This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.To further investigate functions of apoplipoprotein L (ApoL) 2 and 3 in programmed cell death (PCD) and in normal physiology, in the fourth year of this NM-INBRE funded project, we utilized 'Tet-off' inducible gene system in DLD-1 cells and adenovirus (AD)-based system to overexpress L2 or L3 in various cell lines as previously described. Interestingly, unlike their closely related proteins ApoL1 and ApoL6, neither ApoL2 nor ApoL3 induced PCD in cells examined. RT-PCR and immunoblot analyses confirmed the overexpression of L2 or L3 in transfected cells. However, we noticed that both L2 and L3 induce increase generation of autophagic vacuoles (AV), one of the hallmarks of autophagy. Unlike L1 which induces Autophagic cell death, overexpression of L2 or L3 seems to induce autophagic cell survival. It is known that autophagy can be either pro-survival or pro-death. We also noticed that a fraction of L2 might localize in AV. In a related research, we used electron microscopic, immunoblot and immunofluorescence analyses to show that overexpression of ApoL1 induces increase formation of AV, and activating translocation of LC3-II from cytosol to AV, two of the most significant hallmarks in autophagic processes. Inhibitors of class III phosphatidylinostol-3-kinase and autophagy, block ApoL1-induced AuCD. In addition, the BH3 domain deletion construct of ApoL1 failed to induce AuCD, demonstrating that ApoL1 is a bona fide BH3-only pro-death protein. To our knowledge, this is the first BH3-only protein with lipid binding and transport activity that induces AuCD. We will employ the same strategies to further study the subcellular localization and roles of L2 and L3 in the induction of autophagic cell survival. In addition, we plan to employ mass spectrometry (MS)-based proteomic and lipidomic strategies to analyze and characterize the binding partners, both protein and lipid species, of ApoL2 or ApoL3.
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