PDE6D的突变损害了其同源蛋白的功能,磷酸二酯酶6D(PDE6D),在异戊二烯化蛋白质向睫状膜运输中,在小鼠中引起人类纤毛病Joubert综合征(JBTS22)和视网膜变性。在这项研究中,我们通过亲和蛋白质组学纯化PDE6D的异戊烯化货物,以深入了解PDE6D相关的疾病机制。通过这种方法,我们已经确定了一组特定的PDE6D相互作用蛋白,这些蛋白与光感受器的完整性有关,GTP酶活性,核进口,或泛素化。在这些相互作用的蛋白质中,我们鉴定了PDE6D的新型纤毛货物蛋白,包括FAM219A,丝氨酸/苏氨酸蛋白激酶NIM1(NIM1K),泛素样蛋白3(UBL3)。我们证明NIM1K和UBL3以异戊二烯化依赖的方式定位在纤毛内。此外,UBL3也位于纤毛基部周围的囊泡状结构中。通过UBL3的亲和蛋白质组学,我们证实了其与PDE6D的强相互作用及其与调节小细胞外囊泡(sEV)和纤毛发生的蛋白质的关联。此外,我们表明UBL3以异戊二烯化依赖的方式定位于特定的光感受器纤毛区室。因此,我们认为UBL3可能在蛋白质向光感受器外段的分选中发挥作用,进一步解释PDE6D相关视网膜变性的发展。
Mutations in
PDE6D impair the function of its cognate protein, phosphodiesterase 6D (
PDE6D), in prenylated protein trafficking towards the ciliary membrane, causing the human ciliopathy Joubert Syndrome (JBTS22) and retinal degeneration in mice. In this study, we purified the prenylated cargo of
PDE6D by affinity proteomics to gain insight into
PDE6D-associated disease mechanisms. By this approach, we have identified a specific set of
PDE6D-interacting proteins that are involved in photoreceptor integrity, GTPase activity, nuclear import, or ubiquitination. Among these interacting proteins, we identified novel ciliary cargo proteins of
PDE6D, including FAM219A, serine/threonine-protein kinase NIM1 (NIM1K), and ubiquitin-like protein 3 (UBL3). We show that NIM1K and UBL3 localize inside the cilium in a prenylation-dependent manner. Furthermore, UBL3 also localizes in vesicle-like structures around the base of the cilium. Through affinity proteomics of UBL3, we confirmed its strong interaction with PDE6D and its association with proteins that regulate small extracellular vesicles (sEVs) and ciliogenesis. Moreover, we show that UBL3 localizes in specific photoreceptor cilium compartments in a prenylation-dependent manner. Therefore, we propose that UBL3 may play a role in the sorting of proteins towards the photoreceptor outer segment, further explaining the development of PDE6D-associated retinal degeneration.