REGULATION OF CAPSASES BY PHOSPHOINOSITIDES

细胞凋亡发生在多种生理和病理条件下。 它是维持多细胞生物体内平衡的关键，并且不适当的细胞凋亡与许多人类疾病的发病机制有关，包括缺血和癌症。细胞存活通过增殖和凋亡信号之间的平衡来维持。我们发现，I型磷脂酰肌醇磷酸5激酶（PIP5KI），合成一个关键的信号磷酸肌醇脂质，磷脂酰肌醇4，5，二磷酸（PIP2）的酶，位于这些途径之间的十字路口。它合成抑制半胱天冬酶的PIP2，而半胱天冬酶又通过灭活PIP5KI来调节PIP2的产生。我们提出，激活PIP5KI的生长因子通过产生PIP2来保护细胞免受自发的半胱天冬酶激活，并为成熟的半胱天冬酶激活建立高阈值，从而促进细胞存活。这使平衡向存活倾斜，并抑制细胞对低水平外源性凋亡信号的反应。另一方面，强凋亡信号通过自放大级联反应大量激活半胱天冬酶，并淹没PIP2的抑制作用。此外，活化的半胱天冬酶通过破坏合成PIP2的关键酶来降低PIP2水平。这释放了所有半胱天冬酶的抑制，消散了促生存信号，并将平衡完全推向细胞死亡。为了验证这一假设，我们将做以下工作：首先，检查细胞凋亡对磷酸肌醇稳态的影响。如果PTP2合成发生变化，我们将确定变化发生的时间和地点以及它们是如何产生的。将检查与PIP5KI行为和凋亡进展的关系。第二，确定细胞PIP2的操作是否改变细胞凋亡的进展。PIP2水平将通过过表达PIP5KI而增加，并且PIP2可用性将通过过表达PIP2结合PLC δ的普列克底物蛋白同源结构域而降低。将滴定对存活和凋亡之间平衡的影响。第三，在体外表征拟议的监管机制的组成部分，以了解其相互作用的机制和结构基础。将开发特定的工具来分析这些球员在细胞凋亡过程中的复杂环境中的行为。一些可能性是半胱天冬酶PIP2结合突变体或PIP5KI显性负性抑制剂。这些研究可能为不适当的细胞凋亡的治疗干预提供潜在的靶点。

Apoptosis occurs under a variety of physiological and pathological conditions. It is essential for maintaining the homeostasis of multicellular organisms, and inappropriate apoptosis has been implicated in the pathogenesis of many human diseases, including ischemia and cancer. Cell survival is maintained by a balance between proliferative and apoptotic signals. We found that type I phosphatidylinositol phosphate 5 kinase (PIP5KI), the enzyme which synthesizes a key signaling phosphoinositide lipid, phosphatidylinositol 4,5, bisphosphate (PIP2), sits at the crossroad between these pathways. It synthesizes PIP2 which inhibits caspases, and caspases in turn regulates PIP2 generation by inactivating PIP5KI. We propose that growth factors which activate PIP5KI promote cell survival by generating PIP2 to protect against spontaneous caspase activation, and to establish a high threshold for fiill-fledged caspase activation. This tips the balance towards survival and dampens the response of cells to low level adventitious apoptotic signals. On the other hand, strong apoptotic signals massively activate caspases through self-amplifying cascades, and swamp out the dampening effect of PIP2. Furthermore, activated caspases reduces PIP2 level, by destroying the key enzyme that synthesizes PIP2. This releases all caspases from inhibition, dissipates the pro-survival signal and tips the balance completely towards cell death. To test this hypothesis, we will do the following: First, examine the effects of apoptosis on phosphoinositide homeostasis. If there are changes in PTP2 synthesis, we will determine when and where the changes occur, and how they are generated. The relation to PIP5KI behavior and apoptotic progression will be examined. Second, determine if manipulations of cellular PIP2 alter the progression of apoptosis. PIP2 level will be increased by overexpressing PIP5KI, and PIP2 availability will be reduced by overexpressing the PIP2 binding pleckstrin homology domain of PLCdelta. Effects on the balance between survival and apoptosis will be titrated. Third, characterize the components of the proposed regulatory machinery in vitro, in order to understand the mechanistic and structural basis for their interactions. Specific tools will be developed to analyze the behavior of these players in the complex environment of a cell during apoptosis. Some possibilities are caspase PIP2 binding mutants or PIP5KI dominant negative inhibitors. These studies may suggest potential targets for therapeutic intervention of inappropriate apoptosis.