ATP-dependent protein unfolding and translocation by the eukaryotic proteasome

描述(申请人提供)：在所有原核和真核细胞中，蛋白质的降解是高度特异的，并受到依赖能量的间隔性蛋白水解酶的严格调控。这些酶是AAA+ATPase家族的成员，利用ATP水解酶驱动蛋白质底物的机械展开，并将它们转移到隔离的降解室中。真核细胞中主要依赖于ATP的蛋白水解酶是26S蛋白酶体，它通过特异性地降解转录、细胞周期控制、信号转导和细胞凋亡等调节蛋白来控制蛋白质的动态平衡和许多生命过程。大多数蛋白酶体底物通过多聚泛素链的可逆连接而被标记为降解，多泛素链作为底物传递的拴系信号。关于泛素标记和去泛素化系统的大量知识已经可用，但对蛋白酶体控制底物降解的详细机制知之甚少。这项建议的长期目标是了解底物识别的分子基础，依赖于ATP的强力展开和易位，以及蛋白酶体展开机制的去泛素化和微调对其调节。我的实验室已经设计了新的系统，用于在大肠杆菌和昆虫细胞中异源表达蛋白酶体19S碱基，并在体外重建具有功能的26S蛋白酶体。这为我们进行广泛的诱变和前所未有的机制研究提供了强大的工具。我们的目标是结合生化和生物物理方法，1)进一步开发真核细胞的26S蛋白酶体，用于体外定量分析；2)确定协调的ATP水解酶、底物相互作用和去泛素化的分子机制；3)了解机械力-化学耦合和去折叠力的产生。我们预计我们的结果将有助于对依赖于ATP的分子机制、泛素信号转导以及真核细胞中蛋白质周转的调控的总体理解，从而影响生物化学、分子生物学和细胞生物学研究的几个不同领域。鉴于蛋白酶体在许多人类疾病的发病机制中的作用，对底物加工的分子机制的详细了解也具有重要的生物医学意义，并可能有助于开发针对26S蛋白酶体的新型、更特异的药物。

DESCRIPTION (provided by applicant): Degradation of proteins is highly specific and tightly regulated by energy-dependent compartmental proteases in all prokaryotic and eukaryotic cells. These enzymes, members of the AAA+ ATPase family, use ATP hydrolysis to drive the mechanical unfolding of protein substrates and their translocation into a sequestered degradation chamber. The major ATP-dependent protease in eukaryotic cells is the 26S proteasome, which controls protein homeostasis and numerous vital processes by specifically degrading regulatory proteins involved for instance in transcription, cell-cycle control, signal transduction, and apoptosis. Most proteasomal substrates are marked for degradation by the reversible attachment of a poly-ubiquitin chain, which acts as a tethering signal for substrate delivery. Substantial knowledge about ubiquitin-tagging and de-ubiquitinating systems is already available, but only very little is known about the detailed mechanisms that control substrate degradation by the proteasome. The long-term objective of this proposal is to understand the molecular bases for substrate recognition, ATP-dependent forceful unfolding and translocation, and the regulation thereof by de- ubiquitination and fine-tuning of the proteasomal unfolding machinery. My lab has devised novel systems for the heterologous expression of the proteasomal 19S base in E.coli and insect cells, and the reconstitution of functional 26S proteasomes in vitro. This provides us with powerful tools for extensive mutagenesis and unprecedented mechanistic studies. Using a combination of biochemical and biophysical approaches, our goals are to 1) further develop the eukaryotic 26S proteasome for quantitative in-vitro analyses, 2) determine the molecular mechanisms underlying coordinated ATP-hydrolysis, substrate interactions, and the timing of de-ubiquitination, and 3) understand the mechano-chemical coupling and the generation of unfolding force. We anticipate that our results will contribute to the general understanding of ATP-dependent molecular machines, ubiquitin signaling, and the regulation of protein turnover in eukaryotic cells, and thus impact several different areas of biochemistry, molecular biology, and cell-biological research. Given the role of the proteasome in the pathogenesis of numerous human diseases, a detailed knowledge of the molecular mechanisms for substrate processing has also significant biomedical relevance and may aid the development of novel, more specific drugs targeting the 26S proteasome.