published_or_final_version / Botany / Doctoral / Doctor of Philosophy

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No description available.

Sabe-se que a hemoglobina é uma rica fonte de peptídeos antimicrobianos (hemocidinas). A primeira hemocidina derivada da hemoglobina bovina caracterizada em carrapatos foi o peptídeo Hb33-61, que é ativo contra bactérias gram-positivas e fungos. Acredita-se que tais hemocidinas sejam geradas proteoliticamente no intestino do carrapato. Neste trabalho nós caracterizamos bioquimicamente uma catepsina D, designada BmAP. A análise da expressão gênica por qPCR mostrou que ela é expressa predominantemente no intestino. Através de LC-MS/MS, determinamos a especificidade de clivagem da BmAP utilizando Hb bovina, e verificamos que resíduos hidrofóbicos foram preferencialmente clivados nos subsítios P1 e P1. Também investigamos a especificidade de clivagem da catepsina L intestinal BmCL1, utilizando uma biblioteca combinatória de tetrapeptídeos e através de hemoglobinólise in vitro. A BmCL1 preferiu resíduos alifáticos no P2 e polares no P1 e P1. Além disso, hidrolisou a cadeia da Hb bovina entre A63/A64, gerando peptídeos com estrutura primária similar ao Hb 33-61. A hemoglobinólise com a BmAP e/ou BmCL1 resultou na formação de algumas hemocidinas, corroborando a hipótese do seu envolvimento na geração endógena de peptídeos antimicrobianos. / It is known that hemoglobin is a rich source of antimicrobial peptides (hemocidins). The first hemoglobin-derived hemocidin characterized in ticks was the peptide Hb33-61, which is active against Gram-positive bacteria and fungi. It is believed that hemocidins are endogenously generated in the tick gut. In this work we biochemically characterized a cathepsin D, designated BmAP. Expression analysis by qRT-PCR showed that it is expressed predominantly in the gut. Through LC-MS/MS, we determined the cleavage specificity of BmAP using bovine hemoglobin, and we verified that hydrophobic residues were preferentially cleaved at the subsites P1 and P1. We also investigated the cleavage specificity of the intestinal cathepsin L BmCL1, using a positional scanning synthetic combinatorial library and through in vitro hemoglobinolysis. BmCL1 preferred aliphatic residues at P2 and polar residues at P1 and P1. Also, it hydrolysed the subunit of bovine hemoglobin at A63/A64, generating peptides with a primary structure similar to Hb 33-61. Hemoglobinolysis with BmAP and/or BmCL1 resulted in the formation of some hemocidins, corroborating the hypothesis that these proteinases are involved in the endogenous generation of antimicrobial peptides

Thesis (Ph. D.)--University of Chicago, Department of Neurobiology, Pharmacology, and Physiology, 2001. / Includes bibliographical references. Also available on the Internet.

Background and Objectives: Type 1 diabetes (T1D) is an autoimmune disease that results from the immune-mediated destruction of insulin-producing β cells in the islets of Langerhans within the pancreas. A combination of genetic and environmental triggers has been acknowledged to contribute to the development of T1D. However, the detailed mechanisms underlying the initiation and progression of autoimmune diabetes still remain poorly understood. Recent studies have found that the reduction of circulating neutrophils is accompanied by neutrophil infiltration in the pancreas at the onset of T1D, suggesting that neutrophils may be causally involved in the pathogenesis of this disorder. However, further investigations are needed to clarify the precise roles of neutrophils and their cellular components in autoimmune destruction of pancreatic β cells. The objective of this study was to investigate whether neutrophil elastase (NE) and proteinase 3 (PR3), both neutrophil serine proteases stored in neutrophil primary granules, and NETosis, a unique form of cell death of neutrophils characterized by the release of decondensed chromatin and granular contents to the extracellular space, were involved in the pathogenesis of T1D. Key findings: 1) We developed several in-house immunoassays for the measurement of circulating levels of NE, PR3 and their endogenous inhibitor alpha-1 antitrypsin (A1AT), and validated the specificity, precision and sensitivity of these assays in clinical samples; 2) We provided the first clinical evidence demonstrating that both circulating protein levels and enzymatic activities of NE and PR3 were dramatically increased in patients with T1D, especially in those with disease duration less than one year. On the contrary, circulating concentrations of A1AT were significantly decreased in these patients; 3) By measuring circulating levels of myeloperoxidase (MPO)-DNA complexes, we demonstrated that NETosis was evidently increased in T1D patients, and positively correlated with the circulating protein levels as well as enzymatic activities of NE and PR3, suggesting that increased circulating NE and PR3 at least in part attributed to augmented NETosis; 4) Circulating NE and PR3 levels increased progressively with the increase in the positive numbers and titers of autoantibodies against pancreatic β cell antigens, but no significant correlation of NE or PR3 with fasting blood glucose levels was observed, suggesting that elevated NE and PR3 might be causally associated with β-cell autoimmunity, but not glycaemic status, in T1D patients. Furthermore, an obvious elevation of NE and PR3 was detected even in those autoantibody-negative patients, suggesting that circulating NE and PR3 may serve as a novel class of biomarkers for the early diagnosis of T1D. Conclusions: Our present study demonstrated that the drastic elevation of NE and PR3, accompanied by a decrease in the endogenous inhibitor A1AT and the enhancement of NETosis, are closely associated with the β-cell autoimmunity in patients with T1D. Measurement of circulating protein levels of neutrophil serine proteases and/or their enzymatic activities can be used to assist the differential diagnosis of autoimmune diabetes. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

1. A method for the purification of the serine protease,factor D,was developed using conventional chromatographic procedures. The final product was homogeneous as judged by SDS/polyacrylamide gel electrophoresis, its migration as a single component in ion exchange and gel filtration media, and its amino acid sequence analysis. The molecule had an apparent molecular weiyht of 24,000. It contained <1.5% (w/w) reducing sugars as judged by periodic acid/Schiff staining, and existed as a monomer in buffers containing either EDTA or calcium ions. 2. Approximately 84% of the amino acid sequence was established unequivocally by automated sequence analysis of the intact molecule and peptides derived by digestion with CNBr, o-iodosobenzoic acid, trypsin and V8 protease. Carboxypeptidase-Y digestion was used to establish the C-terminal amino acid. The peptides were aligned either by homology with other serine proteases, or by the overlap of sequences obtained from peptides derived by different fragmentation procedures. The molecule nad a typical serine protease-type sequence with isoleucine as the Nterminal amino acid. The active site serine and aspartic acid and the surrounding sequences were conserved as well as the sequence around the position of the active site histidine, although this residue itself was not identified. 3. The possibility of the existence of a factor D̄ zymogen which can be activated by trypsin was reinvestigated, but no evidence for a precursor was found. No enzymic activity towards a number of p-nitroanilide substrates and arginyl and lysyl esters was observed with factor D̄,but it was found to release p-nitrophenol from p-nitrophenyl-p'-guanidinobenzoate. Factor D̄ was inhibited by diisopropylphosphofluoridate and p-nitrophenyl-p'-guanidinobenzoate, but a variety of other non-protein and protein inhibitors including α₂-macroglobulin, c1 inhibitor and inter-α-trypsin inhibitor had no effect on enzymic activity.

Proteases are enzymes that cleave protein amide bonds. Proteases are involved in a myriad of biological processes and are considered good targets for drug design. The proteases described herein are cysteine proteases, which utilize a cysteine residue thiol to attack the amide carbonyl, leading to amide bond cleavage. Irreversible inhibitors of cysteine proteases react with the active site cysteine, forming a covalent bond and rendering the enzyme inactive. The first project involved the design and synthesis of aza-peptide epoxide inhibitors for calpain, a clan CA, ubiquitous, calcium-activated human enzyme involved in neurodegeneration. These inhibitors proved to be poor inactivators of calpain, demonstrating that the aza-peptide epoxide is a warhead specific to clan CD cysteine proteases (caspases, gingipains). Subsequently, a known epoxide inhibitor of calpain was optimized to create a more potent inhibitor. Several of these inhibitors were more potent than the parent, and all were demonstrated to inhibit calpain in a breast cancer cell line which was treated with paclitaxel to spike calpain activity. The second project involved the design and solid phase synthesis of aza-peptide Michael acceptor caspases inhibitors. The two goals of this project were to develop a solid phase method for synthesis of inhibitors that are tedious to synthesize in solution phase, and to use a variety of amino acid residues to determine the optimal interactions in the P3? position for various caspases. The synthesis was successful, and the optimal P3? residues were determined. The third project involved the kinetic evaluation of aza-peptide epoxide and Michael acceptor inhibitor designed for the gingipains. Gingipains K and R are virulence factors in the pathology of Porphyromonas gingivalis involved in gingivitis and periodontal disease. These inhibitors proved to be extremely potent inactivators of gingipains, with some of the highest rates of inhibition measured in the Powers laboratory. Gingipain K preferred larger, aromatic moieties in the P1? position, while gingipain R preferred the Michael acceptor inhibitors, with the P1? substituent having less of an impact on potency.

Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 4 uppsatser.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted.