Rapid degradation of certain short lived "timing" proteins is an effective mechanism for cells to control important regulatory pathways. The mechanisms by which regulatory proteases recognize their substrates are not well understood. Escherichia coli Lon, an energy dependent protease highly conserved in many prokaryotes and eukaryotes provides a model system to study protease/substrate interactions. RcsA, a regulator of capsule synthesis, when present in levels high enough to saturate Lon, cannot protect SulA, a cell division inhibitor, from being degraded. These observations suggest Lon recognizes its different substrates with different affinities. The different affinities of these substrates might relate to the role these substrates play in the cell: stabilization of RcsA leads to a nonlethal phenotype (capsule), while stabilization of SulA leads to lethal filamentation. To further examine protease/substrate interactions, targeted mutagenesis was employed to select for mutations in rcsA which give rise to mutant RcsA protein no longer degraded by Lon protease. Two mutants with an increased half-life in the presence of Lon were identified. Their mutations fall into the C-terminal region of RcsA, supporting the hypothesis that this region is involved in the interaction of RcsA with Lon. Stabilization of RcsA was dependent on its partner RcsB; the interaction of RcsA with RcsB is believed to protect RcsA from Lon dependent degradation. However, it was shown that rcsA expression is enhanced in the presence of RcsB, and RcsA protein cannot be detected in strains mutant for RcsB in the presence or absence of Lon. Furthermore, rcsA expression was shown to be activated by RcsA itself: rcsA::lacZ expression is low in the absence of RcsA. A conserved 25 by motif, designated "RcsA-Box" was identified in the promoter region of the rcsA and capsule (cps) genes. This motif was shown to be a likely candidate for RcsA binding: high level expression of both cps::lacZ and rcsA::lacZ fusions was shown to be dependent on the presence of the "RcsA-Box". These studies expand the understanding of the specific interactions between regulatory proteases and their targets, specifically as they relates to complex regulatory networks. / Graduation date: 1998

No description available.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 175-187). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 175-187). Also available on the Internet.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Assessment of freshness and quality of meat and fish is a major activity of both food regulatory agencies and the food industry. Various methods are used for measuring fish and meat quality, each with its particular advantages and limitations. However, methods based on monitoring the products of proteolysis have received relatively little attention. The objective of the present study was to identify specific protein and peptide products of proteolysis as indicators of freshness and quality during chilled storage of fresh fish and meat. / Samples of meat and fish were subjected to chilled storage; at intervals of 0, 2, 4, 8, 12 and 16 days, samples were subjected to protein and peptide extraction, and separation of individual sarcoplasmic and myofibrillar proteins by SDS and native electrophoresis. These extracted proteins along with acid soluble nitrogen (ASN) were separated by RP-HPLC, fractions were collected and identified by electrospray ionization mass spectrometry (ESI-MS). / RP-HPLC separated at least thirty fractions from the ASN extract of fresh fish. ESI-MS revealed the presence of at least twenty-five polypeptides with molecular weights (MW) ranging from 2 to 32 kDa. The relative area % of the polypeptides with MW 32.8 kDa and 42.8 kDa decreased during the storage while polypeptides of MW of 10.9 kDa and 16.7 kDa increased during storage. Changes in polypeptides of MW 12, 34.2 and 42.8 kDa was also observed. The sarcoplasmic protein extracted from ground and whole meat contained at least 12 polypeptides with MW ranging from 11 to 42 kDa. The relative area % of polypeptide of MW of 35.7 kDa decreased during storage. The results suggest that changes in proteins and polypeptides of MW 10.9, 12, 16.7, 32.8, 34.2 and 42.88 kDa in fish and 35.7 kDa in meat could serve as indicators of spoilage.

Cathepsin L, which has been implicated in many tissue degradative pathologies by virtue of its ability to degrade extracellular matrix components, was isolated by a novel, scaled-up protein purification method and purified to homogeneity in the single-chain form. In addition, the high molecular weight variant of cathepsin L covalently complexed with stefin B was isolated. Both cathepsin L and the complex were stable, in respect of their proteolytic activity, to the chaotropic agent urea, both showing enhanced activity in the presence of urea. Urea did not dissociate the complex. The suitability of cathepsin L for a purported extracellular role was addressed by investigating its pH optimum and pH stability. Cathepsins L and B are affected by ionic strength and so buffers of constant ionic strength (rather than constant molarity, and therefore varying ionic strength) were used in determining their pH optima and stability. Cathepsins L and B had apparent pH optima of pH 6.5 and 7.5, respectively, (measured with synthetic substrates) and, contrary to the previous belief, were substantially stable at physiological pH. In Hanks' balanced salt solution, a model of the extracellular fluid, they were shown to be active and stable, cathepsin L having a half-life of 179 s at pH 7.2 and 657 s at pH 6.8 (the peritumour pH). It was also shown that prior reductive activation of these enzymes increased their stability to extracellular conditions, supporting the hypothesis that the active site thiolate-imidazolium ion pair contributes to their stability. The nature of the bond between cathepsin L and stefin B in the covalent complex was examined, using CNBr cleavage, HPLC and amino acid sequencing. Stefin B was shown to be associated with residues 1-137 of cathepsin L via a reduction sensitive linkage which was deduced to be a thioester bond betwen Asp-71 of cathepsin L and Cys-3 of stefin B. Polyclonal antibodies to cathepsin L and stefin B-complexed cathepsin L were raised in rabbits and chickens, and characterised with respect to their suitability for immunocytochemical localisation of these forms of cathepsin L. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1998.

The protozoan parasites Trypanosoma congolense and T. vivax cause trypanosomosis in cattle. The major lysosomal cysteine proteinase of T. congolense, congopain, may contribute to pathogenesis of the disease, and antibody-mediated inhibition of this enzyme may contribute to mechanisms of trypanotolerance. Oligopeptidase B, a trypanosomal serine peptidase, is also a potential virulence factor in African trypanosomes because it is released into the host circulation by dead or dying parasites, where it retains catalytic activity due to the enzyme's insensitivity to serum protease inhibitors. The vaccine potential of the catalytic domain of congopain, C2, and oligopeptidase B complexed with 0'2-macroglobulin (0'2M) was evaluated by producing antibodies in rabbits. Inhibition of congopain and oligopeptidase B activity by these antibodies was assessed. The oligopeptidase B open reading frame from T. congolense and T. vivax was cloned and expressed in Escherichia coli, from which active recombinant enzymes were purified. These recombinant enzymes exhibited trypsin-like specificity for peptide substrates, cleaving on the carboxy side of basic amino acid residues such as arginine and lysine. Enzymes were found to be optimally active between pH 8 and 10, optimally stable at pH 6, and showed activation by reducing agents and sensitivity to ionic strength. The enzymes showed typical oligopeptidase B-like inhibitor profiles, except that they were not inhibited by thiol sensitive inhibitors such as iodoacetamide and Nethylmaleimide. High yields of bovine and rabbit 0'2M were isolated by a three-step procedure of fractionation by PEG 6000, and zinc chelate and Sephacryl S-300 HR chromatography. Congopain, its catalytic domain C2, papain and cathepsin L all cleaved the bait region of bovine 0'2M and became trapped inside the 0'2M molecule, where their activity against large molecular weight substrates was inhibited. C2 could thus be complexed with 0'2M directly or used to form C2-0'2M-oligopeptidaseB complexes for immunisation purposes. iv The catalytic domain of congopain, C2, was used to immunise rabbits either without adjuvant, as a water-in-oil emulsion with Freund's adjuvant, or in a complex with either bovine or rabbit U2M. Freund's adjuvant elicited the highest anti-C2 antibody response. However, the greatest inhibition, 65%, of C2 activity against Z-Phe-Arg-AMC was obtained with antibodies produced by rabbits receiving C2-U2Mcomplexes. In a second study, C2 and oligopeptidase B were used to immunise rabbits , either in alum, or complexed to bovine U2M. Anti-C2 antibody levels were highest in rabbits immunised with the free proteins in alum, whereas anti-oligopeptidase B antibody levels were comparable for each adjuvant system. Anti-oligopeptidase antibodies produced with alum gave 100% inhibition of oligopeptidase B activity. In contrast, antibodies produced against C2-u2M-oligopeptidase B complexes had little effect on oligopeptidase B activity. However, these antibodies inhibited 55% of C2 activity. Alum was a slightly less efficient adjuvant for C2 and 50% inhibition of C2 activity was observed. It appeared that immunisation of rabbits with C2 complexed to U2M resulted in the production of antibodies that were better able to neutralise the proteolytic activity of C2 and congopain in vitro than that with conventional adjuvants . The immunisation of C2 complexed to bovine u2-macroglobulin therefore has the potential to neutralise parasite congopain in vivo, and may contribute to an anti-disease vaccine against African trypanosomosis. Complexation of oligopeptidase B to u2M offers no benefit, since antibodies produced against this complex are not able to inhibit the activity of oligopeptidase B. Immunisation with oligopeptidase B in alum is sufficient to produce efficient enzyme-inhibiting antibodies in the context of an anti-disease vaccine against African trypanosomosis. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2006.

Cathepsin, matrix metalloproteinase (MMP) enzyme and tissue inhibitor of MMP (TIMP) distribution in J774 mouse macrophages has not been comprehensively studied. The distribution and vesicle regulation, trafficking and release of these is important, possibly suggesting drug targets for the therapeutic regulation of inflammatory disease and phagosomal killing of pathogenic organisms in J774 and other macrophages. Percentage immunofluorescence and ultrastructural enzyme and inhibitor distribution, together with LysoTracker (acidity) and lysosome-associated membrane proteins (LAMPs) colocalisation (both indicating late endosome or “lysosomal” association), western blot estimates of percentage processed- and unprocessed intracellular and secreted enzyme and inhibitor, and vesicle size was used to assign enzyme and inhibitor to “classical” vesicle types. Antibodies against TIMP-1 and TIMP-2 were raised and all antibodies characterised for this purpose. Together these were used to assign cathepsins H, S, D, B and L to possible secretory vesicles (±20 nm, non-acidic, LAMPs-negative, containing precursor enzymes) and identify at least 6 other endosome-“lysosome-like” vesicles. Cathepsin H appears to be present in classical early endosomes (±100 nm, non-acidic, LAMPs-negative) and cathepsin S in late endosomes(±50 nm, acidic, LAMPs-positive) and possibly “lysosomal” (“hybrid” or digestive organelles) (±150-200 nm, acidic, LAMPs-positive). Both cathepsins H and S, however, seem only reliable markers if used together with additional markers. Cathepsin D appearsmainly associated with “lysosomal” (“hybrid” or digestive organelles) (±150-200 nm, acidic, LAMPs-positive), possibly consisting of further subpopulations which requires furtherinvestigation e.g. labelling for LAMP-1 and LAMP-2 and cathepsin D. Cathepsins B and Lmay occur in late endosomes and/or hybrid organelles and “secretory lysosomes” containing cathepsins B, D and L may also exist (±30-50 nm, acidic, LAMPs-positive). The distribution of MMP-9, TIMP-1 and -2 in vesicles (non-acidic, LAMP-2-negative) thatappear novel and distinct from late endosome-“lysosome” vesicles were also demonstrated. In LPS-stimulated cells, the identity of the large (±450 nm), possible recycling endosomes (Rab11-positive, LAMPs-negative), containing colocalised MMP-9 and TIMP-1, needs investigation i.e., requires further verification with triple labelling and EM. Possible cell membrane and recycling endosome localisation of TIMP-2 needs confirmation with labelling of non-permeabilised cells and labelling for MT1-MMP and proMMP-2, respectively. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2007.

Corrigenda pasted onto front fly-leaf. / Bibliography: leaves 247-286. / xv, 286 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis examines an aspect of the pathogenesis of a model extracellular enteric pathogen, the causative agent of human cholera. The export of TcpA (Toxin-Coregulated Pilus) and assembly of the TCP is explored as a paradigm of macromolecular export in Gram negative bacteria. TcpA is examined in detail in an attempt to define strictly conserved regions between species. The TCP of the emergent 0139 (Bengal) serotype is demonstrated to be of El Tor type. The possibily that proteases such as the soluble haemagglutinin (SHA) may have a detachase role centring on TCP dispersal/TcpA degradation is also discussed. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology, 1997