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

泛素介导的蛋白分解是终极通用的细胞开关，几乎控制着生长和发育的方方面面。依赖于ATP的分级的E1-E2-E3酶级联促进特定蛋白质的泛素化。模块化Cullin 1-环E3泛素连接酶(又称SCF)控制着大量的生物过程，并在复杂的组装、靶标募集和Ub向靶标转移的水平上受到精确的调节。尽管泛素化对蛋白质平衡有深远的影响，但我们对SCF的催化和调节以及最初的Ub转移事件对蛋白质靶标的作用知之甚少。球蛋白(GLMN)/异常侧根形成蛋白4(ALF4)是一个跨王国的单拷贝基因，GLMN/ALF4蛋白通过结合和竞争E2对接位点来抑制SCF的活性。GLMN/ALF4基因敲除突变体在哺乳动物和植物中都是致命的，强调了GLMN/ALF4‘S的重要功能。在拟南芥中表达弱alf4等位基因的突变体，虽然是可行的，但显示出生长表型，表明植物激素反应存在缺陷。我们以前发现，ALF4的缺陷破坏了SCF的成分的稳定，从而导致对植物激素反应发生泛素化的蛋白质靶标稳定。目前，我们缺乏关于ALF4为什么、如何以及何时对SCF-E3连接酶和靶向泛素化起抑制作用的信息。在这项提议中，我们计划实施生化和生物物理方法来机械地理解ALF4的作用。我们试图剖析ALF4在体内和体外SCF靶标泛素化过程中的作用，并评估ALF4靶向蛋白质组。此外，通过建立ALF4相互作用网络，我们努力查明ALF4活性是如何调节的。通过我们的研究，我们一定会对E3s的组装以及泛素介导的蛋白质周转有更好的了解。

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.