ATP-dependent protein unfolding and translocation by the eukaryotic proteasome

描述（由申请人提供）：在所有原核和真核细胞中，蛋白质的降解是高度特异性的，并受到能量依赖性区隔蛋白酶的严格调节。这些酶是AAA+ ATP酶家族的成员，利用ATP水解来驱动蛋白质底物的机械展开，并将其转运到一个隔离的降解室。真核细胞中主要的atp依赖蛋白酶是26S蛋白酶体，它通过特异性降解转录、细胞周期控制、信号转导和凋亡等调节蛋白来控制蛋白质稳态和许多重要过程。大多数蛋白酶体底物被标记为可降解的多泛素链的可逆附着，多泛素链作为底物递送的系留信号。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。本提案的长期目标是了解底物识别的分子基础，atp依赖的强制展开和易位，以及通过去泛素化和蛋白酶体展开机制的微调来调节它们。我的实验室设计了新的系统，用于在大肠杆菌和昆虫细胞中异种表达蛋白酶体19S碱基，并在体外重建功能性26S蛋白酶体。这为我们广泛的诱变和前所未有的机制研究提供了强有力的工具。利用生物化学和生物物理方法的结合，我们的目标是1)进一步开发真核26S蛋白酶体进行体外定量分析，2)确定协调atp水解，底物相互作用和去泛素化时间的分子机制，以及3)了解机械-化学耦合和展开力的产生。我们预计我们的结果将有助于对atp依赖的分子机器，泛素信号传导和真核细胞中蛋白质转换的调节的一般理解，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、更特异性的药物。

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.