PATHWAYS REGULATED BY CASPASE CLEAVED BCL XL

拟议的实验将识别和表征受氧化应激调节的蛋白质，因为这种类型的应激在人类衰老过程中增加，经常导致细胞凋亡或细胞死亡。我们拟对Bcl-XL被半胱天冬酶裂解后的活性进行表征。Bcl-XL是一种抗凋亡蛋白，直到它被caspase活性水解。一旦裂解，Bcl-XL成为促凋亡蛋白。从抗凋亡蛋白到促凋亡蛋白的转换是对细胞死亡的不可逆转的承诺。在缺乏相反证据的情况下，一般认为caspases对Bcl-XL的切割是一种“功能丧失”事件。然而，我们的初步数据表明，Bcl-XL的切割是一个“功能获得”事件，因为我们已经发现delta61 Bcl-XL和MEKK3之间存在物理相互作用。我们的假设是，半胱天酶对Bcl-XL的切割产生细胞内配体delta61 Bcl-XL，该配体与丝氨酸/苏氨酸激酶MEKK3相互作用。本提案的具体目标将解决我们的假设：1)识别和表征delta61 Bcl-XL下游的信号通路。我们将从细菌中表达和纯化一种融合蛋白，该融合蛋白由delta61 Bcl-XL和一个细胞可渗透的16个氨基酸序列组成，该序列来源于Antennapedia蛋白的同型蛋白内化结构域。我们将描述il -2依赖性T淋巴细胞系（CTLL-2）中由delta61 Bcl-XL激活的信号通路；II)表征MEKK3在其推定激活剂delta61 Bcl-XL存在下的活性。我们已经确定了MEKK3和delta61 Bcl-XL之间的物理相互作用MEKK3激活了至少3种不同的途径，包括ERK， JNK和NF-kappaB途径。我们将确定delta61 Bcl-XL和MEKK3之间的相互作用是否会影响MEKK3激活这些途径的能力。

The proposed experiments will identify and characterize proteins that are regulated by oxidative stress, as this type of stress increases in human aging and often leads to apoptosis or cell death. We propose to characterize the activity of Bcl-XL after it is cleaved by caspases. Bcl-XL functions as an anti-apoptotic protein until it is proteolyzed by caspase activity. Once cleaved, Bcl-XL becomes a pro-apoptotic protein. The switch from an anti- to a pro-apoptotic protein is an irreversible commitment to cell death. In the absence of evidence to the contrary, it is generally believed that cleavage of Bcl-XL by caspases is a "loss of function" event. However, our preliminary data suggest that cleavage of Bcl-XL is a "gain of function" event, as we have discovered a physical interaction between delta61 Bcl-XL and MEKK3. Our hypothesis is that cleavage of Bcl-XL by caspases produces an intracellular ligand, delta61 Bcl-XL which interacts with the serine/threonine kinase, MEKK3. The specific aims of this proposal that will address our hypothesis are: I) Identify and characterize the signaling pathways that are downstream of delta61 Bcl-XL. We will express and purify a fusion protein from bacteria consisting of delta61 Bcl-XL and a cell permeable 16 amino acid sequence that is derived from the homeoprotein internalization domain of the Antennapedia protein. We will characterize the signaling pathways that are activated by delta61 Bcl-XL in the IL-2-dependent T lymphocyte cell line, CTLL-2; II) Characterize the activity of MEKK3 in the presence of its putative activator, delta61 Bcl-XL. We have identified a physical interaction between MEKK3 and delta61 Bcl-XL MEKK3 activates at least 3 different pathways, including the ERK, JNK, and NF-kappaB pathways. We will determine whether the interaction between delta61 Bcl-XL and MEKK3 affects the ability of MEKK3 to activate any of these pathways.