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Cloning gene of human CPM and expression in HUVEC cells

Grant number: 14/21093-0
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): November 01, 2014
Effective date (End): June 30, 2015
Field of knowledge:Biological Sciences - Biochemistry - Molecular Biology
Principal Investigator:João Bosco Pesquero
Grantee:Carolina Caldas Hoff
Home Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil

Abstract

The kinin-kallikrein system realize actions in inflammation and blood pressure control through blood proteins, paracrine or autocrine manner. Since tissue injury happens, prekallikrein, with the aid of active Hageman factor, becomes proteases are named kallikrein. Through interaction between the kallikrein kininogen, which is a plasma ±-globulin, peptides called kinins are liberated. thesepeptides are substrates of carboxypeptidases, such as carboxypeptidase M (CPM), converting them into desArg-kinins, which are agonists of B1 kinin receptors which are also activated when tissue damage happens. It is known that CPM and B1 kinin receptors are co-localized in rafts in the plasma membrane interacting with each othersuch that the enzyme facilitates signaling of these receptors, increases NO release. Therefore, it is plausible that the idea of the peptide function is related to the location of peptidases, such as CPM. However, no data yet in the literature showing the result of this interaction from the viewpoint of the enzyme.The central objective of this work is to clone the gene for human CPM and transfect in HUVEC cells, creating an artificial cell model for the study, which will be essential to test the hypothesis that the interaction between CPM and kinin B1 receptors can affect the activity of the enzyme . This work will contributes to the general understanding of the kallikrein-kinin system and place, the interaction between these proteins. Expected to have the CPM cloned in expression vector pcDNA3.1TM / Hygro / LacZ, whose analysis of enzyme activity in HUVEC cells show-CPM is increased significantly. Our hypothesis is that the enzymatic activity of CPM cloned in vector pcDNA3.1TM / Hygro / lacZ expression and expressed in HUVEC-B1 cells is significantly increased compared to cells expressing only the enzyme. So this will be the first step in the construction of the essential molecular tools to understand and evaluate the impact of the interaction CPM-B1 receptor in enzyme activity.