE production; and secondly that the effect is specific for EPA as other fatty acids had no effect. Previous studies have suggested the involvement of the signal transduction family NFKB in response to PIF in the liver. It has been demonstrated here that both PIF and 15-HETE increased nuclear translocation of NFKB in the skeletal muscle of tumour bearing mice and that EPA inhibited this process by its ability to prevent the degradation of the NFKB inhibitor protein IKB. When an NFKB inhibitor was added to C2C12 myotubes, prior to the addition of PIF, proteasome activity and protein degradation was inhibited, showing that NFKB is responsible for the increased proteasome activity and muscle catabolism induced by PIF. Taken together this work suggests that 15-hydroxyeicosatetraenoic acid is the intracellular mediator for PIF induced protein degradation in skeletal muscle and that elevated muscle catabolism is accomplished through an increased functioning of the ubiquitin-proteasome pathway, a process possibly mediated through an NFKB dependent mechanism. The anticachectic (and possibly the anti-tumourigenic) effects of EPA appear to be achieved in part by its ability to inhibit the degradation of IKB and possibly by its ability to interfere with 15-HETE production.

No description available.

Pulmonary surfactant is a critical surface-active substance consisting of dipalmitoylphosphatidylcholine (DPPtdCho) and key apoproteins that are produced and secreted into the airspace from alveolar type II epithelial cells. Surfactant deficiency leads to severe lung atelectasis, ventilatory impairment, and gas-exchange abnormalities. These are features of the acute lung injury syndrome, characterized by a strong pro-inflammatory component where cytokines or bacteria infections greatly impair surfactant DPPtdCho biosynthesis. The key enzyme needed to produce surfactant DPPtdCho is a rate-limiting enzyme CTP: phosphocholine cytidylyltransferase (CCTalpha). Calmodulin (CaM), rather than disruption of an NH2-terminal PEST sequence, stabilizes CCTalpha from actions of the proteinase, calpain. Mapping and site-directed mutagenesis of CCTalpha uncovered a motif (LQERVDKVK) harboring a vital recognition site, Q243, whereby CaM directly binds to the enzyme. Mutagenesis of CCTalpha Q243 not only resulted in loss of CaM binding, but also led to complete calpain resistance in vitro and in vivo. These data suggest that CaM, by antagonizing calpain, serves as a novel binding partner for CCTalpha that stabilizes the enzyme under pro-inflammatory stress. We further show that CCTalpha does not undergo polyubiquitination and proteasomal degradation. Rather, the enzyme is monoubiquitinated at a molecular site (K57) juxtaposed near its NLS resulting in disruption of its interaction with importin, nuclear exclusion, and subsequent degradation within the lysosome. Importantly, by using CCTalpha-ubiquitin hybrid constructs that vary in the intermolecular distance between ubiquitin and the NLS, we show that CCTalpha monoubiquitination masks its NLS resulting in cytoplasmic retention. These results unravel a unique molecular mechanism whereby monoubiquitination governs the trafficking of a critical regulatory enzyme in vivo. Last, we identify FBXL2 as a novel F-box E3 ubiquitin ligase that targets CCTalpha for degradation. Interestingly, FBXL2 also interacts with CaM, and CaM directly disrupts CCTalpha and FBXL2 interaction. This study demonstrates in the first time that adenoviral gene transfer of CaM attenuates the deleterious effects of P. aeruginosa infection by improving several parameters of pulmonary mechanics in animal models of sepsis-induced acute pulmonary injury. Collectively, these studies reveal a novel regulatory mechanism for phosphatidylcholine synthesis that may provide important clues to understanding the pathobiology of acute lung injury.

Upon encountering stress conditions, cells must rapidly alter their gene expression and re-model their RNA complement to deal with the changing environment. As a consequence, both new RNA transcription as well as RNA degradation must take place. Accordingly, the RNA degradative machinery may adjust to the changes in RNA metabolism. Thus, a study of the response of the three major degradative exoribonucleases in Escherichia coli, polynucleotide phosphorylase, RNase II, and RNase R, to stress is of significant importance. RNase R, a processive 3' to 5' exoribonuclease, is unique among the known E. coli exoribonucleases in its ability to digest through RNAs containing extensive secondary structure without the aid of a helicase. In vivo, RNase R plays important roles in quality control of stable RNA, decay of mRNA with extensive repetitive extragenic palindromic (REP) sequences, cell-cycle regulated degradation of tmRNA in Caulobacter crescentus, as well as processing of rRNA under low temperature in P. syringae. In this dissertation, RNase R was shown to be unusual among the E. coli exoribonucleases in its dramatic response to a variety of stress conditions. Elevation of RNase R activity by as much as 10-fold was observed in response to entry into stationary phase, starvation and cold shock, and an ~3-fold increase was seen during growth in minimal medium compared to rich medium. The elevation in RNase R activity was associated with an increase in RNase R protein. Phenotypes of rnr mutants were also investigated, and RNase R was found to contribute to cell growth and viability. Further investigation of the regulation of RNase R during stress, primarily in stationary phase, revealed a novel regulation mechanism. Despite the large increase in RNase R protein and activity in stationary phase, rnr message actually decreased to only ~14% of its level in exponential phase. Further study revealed that RNase R is highly unstable in exponential phase and becomes stabilized during stationary phase, cold shock, and in minimal medium. Investigation of proteolysis on the unusual instability of RNase R indicated that both Lon and ClpXP play a role. In the absence of Lon, RNase R stability is increased ~10-fold. Based on these results, I propose that the increase in RNase R during stress is due to its enhanced stability under those conditions.

The objective of this study was to improve canola protein gelation properties with the use of enzymes. Both cross-linking and limited proteolysis were explored. Enzyme treatments were performed prior to heat induced gelation. A texture analyzer, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and scanning electron microscopy were used to characterize the resulting networks. Enzymatic cross-linking with transglutaminase was shown to improve the gelation of canola protein isolate (CPI). To the contrary, proteolysis with trypsin, ficin and bromelin, did not enhance the gelation properties of CPI. / October 2006

The work presented herein details post-translational modifications (PTMs) in Staphylococcus aureus that are involved in mediating the stress response and normal cellular processes. The first PTM that was investigated is regulated intramembrane proteolysis (RIP) for the activation of the ECF sigma factor σS. We achieved this by analyzing the role of the site-1 protease, which we termed “putative regulator of sigmaS” (PrsS), as it is predicted to be the first enzyme in the RIP cascade, leading to the activation of σS. It was determined that the putative site-1 protease, prsS, mimics transcriptional profiles of sigS; with expression low in all strains examined other than in the highly mutagenic strain RN4220. Moreover, up-regulation of the protease was observed in response to cell wall-targeting antibiotics, DNA-damaging agents, and during infection in human serum and RAW 264.7 cells, similar to that previously demonstrated for sigS. It was further determined that prsS mutants, like sigS mutants, are more sensitive to cell wall-targeting antibiotics and DNA-damaging agents, which is explained, in part, by alterations in altered abundance of proteins in the prsS mutant that mediate antibiotic resistance (Pbp2a, FemB, and HmrA) and the response to DNA damage (BmrA, Hpt, and Tag). Importantly, transcriptional analyses of proteins affected in the protease mutant, revealed that their expression is decreased in both prsS and sigS mutants, suggesting that this is a result of sigS-mediated regulation. Lastly, it was determined that PrsS, similar to σS, is required for infection in whole human blood and murine models of virulence. Next, since the abundance of a stress response protease, HtrA1, was altered in prsS mutants, we aimed to assess the roles of this enzyme, and its homolog HtrA2 in S. aureus. Interestingly, we first determined that unlike that previously described for the HtrA enzymes, these proteases do not have a role in Agr-mediated virulence regulation. We attribute this finding to unintended mutations likely introduced during strain construction, which is common for S. aureus strains. We next used transcription profiling of the htrA genes in order to understand their role in the cell, and found that they are moderately expressed under standard conditions, and are up-regulated in response to both in vitro and ex vivo stressors that lead to cell protein, DNA, and cell envelope damage. Further to this, the protease mutants are more sensitive to numerous conditions that affect macromolecular stability, including elevated temperature, alterations in pH, reactive oxygen species, DNA damage, and antimicrobial stress. In order to further explore these sensitivities and gain insight into putative substrates, we employed a yeast-2 hybrid screen, and identified numerous proteins that interact with HtrA1 and HtrA2, including those that mediate the response to stress and normal cellular homeostasis. Taken together, we provide evidence to suggest the HtrA proteases in S. aureus are required both during standard conditions and in stress-inducing environments to mediate protein folding and proteolysis of a broad range of substrates. Finally, we performed the first examination of prenylation in a bacterial organism. Prenylation is a well-studied post-translational modification (PTM) in eukaryotes, wherein a prenyl group is added to a metabolite or the C-terminal “CAAX” motif of a protein. Interestingly, the machinery exists for this PTM in a wide variety of prokaryotic species, thus we set out to investigate its impact in S. aureus. To achieve this, we disrupted prenyl group synthesis by inactivating ispA, the gene encoding a prenyl synthetase. The abrogation of prenylation ensued in striking alterations in the cell, including lack of pigmentation and smaller colony size, similar to small-colony variants (SCVs) of S. aureus. In addition to this, the ispA mutant displayed a growth defect, as a result of lower ATP levels. Moreover, the prenylation mutant displayed alterations in resistance to antibiotics, including increased resistance to aminoglycosides and antimicrobial peptides (AMPs), yet elevated sensitivity to cell wall-targeting antibiotics. These differences in susceptibility to cell envelope targeting antibiotics are a result of alterations in cell envelope architecture, including variations in fatty acid composition and increased membrane fluidity. Collectively, the pleotropic consequences of the disruption of prenylation indicate that this process is key to maintaining cellular homeostasis in S. aureus, and perhaps other bacterial species.

Inactive mutants of the ped gene cause two phenotypes in Drosophila melanogaster: male sterility and the early translation of DHODH within spermatogenesis. Investigation of the PED amino acid sequence revealed an OTU domain and an ubiquitin interacting motif, suggesting that it is a member of the otubain sub-family of de-ubiqutinating enzymes. To test this, the putative active cysteine residue was mutated. Results show that this single cysteine residue is required for ped to confer male fertility. Purified wild type PED was also used to carry out in vitro deubiquitinating assays. These assays failed to show any ability for PED to cut ubiquitin chains of varying length or linkage type. Previously, a translational control element was identified in dhod mRNA which is required for its early translation phenotype in ped mutants. In an attempt to identify additional transcripts that have their translational timing affected by PED, the don juan-like 5′ UTR was inserted into a reporter gene and examined in a ped mutant background. No delay of this reporter gene was observed suggesting that don juan-like mRNA is not under the exact control pathway that dhod is.

The objective of this study was to improve canola protein gelation properties with the use of enzymes. Both cross-linking and limited proteolysis were explored. Enzyme treatments were performed prior to heat induced gelation. A texture analyzer, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and scanning electron microscopy were used to characterize the resulting networks. Enzymatic cross-linking with transglutaminase was shown to improve the gelation of canola protein isolate (CPI). To the contrary, proteolysis with trypsin, ficin and bromelin, did not enhance the gelation properties of CPI.

The objective of this study was to improve canola protein gelation properties with the use of enzymes. Both cross-linking and limited proteolysis were explored. Enzyme treatments were performed prior to heat induced gelation. A texture analyzer, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and scanning electron microscopy were used to characterize the resulting networks. Enzymatic cross-linking with transglutaminase was shown to improve the gelation of canola protein isolate (CPI). To the contrary, proteolysis with trypsin, ficin and bromelin, did not enhance the gelation properties of CPI.