PATHWAYS REGULATED BY CASPASE CLEAVED BCL XL

拟议的实验将确定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中增加，通常导致细胞凋亡或细胞死亡。 我们建议，其特征在于Bcl-XL的活性后，它被半胱天冬酶切割。 Bcl-XL作为抗凋亡蛋白发挥作用，直到其被胱天蛋白酶活性蛋白水解。 一旦切割，Bcl-XL成为促凋亡蛋白。 从抗凋亡蛋白到促凋亡蛋白的转变是对细胞死亡的不可逆承诺。 在没有相反证据的情况下，通常认为胱天蛋白酶对Bcl-XL的切割是“功能丧失”事件。 然而，我们的初步数据表明，Bcl-XL的裂解是一个“功能获得”事件，因为我们已经发现了delta 61 Bcl-XL和MEKK 3之间的物理相互作用。我们的假设是，通过半胱天冬酶切割Bcl-XL产生细胞内配体，delta 61 Bcl-XL，其与丝氨酸/苏氨酸激酶MEKK 3相互作用。 该提案的具体目标将解决我们的假设是：I）识别和表征delta 61 Bcl-XL下游的信号通路。 我们将从细菌中表达和纯化由delta 61 Bcl-XL和细胞可渗透的16个氨基酸序列组成的融合蛋白，该序列来源于Apneapedia蛋白的同源异型蛋白内化结构域。 我们将表征在IL-2依赖性T淋巴细胞系CTLL-2中由delta 61 Bcl-XL激活的信号传导途径; II）表征MEKK 3在其假定激活剂delta 61 Bcl-XL存在下的活性。 我们已经确定了MEKK 3和delta 61 Bcl-XL之间的物理相互作用MEKK 3激活至少3种不同的途径，包括ERK，JNK和NF-κ B途径。 我们将确定delta 61 Bcl-XL和MEKK 3之间的相互作用是否影响MEKK 3激活任何这些途径的能力。

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.