Studying the biochemical basis for the action of ABERRANT LATERAL ROOT FORMATION4, a putative ubiquitylation tuner

泛素介导的蛋白水解是最终的通用细胞开关，控制生长和发育的几乎每个方面。一个分层的ATP依赖性E1-E2-E3酶级联促进特异性蛋白质泛素化。模块化Cullin 1-RING E3泛素连接酶（又称SCF）控制过多的生物过程，并且在复合物组装、靶募集和Ub转移至靶的水平上进行精确调节。尽管泛素化对蛋白质稳定有着深远的影响，但我们对SCF的催化和调节以及蛋白质靶点上的初始Ub转移事件如何发生知之甚少。球蛋白（GLMN）/畸变侧根形成4（ALF 4）是跨界的单拷贝基因，并且GLMN/ALF 4蛋白抑制SCF活性，因为它结合并竞争E2对接位点。敲除glmn/alf 4突变体在哺乳动物和植物中都是致命的，强调了GLMN/ALF 4的基本功能。 在拟南芥中表达弱alf 4等位基因的突变体，虽然可行，显示生长表型，表明植物激素反应的缺点。我们以前表明，ALF 4的缺陷使SCF的组分不稳定，从而导致蛋白质靶点的稳定，这些蛋白质靶点在植物激素的作用下发生泛素化。目前，我们缺乏关于ALF 4为什么、如何以及何时对SCF-E3连接酶和靶向泛素化发挥抑制作用的信息。在这项提案中，我们计划实施生物化学和生物物理方法来机械地理解ALF 4的作用。我们试图在体内和体外解剖SCF靶点泛素化过程中ALF 4的作用，并评估ALF 4靶向蛋白质组。此外，通过建立ALF 4相互作用网络，我们努力查明ALF 4活性是如何调节的。通过我们的研究，我们肯定会更好地了解E3的组装和泛素介导的蛋白质周转。

Ubiquitin-mediated proteolysis is the ultimate universal cellular switch controlling almost every aspect of growth and development. A hierarchical ATP-dependent E1-E2-E3 enzymatic cascade facilitates specific protein ubiquitylation. Modular Cullin 1–RING E3 ubiquitin ligases (a.k.a SCFs) control a plethora of biological processes, and are subjected to precise tuning at the level of complex assembly, target recruitment, and Ub transfer to the targets. Despite the profound impact of ubiquitylation on proteostasis, we know little about how the catalysis and regulation of SCFs and the initial Ub transfer event on protein targets takes place. Glomulin (GLMN)/ABERRANT LATERAL ROOT FORMATION4 (ALF4) is a single copy gene across kingdoms, and the GLMN/ALF4 protein inhibits SCF activity, as it binds and competes for the E2 docking site. Knockout glmn/alf4 mutants are lethal in both mammals and plants, emphasizing GLMN/ALF4’s essential function. Mutants expressing weak alf4 alleles in Arabidopsis, although viable, show growth phenotypes that suggest shortcomings in plant hormone responses. We previously showed that defects in ALF4 destabilize components of SCFs, thereby causing stabilization of protein targets that undergo ubiquitylation in response to phytohormones. Currently, we lack information about why, how, and when ALF4 exerts its inhibitory effect on SCF-E3 ligases and target ubiquitylation. In this proposal, we plan to implement biochemical and biophysical approaches to mechanistically understand ALF4 action. We seek to dissect the effect of ALF4 during ubiquitylation of SCF targets in vivo and in vitro and assess the ALF4-targeted proteome. Also, by building up an ALF4 interaction network we strive to pinpoint how ALF4 activity is regulated. With our research, we certainly will get a better understanding of E3s assembly, and ubiquitin-mediated turnover of proteins.