Regulation of mitochondria morphology

我们的研究小组一直对了解线粒体形态变化的分子机制感兴趣。我们专注于特定的分子相互作用，这些分子相互作用调节这些线粒体在细胞死亡(凋亡)启动后的变化。我们正在集中研究Bcl-2蛋白家族，特别是Bax蛋白，它代表着一种不可逆转的细胞凋亡触发因素。在研究BAX方面有许多挑战需要克服。这是由于其在启动细胞凋亡后与线粒体膜结合，可形成多组分复合体，以及随细胞凋亡信号而发生的构象和生理特性随时间的变化。这就需要新的核磁共振方法和途径，以便我们能够在详细的分子水平上高效地研究这种蛋白质的各种生理形式。 我们最近确定了Bax和Bim-BH3多肽复合体的结构。我们的结构显示了Bim-BH3对Bax的明显起始位置，挑战了目前已经存在了十多年的Bcl-2蛋白调控模型。基于我们的模型，我们设计了各种基于体外线粒体和体内细胞的检测方法，以明确证实我们的发现。此外，我们还启动了对线粒体裂变的研究。我们解决了酵母和人类Fis1蛋白的结构，Fis1蛋白是一种招募裂变机器其他成员的成分。我们的结构显示，与人类相比，酵母中Fis1的调控机制有所不同。这些研究为研究调节酵母和人类线粒体分裂的蛋白质复合体提供了一个起点。 我们开发了几种新的核磁共振方法，以提高获得结构和松弛数据的效率，并提高精度。我们评估了主干15N和13C驰豫数据的温度依赖性，表明主干关联运动不存在。这对于解释涉及这些核的结构信息的核磁共振测量是重要的。我们举例说明了可能影响标量耦合测量结果的非平凡核磁共振自旋弛豫干扰的存在。然后我们展示了人们可以利用13C驰豫数据来精炼核磁共振结构。此外，我们发展了一种分析偶极耦合数据的新方法，并提出了一种在一次实验中同时获得不同驰豫时间的方法。为了评估我们的核磁共振弛豫分析的有效性，我们开发了慢域运动的随机模拟。我们已经成功地利用分子比对确定了蛋白质在溶液中的残基特异性15N和13C化学位移各向异性(CSA)。这使我们能够将CSA张量的各种分量与特定的结构元素相关联。在这个过程中，我们不得不通过使用I型胶原基质来创造一种替代的蛋白质比对方法。 我们开发了一种利用溶剂和蛋白质相互作用信息作为结构约束的新方法。我们表明，这种方法增强了我们的核磁共振结构计算的收敛。此外，我们还说明了这些信息可以用于验证核磁共振结构。这项技术在确定蛋白质复合体的结构方面非常有用。 我们正在最终确定Bax和逆转录病毒蛋白VMIA中的一个肽之间的复合体的结构。我们的结构是独特的，有助于解释Bax在细胞凋亡途径中被激活的原因。这也间接暗示了Bax可能的结构区域，这对膜插入和膜聚合是重要的。与此同时，我们与尤勒的实验室合作，证明了Cys突变体可以被设计成在细胞的胞浆中保留Bax的结构。核磁共振分析表明，突变蛋白在溶液中的构象与野生型相同。这种突变的蛋白质实际上有助于表明Bax实际上处于胞浆和线粒体膜之间的平衡状态。通过蛋白质之间的相互作用改变这种平衡，可以启动细胞凋亡。此外，我们还开始了对另一种在诱导细胞凋亡之前被切割的BID蛋白的研究。我们正在膜环境中研究截短形式的Bid(t-Bid)以确定其结构，并希望它将有助于揭示它在细胞凋亡过程中如何与线粒体膜上的其他Bcl2蛋白：Bax和Bclxl相互作用。

Our research group has a continuing interest in understanding the molecular mechanisms responsible for morphologic changes in mitochondria. We are focusing on specific molecular interactions that regulate these mitochondria changes following a cell death (apoptotic) initiation. We are concentrating our study on the Bcl-2 family of proteins, in particular the Bax protein, which represents a non-reversible trigger for apoptosis. There are many challenges in studying Bax that have to be overcome. This is due to its mitochondria membrane association after apoptosis initiation, multi-component complexes that it can form, and the time dependent changes in its conformation and physiological properties following the apoptosis signal. This requires new methodologies and approaches in Nuclear Magnetic Resonance in order to allow us to study this protein in its various physiological forms at a detailed molecular level and with a high efficiency. We recently determined the structure of Bax and the Bim-BH3 peptide complex. Our structure showed a distinct initiation site of Bax by Bim-BH3, challenging the current model of Bcl-2 protein regulation that has been in place for more than a decade. Based on our model we designed various in vitro mitochondria and in vivo cell based assays to unequivocally confirm our findings. In addition we initiated studies of mitochondria fission. We solved the structures of yeast and human Fis1 protein, a component that recruits other members of the fission machinery. Our structures showed variation in regulatory mechanism of Fis1 in yeast compared to human. These studies provided a starting point to investigate protein complexes that regulate mitochondria fission in both yeast and human. We have developed several new NMR methods to improve efficiency in obtaining structural and relaxation data with increased precision. We evaluated the temperature dependence of backbone 15N and 13C relaxation data to show that backbone correlated motion does not exist. This is important in interpreting NMR measurements involving these nuclei for structural information. We illustrated the presence of non-trivial NMR spin relaxation interferences that could affect the outcome of scalar coupling measurements. We then showed that one could take advantage of 13C relaxation data to refine NMR structure. In addition we developed a new way to analyze dipolar coupling data and proposed a way to acquire different relaxation times simultaneously in a single experiment. In order to evaluate the validity of our NMR relaxation analysis, we developed stochastic simulation of slow domain motion. We have successfully determined residue specific 15N and 13C chemical shift anisotropy (CSA) of a protein in solution using molecular alignment. This allowed us to correlate various components of the CSA tensors to specific structural elements. In the process we had to create an alternative means of protein alignment by the use of type I collagen matrix. We developed a new method in using solvent and protein interaction information as structural restraints. We showed that this approach enhanced the convergence of our NMR structure calculation. Furthermore, we also illustrated that this information can be used to validate NMR structures. This technology is very useful in determining structures of protein complexes. We are finalizing our structure of the complex between Bax and a peptide from a retrovirus viral protein vMIA. Our structure is unique and helps to explain the sequestration of Bax from activation in the apoptosis pathway. It also indirectly suggests the possible structural regions of Bax which is important for membrane insertion and its polymerization. At the same time, in collaboration with Youle's lab we showed that cys mutants could be designed to preserve the structure of Bax in the cytosol of the cell. We used NMR to show that the conformation of the mutant protein is the same as the wild type in solution. This mutant protein actually helped in showing that Bax is actually in equilibrium between cytosol and mitochondria membrane. By changing this equilibrium thru protein-protein interaction, apoptosis can be initiated. In addition, we have initiated a study of another Bcl-2 protein Bid which is cleaved prior to apoptosis induction. We are studying the truncated form of Bid (t-Bid) in a membrane environment to determine its structure and hope that it will shed lights on how it interacts with other Bcl-2 proteins: Bax and Bcl-xL on the mitochondria membrane during apoptosis.