REGULATION OF CASPASES BY PHOSPHOINOSITIDES

细胞凋亡发生在多种生理和病理条件下。它对维持多细胞生物的体内平衡至关重要，不适当的凋亡与许多人类疾病的发病机制有关，包括缺血和癌症。细胞存活是通过增殖和凋亡信号之间的平衡来维持的。我们发现I型磷脂酰肌醇磷酸5激酶（PIP5KI）位于这些途径之间的十字路口，PIP5KI是合成关键信号磷脂酰肌醇脂的酶，磷脂酰肌醇4,5二磷酸（PIP2）。它合成抑制caspase的PIP2，而caspase反过来通过使PIP5KI失活来调节PIP2的生成。我们提出激活PIP5KI的生长因子通过产生PIP2来促进细胞存活，以防止自发的caspase激活，并为成熟的caspase激活建立一个高阈值。这就打破了生存的平衡，抑制了细胞对低水平的非定式凋亡信号的反应。另一方面，强烈的凋亡信号通过自扩增级联大量激活caspases，淹没了PIP2的抑制作用。此外，激活的半胱天冬酶通过破坏合成PIP2的关键酶来降低PIP2水平。这将所有的半胱天冬酶从抑制中释放出来，使促生存信号消散，使平衡完全向细胞死亡倾斜。为了验证这一假设，我们将做以下工作：首先，研究细胞凋亡对磷酸肌苷稳态的影响。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。我们将研究其与PIP5KI行为和凋亡进展的关系。其次，确定操作细胞PIP2是否会改变细胞凋亡的进程。过表达PIP5KI会增加PIP2水平，过表达PIP2结合PLCdelta的pleckstrin同源结构域会降低PIP2的可用性。对存活和凋亡平衡的影响将被滴定。第三，描述拟议的体外调控机制的组成部分，以便了解它们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参与者在细胞凋亡过程中复杂环境中的行为。一些可能性是caspase PIP2结合突变体或PIP5KI显性阴性抑制剂。这些研究可能提示不适当细胞凋亡治疗干预的潜在靶点。

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.