Hypertension is a global health problem, and angiotensin I (ANG I)-converting enzyme (ACE) inhibitors are largely used to control this pathology. Recently, it has been shown that ACE can also act as a transducer signal molecule when its inhibitors or substrates bind to it. This new role of ACE could contribute to understanding some of the effects not explained by its catalytic activity only. In this study, we investigated signaling pathway activation in Chinese hamster ovary (CHO) cells stably expressing ACE (CHO-ACE) under different conditions. We also investigated gene modulation after 4 h and 24 h of captopril treatment. Our results demonstrated that CHO-ACE cells when stimulated with ANG I, ramipril, or captopril led to JNK and ERK1/2 phosphorylation. To verify any physiological role at the endogenous level, we made use of primary cultures of mesangial cells from spontaneously hypertensive rats (SHR) and Wistar rats. Our results showed that ERK1/2 activation occurred mainly in primary cultures of mesangial cells from SHR rats upon captopril stimulation, suggesting that this signaling pathway could be differentially regulated during hypertension. Our results also showed that captopril treatment leads to a decrease of cyclooxygenase 2, interleukin-1β, and β-arrestin2 and a significant increase of AP2 gene expression levels. Our findings strengthen the fact that, in addition to the blockage of enzymatic activity, ACE inhibitors also trigger signaling pathway activation, and this may contribute to their beneficial effects in the treatment of hypertension and other pathologies.

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Agradeço primeiramente à minha orientadora Dra. Regina Affonso, pelos ensinamentos de Biologia Molecular, pela amizade, por todo incentivo, apoio e paciência, principalmente nas horas mais difíceis, por ter confiado em mim e por todos os ensinamentos que ultrapassaram a Biologia Molecular. À Dra. Ligia Ely Morganti Ferreira Dias, pelo uso de seu laboratório, pela colaboração na expressão da proteína QM com suas enriquecedoras sugestões e por todas as discussões sobre o trabalho. A Dra. Dulce E. Casarini, da Universidade Federal de São Paulo (UNIFESP), por permitir utilizar toda a infra-estrutura de seu laboratório, o que foi essencial para a obtenção dos resultados. Ao Dr. Patrick Jack Spencer, pelos ensinamentos, pela paciência e por toda ajuda com a purificação da proteína. À Dra. Maria Teresa C. P. Ribela, pela ajuda e sugestões com a purificação da proteína. À Dra. Nanci do Nascimento, gerente do Centro de Biotecnologia, pelo apoio. Ao grupo de Hormônios do Centro de Biotecnologia pela utilização de toda infraestrutura. À Dra. Nélida, do Instituto de Química -USP, por toda ajuda com os ensaios de dicroísmo circular. À Dani Aragão, do laboratório da Dra. Dulce, da UNIFESP, pela ajuda com a exclusão molecular.À amiga Natália Malavasi, pela amizade conquistada, risadas, conversas na copa, por toda ajuda no laboratório, por todas as discussões científicas, pela paciência e por me ajudar, especificamente, nos ensaios de fluorescência. À amiga Karina Corleto, pela amizade, por todos os momentos bons, almoços, conversas, risadas e ajuda no laboratório e, especificamente, por me ajudar na purificação da proteína.

ACE is responsible for generating the vasoconstrictor peptide angiotensin II of the Renin Angiotensin Aldosterone System, and blockage of this system with the use of ACE inhibitors and antagonists of angiotensin II receptors are strategies for treatment of hypertension. Studies demonstrated that the use of site‐directed mutagenesis and the construction of chimeric enzymes can assist in the identification of the amino acids responsible for the interaction of enzyme/substrate or enzyme‐inhibitor complexes. We investigated the role of amino acid residues targets located in the N‐domain of ACE, Ala361, Thr378 and Phe467, strong candidates for the interaction of the enzyme with its inhibitors. The enzyme mutants were expressed in CHO cells, purified and characterized by enzymatic activity using specific substrates, inhibition assays with captopril, lisinopril and enalapril. After plasmid expression of wild type ACE (WT) cDNA in mammalian cells, PCR was performed to insert mutations 1 to 9 (M1 to M9), followed by electrophoresis, bacterial transfection, amplification of plasmids and sequencing. Only mutations M4 (A361W), M7 (T378A) and M8 (F437A) showed satisfactory results. M4, M7, M8 and WT were purified by a Superdex 200 column coupled to the FPLC system. The purified enzymes were characterized biochemically through enzymatic assays using the specific substrates for ACE, Hippuryl‐His‐Leu (HHL) and Z‐Phe‐His‐Leu (z‐FHL). WT, M4 and M7 showed high specific activity values. Analyzing the catalytic efficiency (kcat/KM), we observed that mutant enzymes better hydrolyzed the substrate z‐FHL when compared to HHL. M7 showed higher specificity (0.39 mM) and catalytic velocity (1.02 s‐1) for the substrate z‐FHL. The one with the best specificity for the HHL substrate (1.35 mM) and catalytic velocity (0.54 s‐1) was M4, compared to the WT. All enzymes showed an optimum pH around 8.0 with both substrates. Captopril inhibited the enzymes most efficiently using HHL as substrate, while Lisinopril was the one that best inhibited the enzymes using z‐FHL. It suggests that there is a negative cooperation between the ACE catalytic sites by the substrate. For both inhibitors and substrates, the best inhibition occurred with M7, suggesting that this mutation may have altered the interaction of the enzyme active site. The results obtained in this work can lead to a better understanding of the importance of these amino acids residues for the activity/functionality of the enzyme, seeking the identification of new molecules that can inhibit ACE.

Hypertension is a worldwide health problem considered to be one of the greatest public health challenges. Since hypertension is a major risk factor for cardiovascular and renal disease, many studies have focused on the Renin‐Angiotensin System (RAS), an endocrine system where renin cleaves angiotensinogen and releases Angiotensin I (AI) which is converted into Angiotensin II (AII) by the action of Angiotensin Converting Enzyme I (ACE). The ACE has a large physiological importance being a target of several therapeutic studies. This work aims to investigate the role of amino acids residues Asp140, Gln259, Ala332, Ser 333, Gln 355, Thr 358, Phe435 and Arg 500, in the interaction with the ACE inhibitor lisinopril as well as other ones.. An analysis of sequence homology to the ACEs of Xenopus laevis, mouse, rat and human, human isoform N‐domain (ND) and isoforms of 65 and 90 kDa of rat showed a high conservation of amino acid residues proposed to be mutated. Primers containing the mutation were synthesized. Following, we used a vector containing the cDNA with the human ACE gene (pACE) to perform PCR reactions; then the products obtained from PCR reactions were digested with the enzyme DpnI and transformed in E. coli DH5α. Positive colonies had the plasmid extracted, sequenced and analyzed concerning the desired mutation. The ACE mutants will be analyzed related to enzyme‐substrate interaction as well as enzyme activity.