Institut de Biotecnologia i Biomedicina, Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Barcelona, Spain.
iRIP Unit, Laboratory of Microbiology, Department of Biochemistry and Microbiology, Ghent University, K. L. Ledeganckstraat 35, 9000 Ghent, Belgium.
Int J Mol Sci. 2023 Jan 9;24(2):1273. doi: 10.3390/ijms24021273.
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.
细胞质羧肽酶 6（CCP6）催化多谷氨酸侧链的脱谷氨酸化，这是一种影响微管蛋白或核小体组装蛋白等蛋白质的翻译后修饰。CCP6 参与多种细胞过程，如精子发生、抗病毒活性、胚胎发育和肾腺癌等病理学。在本工作中，通过生物 ID（一种用于映射生理相关蛋白-蛋白相互作用（PPIs）和诱饵近端蛋白的接近标记方法）评估了 CCP6 的细胞作用。我们使用稳定表达 CCP6-BirA*的 HEK 293 细胞来鉴定该酶的 37 个潜在相互作用蛋白。该 CCP6 近端蛋白列表显示富含与中心体和中心粒卫星相关的蛋白质，表明 CCP6 可能存在于中心粒周围物质中。此外，我们鉴定出与纤毛组装相关的蛋白质是 CCP6 的潜在相互作用蛋白。此外，CCP6 近端伴侣列表包括与杰特综合征相关的五个蛋白，这是一种与多谷氨酸化缺陷相关的纤毛病。使用接近连接测定（PLA），我们表明 PCM1、PIBF1 和 NudC 是 CCP6 的真正物理相互作用蛋白。因此，生物 ID 方法证实了 CCP6 在中心体和中心粒中的位置和可能的功能作用，以及初级纤毛的形成和维持。
