National Institute of Child Health and Human Development, Rockville, MD, 20847, USA.
Department of Animal Biology, University of Hyderabad, Hyderabad, 500046, India.
Reprod Sci. 2023 Dec;30(12):3520-3528. doi: 10.1007/s43032-023-01292-y. Epub 2023 Jul 19.
Uroplakins (UPKs) are specialized proteins that plan an important role in protecting the epithelium of the bladder from toxic waste. We recently demonstrated the expression pattern of UPKs in the male reproductive tract and their importance in sperm function in murine models. However, the exact mechanisms through which UPKs affect spermatogenesis are not reported. In this study, using yeast two-hybrid screening was conducted to determine the interaction partners of Uroplakin 1a (UPK1A). Y2H Gold yeast strain overexpressing UPK1A was mated with Y187 yeast strain overexpressing human testis cDNA library and the mutants were plated on SD agar plates containing selection media. Colonies that grew on SD/-Trp, SD/-Leu, SD/-His, and SD/-Ade plates were isolated and evaluated to identify the interacting partners of UPK1A. Regucalcin (RGN) and proteasome subunit beta 1 (PSMB1) were identified as potential interaction partners. Using HEK cells that overexpress UPK1A and RGN or PMSB1, the co-localization and interaction were estimated with high-resolution microscopy and Pearson's coefficient. In light of the fact that UPK1A knockout caused subfertility and that the role of RGN and PSMB1 in spermatogenesis is documented, an interaction between UPK1A and RGN or PSMB1 could be required for spermatogenesis.
尿路上皮蛋白（UPKs）是一类具有特殊功能的蛋白，在保护膀胱上皮免受有毒废物侵害方面发挥着重要作用。我们最近在雄性生殖系统中证实了 UPKs 的表达模式及其在精子功能中的重要性。然而，UPKs 影响精子发生的确切机制尚未报道。在这项研究中，我们使用酵母双杂交筛选来确定 Uroplakin 1a（UPK1A）的相互作用伙伴。过表达 UPK1A 的 Y2H Gold 酵母菌株与过表达人睾丸 cDNA 文库的 Y187 酵母菌株交配，然后将突变体接种到含有选择培养基的 SD 琼脂平板上。在 SD/-Trp、SD/-Leu、SD/-His 和 SD/-Ade 平板上生长的菌落被分离并进行评估，以鉴定 UPK1A 的相互作用伙伴。Regucalcin（RGN）和蛋白酶体亚基β1（PSMB1）被鉴定为潜在的相互作用伙伴。使用过表达 UPK1A 和 RGN 或 PSMB1 的 HEK 细胞，通过高分辨率显微镜和 Pearson 系数估计共定位和相互作用。鉴于 UPK1A 敲除导致生育力下降，以及 RGN 和 PSMB1 在精子发生中的作用已有文献记载，UPK1A 与 RGN 或 PSMB1 之间的相互作用可能是精子发生所必需的。
