ABSTRACT
Cysteine and aspartic proteases possess high elastolytic activity and might contribute to the degradation of the abdominal aortic aneurysm (AAA) wall. The aim of this study was to analyze, in detail, the proteases (cathepsins B, D, K, L and S, and inhibitor cystatin C) found in human AAA and healthy aortic tissue samples. The vessel walls from AAA patients (n=36) and nonaneurysmal aortae (n=10) were retrieved using conventional surgical repair and autopsy methods. Serum samples from the same AAA patients and 10 healthy volunteers were also collected. Quantitative expression analyses were performed at the mRNA level using real-time reverse transcriptase-PCR (RT-PCR). Furthermore, analyses at the protein level included western blot and immunoprecipitation analyses. Cellular sources of cysteine/aspartic proteases and cystatin C were identified by immunohistochemistry (IHC). All cysteine/aspartic proteases and cystatin C were detected in the AAA and control samples. Using quantitative RT-PCR, a significant increase in expression was observed for cathepsins B (P=0.021) and L (P=0.018), compared with the controls. Cathepsin B and cystatin C were also detected in the serum of AAA patients. Using IHC, smooth muscle cells (SMCs) and macrophages were positive for all of the tested cathepsins, as well as cystatin C; in addition, the lymphocytes were mainly positive for cathepsin B, followed by cathepsins D and S. All cysteine/aspartic proteases analyzed in our study were detected in the AAA and healthy aorta. The highest expression was found in macrophages and SMCs. Consequently, cysteine/aspartic proteases might play a substantial role in AAA.
Subject(s)
Aged , Humans , Middle Aged , Aorta/enzymology , Aortic Aneurysm, Abdominal/enzymology , Aspartic Acid Proteases/genetics , Case-Control Studies , Cathepsins/genetics , Cysteine Proteases/genetics , Lymphocytes/enzymology , Macrophages/enzymology , Myocytes, Smooth Muscle/enzymology , RNA, Messenger/geneticsABSTRACT
La cruzipaína es la cisteína proteinasa principal del parásito causante de la enfermedad de Chagas, Trypanosoma cruzi. La enzima está codificada por un número grande de genes (hasta 130 en la cepa Tul2) dispuestos en tandem cabeza-cola, y ubicados en dos a cuatro cromosomas diferenes, según el clon o cepa del parásito. La expresión simultánea de varios genes diferentes lleva a la producción de una mezcla compleja de isoformas. Las isoformas englobadas en el término "cruzipaína 1" difieren esencialmente en el dominio C-terminal, en su secuencia de aminoácidos y en su patrón de N-glicosilación. Se ha determinado la presencia de una forma que difiere más marcadamente, particularmente en la región catalítica, la cruzipaína 2.
Subject(s)
Animals , Cysteine Proteases/genetics , Trypanosoma cruzi/enzymology , Amino Acid Sequence , Cysteine Proteases/metabolism , Genome, ProtozoanABSTRACT
New opportunities to develop structure-based strategies for design of antiparasite drugs have emerged from studies of a family of structurally homologous cysteinyl proteinases, recently identified in several pathogenic parasites. Research on the major cysteinyl proteinase from T. cruzi (designated as cruzain, cruzipain or GP57/51) has rapidly progressed, due to independent studies conducted in Buenos Aires, Rio de Janeiro and San Francisco. The biochemical heterogeneity of this developmentally regulated group of glycoproteins is at least in part determined by genetic polymorphism. Distinguished from mammalian lysosomal cathepsins (L/S/B) by the presence of a long COOH-terminus, these single chained proteins are encoded by multiple copies of polymorphic genes. Nonconservative amino acid substitutions appear to concentrate in the papain-like catalytic domain, and may impart different substrate specificity properties to cruzipain variants expressed at the various stages of parasite development. Notably, the replication and differentiation of T. cruzi in infected cells is critically dependent on the activity of cathepsin L-like cruzipains, expressed by intracellular amastigotes. The goal of improving target selectivity of cruzipain inhibitors may be now pursued, based on clues derived from the recently elucidated crystal structure of recombinant cruzain. Concurrent with progress made in the study of cruzipain's role in parasite physiology, these proteinases have beem recognized as major antigens in chronically infected patients. By stimulating antigen specific T cells to release gamma-interferon to host tissues, cruzipain may further promote inflammation thereby contributing to pathology. Ongoing studies should determine if the proteolytic activity of cruzipain can also contribute to parasite-induced tissue damage or to the immunoregulatory abnormalities associated with Chagas'disease.