TSPYL5通过p53信号通路调控精原细胞自我更新与分化的分子机制研究

Both self-renewal and differentiation are the fundamental phenotypes of spermatogonia. It is crucial to keep the balance of the two cell mitosis and proliferation-related phenotypes to maintain spermatogonial pool homeostasis and robust spermatogenesis. Recent studies have suggested an important regulation of spermatogonial phenotype by p53, while our understanding for the molecular mechanism underlying the function of p53 in spermatogonia cell is greatly limited. Our previous works, for the first time, suggested that TSPYL5, as a member of TTSN protein superfamily with highly conserved amino acid sequence among species, was a spermatogonia-specific suppressor of p53 function in sexual maturity males. Notably, the knockdown of the protein caused severe spermatogenic failure in mouse. The finding provides us a breakthrough point to study intensively p53 function in spermatogonia cells. For this, using the testicular biopsy tissues that were obtained from the patients with obstructive/non-obstructive azoospermia and mouse models with wild-type and knockout-type of Tspyl5 gene as primary study subjects, we will screen the key signaling pathway and key proteins through which TSPYL5/Tspyl5 regulates the spermatogonial phenotypes via p53-mediated pathway by the pull-down and protein mass spectrometry, quantitative proteome mass spectrometry and RNA-seq. The recovery of Tspyl5 knockout-related function and phenotype abnormalities in testicular tissue and spermatogonia cell by human TSPYL5 will be investigated by using constructed TSPYL5-transgenic Tspyl5-knockout mouse model. Moreover, the regulation mechanism of TSPYL5 gene expression by epigenetic modification, miRNA, androgen and its acceptor will be analyzed and the contribution of TSPYL5 to spermatogenic failure will be investigated. This study will illustrate the action mechanism underlying the regulation of spermatogonial self-renewal and differentiation by TSPYL5 via p53 signaling pathway. The works will be also helpful for better understanding the molecular system regulating spermatogonial function, developing spermatogonia preservation and transplantation system and improving the ability of diagnosis and treatment of spermatogenic failure.

精原细胞自我更新与分化的平衡维持着精原细胞池的稳态，是正常生精的必要条件之一，最新的研究提示p53在其中发挥着重要的调控作用，然而对其机制缺乏了解。前期研究中，我们首次发现TTSN蛋白超家族成员TSPYL5是一个高度保守的性成熟期精原细胞特异的p53功能抑制分子，Tspyl5敲减导致小鼠发生严重生精障碍，提示TSPYL5可能通过调控精原细胞中p53水平发挥重要作用。申请人拟以无精症患者睾丸穿刺组织与Tspyl5敲除小鼠为主要材料，筛选与验证TSPYL5通过p53信号调控精原细胞表型的关键通路与关键蛋白，调查人TSPYL5对Tspyl5缺陷小鼠异常生殖表型的恢复作用，分析TSPYL5基因的表达调控机制及其异常对生精障碍的贡献度。通过阐明TSPYL5调控精原细胞表型的信号网络及分子机制，本研究将有助于深入认识精原细胞功能调控的分子系统，发展完善精原细胞的保存与移植体系及提高生精障碍的诊疗水平。

该项目系统地探究了TSPYL5(TSPY like 5)通过TP53(tumor protein p53)信号通路调控细胞干性的分子机制，以及Tspyl5调控Pcna介导的DNA复制促进小鼠精原干细胞增殖的分子机制。我们首次发现TSPYL5的过表达可以基本关闭p53作为转录因子的功能。进一步的分子机制研究提示，TSPYL5促成了p53在胞质的滞留，一方面TSPYL5与去泛素化酶USP10(ubiquitin specific peptidase 10)互作，占据后者的核心催化结构域，削弱了USP10对TP53的去泛素化作用，显著增加了TP53泛素化与胞质聚集；更重要的是，TSPYL5与G3BP1(G3BP stress granule assembly factor 1)相互作用，促进了G3BP1向核膜转移与聚集，促进了G3BP1与E3苏木连接酶RanBP2(RAN binding protein 2)的结合与TP53的SUMO化，导致TP53-SUMO大量出核。该研究揭示了一个全新的TSPYL5蛋白相关干细胞功能及其分子机制，并为TP53功能调控领域增加了一个新的成员，这对TSPYL5在精原干细胞中的功能有重要提示作用。Tspyl5在小鼠睾丸组织优势表达，其敲出明显降低了老年小鼠的生殖能力，在机制研究中，我们分离富集了Tspyl5-/-小鼠的精原干细胞，发现全转录组差异表达基因显著富集于DNA复制通路，Tspyl5敲出使精原干细胞中该通路相关分子的表达出现显著下调；Tspyl5缺陷小鼠精原干细胞与小鼠精原细胞系GC-1中过表达Tspyl5能显著提高细胞增殖相关的Pcna(proliferating cell nuclear antigen)通路分子的表达水平；进一步的研究发现，Tspyl5可通过抑制 mir-185-5p 的表达，上调Pcna的mRNA水平；Tspyl5还可通过增加 Trp53蛋白泛素化降解水平，降低Trp53对Pcna表达抑制作用，促进Pcna的表达，进而促进精原干细胞增殖。这些发现为Tspyl5通过Pcna促进精原干细胞增殖及维持雄性生殖潜力提供了生物学机制证据，并为阐释人TSPYL5的相关生殖功能及其机制提供了新线索。