Structure and function of small heat shock proteins

描述（由申请人提供）：由压力、疾病或突变引起的蛋白质聚集对所有细胞构成主要威胁。蛋白质聚集造成的损伤是有限的，并通过由伴侣蛋白和蛋白酶组成的细胞蛋白质质量控制网络进行修复。提出的研究调查了这种蛋白质质量控制网络的一个组成部分，小热休克蛋白（sHSPs），一种普遍存在的分子伴侣。sHSPs的表达和/或突变与多种蛋白质错误折叠疾病有关，包括神经退行性疾病、肌病和白内障。因此，sHSP伴侣作用和与底物相互作用的机制对理解细胞应激和疾病过程具有广泛的意义，但仍未明确定义。此外，sHSPs是研究蛋白质动力学在蛋白质-蛋白质识别中的重要性的一个很好的模型系统。我们假设，二级、三级和四级动力学是sHSPs识别和结合各种变性蛋白的有效性的基础。目的1将使用交联和先进的质谱技术来确定shsp结合的底物位点的性质以及体外形成的shsp -底物配合物的组织结构。细胞环境如何影响底物识别将在大肠杆菌体内进行测试。目的2将通过分子动力学模拟和突变sHSP活性的体外分析，确定sHSP n端臂的构象灵活性如何影响sHSP底物保护效率。NMR将用于获得sHSP与底物之间动态相互作用的氨基酸分辨率。这些互补的方法将允许交叉验证结果。具有已知高分辨率结构的sHSPs和底物，或易于通过同源性建模，将用于目标1和2。第三个也是最后一个Aim采用模型遗传系统来测试从体外研究中得出的原理，并确定shsp -底物相互作用如何改变细胞中蛋白质的命运。总之，所提出的实验不仅将定义sHSPs如何识别底物并影响其代谢，而且还将定义蛋白质识别和聚集形成的新方面，这对理解许多疾病状态至关重要。

DESCRIPTION (provided by applicant): Protein aggregation resulting from stress, disease or mutation poses a major threat to all cells. Damage due to protein aggregation is limited and repaired by a cellular protein quality control network consisting of chaperones and proteases. The proposed research investigates one component of this protein quality control network, the small heat shock proteins (sHSPs), a ubiquitous class of molecular chaperones. Expression and/or mutation of sHSPs are linked to multiple diseases of protein misfolding, including neurodegenerative diseases, myopathies and cataract. The mechanism of sHSP chaperone action and interaction with substrates, therefore, has wide-ranging implications for understanding cellular stress and disease processes, but remains poorly defined. Furthermore, sHSPs are an excellent model system for investigating the importance of protein dynamics in protein-protein recognition. We hypothesize that secondary, tertiary and quaternary dynamics underlie the effectiveness of sHSPs in recognizing and binding diverse denaturing proteins. Aim 1 will use crosslinking and advanced mass spectrometry to define the nature of substrate sites bound by sHSPs and the organization of sHSP-substrate complexes formed in vitro. How the cellular environment impacts substrate recognition will then be tested in vivo in E. coli. Aim 2 will determine how conformational flexibility of the sHSP N-terminal arm effects efficiency of sHSP substrate protection using iterations of molecular dynamics simulations and in vitro assays of the activity of mutant sHSPs. NMR will be used to obtain amino acid resolution of the dynamic interactions between sHSP and substrate. These complementary approaches will allow cross validation of results. sHSPs and substrates with known high resolution structures, or readily modeled by homology, will be used in Aims 1 and 2. The third and final Aim employs a model genetic system to test principles derived from the in vitro studies and to establish how sHSP-substrate interactions alter protein fate in the cell. In total, the proposed experiments will define not only how sHSPs recognize substrates and impact their metabolism, but also define new aspects of protein recognition and aggregate formation, which are critical to understanding many diseased states. PUBLIC HEALTH RELEVANCE: All cells contain machinery to protect and repair proteins, the major workhorses of cells. Defects in this "protein quality control network" result in diseases of protein folding, including neurodegenerative disease, myopathies and cataract. Decline in this machinery is also associated with aging. The proposed research involves detailed biochemical studies of an important protein component of this protective network.