Regulation of mitochondria morphology

我们的研究小组对理解线粒体形态变化的分子机制有着持续的兴趣。我们专注于特定的分子相互作用，调节这些线粒体的变化后，细胞死亡（凋亡）的启动。我们的研究集中在Bcl-2蛋白家族，特别是Bax蛋白，它代表了细胞凋亡的不可逆触发因素。研究Bax有许多挑战需要克服。这是由于其在凋亡起始后的线粒体膜缔合、其可以形成的多组分复合物以及在凋亡信号后其构象和生理特性的时间依赖性变化。这需要核磁共振的新方法和途径，以便我们能够在详细的分子水平上以高效率研究这种蛋白质的各种生理形式。 我们最近确定了Bax和Bim-BH 3肽复合物的结构。我们的结构显示Bim-BH 3的Bax的独特起始位点，挑战了已经存在十多年的Bcl-2蛋白调节的当前模型。基于我们的模型，我们设计了各种体外线粒体和体内基于细胞的测定来明确证实我们的发现。此外，我们开始了线粒体分裂的研究。我们解决了酵母和人类Fis 1蛋白的结构，Fis 1蛋白是一种招募裂变机器其他成员的成分。我们的结构表明，与人类相比，酵母中Fis 1的调控机制存在差异。这些研究为研究酵母和人类中调节线粒体分裂的蛋白质复合物提供了起点。 我们已经开发了几种新的核磁共振方法，以提高效率，获得结构和弛豫数据，提高精度。我们评估了主链15 N和13 C弛豫数据的温度依赖性，以表明主链相关运动不存在。这在解释涉及这些核的结构信息的NMR测量中是重要的。我们说明了存在的非平凡的NMR自旋弛豫干扰，可能会影响标量耦合测量的结果。然后，我们表明，可以利用13 C弛豫数据来细化NMR结构。此外，我们开发了一种新的方法来分析偶极耦合数据，并提出了一种方法，同时获得不同的弛豫时间在一个单一的实验。为了评估我们的NMR弛豫分析的有效性，我们开发了慢畴运动的随机模拟。我们已经成功地确定了残基特定的15 N和13 C化学位移各向异性（CSA）的蛋白质在溶液中使用分子对齐。这使我们能够将CSA张量的各个组成部分与特定的结构元素相关联。在这个过程中，我们必须通过使用I型胶原基质来创建蛋白质对齐的替代方法。 我们发展了一种新的方法，利用溶剂和蛋白质相互作用的信息作为结构约束。我们表明，这种方法增强了我们的NMR结构计算的收敛性。此外，我们还说明了这些信息可以用来验证NMR结构。该技术在确定蛋白质复合物的结构方面非常有用。 我们正在确定Bax和逆转录病毒蛋白质vMIA的肽之间的复合物的结构。我们的结构是独特的，并有助于解释从激活的凋亡途径中的Bax的隔离。这也间接表明了Bax的可能结构区域，这是重要的膜插入和聚合。与此同时，在与Youle实验室的合作中，我们表明可以设计cys突变体来保留细胞胞质溶胶中Bax的结构。我们用核磁共振表明突变蛋白在溶液中的构象与野生型相同。这种突变蛋白实际上有助于表明Bax实际上在细胞质和线粒体膜之间处于平衡状态。通过蛋白质-蛋白质相互作用改变这种平衡，可以启动细胞凋亡。此外，我们已经开始了另一个Bcl-2蛋白Bid的研究，这是细胞凋亡诱导前裂解。我们正在膜环境中研究Bid的截短形式（t-Bid），以确定其结构，并希望它将揭示它如何与其他Bcl-2蛋白相互作用：凋亡期间线粒体膜上的Bax和Bcl-xL。

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.