Xiao Yuxuan, Pollack Daniel, Andrusier Miriam, Levy Avi, Callaway Myrasol, Nieves Edward, Reddi Prabhakara, Vigodner Margarita
Department of BiologyStern College, Yeshiva University, New York, New York, USALaboratory for Macromolecular Analysis and ProteomicsAlbert Einstein College of Medicine, Bronx, New York, USADepartment of BiologyStern College, Yeshiva University, 245 Lexington Avenue, New York, New York 10016, USADepartment of Developmental and Molecular BiologyAlbert Einstein College of Medicine, Bronx, New York, USADepartment of PathologyUniversity of Virginia, Charlottesville, Virginia, USA.
Department of BiologyStern College, Yeshiva University, New York, New York, USALaboratory for Macromolecular Analysis and ProteomicsAlbert Einstein College of Medicine, Bronx, New York, USADepartment of BiologyStern College, Yeshiva University, 245 Lexington Avenue, New York, New York 10016, USADepartment of Developmental and Molecular BiologyAlbert Einstein College of Medicine, Bronx, New York, USADepartment of PathologyUniversity of Virginia, Charlottesville, Virginia, USA Department of BiologyStern College, Yeshiva University, New York, New York, USALaboratory for Macromolecular Analysis and ProteomicsAlbert Einstein College of Medicine, Bronx, New York, USADepartment of BiologyStern College, Yeshiva University, 245 Lexington Avenue, New York, New York 10016, USADepartment of Developmental and Molecular BiologyAlbert Einstein College of Medicine, Bronx, New York, USADepartment of PathologyUniversity of Virginia, Charlottesville, Virginia, USA
Reproduction. 2016 Feb;151(2):149-66. doi: 10.1530/REP-15-0239.
Recent findings suggest diverse and potentially multiple roles of small ubiquitin-like modifier (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 (KAP1 and 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.
最近的研究结果表明，小泛素样修饰物（SUMO）在睾丸功能和精子发生中具有多种潜在作用。然而，由于睾丸组织复杂的多细胞性质、无法在体外维持和操纵精子发生，以及鉴定低丰度内源性SUMO靶标的技术挑战，SUMO靶标在睾丸中仍未得到表征。在本研究中，我们使用从新鲜纯化的精母细胞和精子细胞中制备的去SUMO化抑制剂浓缩细胞裂解物，对SUMO化蛋白进行细胞特异性鉴定。在精母细胞和/或精子细胞组分中独特地鉴定出120种蛋白质。所鉴定的蛋白质参与转录调控、应激反应、微小RNA生物合成、主要酶途径调控、核质运输、细胞周期控制、顶体生物合成及其他过程。选择了几种在精子发生过程中起重要作用的蛋白质，通过免疫共沉淀、共定位和体外SUMO化研究进行进一步表征。使用GPS-SUMO软件鉴定蛋白质氨基酸序列中的共有和非共有SUMO化位点。分析证实了几种新的、以前未表征的SUMO靶标的细胞特异性SUMO化和/或SUMO相互作用，如细胞周期蛋白依赖性激酶1（CDK1）、RNA聚合酶II（RNAP II）、细胞分裂周期蛋白5（CDC5）、小鼠piRNA簇蛋白MILI、解旋酶DDX4、TDP-43蛋白（TDP-43）和丝氨酸/苏氨酸蛋白激酶31（STK31）。此外，一些先前在体细胞中被鉴定为SUMO靶标的蛋白质（KAP1和MDC1）在生殖细胞中也被鉴定为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作用。这项研究为在单个靶标水平上进一步研究SUMO开辟了一条新途径。
