胞质羧肽酶6(CCP6)催化聚谷氨酸侧链的去谷氨酰胺化,影响蛋白质如微管蛋白或核小体组装蛋白的翻译后修饰。CCP6参与多个细胞过程,比如精子发生,抗病毒活性,胚胎发育,和肾腺癌等病理。在目前的工作中,CCP6的细胞作用已通过BioID评估,一种通过质谱法绘制生理相关蛋白质-蛋白质相互作用(PPI)和诱饵近端蛋白质的邻近标记方法。我们使用稳定表达CCP6-BirA*的HEK293细胞来鉴定该酶的37种推定的相互作用物。这份CCP6近端蛋白列表显示了与中心体和中心体卫星相关的蛋白质的富集,表明CCP6可能存在于中心周围物质中。此外,我们确定纤毛组装相关蛋白是CCP6的推定相互作用蛋白.此外,CCP6近端伴侣列表包括与Joubert综合征相关的五种蛋白质,与多谷氨酰缺陷有关的纤毛病。使用邻近连接测定(PLA),我们证明PCM1、PIBF1和NudC是真正的CCP6物理交互因子。因此,BioID方法证实了CCP6在中心体和中心粒的位置和可能的功能作用,以及初级纤毛的形成和维持。
The cytosolic carboxypeptidase 6 (CCP6) catalyzes the deglutamylation of polyglutamate side chains, a post-translational modification that affects proteins such as tubulins or nucleosome assembly proteins. CCP6 is involved in several cell processes, such as spermatogenesis, antiviral activity, embryonic development, and pathologies like renal adenocarcinoma. In the present work, the cellular role of CCP6 has been assessed by BioID, a proximity labeling approach for mapping physiologically relevant protein-protein interactions (PPIs) and bait proximal proteins by mass spectrometry. We used HEK 293 cells stably expressing CCP6-BirA* to identify 37 putative interactors of this enzyme. This list of CCP6 proximal proteins displayed enrichment of proteins associated with the centrosome and centriolar satellites, indicating that CCP6 could be present in the pericentriolar material. In addition, we identified cilium assembly-related proteins as putative interactors of CCP6. In addition, the CCP6 proximal partner list included five proteins associated with the Joubert syndrome, a ciliopathy linked to defects in polyglutamylation. Using the proximity ligation assay (PLA), we show that PCM1, PIBF1, and NudC are true CCP6 physical interactors. Therefore, the BioID methodology confirms the location and possible functional role of CCP6 in centrosomes and centrioles, as well as in the formation and maintenance of primary cilia.