Dissecting the Molecular Pathway of Aponecrosis

这项建议的长期目标是阐明细胞坏死的分子机制。坏死被定义为由死亡受体如Fas和TNFalpha受体诱导的caspase非依赖性细胞死亡。在存在泛半胱天冬酶抑制剂的情况下，用Fas或TNFalpha诱导某些类型的细胞发生凋亡性细胞死亡，缺乏典型的凋亡特征，如细胞质和细胞核凝聚以及DNA切割，并且与半胱天冬酶激活无关。相反，坏死细胞死亡表现为细胞核和细胞质肿胀，这通常与坏死有关。为了说明这种受体介导的caspase非依赖性坏死途径，我们开发了一种高通量筛选caspase非依赖性细胞死亡的小分子抑制剂。这样的筛选使我们能够鉴定出三种小分子，MTHtrp， U2和U3，它们有效地阻断fas介导的caspase非依赖性细胞死亡。具体来说，MTHtrp阻断TNF/zVAD和Fas/zVAD诱导的所有细胞类型的细胞死亡；而U2阻断TNF/zVAD和Fas/zVAD仅在某些细胞类型中诱导细胞死亡。因此，MTHtrp可能定义了坏死的共同调节因子，而U2定义了细胞类型/途径特异性控制点。这一建议是使用细胞，分子和化学遗传学的方法来表征分子途径的脂肪坏死。这项工作是波士顿大学有机化学家John Porco和哈佛医学院细胞生物学家袁俊英的密切合作。特异性目的1是评估Bcl-xL、caspases和应激激酶在坏死细胞死亡中的作用。抗凋亡Bcl-2家族成员通过防止线粒体损伤抑制细胞凋亡；但它们在肺坏死中的作用尚不清楚。我们的初步数据表明，caspase通常可以抑制caspase不依赖的坏死途径。我们将通过表达细胞caspase抑制剂、显性阴性突变体和caspase敲除EF细胞来确定所涉及的caspase的身份。我们在初步结果中表明p38可能是一种细胞类型/信号特异性坏死介质。我们想进一步表征p38和其他应激激酶在调节脂肪坏死中的作用。具体目标2是利用化学遗传学方法确定参与调节肺坏死的关键分子。我们将合成MTHtrp的亲和试剂，以鉴定其参与调节细胞坏死的细胞靶点。我们将使用平行合成来产生MTHtrp的衍生物，以确定更有效的脂肪坏死抑制剂。作为一种替代方法，我们将使用组合方法生成U2的衍生物，以开发更好的细胞类型/途径特异性坏死抑制剂，并合成U3以分析其活性。特异性目的3是鉴定不依赖caspase的细胞死亡的关键介质。我们将描述细胞MTHtrp靶标。我们将验证半胱天冬酶可能切割某些关键调节因子以促进细胞凋亡和抑制坏死的假设。我们将分析正常BalbC3T3细胞和抗坏死/凋亡敏感亚群基因表达模式的差异。我们的工作可能会导致鉴定出凋亡的关键调节因子，它在细胞凋亡中起着类似的作用。

The long term objective of this proposal is to illustrate the molecular mechanism of aponecrosis. Aponecrosis is defined as caspase-independent cell death induced by the death receptors such as Fas and TNFalpha receptors. Aponecrotic cell death, which occurs in certain cell types when induced with Fas or TNFalpha in the presence of pan caspase inhibitors, lacks the typical apoptotic feature such as cytoplasm and nuclear condensation and DNA cleavage, and is not associated with caspase activation. Instead, aponecrotic cell death exhibit nuclear and cytoplasm swelling which is typically associated with necrosis. To illustrate this receptor-mediated caspase-independent necrosis pathway, we developed a high throughput screen for small molecule inhibitors of caspase-independent cell death. Such screens allowed us to identify three small molecules, MTHtrp, U2 and U3, which effectively block Fas-mediated caspase-independent cell death. Specifically, MTHtrp blocks TNF/zVAD and Fas/zVAD induced cell death in all cell types tested; whereas U2 blocks TNF/zVAD and Fas/zVAD induced cell death only in certain cell types. Thus, MTHtrp may define a common regulator of necrosis whereas U2 defines a cell -type/pathway-specific control point. This proposal is to use cellular, molecular and chemical genetic approaches to characterize the molecular pathway of aponecrosis. This work is a close collaboration between John Porco, an organic chemist at Boston University, and Junying Yuan, a cell biologist at Harvard Medical School. The Specific Aim 1 is to evaluation of the roles of Bcl-xL, caspases and stress kinases in necrotic cell death. Antiapoptotic members of the Bcl-2 family inhibit apoptosis by preventing mitochondrial damage; but their roles in aponecrosis are not well characterized. Our preliminary data indicate that caspases may normally act to inhibit the caspase- independent necrosis pathway. We would determine the identity of the caspases involved by expressing cellular caspase inhibitors, dominant negative mutants and caspase knockout EF cells. We showed in our preliminary results that p38 may be a cell type/signal-specific necrosis mediator. We would like to further characterize the roles of p38 and other stress kinases in regulating aponecrosis. The Specific Aim 2 is to use chemical genetic approach to identify the key molecules involved in regulating aponecrosis. We would synthesize affinity reagent of MTHtrp to identify its cellular target involved in regulating aponecrosis. We would use parallel synthesis to generate derivatives of MTHtrp to identify more effective inhibitors of aponecrosis. As an alternative approach, we would generate derivatives of U2 using combinatorial method to develop better cell type/pathway-specific inhibitors of necrosis and synthesize U3 to analyze its activity. The Specific Aim 3 is to identify critical mediators of caspase-independent cell death. We will characterize the cellular MTHtrp target. We will test the hypothesis that caspases may cleave certain critical regulators to promote apoptosis and inhibit necrosis. We will analyze the difference in gene expression pattern of normal BalbC3T3 cells and the necrosis-resistant/apoptosis-sensitive subline. Our work may lead to the identification of critical regulators of aponecrosis which plays a similar role as that of caspases in apoptosis.