Huntington's disease (HD) is caused by an expanded plyglutamine repeat in the huntingtin protein. In this study, I focused on the effect of the mutant huntingtin protein (mhtt) on the subcellular localization of glutamic acid decarboxylase (GAD), the enzyme responsible for synthesizing gama-aminobutyric acid (GABA). Subcellular distribution of GAD65 is significantly altered in two neuronal cell lines that express either the N-terminus or full length mhtt. GAD65 is predominantly associated with the Golgi membrane in cells expressing normal huntingtin (Htt). However, it diffuses in the cytosol of cells expressing mhtt. Palmitoylation of GAD65 is required for GAD65 trafficking, and I demonstrated the palmitoylation of GAD65 is reduced in the HD model. Overexpression of huntingtin-interacting protein 14 (HIP14), the enzyme that palmitoylates GAD65, rescues GAD65 palmitoylation and vesicle-associated trafficking. This data suggests that impairment of GAD65 palmitoylation by mhtt may alter its localization and lead to altered inhibitory neurotransmission in HD. / by Daniel Rush. / Thesis (M.S.)--Florida Atlantic University, 2012. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2012. Mode of access: World Wide Web.

Molecular chaperones guide peptide fold conformation throughout the lifetime of the peptide. One network of chaperone proteins involved in this activity, Heat shock protein 70s (Hsp70s), are well characterized at restoring peptide fold, utilizing J-domain containing protein chaperone cofactors to activate Hsp70 activity. DnaJ (Hsp40) homolog, subfamily C, member 25 (DNAJC25) is a class III transmembrane J-domain containing protein that to date is underrepresented in the literature. Recently, Hejtmancik et al. 2012. (unpublished data) have revealed that missense mutation to DNACJ25 at Pro90Leu (P90L) is strongly correlated with inherited Closed-Angle Glaucoma. Inherited mutations are well characterized for Open-Angle Glaucoma, however, prior to this finding, were unknown for Closed-Angle Glaucoma. In this report, analysis of the in vitro chaperone activity of DNAJC25 w+ and P90L is assessed utilizing an Hsp70 mediated Glucose-6-Phosphate Dehydrogenase refolding system, SWISS-MODEL predictions are performed for the J-domain structure of DNAJC25 w+ and P90L with consequent analysis of DNAJC25 Pro90 conservation relative to other type I, II, and III J-domain containing proteins. DNAJC25 P90L demonstrated decreased chaperone activity in vitro compared to w+ DNAJC25. / by Daniel C. Chauss. / Vita. / Thesis (M.S.)--Florida Atlantic University, 2012. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2012. Mode of access: World Wide Web.

Tissue inhibitors of metalloproteinases (TIMPs) comprise a family of four proteins in humans that modulate the turnover of the extracellular matrix by regulating the activities of endopeptidases that catalyze its degradation, especially the matrix metalloproteinases (MMP). In general, the four TIMPs are broad-spectrum tight binding inhibitors of MMPs with individual differences in specificity. In this study, we attempted to understand the basis of such variation by using membrane type-1 MMP (MT1-MMP) as a model, since it is inefficiently inhibited by TIMP-1 in contrast with the other TIMPs. We designed and engineered mutations in the N-domain of TIMP-1, based on current knowledge of TIMP interactions. By measuring inhibition levels of each mutant against several MMPs, including MT1-MMP, we were able to obtain a triple mutant with an vii improved affinity for MT1-MMP. / Our results, along with previous data, confirm that multiple residues in the critical interface segments between TIMPs and MMPs, namely at positions 2, 4, 5, 6, and 98, are key in determining the basic interaction between the two molecules. The second part of this work focused on naturally occurring mutations in TIMP-3 which cause an early form of macular degeneration called Sorsby's Fundus Dystrophy (SFD). The TIMP-3 mutants identified so far share certain features but the mechanism by which they result in macular disease is not yet understood. As an initial step, we expressed recombinant TIMP-3 carrying a truncation mutation, glutamic acid 139 to a stop codon (E139X), and assessed its activity towards representative MMPs and tumor necrosis factor-(Sa (Bconverting enzyme, another metalloproteinase normally inhibited by TIMP-3. Our results indicate that this mutation does not impair the inhibitory activity of TIMP-3. / Expression of this mutant in mammalian retinal cells revealed a difference in localization between wild-type and E139X mutant TIMP-3. Therefore, we concluded that the SFD mutations may actually influence the processing and/or binding properties of TIMP-3 in the retina. / by Asmaa Bilal Hamze. / Vita. / Thesis (Ph.D.)--Florida Atlantic University, 2008. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2008. Mode of access: World Wide Web.

During the activation and transferring process, E1 and E2 form a thioester-linkage with SUMOs. By using an in vitro assay, it is demonstrated that SENP1 is able to cleave the thioester-linkage between SUMO-1/SUMO-3 and E1/E2. This finding suggests that SUMO proteases regulate the sumoylation pathway, not only during maturation and deconjugation, but also in the E1 activation and E2 conjugation processes. / Recently, reactive oxygen species have been demonstrated to influence the equilibrium of sumoylation-desumoylation. Here, by in vitro assay, it is shown that H2O2 induces formation of inter-molecular disulfide linkage of human SUMO protease SENP1, via the active-site Cys 603 and a unique residue Cys 613. Such reversible modification confers higher enzyme activity recovery which is also observed in yeast Ulp1, but not in human SENP2, suggesting its protective role against irreversible sulfhydryl oxidation. The physiological relevance of the disulfide-linked dimer of SENP1 is also detected in cultured cells upon oxidative stress. The modifications are further verified by the crystal structures of Ulp1 with catalytic cysteine oxidized to sulfenic, sulfinic and sulfonic acids. The current findings suggest that, in addition to SUMO conjugating enzymes, SUMO proteases may act as redox sensors and effectors, which modulate the desumoylation pathway and allow immediate specific cellular responses to oxidative stress. / SUMO (small ubiquitin-related modifier) is a member of the ubiquitin-like protein family that is highly conserved in all eukaryotic organisms and regulates cellular function of a variety of target proteins. SUMO proteins are expressed in their precursor forms and precursor processing involves cleavage of the residues after the conserved 'GG' region by the hydrolytic activity of SUMO-specific protease. The exposed second glycine then forms a covalent bond with the epsilon-amino group of a substrate lysine residue at the psiKxE motif by a cascade of SUMO El, E2 and E3 ligases. As a reversible modification, SUMO proteases can cleave SUMOs from their substrates during de-conjugation process. / To date, four SUMO family members, SUMO-1, -2, -3 and -4 and six SUMO proteases, SENP1--3 and 5-7 (where SENP stands for sentrin-specific protease) have been identified in human. By characterizing the maturation reactions of SUMO-1, -2 and -3 catalyzed by SENP1, it is demonstrated that SENP1 contains the highest maturation efficiency for SUMO-1, followed by SUMO-2 and SUMO-3. By mutagenesis study, it is further identified that the two amino acids immediately after GG motif could influence the maturation efficiency of SENP1. By comparison with another investigation which showed the preference of the maturation reaction of SUMO-2 by SENP2, the results suggest that SUMO proteases with specific tissue distribution control the availability of different mature SUMOs in human. / To gain a deeper insight into the molecular basis of maturation and de-conjugation processes catalyzed by SENP1, it has been determined, at 2.8 A resolution, the X-ray structure of a complex between the catalytic domain of SENP1C(C603S) and matured SUMO-1. The structure shows that the substituted serine residue does not undergo any local structural rearrangements at the active site as observed in the previously solved SENP2/SUMO-1 complex structure. This finding suggests that SUMO proteases require a self-conformational change prior to the cleavage reaction, and further disclose the cleavage mechanism of the hydrolytic reactions catalyzed by SUMO proteases. Moreover, analysis of the interface of SENP1 and SUMO1 has identified four amino acids that are unique in SENP1 sequence and facilitate the interaction of SENP1 and SUMO-1. / Xu, Zheng. / "July 2007." / Advisers: Shannon Au Wing Ngor; Tzi-Bun Ng. / Source: Dissertation Abstracts International, Volume: 69-01, Section: B, page: 0125. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 181-194). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

The effect of proteolytic enzymes on the transformation and transfection of Streptococcus lactis LM2301 protoplasts was examined in an attempt to eliminate the variability observed. By using both chymotrypsin and mutanolysin to form protoplasts followed by spread plating, consistent frequencies of 104 to 105 transformants per μg of pGB301 DNA, and 105 transfectants per μg of c2 bacteriophage DNA where achieved. Optimum transformation and transfection frequencies were obtained when 16 h cultures were treated simultaneously with 25 U/ml mutanolysin and 1.25 U/ml chymotrypsin for 15 min. Trypsin and pronase in conjunction with mutanolysin also increased transformation frequencies higher than when mutanolysin was used alone, but pronase was not as effective as chymotrypsin or trypsin. These results may explain the variability in transformation of mutanolysin-treated cells of S. lactis since commercial sources of mutanolysin contain varying amounts of proteolytic enzyme activity. Transformation of Streptococcus cremoris CS224 at low frequency (5 transformants per μg of pGB301 DNA) was achieved. Plasmid pGB301 was able to replicate and express antibiotic resistance in the resultant transformant (designated S. cremoris SW301). The presence of pGB301 in S. cremoris SW301 was confirmed by agarose gel electrophoresis.

Listeriosis has the highest fatality and hospitalization rate among foodborne illnesses. Listeria monocytogenes causes listeriosis and is a difficult bacterium for ready-to-eat food processors to eliminate because of its ability to grow in the absence of oxygen and under refrigeration. Recently, milk and its proteins have gained recognition as the largest source of biologically active peptides, and, it stands reason that several antimicrobial peptides (AMP) can be released from casein as it is the most abundant milk protein. AMPs are commonly obtained by cutting the whole protein into peptide fragments using enzymes or by acidification. The objective of this study was to predict potential AMPs through computer aided tools, improve hydrolysate preparation, and determine trypsin and pepsin-casein hydrolysate antimicrobial activity in growth media and on frankfurters against two strains of Listeria monocytogenes (Scott A and 310) and Escherichia coli O157:H7 (Salami strain). The prediction study procedure was to identify the most common variants of primary peptide sequences. The sequences were analyzed for greatest possible enzyme cuts on the protein, peptide masses, isoelectric point, net charge and percent hydrophilic residues using online proteomics programs. The fragments were explored for AMP commonalities: fragment length of 3 to 50 amino acids, positive (cationic) net charge, and hydrophilic residues between 25 and 50%. This technique identified 16 potential AMPs which proved that it is possible to screen for AMPs. The method used to determine the trypsin-casein hydrolysate (TCH) and pepsin-casein hydrolysate (PCH) antimicrobial activity was to hydrolyze sodium caseinate with pepsin or trypsin. L. monocytogenes (strains Scott A and 310) were incubated in 0, 10, 20, and 40% PCH and 0 and 50% TCH concentrations over a 24 hour period. PCH suppressed growth of L. monocytogenes Scott A by 1.76 log CFU/mL and reduced initial populations of L. monocytogenes 310 and E. coli O157:H7 by 0.52 and 0.62 log CFU/ml, respectively. TCH had little or no effect on growth suppression of any of the three test organisms. The frankfurter study was conducted by spot inoculating frankfurters with L. monocytogenes Scott A and then dipping frankfurters into one of five treatments (deionized water, pH 2.7 buffer, pH 5.1 buffer, pH 2.7 PCH, and pH 5.1 PCH) for 30 seconds; inoculated frankfurters that were not dipped served as controls. Frankfurters were incubated at 32°C for seven days. The results showed that there was no significant difference (p>0.05) in antimicrobial effectiveness among the treatments and control. This study demonstrated that enzymatically derived casein hydrolysates somewhat inhibit growth of L. monocytogenes and E. coli O157:H7 in culture media, but were ineffective when applied to frankfurters. Casein hydrolysate solutions can be easily made in a processing facility for application in fluid systems such as an antimicrobial spray on beef carcasses and in milk, juice, sports drinks, soda, soups, and yogurt. It also could be used in solid systems such as frankfurters, cheese, ground beef, and processed or RTE foods.

The Best PhD Thesis in the Faculties of Architecture, Arts, Business &Economics, Education, Law and Social Sciences (University of HongKong), Li Ka Shing Prize, 2008-2009 / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Subcellular fractionation (together with immunocytochemical localisation studies) showed that the parasite Trypanosoma brucei brucei possesses a multicatalytic protease complex (MCPTb). This complex is predominantly cytosolic but some activity is also present in the nuclear fraction. MCP-Tb was isolated from T. b. brucei and compared to the properties of other proteasomes reported in the literature and to the 20S MCP isolated from bovine red blood cells (MCP-rbc). The isolation procedure employed four-steps: anion exchange chromatography on Q-Sepharose, adsorption chromatography on HA-Ultrogel, molecular exclusion chromatography on Sephacryl S-300 and glycerol density gradient sedimentation. The molecular mass of intact MCP-Tb was shown to be smaller than that of MCP-rbc. Separation of the different proteasome subunits by 2D-PAGE showed that MCP-Tb has 12 different polypeptide components compared to the 28 different polypeptide components of MCP-rbc. The N-terminal sequence of an MCP-Tb subunit showed that this subunit did not have any obvious sequence homology with the subunits of proteasomes from other cells. Furthermore, anti-MCP-Tb antibodies (which exhibited the in vitro inhibitory activity of MCP-Tb) did not cross-react with MCP-rbc showing that MCP-Tb and MCP-rbc are antigenically distinct. The basic enzymatic properties of MCP-Tb were fairly typical of other 20S proteasomes. MCP-Tb had multiple peptidase activities (identified as chymotrypsin-like, trypsin-like and peptidyl glutamylpeptide hydrolase activities) that are characteristic of proteasomes. Furthermore, the characteristics of inhibition by a variety of inhibitors were similar to those of other proteasomes, including MCP-rbc. The activities of 20S proteasomes from most cell types are activated by endogenous high molecular mass complexes such as the bovine 19S complex called PA700. These complexes form end-on associations with the 20S proteasome. However, no endogenous MCP-activator was found in T. b. brucei. Nevertheless, MCP-Tb was activated in an ATP-dependent manner by bovine PA700. Inhibition of the intrinsic phosphatase activity of PA700 inhibited the protease enhancing effect of PA700. Electron microscopic examination of negatively stained MCP-Tb and MCP-rbc showed particles that were morphologically indistinguishable. However, the MCP-Tb also exhibited unique end-on associations between individual units forming long (up to 200 nm) ribbon-like chains. Since access to the active sites of proteasomes occurs through the pores at the end of the complexes, this end-on association, when coupled to our observation of an apparent lack of an endogenous activator, suggests that T. b. brucei may have evolved an alternate mechanism for controlling their proteasome activity. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1999.

The protozoan parasite Trypanosoma brucei brucei causes nagana in cattle and is a widely used model for human sleeping sickness. The major lysosomal cysteine proteinases (trypanopains) of African trypanosomes may contribute to pathogenesis by degrading proteins in the mammalian bloodstream and also appear to be essential for the viability of T. cruzi and T. congolense. This study describes the first purification to electrophoretic homogeneity of trypanopain-Tb from T. b. brucei and the first reported characterisation of its enzymatic properties. Trypanopain-Tb was purified from bloodstream forms of T. b. brucei by a combination of three phase partitioning (between ammonium sulfate and tertiary butanol), and chromatography on quaternary amine or pepstatin A-Sepharose resins. Trypanopain-Tb was found to be a typical cysteine proteinase, in that it is inhibited by typical cysteine proteinase inhibitors and requires reducing agents for full activity. Trypanopain has cathepsin L-like specificity for synthetic substrates and readily degrades various proteins. In vitro analysis of the kinetics of trypanopain interaction with cystatins suggested that these are likely to inhibit any trypanopain released into the mammalian bloodstream. Furthermore, no trypanopain-like activity was detectable in the blood of infected hosts, so it appears that trypanopain is unlikely to contribute directly to pathogenesis by degrading bloodstream host proteins. Antibodies against a peptide corresponding to a region of the trypanopain active site were produced in rabbits and chickens. Both enzyme activity-enhancing and enzyme activity inhibiting antibodies were produced and these effects varied with the substrate tested. Thus, the in vivo effects of anti-trypanopain antibodies will only become clearly understood once the physiological substrates of trypanopain have been identified. Various cysteine proteinase inhibitors, including peptidyl diazomethylketones, killed cultured bloodstream forms of T. b. brucei. Use of biotinylated derivatives of peptidyl diazomethylketone and fluoromethylketone inhibitors suggested that trypanopain is the likely intracellular target of these inhibitors, indicating that the enzyme is essential for parasite viability. Furthermore, chalcones (a class of reversible cysteine proteinase inhibitors) killed in vitro cultured parasites and also prolonged the life of T. b. brucei-infected mice. Thus, trypanopain-Tb seems to be a possible target for new anti-trypanosomal drugs. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1997.

The collagenolytic proteinases, type IV collagenase and cathepsins Land H, have been implicated in tumour invasion and metastasis, by virtue of their degradative action on the extracellular matrix barriers traversed by migrating tumour cells. Type IV collagenase was isolated from human leucocytes using anti-peptide antibody immunoaffinity chromatography. The highly specific targeting of both native and denatured forms of human type IV collagenase by these anti-peptide antibodies holds much promise for immunolocalisation studies in human tumour tissue. Cathepsin L was purified in both a free; single-chain form from sheep liver, and as complexes with the endogenous cysteine proteinase inhibitor, stefin B. These complexes comprised mixtures of the usual tight-binding non-covalent, inhibitory complexes, and novel, proteolytically active, covalent cathepsin L/stefin B complexes. The latter form spontaneously in a pH-dependent manner in vitro from purified, active constituents. The primary structures of these complexing moieties from sheep liver are reported here for the first time, and showed a high degree of sequence homology with their human counterparts. Single-chain cathepsin L, both in the free, and novel, covalently complexed forms, manifested stability and increased activity at neutral pH, thus suggesting a role in extracellular tissue destruction. This potential involvement in tumour invasion was strengthened by demonstrating that the single-chain form of the enzyme, and similar covalent complexes, active under physiological conditions, could be isolated from liver tissue homogenates of higher primates, baboon (Papio ursinus) and man. A battery of versatile polyclonal anti-sheep cathepsin L and anti-human cathepsins L and H peptide antibodies were raised in chickens and rabbits. The chicken egg yolk antibodies were often of a higher titre than the corresponding rabbit serum antibodies, and additionally manifested unique immunoinhibitory properties. In the case of the polyclonal chicken anti-sheep cathepsin L antibodies, this was derived from their ability to target a peptide located in the active site of cathepsin L. The chicken anti-human cathepsins L and H peptide antibodies constitute the immunological probes of choice for immunolocalisation and in vitro tumour invasion studies to elucidate the relative contributions of these collagenolytic cathepsins to tumour invasion, and could ultimately find application in tumour immunotherapy. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1992.