Instituto de Histologia y Embriologia de Mendoza (IHEM) Dr. Mario H. Burgos-CONICET, Universidad Nacional de Cuyo, casilla de correo 56, 5500 Mendoza, Argentina.
Instituto de Histologia y Embriologia de Mendoza (IHEM) Dr. Mario H. Burgos-CONICET, Universidad Nacional de Cuyo, casilla de correo 56, 5500 Mendoza, Argentina.
Cell Signal. 2018 Apr;44:72-81. doi: 10.1016/j.cellsig.2018.01.010. Epub 2018 Jan 11.
The acrosome reaction is the regulated exocytosis of mammalian sperm's single secretory granule, essential for fertilization. It relies on small GTPases, the cAMP binding protein Epac, and the SNARE complex, among other components. Here, we describe a novel tool to investigate Rab27-related signaling pathways: a hybrid recombinant protein consisting of human Rab27A fused to TAT, a cell penetrating peptide. With this tool, we aimed to unravel the connection between Rab3, Rab27 and Rap1 in sperm exocytosis and to deepen our understanding about how isoprenylation and guanine nucleotides influence the behaviour of Rab27 in exocytosis. Our results show that TAT-Rab27A-GTP-γ-S permeated into live sperm and triggered acrosomal exocytosis per se when geraylgeranylated but inhibited it when not lipid-modified. Likewise, an impermeant version of Rab27A elicited exocytosis in streptolysin O-permeabilized - but not in non-permeabilized - cells when geranylgeranylated and active. When GDP-β-S substituted for GTP-γ-S, isoprenylated TAT-Rab27A inhibited the acrosome reaction triggered by progesterone and an Epac-selective cAMP analogue, whereas the non-isoprenylated protein did not. Geranylgeranylated TAT-Rab27A-GTP-γ-S promoted the exchange of GDP for GTP on Rab3 and Rap1 detected by far-immunofluorescence with Rab3-GTP and Rap1-GTP binding cassettes. In contrast, TAT-Rab27A lacking isoprenylation or loaded with GDP-β-S prevented the activation of Rab3 and Rap1 elicited by progesterone. Challenging streptolysin O-permeabilized human sperm with calcium increased the population of sperm with Rap1-GTP, Rab3-GTP and Rab27-GTP in the acrosomal region; pretreatment with anti-Rab27 antibodies prevented the activation of all three. The novel findings reported here include: the description of membrane permeant TAT-Rab27A as a trustworthy tool to unveil the regulation of the human sperm acrosome reaction by Rab27 under physiological conditions; that the activation of endogenous Rab27 is required for that of Rab3 and Rap1; and the connection between Epac and Rab27 and between Rab27 and the configuration of the SNARE complex. Moreover, we present direct evidence that Rab27A's lipid modification, and activation/inactivation status correlate with its stimulatory or inhibitory roles in exocytosis.
顶体反应是哺乳动物精子单一分泌颗粒的调节性胞吐作用，对受精至关重要。它依赖于小 GTP 酶、cAMP 结合蛋白 Epac 和 SNARE 复合物等成分。在这里，我们描述了一种研究 Rab27 相关信号通路的新工具：一种由人 Rab27A 与 TAT（一种穿透肽）融合而成的杂交重组蛋白。使用该工具，我们旨在揭示 Rab3、Rab27 和 Rap1 在精子胞吐作用中的联系，并深入了解异戊烯化和鸟嘌呤核苷酸如何影响 Rab27 在胞吐作用中的行为。我们的结果表明，TAT-Rab27A-GTP-γ-S 穿透到活精子中，并在 geraylgeranylated 时本身触发顶体反应，但在非脂质修饰时抑制其反应。同样，不可渗透的 Rab27A 版本在链霉蛋白酶 O 通透化的 - 但不是非通透化的 - 细胞中引发 geranylgeranylated 和活性时引发胞吐作用。当 GDP-β-S 取代 GTP-γ-S 时，异戊烯化的 TAT-Rab27A 抑制了孕酮和 Epac 选择性 cAMP 类似物触发的顶体反应，而非异戊烯化的蛋白质则没有。 geranylgeranylated TAT-Rab27A-GTP-γ-S 促进了 Rab3 和 Rap1 上 GDP 与 GTP 的交换，这是通过远免疫荧光用 Rab3-GTP 和 Rap1-GTP 结合盒检测到的。相比之下，缺乏异戊烯化或加载 GDP-β-S 的 TAT-Rab27A 阻止了孕酮引发的 Rab3 和 Rap1 的激活。用钙挑战链霉蛋白酶 O 通透化的人精子会增加顶体区域中具有 Rap1-GTP、Rab3-GTP 和 Rab27-GTP 的精子群体；用抗 Rab27 抗体预处理可防止所有三种蛋白的激活。这里报道的新发现包括：描述膜可渗透的 TAT-Rab27A 作为一种可靠的工具，用于在生理条件下揭示 Rab27 对人精子顶体反应的调节作用；内源性 Rab27 的激活对于 Rab3 和 Rap1 的激活是必需的；以及 Epac 和 Rab27 之间以及 Rab27 和 SNARE 复合物构象之间的联系。此外，我们提供了直接证据，证明 Rab27A 的脂质修饰及其激活/失活状态与它在胞吐作用中的刺激或抑制作用相关。
