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Universiẗat, Diss.--Bonn, 1996.

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Tissue inhibitor of metalloproteinase-3 (TIMP-3) is a protein with multiple functions that include regulating the turnover of the extracellular matrix (ECM) by inhibiting members of the metzincin family. Extracellular levels of soluble TIMP-3 are low, reflecting its binding to components of the ECM including sulfated glycosaminoglycans (SGAGs) and its endocytosis by low density lipoprotein receptor-related protein 1. Because TIMP-3 inhibits ECM-degrading enzymes, the ability of SGAG mimetics to elevate extracellular concentrations of TIMP3 is of interest for osteoarthritis treatment. However, previous studies of such interactions have utilized immobilized forms of the protein or ligands. Here we have quantified the thermodynamics of the interactions of the inhibitory domain of TIMP-3 with chondroitin sulfate (CS), pentosan polysulfate (PPS) and suramin in solution using isothermal titration calorimetry. All three interactions are driven by a (favorable) negative enthalpy ychange combined with an unfavorable decrease in entropy. The heat capacity change (ΔCp) for the interaction of N-TIMP-3 with CS, PPS, or suramin is essentially zero, indicating an insignificant contribution from the hydrophobic effect. Based on the effects of ionic strength on the interaction of N-TIMP-3 with suramin, their interaction appears to be driven by electrostatic interactions. Modeling supports the view that the negatively charged sulfates of CS, PPS, and suramin interact with a cationic region on N-TIMP-3 that includes Lys -26, -27, -30, and -possibly 76 on the opposite face of TIMP-3 from its reactive site for metalloproteases. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2019. / FAU Electronic Theses and Dissertations Collection

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Orientador: Maria Julia Marques / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T17:20:57Z (GMT). No. of bitstreams: 1 Moreira_DrielendeOliveira_D.pdf: 1797583 bytes, checksum: 467851661491c4671f42d059f14dbd53 (MD5) Previous issue date: 2015 / Resumo: A Distrofia Muscular de Duchenne (DMD) é uma doença muscular progressiva que causa falência respiratória e cardíaca resultando em morte por volta da terceira década de vida. A ausência da distrofina na DMD e no camundongo mdx, modelo experimental da doença, leva a instabilidade do sarcolema e aumento do influxo de cálcio seguido de mionecrose e fibrose. Receptores purinérgicos estão envolvidos na patogênese das distrofinopatias. A ativação destes receptores por ATP deflagra vias de sinalização secundárias que promovem a entrada de cálcio e ativam a formação de fibrose. No presente estudo, verificamos se a suramina, droga anti-fibrótica e antagonista de receptores purinérgicos, modifica o cálcio e proteínas a ele relacionadas, bem como afeta metaloproteinases e seus inibidores teciduais, no coração e no músculo diafragma de camundongos mdx, na fase mais tardia da doença (11 meses de idade). Análise de Western blotting revelou níveis elevados do receptor purinérgico P2Y2 em ambos os músculos estudados. A terapia com suramina diminui o nível deste receptor significativamente. Concomitantemente, observamos redução dos níveis do canal de cálcio ativado por estiramento (TRPC1) e do cálcio total, o que no músculo cardíaco pode explicar a redução da mionecrose (diminuição da creatina quinase cardíaca no plasma e da troponina I no miocárdio). A atividade e o nível da metaloproteinase-9 (MMP-9), principal envolvida na remodelação da matriz extracelular e formação de fibrose, estavam elevados no coração distrófico em relação ao coração normal. A suramina promoveu aumento adicional da atividade da MMP-9 e dos níveis do seu inibidor tecidual, a TIMP-1. A suramina preveniu a redução da beta-distroglicana, componente do complexo distrofina-proteínas que encontra-se reduzido no coração distrófico. De maneira geral, a suramina promoveu efeitos semelhantes no diafragma distrófico, no que se refere ao cálcio total, TRPC1, MMP-9 e TIMP-1. Estes dados sugerem que, tanto no coração quanto no diafragma distróficos, o receptor purinérgico P2Y2 desempenha papel importante em vias de sinalização relacionadas ao cálcio (através do TRPC1) e da modulação da matriz extracelular (através da MMP-9 e TIMP-1). A suramina, por ser utilizada em outras doenças humanas, com doses e efeitos colaterais já estabelecidos em humanos, tem potencial para a terapia da cardiomiopatia na DMD, merecendo atenção em estudos clínicos desta doença / Abstract: Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease that causes respiratory and cardiac failure and results in death at about 30 years of age. The lack of dystrophin in mdx mice, the experimental model of DMD, causes sarcolemmal breakdown and increased calcium influx followed by myonecrosis and fibrosis. Purinergic receptors are involved in the pathogenesis of dystrophinopathies. Activation of these receptors by ATP triggers secondary signaling pathways that promote calcium entry and activates the formation of fibrosis. In the present study, we investigated if suramin, an anti-fibrotic drug and an antagonist of purinergic receptors, modifies total calcium and calcium related-proteins and affects metalloproteinases and their tissue inhibitors in the heart and diaphragm muscle of dystrophic mice, in the later stage of the disease (11 months of age). Western blotting analysis indicated increased levels of P2Y2 purinergic receptor in the both muscles studied, which were significantly decreased by suramin. Concomitantly, suramin lead to a reduction in total calcium and in the levels of the stretch-activated calcium channel TRPC1, which may explain the reduction of cardiac necrosis (decreases heart creatine kinase in plasma and troponin I in the myocardium). The activity and level of metalloproteinase-9 (MMP-9), the main involved in remodeling of the extracellular matrix and formation of fibrosis, were elevated in the dystrophic heart compared to normal heart. Suramin promoted further increase in MMP-9 activity and in the levels of MMP-9 inhibitor TIMP-1. Suramin prevented the reduction of beta-dystroglycan, one of the main components of the dystrophin-protein complex usually reduced in dystrophic heart. In general, suramin promoted similar effects in the dystrophic diaphragm, with respect to total calcium, TRPC1, MMP-9 and TIMP-1. These data suggest that P2Y2 purinergic receptor plays an important role in signaling pathways involved in calcium regulation (via TRPC1) and modulation of extracellular matrix (via MMP-9 and TIMP-1), in cardiac and respiratory dystrophic muscles. Suramin may be a potential alternative to treat dystrophic cardiomyopathy deserving attention in clinical trials / Doutorado / Anatomia / Doutora em Biologia Celular e Estrutural

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