Mitochondrial outer membrane permeabilization

线粒体外膜透化

• 批准号: 6919589
• 负责人: TOMOMI KUWANA
• 金额: $ 14.75万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-06-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6919589
• 关键词: BCL2 gene /protein; Xenopus; apoptosis; electron microscopy; laboratory mouse; lipids; liposomes; membrane permeability; membrane proteins; mitochondrial membrane; molecular biology; molecular cloning; phosphorylation; protein reconstitution; protein structure function; two dimensional gel electrophoresis

DESCRIPTION (provided by applicant): Apoptosis is a fundamental biological mechanism of the cell, by which it executes its own death upon receiving appropriate signals. Our long-term objective is to understand the molecular mechanisms of this process to develop effective therapeutic strategies for a number of pathological conditions including cancer, neurodegenerative diseases or aging, where apoptosis plays a key role. Death signals appear to impinge on mitochondria in many cases, resulting in the release of key effector proteins from the intermembrane space. We have shown that Bcl-2 family proteins permeabilize the outer membrane directly using in vitro systems such as outer membrane vesicles and cardiolipin containing liposomes. However, cardiolipin appears not to be abundant in the outer membrane of mitochondria. Therefore, we hypothesize that components in the outer membrane are required for Bcl-2 family proteins to permeabilize it. We will propose a biochemical approach to test this hypothesis. The specific aims are to: 1. Investigate which components, either lipids or proteins, or both, in the outer membrane are required for permeabilization. We will examine this by loading detergent extracted components from isolated outer membrane vesicles into protein-free lipid vesicles. We will treat extracted components of the outer membrane with proteases and reconstitute them into liposomes to see permeabilization of these proteo-liposomes is compromised. If so, it is likely that proteins are responsible for permeabilization induced by Bax/Bid. 2. Fractionate and identify factor(s) that is co-operating with Bax/Bid or Bid alone to permeabilize the outer membrane. We will fractionate detergent extracted outer membrane proteins to identify the factor(s) that confer the activity to liposomes when reconstituted. We will also test if a membrane incorporated proapoptotic multi-domain Bcl-2 family member, such as Bax or Bak, is sufficient for Bid to permeabilize the membrane by incorporating them into liposomes with low levels of cardiolipin.

描述（由申请人提供）：凋亡是细胞的基本生物学机制，在收到适当的信号时，它可以执行自己的死亡。 我们的长期目标是了解该过程的分子机制，以针对多种病理状况，包括癌症，神经退行性疾病或衰老，在其中凋亡起着关键作用的许多病理状况。 在许多情况下，死亡信号似乎会影响线粒体，从而导致膜间空间释放关键效应蛋白。 我们已经表明，Bcl-2家族蛋白使用体外系统（例如外膜囊泡和含有脂质体的心脂蛋白）直接将外膜通透。 然而，在线粒体的外膜中，心氨基脂蛋白似乎并不丰富。 因此，我们假设Bcl-2家族蛋白需要将其透化需要外膜中的成分。 我们将提出一种生化方法来检验该假设。 具体目的是： 1。研究哪些成分，脂质或蛋白质，或两者在外膜中都是透化需要的。 我们将通过将清洁剂从分离的外膜囊泡中萃取的清洁剂提取的成分加载到无蛋白质脂质囊泡中进行研究。 我们将用蛋白酶对外膜的提取成分进行处理，并将其重新构成脂质体，以观察这些蛋白脂质体的透化受到损害。 如果是这样，蛋白质可能是由BAX/BID诱导的透化负责。 2。分馏和识别与Bax/BID或仅出价合作以透化外膜的因子。 我们将分馏清洁剂提取外膜蛋白，以确定重构后将活性赋予脂质体的因子。 我们还将测试膜掺入促凋亡的多域Bcl-2家族成员（例如Bax或bak）足以使膜通过将膜掺入较低的心脂蛋白水平的脂质体中，以使膜渗透。