Molecular level regulation of BIP, a central molecular chaperone in the ER

分子伴侣BIP(结合免疫球蛋白，或GRP78，或Hsp5A)是内质网(ER)中唯一的Hsp70伴侣，内质网是一个细胞器，充当三分之一的细胞蛋白(包括大多数分泌和膜蛋白)的制造和包装场所。BIP是内质网中的中心伴侣，协助内质网中蛋白质的合成、折叠、成熟和降解。越来越多的证据表明，调节BIP活性可以为与蛋白质折叠问题相关的疾病(例如阿尔茨海默氏症和帕金森氏症、糖尿病和心血管疾病)带来治疗益处。此外，许多癌细胞对BIP上瘾，可以通过取消BIP活性来治疗，这表明调节BIP活性是治疗和/或防止几个破坏性病理过程的合理和极具吸引力的方法。如何调节BIP的伴侣活性是一个关键的悬而未决的问题，对于我们从根本上理解这个伴侣系统和未来药理工具的发展是至关重要的。BIP是一种依赖于ATP的机器，它持续地结合和实现未折叠(或错误折叠)的蛋白质，以将它们从聚集中拯救出来，并促进正确的折叠。70 kDa BIP由两个结构域组成：核苷酸结合域(NBD)和底物结合域(SBD)，它们相互联系，相互调节底物结合和ATP水解酶。这种交流是如何进行的还有待披露。为了获得对这种依赖于ATP的伴侣机器及其域间通信的详细机制理解，我们将使用生物分子核磁共振(NMR)光谱的尖端进展，从而能够对伴侣结构和动力学的变化进行特定部位的表征。我们还将利用为这些变化提供热力学特征的等温滴定热计(ITC)和便于分析和解释实验数据的计算分子动力学(MD)。这些最先进的技术的协同作用将导致对负责调节ATP水解和底物结合亲和力的独特伴侣结构和动态特征的详细描述。接下来，我们将描述生理因素如何控制和微调BIP ATPase活性以及底物结合和释放的机制，以实现在不断变化的ER环境中最有效的蛋白质折叠。我们将利用核磁共振、ITC和MD来阐明钙离子浓度的变化和翻译后修饰如何影响BIP的结构和动力学，以及这些结构和动态扰动如何与BIP ATPase活性和底物结合的变化相结合。我们将进一步利用电喷雾质谱结合离子迁移率光谱(ESI-IMS-MS)、DMSO猝灭的核磁共振氢/D交换和实时甲基核磁共振来研究BIP齐聚的分子机制--BIP齐聚是一种独特的生理过程，可在未折叠蛋白质和/或ATP浓度波动时对BIP活性进行可逆调节。我们将表征寡聚BIP物种的大小、形状和结构组织，并解释为什么以及如何底物和ATP结合稳定BIP的活性单体形式。为了在活细胞中发挥其功能，BIP总是与两种类型的辅伴侣--J-结构域蛋白和核苷酸交换因子协同工作，从而显著增强BIP的活性。为了阐明BIP辅助伴侣的作用，在项目的最后部分，我们将利用甲基核磁共振来监测BIP，由它的两个辅助伴侣(ERj3和Grp170)控制，如何结合和释放其真正的蛋白质，一个内在无序的抗体CH1结构域。因此，我们将使用原子分辨率实时‘观察’BIP陪伴机制的运行情况。

The molecular chaperone BIP (Binding Immunoglobulin Protein, or Grp78, or Hsp5A) is the only Hsp70 chaperone in the endoplasmic reticulum (ER), a cellular organelle acting as a manufacturing and packaging site for one-third of cellular proteins (including the majority of secreted and membrane proteins). BIP is a central chaperone in the ER, which assists in protein synthesis, folding, maturation and degradation in the ER. Growing evidence suggests that the regulation of BIP activity can result in therapeutic benefits for diseases associated with problems in protein folding (e.g., Alzheimer's and Parkinson's diseases, diabetes, and cardiovascular diseases). Moreover, many cancer cells are addicted to BIP and can be treated by withdrawing BIP activity, suggesting that the regulation of BIP activity is a rational and highly attractive way to treat and/or prevent several devastating pathological processes.How to regulate the chaperone activity of BIP is a crucial unresolved question that is essential for our fundamental understanding of this chaperone system and the future development of pharmacological tools. BIP is an ATP dependent machine that continuously binds and realizes unfolded (or misfolded) proteins to rescue them from aggregation and promote correct folding. 70 kDa BIP consists of two domains: nucleotide-binding domain (NBD) and substrate-binding domain (SBD), which communicate with each other to mutually regulate substrate binding and ATP hydrolysis. How this communication occurs has yet to be exposed. To obtain a detailed mechanistic understanding of this ATP-dependent chaperone machine and its interdomain communication, we will use cutting-edge advances in biomolecular nuclear magnetic resonance (NMR) spectroscopy, thereby allowing site-specific characterization of changes in chaperone structure and dynamics. We will also utilize isothermal titration calorimetry (ITC), which provides thermodynamic features for these changes and computational molecular dynamics (MD), which facilitate the analysis and interpretation of experimental data. A synergy of these state-of-the-art techniques will result in detailed characterization of unique chaperone structural and dynamic features responsible for the regulation of ATP hydrolysis and the affinity of substrate binding. We will next characterize the mechanistic basis on how physiological factors control and fine-tune BIP ATPase activity and substrate binding and release to achive the most effective protein folding in the constantly changing ER environment. We will utilize NMR, ITC and MD to elucidate how changes in the Ca2+ concentration and post-translational modifications affect BIP structure and dynamics and finally, how these structural and dynamic perturbations are coupled with changes in BIP ATPase activity and substrate binding. We will further exploit electrospray ionisation-mass spectrometry coupled with ion mobility spectrometry (ESI-IMS-MS), DMSO-quenched NMR H/D exchange and real-time methyl NMR to examine the molecular mechanisms of BIP oligomerization-a unique physiological process for the reversible regulation of BIP activity upon fluctuations in concentrations of unfolded proteins and/or ATP. We will characterize size, shape and structural organisation of oligomeric BIP species and elucidate why and how substrate and ATP binding stabilize the active monomeric form of BIP. To perform its functions in the living cells, BIP always collaborates with two types of co-chaperones, J-domain proteins and a nucleotide exchanges factors, which significantly enhance BIP activity. To elucidate the role of BIP co-chaperones, in the last part of the project, we will utilize methyl NMR to monitor how BIP, governed by its two co-chaperones (ERj3 and Grp170), binds and releases its authentic protein, an intrinsically disordered CH1 domain of antibodies. We will thus 'watch' the BIP chaperone machinery in action in real time with the atomic resolution.