Department of Biochemistry & Molecular Biology, University of Georgia, Athens, Georgia, USA; Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia, USA.
Department of Biochemistry & Molecular Biology, University of Georgia, Athens, Georgia, USA; Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia, USA; Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, Georgia, USA.
J Biol Chem. 2022 Sep;298(9):102305. doi: 10.1016/j.jbc.2022.102305. Epub 2022 Aug 4.
E3-SCF (Skp1/cullin-1/F-box protein) polyubiquitin ligases activate the proteasomal degradation of over a thousand proteins, but the evolutionary diversification of the F-box protein (FBP) family of substrate receptor subunits has challenged their elucidation in protists. Here, we expand the FBP candidate list in the social amoeba Dictyostelium and show that the Skp1 interactome is highly remodeled as cells transition from growth to multicellular development. Importantly, a subset of candidate FBPs was less represented when the posttranslational hydroxylation and glycosylation of Skp1 was abrogated by deletion of the O-sensing Skp1 prolyl hydroxylase PhyA. A role for this Skp1 modification for SCF activity was indicated by partial rescue of development, which normally depends on high O and PhyA, of phyA-KO cells by proteasomal inhibitors. Further examination of two FBPs, FbxwD and the Jumonji C protein JcdI, suggested that Skp1 was substituted by other factors in phyA-KO cells. Although a double-KO of jcdI and its paralog jcdH did not affect development, overexpression of JcdI increased its sensitivity to O. JcdI, a nonheme dioxygenase shown to have physiological O dependence, is conserved across protists with its F-box and other domains, and is related to the human oncogene JmjD6. Sensitization of JcdI-overexpression cells to O depended on its dioxygenase activity and other domains, but not its F-box, which may however be the mediator of its reduced levels in WT relative to Skp1 modification mutant cells. The findings suggest that activation of JcdI by O is tempered by homeostatic downregulation via PhyA and association with Skp1.
E3-SCF（Skp1/cullin-1/F-box 蛋白）多泛素连接酶可使超过 1000 种蛋白质发生蛋白酶体降解，但底物受体亚基 F-box 蛋白（FBP）家族的进化多样化，使得在原生动物中阐明其功能具有挑战性。在这里，我们在社会性粘菌 Dictyostelium 中扩展了 FBP 候选名单，并表明 Skp1 互作组在细胞从生长到多细胞发育的转变过程中发生了高度重塑。重要的是，当通过缺失 O 感应 Skp1 脯氨酰羟化酶 PhyA 来消除 Skp1 的翻译后羟化和糖基化时，候选 FBP 的一部分代表减少。Skp1 修饰对 SCF 活性的作用是通过蛋白酶体抑制剂部分挽救通常依赖高 O 和 PhyA 的 phyA-KO 细胞的发育来指示的。对两个 FBP，FbxwD 和 Jumonji C 蛋白 JcdI 的进一步研究表明，Skp1 在 phyA-KO 细胞中被其他因子取代。尽管 jcdI 和其 paralog jcdH 的双 KO 不影响发育，但 JcdI 的过表达增加了其对 O 的敏感性。JcdI 是一种非血红素双加氧酶，具有生理 O 依赖性，在具有其 F-box 和其他结构域的原生动物中保守，与人类癌基因 JmjD6 相关。JcdI 过表达细胞对 O 的敏感性依赖于其双加氧酶活性和其他结构域，但不依赖于其 F-box，而 F-box 可能是其在 WT 细胞中相对于 Skp1 修饰突变细胞的水平降低的介导物。这些发现表明，O 通过 PhyA 及其与 Skp1 的关联来下调，从而调节 JcdI 的激活。
