SERBP1 is a component of the Liver Receptor Homolog-1 transcriptional complex

Liver receptor homolog-1 (NR5A2) is an orphan nuclear receptor whose expression is essential for normal liver, intestine, and pancreas function. Nuclear receptors are ligand regulated transcription factors and NR5A2 has been shown to play a role in the transcriptional regulation of pathways involved in cancer. Elucidating the components of the NR5A2 transcriptional complex to better understand endogenous regulation of the receptor as well as its role in cancer remains a high priority. A sub-cellular enrichment strategy coupled with proteomic approaches was employed to identify putative NR5A2 coregulators. Nuclear fractionation protocol was essential for detection of NR5A2 peptides by MS, with most peptides being observed in the insoluble fraction (receptor bound to DNA). Duplicate data-dependent MS/MS runs using an inclusion/exclusion list improved the number of NR5A2 peptide identifications, including isoform-specific and fusion-specific peptides. Moreover, increased NR5A2 sequence coverage and a greater number of protein hits were observed with on-bead trypsin digestion of affinity captured protein. SERBP1 and ILF3 were identified as NR5A2 interacting partners. Both western blot and MS/MS analysis of endogenous receptor confirmed these binding partners are present in the NR5A2 transcriptional complex in established cell lines. Receptor knockdown by siRNA showed an increased in SERBP1 expression while ILF3 expression was unchanged. In contrast, receptor overexpression decreased only SERBP1 mRNA levels. Consistent with these data, in a promoter:reporter assay, binding of NR5A2 to the promoter region of SERBP1 resulted in a decrease in the expression level of the reporter gene, and subsequently, inhibiting transcription. In conclusion, using a sub-cellular enrichment strategy coupled with proteomic approaches, functionally relevant NR5A2 coregulators were identified. Given the receptor’s role in cancer progression, the study here elucidates additional transcriptional machinery involved in NR5A2 signaling and potentially provides new targets for therapeutics development.

肝受体同源物1（Liver receptor homolog-1, NR5A2）是一种孤儿核受体（orphan nuclear receptor），其表达对于维持肝脏、肠道与胰腺的正常生理功能不可或缺。核受体是一类配体调控型转录因子，已有研究证实NR5A2参与调控癌症相关通路的转录过程。解析NR5A2转录复合物的组成成分，以深入理解该受体的内源性调控机制及其在癌症发生发展中的作用，仍是当前的核心研究方向。本研究采用亚细胞富集（sub-cellular enrichment）策略结合蛋白质组学方法，对推定的NR5A2共调控因子进行鉴定。细胞核分级分离（nuclear fractionation）实验方案是通过质谱（Mass Spectrometry, MS）检测NR5A2肽段的必要条件，绝大多数肽段均在不溶性组分（与DNA结合的受体）中被检出。采用包含/排除列表的重复数据依赖性质谱联用（MS/MS）分析，提升了NR5A2肽段的鉴定数量，包括亚型特异性肽段与融合特异性肽段。此外，对亲和捕获的蛋白质进行磁珠胰蛋白酶（trypsin）酶解后，可获得更高的NR5A2序列覆盖度，且鉴定到的蛋白质数目显著增加。研究人员鉴定出SERBP1与ILF3为NR5A2的互作伴侣蛋白。对内源性NR5A2受体进行蛋白质印迹（western blot）与MS/MS分析的结果均证实，在已建立的细胞系中，这些结合伴侣蛋白存在于NR5A2转录复合物内。通过小干扰RNA（small interfering RNA, siRNA）敲低NR5A2受体后，SERBP1的表达水平显著上调，而ILF3的表达水平无明显变化。与之相反，过表达NR5A2受体仅会降低SERBP1的mRNA水平。与上述实验结果一致，在启动子-报告基因（promoter:reporter）检测实验中，NR5A2结合至SERBP1的启动子区域后，会导致报告基因的表达水平下降，进而抑制转录过程。综上，本研究通过亚细胞富集策略结合蛋白质组学方法，鉴定出了功能相关的NR5A2共调控因子。鉴于该受体在癌症进展中的关键作用，本研究阐明了参与NR5A2信号通路的额外转录调控机制，有望为抗肿瘤治疗的靶点开发提供新的方向。