Identification of cell-specific targets of sumoylation during mouse spermatogenesis.
Identification of cell-specific targets of sumoylation during mouse spermatogenesis.
复制标题
DOI：
10.1530/rep-15-0239
复制
发表时间：
2016-02
期刊：
影响因子：
--
通讯作者：
Vigodner M
中科院分区：
文献类型：
--
作者：
Xiao Y;Pollack D;Andrusier M;Levy A;Callaway M;Nieves E;Reddi P;Vigodner M
最近的发现表明，Sumo在睾丸功能和精子发生中的各种多种作用。然而，由于睾丸组织的复杂多细胞性质，无法在体外维持和操纵精子发生以及识别低增加无丰度的内源性SUMO靶标所带来的技术挑战，因此在睾丸中仍然没有表征。在这项研究中，我们使用浓缩的细胞裂解物对新鲜纯化的精子细胞和精子抑制剂制备的浓缩细胞裂解物对Sumoylated蛋白进行了细胞特异性鉴定。一百二十种蛋白在精子细胞和/或精子级分中被唯一鉴定。所鉴定的蛋白质参与了转录，压力反应，microRNA生物发生，主要酶促途径的调节，核质质转运，细胞周期控制，Adrosome生物发生和其他过程。选择了多种在精子发生过程中具有重要作用的蛋白质，以通过共免疫沉淀，共定位和体外sumoylation研究进一步表征。 GPS-SUMO软件用于鉴定蛋白质氨基酸序列中的共识和非共识SUMOYLATION位点。该分析证实了几种新型的，以前未表征的SUMO靶标，例如CDK1，RNAP II，CDC5，MILI，DDX4，TDP-43和STK31的细胞特异性Sumoylation和/或Sumo相互作用。此外，在体细胞中以前被鉴定为SUMO靶标的几种蛋白质（例如KAP1，MDC1）被鉴定为生殖细胞中的SUMO靶标。这些蛋白质中的许多在精子发生和减数分裂进程中具有独特的作用。这项研究为在个体目标水平上进行进一步研究开辟了一条新颖的途径。
Recent findings suggest diverse and potentially multiple roles of SUMO in testicular function and spermatogenesis. However, SUMO targets remain uncharacterized in the testis due to the complex multicellular nature of testicular tissue, the inability to maintain and manipulate spermatogenesis in vitro, and the technical challenges involved in identifying low-abundance endogenous SUMO targets. In this study, we performed cell-specific identification of sumoylated proteins using concentrated cell lysates prepared with de-sumoylation inhibitors from freshly purified spermatocytes and spermatids. One-hundred and twenty proteins were uniquely identified in the spermatocyte and/or spermatid fractions. The identified proteins are involved in the regulation of transcription, stress response, microRNA biogenesis, regulation of major enzymatic pathways, nuclear-cytoplasmic transport, cell cycle control, acrosome biogenesis, and other processes. Several proteins with important roles during spermatogenesis were chosen for further characterization by co-immunoprecipitation, co-localization and in-vitro sumoylation studies. GPS-SUMO software was used to identify consensus and non-consensus sumoylation sites within the amino acid sequences of the proteins. The analyses confirmed the cell-specific sumoylation and/or SUMO interaction of several novel, previously uncharacterized SUMO targets such as CDK1, RNAP II, CDC5, MILI, DDX4, TDP-43 and STK31. Furthermore, several proteins that were previously identified as SUMO targets in somatic cells (e.g., KAP1, MDC1) were identified as SUMO targets in germ cells. Many of these proteins have a unique role in spermatogenesis and during meiotic progression. This research opens a novel avenue for further studies of SUMO at the level of individual targets.