Caracterização funcional das isoformas de splicing do gene ADAM23 / Functional characterization of ADAM23 gene splicing isoforms

A ADAM23 é uma glicoproteína transmembrana pertencente à família ADAM (A Disintegrin and Metalloprotease) que apresenta a estrutura protéica típica dos membros desta família, mas não possui atividade de metaloprotease. O gene ADAM23 apresenta três isoformas de splicing, α, ß e γ, que codificam proteínas com porções C-terminais distintas. As isoformas α e ß codificam proteínas com domínios transmembranas diferentes, enquanto γ provavelmente consiste em uma isoforma secretada ou citoplasmática de ADAM23. Foi demonstrado que o gene ADAM23 está epigeneticamente silenciado em tumores de mama de estágios mais avançados e que seu silenciamento está associado a um maior risco de desenvolvimento de metástases e a um pior prognóstico. Recentemente, foi descrito que a proteína ADAM23 interage diretamente com a integrina αVß3 na linhagem tumoral de mama MDA-MB-435, sendo capaz de modular seu estado conformacional, controlando sua ativação. Utilizando RNAi, observou-se que o silenciamento completo do gene ADAM23 (i.e., as três isoformas) aumenta os níveis de αVß3 em conformação ativa na superfície das células MDA-MB-435, promovendo um incremento de sua capacidade migratória e adesiva. No presente trabalho, avaliamos por reações de amplificação em tempo real o perfil de expressão das três isoformas de splicing do gene ADAM23 em cinco tecidos normais (mama, cólon, cérebro, próstata e pâncreas) e em doze linhagens tumorais derivadas destes tecidos. Observamos diferenças nos níveis de expressão das isoformas em todas as amostras avaliadas, tanto dentro de uma determinada amostra, como quando comparamos tecidos normais entre si ou com linhagens tumorais. A isoforma γ é a mais expressa em todos os tecidos normais (exceto em cérebro) e em todas as linhagens tumorais. Em tecido normal de mama e de próstata e nas doze linhagens tumorais, ADAM23α é a segunda isoforma mais expressa, sendo ß a menos expressa. Constatamos também que a fração representada por cada isoforma, em relação à expressão total do gene ADAM23, está alterada nas linhagens tumorais, em comparação aos tecidos normais correspondentes. Com o intuito de elucidar a função das isoformas de ADAM23 separadamente, utilizamos shRNAs (short hairpin RNAs) para reduzir a expressão de cada isoforma de modo individual e específico na linhagem tumoral MDA-MB-435, e avaliamos seu efeito na proliferação, na morfologia, na adesão e no espraiamento celular. Verificamos que a redução da expressão da isoforma γ aumentou significativamente a taxa de proliferação das células MDA-MB-435 cultivadas em modelo tridimensional. Demonstramos também que ADAM23γ participa da regulação da morfologia e da capacidade de espraiamento das células MDA-MB-435 em condições padrão de cultivo (i.e., meio de cultura completo e placas não-sensibilizadas com substratos) e em componentes específicos da matriz extracelular, como fibronectina, colágeno I e matrigel. A isoforma α também está envolvida no controle da morfologia e do espraiamento da linhagem MDA-MB-435, porém, de modo distinto da isoforma γ. Já ADAM23ß não interfere na morfologia das células MDA-MB-435 e tem efeito marginal no espraiamento celular apenas em condições padrão de cultivo. Em conjunto, nossos resultados demonstram que as isoformas de ADAM23 são diferencialmente expressas em tecidos normais e tumorais, e exercem funções biológicas distintas

ADAM23 is a transmembrane glycoprotein that belongs to the ADAM (A Disintegrin and Metalloprotease) family of proteins and exhibits the typical protein structure of the family members, but it doesn't have metalloprotease activity. The ADAM23 gene has three splicing isoforms, α, ß and γ, that code for proteins with different C-terminal regions. Isoforms α and ß code for proteins with different transmembrane domains, while γ probably constitute a secreted or cytoplasmatic isoform of ADAM23. It has been demonstrated that the ADAM23 gene is epigenetically silenced in advanced stage breast tumors and that its silencing is associated with a higher risk of developing metastases and with a worse prognosis. Recently, it was described that ADAM23 protein interacts directly with αVß3 integrin in the breast tumor cell line MDA-MB-435, modulating its conformational state and controlling its activation. Using RNAi, it was observed that the complete silencing of ADAM23 gene (the three isoforms) raises the levels of αVß3 in its active conformation in the surface of MDA-MB-435 cells, promoting an increase in its migratory and adhesive capacity. In the present work, we evaluated by real time PCR the expression pattern of the three splicing isoforms of ADAM23 gene in five normal tissues (breast, colon, brain, prostate and pancreas) and in twelve tumor cell lines derived from these tissues. We observed differences in the expression levels of the three isoforms in all samples, either within a specific sample or comparing normal tissues among them or with tumor cell lines. Isoform γ has the highest expression in all normal tissues (except for brain) and in all tumor cell lines evaluated. In breast and prostate normal tissues and in all tumor cell lines, ADAM23α is the second most expressed isoform, while ß is the less expressed. We also noticed that the ratio represented by each isoform, relative to the total expression of ADAM23 gene, is altered in the tumor cell lines, compared to the corresponding normal tissues. With the aim to elucidate the function of ADAM23 isoforms separately, we used shRNAs (short hairpin RNAs) to reduce the expression of each isoform specifically in the MDA-MB-435 tumor cell line, and studied its effects in proliferation, morphology, adhesion and cell spreading. We observed that the reduced expression of isoform γ significantly increased the proliferation rate of MDA-MB-435 cells cultivated in tridimensional system. Also, we demonstrated that ADAM23γ participates in the regulation of cell morphology and spreading of MDA-MB-435 cells, both in standard culture conditions (cell culture media with fetal serum and in plates not sensitized with substrates) and in specific components of extracellular matrix, such as fibronectin, collagen type I and matrigel. Isoform α is also involved in the control of morphology and spreading of MDA-MB-435 cell line, although in a distinct manner from isoform γ. ADAM23ß doesn't interfere in the morphology of MDA-MB-435 cells and plays a discrete role in cell spreading only under standard culture conditions. Together, our results demonstrate that ADAM23 isoforms are differently expressed in normal and tumoral tissue, and play distinct biological roles

Establishment of a heterologous system for the expression of Canavalia brasiliensis lectin: a model for the study of protein splicing

During its biosynthesis in developing Canavalia brasiliensis seeds, the lectin ConBr undergoes a form of protein splicing in which the order of the N- and C-domains of the protein is reversed. To investigate whether these events can occur in other eukaryotic organisms, an expression system based on Pichia pastoris cells was established. A DNA fragment encoding prepro-ConBr was cloned into the vector pPICZB, and the recombinant plasmid was transformed in P. pastoris strain GS115. Ten clones were screened for effective recombinant protein production. Based on Western blot analysis of the two clones with the highest level of protein expression: 1) diffuse high-molecular mass immunoreactive bands were produced as early as 24 h after induction; 2) a single-, high-molecular mass protein was secreted into the medium, and 3) a significant fraction of the recombinant polypeptides that cross-reacted with anti-ConBr antibodies comprised a band of approximately 34.5 kDa. Diffuse protein bands with high molecular masses are attributed to hyperglycosylation at the single potential N-glycosylation site located in the linker peptide of prepro-ConBr. In contrast, native ConBr is made up of three polypeptides, the intact alpha chain (aa 1-237) and the fragments beta (aa 1-118) and gamma (aa 119-237), which have apparent molecular masses of 30, 16 and 12 kDa, respectively. Apparently, the yeast P. pastoris is not able to carry out all the complex post-translational proteolytic processing necessary for the biosynthesis of ConBr.