UC Berkeley

Based on the hydrophobic tendencies exhibited by certain groups of molecules, the primary focus of this dissertation has been to create environments enriched in lipids and amphipathic molecules to explore the effect on their biological behavior. To this effect, ternary complexes of phospholipid, an apolipoprotein scaffold and a hydrophobic bioactive MDM2 inhibitor, nutlin-3a were used to form nanoassemblies of nutlin-3a nanodisks (ND). Nutlin-3a, as a part of ND was conferred with aqueous solubility, formed a homogeneous population of ND particles and associated reversibly with the ND. Biological activity of nutlin-3a ND was examined in three distinct glioblastoma cell lines, U87MG, SF763 and SF767. An overall robust decrease in cell viability, increase in apoptosis, increase in protein levels of p53 and MDM2 was observed in U87MG cells in response to nutlin-3a ND incubation while the other two cell lines remained mostly unresponsive. The nanoscale size of the formulation particles, their facile assembly and nutlin-3a solubilization capability suggest ND represent a potentially useful vehicle for in vivo administration of this anti-tumor agent.

“Wnt” family of signaling molecules regulates cell fate and proliferation in tissues of many multicellular organisms. Structure function studies of Wnts have been impeded due to the high propensity of isolated Wnts to self-associate. Stably transfected Drosophila S2 cells were used to improve recovery of recombinant murine Wnt3a. As a part of Wnt3a characterization, modulators of Wnt3a signaling were evaluated. Based on the central role played by low density lipoprotein receptor related protein 5 or 6 (LRP5/6) in Wnt3a signaling and apolipoprotein (apo)E3 lipoprotein metabolism, the ability of apoE3 to modulate Wnt signaling was evaluated. Wnt3a canonical signaling was down regulated in the presence of lipid-free apoE3 N-terminus however, this interaction appeared to be mediated through apoE3 binding with Wnt3a rather than LRP5/6. Additionally, while dissecting the supporting scaffolds of Wnt3a using thrombin mediated limited proteolysis, a site-specific cleavage within the N-terminal domain of Wnt3a was identified. Within the N-terminal (NT) domain there exists a motif that is superimposable upon saposin-like protein (SAPLIP) family members. SAPLIPs possess lipid surface seeking activity. Further inspection into the Wnt3a saposin-like sub-domain (SLD) revealed that neighboring structural elements within full-length Wnt3a affect SLD conformational stability. Overall, SLD function(s) in Wnt proteins appear to have evolved away from those commonly attributed to SAPLIP family members.