Estudo da proteína espermática EPPIN: aspectos celulares e estruturais
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Data
2023-02-08
Autores
Santos, Natália Calixto Miranda
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Universidade Estadual Paulista (Unesp)
Resumo
Em um contexto global, aproximadamente metade das gestações anuais não são planejadas pelos casais, e em 50% dos casos culminam em aborto eletivo. Novas estratégias contraceptivas masculinas promoverão planejamento familiar e o compartilhamento da responsabilidade do controle de natalidade entre os parceiros. Eventos associados à função espermática, como a motilidade e a capacitação emergem como fontes de alvos para contracepção masculina não-hormonal. A proteína espermática EPPIN (Epididymal protease inhibitor), estruturada em dois domínios inibidores de protease (WFDC e Kunitz), atua como centro para interações proteína-proteína (IPPs) na superfície do espermatozoide. Por exemplo, a EPPIN interage com a proteína do plasma seminal SEMG1 (seminogenina-1), resultando na inibição temporária da motilidade espermática após a ejaculação. Além disso, pequenos compostos experimentais orgânicos, EP055 e EP012, ao interagir com a EPPIN promoveram a redução da motilidade espermática. Esses resultados elevaram a EPPIN a um dos mais promissores alvos farmacológicos para contracepção masculina. Estudos recentes identificaram as proteínas espermáticas AKAP4 (proteína âncora 4 da proteína quinase A) e GAPDHS (gliceraldeído 3-fosfato desidrogenase) como novas potenciais parceiras da EPPIN, ampliando o leque de funções de suas IPPs. Entretanto, pouco se conhece sobre a distribuição e colocalização da EPPIN com a AKAP4 e GAPDHS durante a maturação espermática pós-testicular, além de ser pouco explorada do ponto de vista estrutural com potenciais ligantes endógenos ou exógenos. Sendo assim, o objetivo deste estudo foi investigar potenciais IPPs da EPPIN e seus sítios de ligação passíveis de intervenção farmacológica, visando aplicações na contracepção masculina. Utilizando ensaios de imunofluorescência, observamos a presença da EPPIN, GAPDHS e AKAP4 no espermatozoide murino durante a maturação pós-testicular in vivo e in vitro. Observamos a colocalização da EPPIN e com a GAPDHS e AKAP4 essencialmente na peça intermediária e principal do flagelo do espermatozoide, indicando um potencial papel em eventos que governam a motilidade espermática. No estudo estrutural, aplicamos técnicas de modelagem molecular para explorar diferentes conformações da EPPIN. Apesar da baixa variação dos ângulos entre os domínios WFDC (N-terminal) e Kunitz (C-terminal), a EPPIN apresentou conformações abertas, intermediárias e fechadas bem definidas. Os ensaios de ancoragem molecular demonstraram que a conformações intermediárias apresentaram melhor acomodação do EP055 no sítio de ligação da EPPIN, ressaltando resíduos dos domínios WFDC e Kunitz na interface de contatos do complexo. Em conjunto, nosso estudo esclarece que a EPPIN e suas proteínas parceiras permanecem associadas ao espermatozoide durante seu percurso até a fertilização. Ainda, sugerimos que a estrutura da EPPIN apresenta estado conformacional favorável para a interação de ligantes em seu sítio de ligação, sustentando a hipótese de que essas proteínas e ligantes possuem papel essencial na fertilidade.
In a global context, approximately half pregnancies per year are unplanned by couples, and in 50% of cases they culminate in elective abortion. New male contraceptive strategies will encourage family planning and the sharing of birth control responsibility between partners. Events associated with sperm function, such as sperm motility and capacitation, emerge as sources of targets for non-hormonal male contraception. The sperm protein EPPIN (Epididymal protease inhibitor), structured in two protease inhibitor domains (WFDC and Kunitz), acts as a central hub for protein-protein interactions (PPIs) on the sperm surface. For example, EPPIN interacts with the seminal plasma protein SEMG1 (seminogenin-1), resulting in temporary inhibition of sperm motility after ejaculation. In addition, small organic experimental compounds, EP055 and EP012, when interacting with EPPIN promote sperm motility reduction. These results have elevated EPPIN to one of the most promising pharmacological targets for male contraception. Recent studies have identified the sperm proteins AKAP4 (A-kinase anchor protein 4) and GAPDHS (glyceraldehyde 3-phosphate dehydrogenase) as new potential partners of EPPIN, expanding PPIs functions. However, little is known about the distribution and colocalization of EPPIN with AKAP4 and GAPDHS during post-testicular sperm maturation from a structural point of view, as well as their potential endogenous or exogenous ligands. Therefore, this study aimed to investigate potential EPPIN PPIs and their binding sites used in pharmacological intervention, purposing applications in male contraception. Using immunofluorescence assays, we observed the presence of EPPIN, GAPDHS and AKAP4 in murine spermatozoa during post-testicular maturation in vivo and in vitro. We observed colocalization of EPPIN with GAPDHS and AKAP4 essentially in the midpiece and main piece of the sperm flagellum, indicating a potential role in events that regulate sperm motility. In the structural study, we applied molecular modeling strategies to explore different EPPIN conformations. Despite the low angle variation between the WFDC (N-terminal) and Kunitz (C-terminal) domains, EPPIN presented well-defined open, intermediate, and closed conformations. Molecular docking assays showed intermediate conformations as better accommodation of EP055 in the EPPIN binding site, emphasizing residues from the WFDC and Kunitz domains in the contact interface of the complex EPPIN/EP055. Altogether, our study clarifies that EPPIN and its partner proteins remain associated with spermatozoa during its journey to fertilization. Furthermore, we suggest that the EPPIN structure presents a favorable conformational state for the interaction of the ligands in its binding site, supporting the hypothesis that these proteins and ligands play an essential role in fertility.
In a global context, approximately half pregnancies per year are unplanned by couples, and in 50% of cases they culminate in elective abortion. New male contraceptive strategies will encourage family planning and the sharing of birth control responsibility between partners. Events associated with sperm function, such as sperm motility and capacitation, emerge as sources of targets for non-hormonal male contraception. The sperm protein EPPIN (Epididymal protease inhibitor), structured in two protease inhibitor domains (WFDC and Kunitz), acts as a central hub for protein-protein interactions (PPIs) on the sperm surface. For example, EPPIN interacts with the seminal plasma protein SEMG1 (seminogenin-1), resulting in temporary inhibition of sperm motility after ejaculation. In addition, small organic experimental compounds, EP055 and EP012, when interacting with EPPIN promote sperm motility reduction. These results have elevated EPPIN to one of the most promising pharmacological targets for male contraception. Recent studies have identified the sperm proteins AKAP4 (A-kinase anchor protein 4) and GAPDHS (glyceraldehyde 3-phosphate dehydrogenase) as new potential partners of EPPIN, expanding PPIs functions. However, little is known about the distribution and colocalization of EPPIN with AKAP4 and GAPDHS during post-testicular sperm maturation from a structural point of view, as well as their potential endogenous or exogenous ligands. Therefore, this study aimed to investigate potential EPPIN PPIs and their binding sites used in pharmacological intervention, purposing applications in male contraception. Using immunofluorescence assays, we observed the presence of EPPIN, GAPDHS and AKAP4 in murine spermatozoa during post-testicular maturation in vivo and in vitro. We observed colocalization of EPPIN with GAPDHS and AKAP4 essentially in the midpiece and main piece of the sperm flagellum, indicating a potential role in events that regulate sperm motility. In the structural study, we applied molecular modeling strategies to explore different EPPIN conformations. Despite the low angle variation between the WFDC (N-terminal) and Kunitz (C-terminal) domains, EPPIN presented well-defined open, intermediate, and closed conformations. Molecular docking assays showed intermediate conformations as better accommodation of EP055 in the EPPIN binding site, emphasizing residues from the WFDC and Kunitz domains in the contact interface of the complex EPPIN/EP055. Altogether, our study clarifies that EPPIN and its partner proteins remain associated with spermatozoa during its journey to fertilization. Furthermore, we suggest that the EPPIN structure presents a favorable conformational state for the interaction of the ligands in its binding site, supporting the hypothesis that these proteins and ligands play an essential role in fertility.
Descrição
Palavras-chave
Contracepção masculina, Espermatozoides, Motilidade espermática, Interação ligante-receptor, Modelagem molecular