Anaphase Promoting Complex-mediated Proteolysis

描述（由申请人提供）：有丝分裂过程中的一个关键事件是后期促进复合体（APC，也称为环体）对细胞周期蛋白和其他关键蛋白的泛素依赖性降解。APC底物含有降解基序，如破坏盒和KEN盒，它们是其泛素化和与APC激活剂Cdc20和Cdh1结合所必需的。这两个基序都是有效降解所必需的，但任何一个基序都足以与Cdh1p结合。我们提出了apc -Cdh1p-底物复合物的组装途径，其中无apc的Cdh1p首先结合底物。Cdh1p参与底物破坏盒刺激Cdh1p与APC的结合，可能是通过Cdh1p的构象改变。目前的研究旨在进一步加深我们对芽殖酵母中apc介导的蛋白质水解的理解。特别是，我们希望鉴定新的APC底物，了解APC- cdh1p / cdc20p -底物复合物如何组装和拆卸，并了解纺锤体组装检查点如何利用KEN盒在染色体未正确附着于有丝分裂纺锤体时关闭APC介导的蛋白质水解。为了实现这些目标，我提出了以下具体目标：1)鉴定新的APC底物2)评估APC-Cdh1p-底物组装途径的普遍性3)确定D-Box结合如何刺激Cdh1p与APC结合。4)确定保守的Cdh1p基序和磷酸化在apc -Cdh1p-底物组装中的作用5)确定apc -Cdh1p-底物复合物的拆卸是否是一个主动过程6)确定MadSp KEN Box在纺锤体组装检查点中的功能。关键蛋白质的降解对细胞分裂至关重要。因此，了解这种降解对于理解正常细胞增殖以及这一过程在疾病状态（尤其是癌症）中如何出错非常重要。

DESCRIPTION (provided by applicant): A key event in the progression through and exit from mitosis is the ubiquitin-dependent degradation of cyclins and other key proteins by the Anaphase Promoting Complex (APC; also called the cyclosome). APC substrates contain degradation motifs such as the Destruction Box and the KEN Box that are required for their ubiquitination and for their binding to the APC activators, Cdc20 and Cdh1. Both motifs are required for efficient degradation, but either one alone suffices for binding to Cdh1p. We have proposed a pathway for the assembly of the APC-Cdh1p-substrate complex in which APC-free Cdh1p first binds a substrate. Engagement of the substrate Destruction Box by Cdh1p stimulates the binding of Cdh1p to the APC, presumably via a conformation change in Cdh1p. The current studies are aimed at furthering our understanding of APC-mediated proteolysis in budding yeast. In particular, we wish to identify novel APC substrates, to understand how APC-Cdh1p/Cdc20p-substrate complexes are assembled and disassembled, and to understand how the spindle assembly checkpoint makes use of a KEN box to turn off APC-mediated proteolysis when chromosomes are not properly attached to the mitotic spindle. To achieve these goals, I propose the following Specific Aims: 1) To Identify Novel APC Substrates. 2) To Assess the Generality of the APC-Cdh1p-Substrate Assembly Pathway. 3) To Determine How D-Box Engagement Stimulates Cdh1p Binding to the APC: 4) To Determine the Roles of Conserved Cdh1p Motifs and of Phosphorylation in APC-Cdh1p-Substrate Assembly. 5) To Determine Whether Disassembly of the APC-Cdh1p-Substrate Complex is an Active Process. 6) To Determine the Function of the MadSp KEN Box in the Spindle Assembly Checkpoint. Degradation of key proteins is essential for cell division. Understanding this degradation is thus important for understanding normal cell proliferation, and how this process goes awry in disease states, particularly cancers.