Multiple Forms of cPLA2

nuclear cPLA2;apoptosis;inflammation

Perera, Pubuduni Sudarika ; Scott M Laster, Committee Chair,Perera, Pubuduni Sudarika ; Scott M Laster ; Committee Chair

University:North Carolina State University

关键词: nuclear cPLA2;    apoptosis;    inflammation;   

Others  :  https://repository.lib.ncsu.edu/bitstream/handle/1840.16/2308/etd.pdf?sequence=1&isAllowed=y

美国|英语

来源: null

【 摘 要 】

Cytosolic Phospholipase A₂α is an important inflammatory molecule that preferentially hydrolyzes phospholipids containing arachidonic acid at the sn-2 position, which in turn is processed to generate lipid mediators of inflammation such as prostaglandins, leukotrienes and platelet activating factor. Hence, cPLA₂α plays a vital role in the pathophysiology of inflammatory and degenerative diseases. cPLA₂α is also known to mediate apoptosis via arachidonic acid, which causes mitochondrial dysfunction leading to cell death. The focus of this investigation was the identification and characterization of a possible secondary form of cPLA₂α selectively associated with the nucleus. Gaining knowledge about the identity of a possible secondary form may give new insight to the mechanism of activation of cPLA₂α, which can then lead to the development of novel therapeutic agents that can act as inhibitors of cPLA₂α and its physiological effects. Two lines of questioning were employed within the scope of this study; (1) what portion of the full-length cPLA₂α is forming the possible secondary form? (2) what is the mechanism of generation of the secondary form? Two methods were utilized to address the above questions: (1) A panel of protease inhibitors targeting several major classes of proteases was employed to selectively inhibit each group independently in order to determine their effect on the generation of the nuclear species and (2) Performing structural analysis of cPLA₂α using a panel of antibodies with varying recognition sites along the length of the protein in order to generate a putative map of the nuclear forms. It was observed that nuclei contain three distinct forms of nuclear cPLA₂α ranging in molecular weight from 103kDa, 78kDa to 70kDa. The structural analysis revealed that the 78kDa and the 70kDa nuclear-associated proteins are both amino-terminal fragments of the full-length cPLA₂α where the 78kDa protein may potentially encompass residues 42-444 of the full-length cPLA₂α, while the 70kDa may share the majority of its residues with the 78kDa protein. Furthermore, the protease inhibitor assays have indicated that the generation of the nuclear proteins may be due to a proteolytic cleavage event that is mediated by the concerted action of a cathepsin protease, a serine protease and caspase-3. Taken together, these results indicate that gaining knowledge about the identity of a possible secondary form of cPLA₂α and hence the activation of the protein would lend immensely to the development of therapeutic agents in the treatment of inflammatory diseases.