CAROLINE CRISTINA ELIAS
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Resumo IPEN-doc 26944 A new approach to obtain the catalytic sites region of human sACE with correct fold and activity2017 - AFFONSO, REGINA; SAMPAIO, SUELEN de B.; JANUARIO, FAGNER S.; PEREIRA, LARISSA M.; ARAGÃO, DANIELLE S.; CASARINI, DULCE E.; ELIAS, CAROLINE C.Angiotensin-converting enzyme I (ACE) is a membrane-bound that catalyzes the conversion of angiotensin I to the potent vasopressor angiotensin II. ACE is a key part of the renin-angiotensin system, which regulates blood pressure and is widely distributed throughout the body. There are two isoforms of human ACE, including the somatic ACE (sACE) present in somatic tissue and the testicular ACE (tACE) present in male germinal cells. The sACE possesses two domains, N- C- domains, with catalytic sites which exhibit 60% sequence identity. These domains differ in terms of chloride-ion activation profiles, rates of peptide hydrolysis of angiotensin I, bradykinin, Goralatide, Luliberin, substance P, angiotensina, beta-amyloid peptide and sensitivities to various inhibitors. A more detailed analysis shows that these regions are composed of HEMGH and EAIGD sequences that bind zinc ions to facilitate catalytic activity (Fig. 1). Our question is: If the synthesis of catalytic sites with corrects structure and activity could be a good model per si to study new drugs. The objective was to obtain the Ala361 a Gli468 and Ala959 to Ser1066 catalytic regions sACE in a structural conformation that resembles its native form. The catalytic regions were obtained from bacterial system; the expression of this protein in soluble form enables completion of the solubilization/purification steps without the need for refolding. The characterization of Ala959 to Ser1066 region shows that this has an α-helix and β-strand structure, Fig. 1b, which zinc ion (essential for its activity) binds to, and with enzymatic activity. Our conclusion is that the strategy used to obtain the Ala959 to Ser1066 region in the correct structural conformation and with activity was successful.Artigo IPEN-doc 24238 Structural characterization and enzymatic activity of the recombinant Ala959 to Ser1066 region of human ace2017 - ELIASA, CAROLINE C.; PEREIRA, LARISSA M.; ARAGAO, DANIELLE S.; CASARINI, DULCE E.; AFFONSO, REGINAAngiotensin-converting enzyme catalyzes the conversion of angiotensin I to the vasoconstrictor angiotensin II and the hydrolysis of bradykinin (BK). Human somatic angiotensin-converting enzyme has two homologous domains (N and C) that share 60% identity. Although these two regions have high homology, the catalytic site of the C-domain exhibits three-fold greater activity than the N-domain in the hydrolysis of angiotensin I in vivo. The present study aimed to obtain the Ala959 to Ser1066 catalytic region of the C-domain of angiotensin-converting enzyme in a structural conformation that resembles its native form. We amplified the 324-bp sequence corresponding to the catalytic site of C-domain of angiotensin-converting enzyme and cloned this sequence into a pET28 vector. The catalytic site of C-domain of angiotensin-converting enzyme peptide was expressed in a bacterial system, and its purification was performed in one step using a His-tag affinity column. Structural analysis by circular dichroism and fluorescence confirmed that the purified protein is correctly folded, and catalytic site of C-domain of angiotensin-converting enzyme possesses enzymatic activity and is inhibited by lisinopril. This peptide can be used to test new inhibitors and C-domain of angiotensin-converting enzyme substrates because this peptide is easy to produce and this has proven efficient link with these molecules.Resumo IPEN-doc 22462 Expression, characterization and enzymatic activity of the C-catalytic site of the angiotensin-converting enzyme I2015 - ELIAS, C.C.; PEREIRA, L.M.; SANTANA, F.; SAMPAIO, S.B.; ARAGAO, D.S.; CASARINI, D.E.; AFFONSO, R.Dissertação IPEN-doc 21192 Obtenção, caracterizações estruturais e atividade enzimática do sítio C-catalítico da enzima conversora de angiotensina I - região ALAsup(959) até SERsup(1066)2015 - ELIAS, CAROLINE C.A enzima conversora de angiotensina (ECA) catalisa a conversão de angiotensina I (Ang I) no vasoconstritor angiotensina II (Ang II) e hidrolisa a bradicinina (BK). ECA somática (sECA) possui dois domínios homólogos (N e C) que têm 60% de identidade. Embora estas duas regiões tenham homologia grande, o sítio catalítico C-domínio exibe uma atividade três vezes maior do que o N-domínio na hidrolise de Ang I in vivo. Este fato torna interessante o desenvolvimento de novos estudos de inibidores ou a melhoria dos já existentes. O objetivo deste estudo foi obter a região Ala959 até Ser1066 do Cdomínio da sECA (c-sECA), em uma estrutura conformacional semelhante à estrutura nativa. Nós amplificamos a sequência correspondente ao sítio catalítico da c-sECA com 324pb e clonamos esta sequência no vetor pET 28a(+). O segmento (nomeado de pET28_c-sECA) foi expresso em sistema bacteriano. A proteína foi expressa na forma solúvel e a purificação foi feita em uma única etapa utilizando a coluna de afinidade His-tag, a qual produziu a proteína pura. Análises estruturais por dicroísmo circular e fluorescência confirmaram que a proteína recombinante estava na conformação correta, e os ensaios de atividade mostraram que a c-sECA possui atividade enzimática e é inibida por lisinopril.