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

BIP(ER 中的中心分子伴侣)的分子水平调控

基本信息

  • 批准号:
    BB/M021874/1
  • 负责人:
  • 金额:
    $ 61.55万
  • 依托单位:
  • 依托单位国家:
    英国
  • 项目类别:
    Research Grant
  • 财政年份:
    2015
  • 资助国家:
    英国
  • 起止时间:
    2015 至 无数据
  • 项目状态:
    已结题

项目摘要

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.
分子伴侣BIP(结合免疫球蛋白蛋白,或Grp78,或Hsp5A)是内质网(ER)中唯一的Hsp70伴侣,内质网是一种细胞器,作为三分之一的细胞蛋白(包括大多数分泌蛋白和膜蛋白)的制造和包装位点。BIP是内质网的中心伴侣,它协助内质网的蛋白质合成、折叠、成熟和降解。越来越多的证据表明,对BIP活性的调节可以对与蛋白质折叠问题相关的疾病(如阿尔茨海默病和帕金森病、糖尿病和心血管疾病)产生治疗益处。此外,许多癌细胞对BIP上瘾,可以通过停止BIP活性来治疗,这表明调节BIP活性是治疗和/或预防几种破坏性病理过程的一种合理且极具吸引力的方法。如何调节BIP的伴侣活性是一个关键的未解决的问题,这对我们对这种伴侣系统的基本认识和未来药理工具的发展至关重要。BIP是一种ATP依赖的机器,它持续结合和实现未折叠(或错误折叠)的蛋白质,以使它们免于聚集并促进正确折叠。70 kDa BIP由两个结构域组成:核苷酸结合结构域(NBD)和底物结合结构域(SBD),它们相互通信,相互调节底物结合和ATP水解。这种交流是如何发生的还不得而知。为了获得对这种依赖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)光谱学的最新进展,从而允许对伴侣结构和动力学的特定位点变化进行表征。我们还将利用等温滴定量热法(ITC)和计算分子动力学(MD),前者提供了这些变化的热力学特征,后者有助于分析和解释实验数据。这些最先进技术的协同作用将导致对ATP水解调节和底物结合亲和力的独特伴侣结构和动态特征的详细表征。接下来,我们将描述生理因素如何控制和微调BIP atp酶活性和底物结合和释放的机制基础,以在不断变化的内质网环境中实现最有效的蛋白质折叠。我们将利用NMR, ITC和MD来阐明Ca2+浓度的变化和翻译后修饰如何影响BIP结构和动力学,最后,这些结构和动力学扰动如何与BIP atp酶活性和底物结合的变化相耦合。我们将进一步利用电喷雾电离-质谱联用离子迁移谱(ESI-IMS-MS)、dmso猝灭核磁共振H/D交换和实时甲基核磁共振来研究BIP寡聚化的分子机制——这是一种独特的生理过程,可以在未折叠蛋白质和/或ATP浓度波动时可逆调节BIP活性。我们将描述低聚BIP物种的大小,形状和结构组织,并阐明底物和ATP结合为什么以及如何稳定活性BIP的单体形式。为了在活细胞中发挥其功能,BIP总是与两种类型的共伴侣,j结构域蛋白和核苷酸交换因子协同作用,从而显著增强BIP的活性。为了阐明BIP共同伴侣的作用,在项目的最后一部分,我们将利用甲基核磁共振来监测BIP是如何由它的两个共同伴侣(ERj3和Grp170)控制的,结合并释放其真正的蛋白质,一个内在无序的抗体CH1结构域。因此,我们将以原子分辨率实时“观察”BIP伴侣机制的作用。

项目成果

期刊论文数量(3)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Protein folding by NMR.
Allosteric fine-tuning of the conformational equilibrium poises the chaperone BiP for post-translational regulation.
  • DOI:
    10.7554/elife.29430
  • 发表时间:
    2017-10-24
  • 期刊:
  • 影响因子:
    7.7
  • 作者:
    Wieteska L;Shahidi S;Zhuravleva A
  • 通讯作者:
    Zhuravleva A
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Anastasia Zhuravleva其他文献

Anastasia Zhuravleva的其他文献

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{{ truncateString('Anastasia Zhuravleva', 18)}}的其他基金

Targeting SurA Dynamics: An Achilles Heel in Bacterial Outer Membrane Biogenesis
靶向 SurA 动力学:细菌外膜生物发生的致命弱点
  • 批准号:
    BB/T000635/1
  • 财政年份:
    2019
  • 资助金额:
    $ 61.55万
  • 项目类别:
    Research Grant
Enabling ultra-high resolution hydrogen/deuterium exchange for challenging biomedical systems
为具有挑战性的生物医学系统实现超高分辨率氢/氘交换
  • 批准号:
    EP/P012701/1
  • 财政年份:
    2017
  • 资助金额:
    $ 61.55万
  • 项目类别:
    Research Grant

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