Structure and function of small heat shock proteins

描述(申请人提供)：应激、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，并通过由伴侣和蛋白酶组成的细胞蛋白质质量控制网络进行修复。这项拟议的研究调查了这个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分子伴侣的作用和与底物的相互作用机制对于理解细胞应激和疾病过程具有广泛的意义，但仍不清楚。此外，sHSP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识别和结合不同变性蛋白的有效性的基础。目标1将使用交联和先进的质谱学来确定与sHSP结合的底物位点的性质以及在体外形成的SHSP-底物复合体的组织。细胞环境是如何影响底物识别的，然后将在大肠杆菌中进行体内测试。目的2通过反复分子动力学模拟和体外分析突变型sHSP的活性，确定SHSP N末端臂的构象灵活性如何影响SHSP底物保护的效率。核磁共振将被用来获得SHSP与底物之间动态相互作用的氨基酸解析。这些相辅相成的办法将允许交叉验证结果。目标1和目标2将使用具有已知的高分辨率结构或容易通过同源建模的热休克蛋白和底物。第三个也是最终的目标使用模型遗传系统来测试来自体外研究的原理，并建立热休克蛋白与底物相互作用如何改变细胞中蛋白质的命运。总之，拟议的实验不仅将定义sHSP如何识别底物并影响其新陈代谢，还将定义蛋白质识别和聚集形成的新方面，这对了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.