Barth Syndrome (BTHS) is a rare genetic disorder caused by mutations in the gene that encodes tafazzin, a protein whose sole function is to remodel the mitochondrial phospholipid cardiolipin (CL). Despite comprising a small proportion of the body’s total phospholipid pool, the critical role CL plays in the structure and function of the mitochondrial inner membrane means that defects in CL composition lead to profound mitochondrial impairment. These mitochondrial defects, in turn, lead to the failure of high energy-demand systems, resulting in neutropenia, skeletal muscle weakness, and cardiomyopathy. While some researchers have suggested gene therapy or protein replacement as potential future treatments, it is conceivable that, if CL could be delivered to the defective mitochondria, the need for a functional tafazzin could be bypassed entirely. In the current study, we use the amphipathic plasma protein apolipoprotein A-I to solubilize CL into discoidal nanoparticles resembling HDL. These CL nanodisks (CLND) were incubated with a cell culture model of the neutropenia seen in BTHS. CL-ND treatment increased cellular CL levels and ameliorated the BTHS phenotype. To extend these findings to whole animals, a 10 week study was carried out using a mouse model of BTHS. However, at the end of the study, while the BTHS mice developed significant differences in CL composition characteristic of the disease, CL-ND treatment had no effect on CL content or composition. Thus, while CL-ND may eventually prove to be a useful therapy for BTHS, the current study highlights the difficulty of translating cell culture results to intact animals.

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.

Coronary heart disease is the leading cause of death in the United States, and epidemiological studies have shown that an elevated plasma triglyceride level is an independent risk factor for cardiovascular disease. In 2001, a new member of the exchangeable apolipoprotein (apo) family, apoA-V, was discovered using comparative genomics. Studies with transgenic mice over-expressing human apoA-V and apoA-V knock-out mice have shown that there exists a negative correlation between apoA-V levels and plasma triglyceride concentrations. Such findings have also been observed in humans, where single nucleotide polymorphisms within the APOA5 gene have been associated with hypertriglyceridemia. These physiological findings suggest that apoA-V plays a major role in the regulation of triglyceride metabolism, and elucidating the mechanism by which apoA-V functions could yield therapeutic effects.

Several features of apoA-V, including an extremely low plasma concentration, a lack of correlation with cholesterol levels despite its association with high density lipoprotein, and its insolubility at neutral pH in the absence of lipid, are unlike other exchangeable apolipoproteins. Findings indicate apoA-V is comprised of two independently folded structural domains, with the amino-terminal domain forming an amphipathic alpha-helix bundle in the absence of lipid. In the presence of lipid, the helix bundle motif is postulated to unfurl, exposing hydrophobic residues for lipid interaction. The carboxy-terminal region plays a key function in apoA-V lipid binding, consistent with its known association with plasma lipoproteins.

Apolipoprotein (apo) E is an exchangeable apolipoprotein that is critical for the trafficking of lipid and cholesterol nutrients in the brain and peripheral circulation. ApoE is a 299 amino acid (37 kDa) protein comprised of two independently folded functional domains, the carboxy-terminal lipid-binding domain and the receptor-binding amino-terminal (NT) domain that only displays receptor competent activity upon association with lipid. In the absence of lipid, the isolated NT domain (residues 1-183) of apoE adopts an amphipathic four alpha-helix bundle architecture that is characteristic of several other related apolipoproteins.

Various models have been advanced that describe the predicted conformational change of the protein upon lipid binding. Experiments have shown that the alpha-helical secondary structure is preserved if not enhanced upon lipid binding and yet it is known that the protein undergoes a dramatic conformational change in the transition to the lipid-bound state. Low-resolution experiments have provided insight into the mechanism and possible path of this transition, but a high-resolution determination of the lipid-bound conformation of apoE has not been accomplished. Using a combination of unique protein engineering methods and nuclear magnetic resonance (NMR) spectroscopy, this thesis advances the understanding of the lipid-induced conformational change of the apoE N-terminus. Recombinant apoE NT (residues 1-183) is a representative model for apolipoprotein helix bundle conformational flexibility in the presence of lipid and on the surface of lipoprotein particles. The 22 kDa domain is predominantly alpha-helical, monomeric, and comparably stable relative to the native protein. This domain readily forms discoidal particles in the presence of phospholipids, which imparts low-density lipoprotein (LDL) receptor activity to the protein.

A protein engineering approach was used to further define the structural determinants of apoE NT that are necessary for lipid binding. A short helix connecting helix 1 and 2 in the four-helix bundle was replaced by a sequence predicted to adopt a beta-turn. The resulting stable recombinant protein was not compromised in its ability to function as a ligand for the LDL receptor, yet the protein displayed greatly enhanced binding affinity for lipid as assessed by phospholipid solubilization studies, a lipophilic fluorescent dye binding assay, and protection against phospholipase induced aggregation of human LDL fractions.

In order to define the detailed lipid-induced conformational change in apoE, a protein engineering approach termed segmental isotope labeling was deemed necessary to simplify the system for analysis by NMR. Using expressed protein ligation (EPL) methodology, a hybrid apolipoprotein was constructed from two independently generated fragments, apoE residues 1-111 and a 91 amino acid apolipophorin protein fragment. This protein ligation experiment tested the novel use of a pelB leader sequence for the generation of an N-terminal cysteine-containing protein fragment required for the joining of protein fragments by EPL.

Expressed protein ligation techniques were alternatively adapted to create an intact, semisynthetic apoE NT domain using apoE(1-111) and apoE(112-183) protein fragments. This semisynthetic protein displayed nearly identical structural and function properties as wild-type apoE NT by circular dichroism spectroscopy, guanidine denaturation studies, and functional lipid and LDL receptor binding studies. Stable isotope-labeled (15N) apoE(112-183) was produced and ligated to unlabeled apoE 1-111 protein to create a segmental isotope-labeled protein. NMR experiments of the segmental protein further confirmed a structural and functional correspondence between wild-type, fully 15N-labeled and segmental isotope-labeled apoE NT while affirming that the segmental system dramatically simplified the NMR system for examining the protein region containing the LDL receptor recognition sequence.

According to the definition from the National Nanotechnology Initiative, nanotechnology refers to research conducted at a nano-scale level which is about 1 to 100 nanometers (nm). Recent years have seen widespread application of nanotechnology in the field of drug delivery. Using nanotechnology, it is possible to package drugs at a nano-scale level to improve bioavailability, increase plasma circulation time and achieve tissue specific drug delivery with concomitant reduction in possible adverse side effects.

Nanodisks (ND) are self assembled nanoparticles comprised of a phospholipid bilayer stabilized by an apolipoprotein "scaffold". ND can be formulated with significant amounts of bioactive agents. The ND architecture is reminiscent of nascent high density lipoprotein (HDL) particles. In the physiological context, nascent HDL picks up excess cholesterol from peripheral tissues and transports it to the liver for excretion or re-use. In vitro, nascent HDL particles can be reconstituted by incubating phospholipid vesicles with apolipoprotein molecules. In reconstituted HDL (rHDL) the phospholipids arrange as a disk-shaped bilayer with two or more apolipoprotein molecules circumscribing the edge of the disk. In recent years rHDL have emerged as a platform for incorporation and transport of hydrophobic and amphipathic biomolecules. To distinguish rHDL loaded with a bioactive agent from physiological HDL we have coined the terminology ND. The focus of my research has been to evaluate ND's ability to function as a vehicle for two different bioactive agents- curcumin, a polyphenol with anti-cancer properties and small interfering RNA (siRNA), novel nucleic acid molecules with enormous therapeutic potential.

Curcumin is water insoluble and oral administration results in poor systemic bioavailability. Hence, the development of a suitable intravenous delivery vehicle is crucial for curcumin's clinical success. Incorporation of curcumin into ND generates a water soluble formulation suitable for intravenous delivery. Compared to free curcumin, curcumin-ND displayed improved cellular uptake of curcumin, which translated into potent biological effects in established tumor cell lines. Strategic use of an apolipoprotein scaffold, known to bind receptors over-expressed on certain tumor cells, caused further improvement in ND mediated curcumin delivery. In other studies, synthetic siRNA mediated silencing of validated disease targets has been shown to improve clinical outcomes in relevant disease models. However, successful therapeutic application of siRNA relies upon the development of a carrier that protects the siRNA molecules from serum nucleases and facilitates their efficient delivery to target tissues, minimizing off-target effects. In my studies I show that an optimum mole percent of a bilayer forming cationic lipid can be included in the ND formulation generating intact nanometer sized positively charged ND (i.e. cationic lipid ND). Cationic lipid ND stably bound 23-mer double stranded oligonucleotides and experiments showed that cationic lipid ND-bound siRNA efficiently knocked down a target gene in hepatocarcinoma cells. Results indicate that cationic lipid ND have the potential to function as a siRNA carrier.

As a delivery vehicle ND possess several advantages. ND components are biocompatible and their assembly is facile. The potential to engineer the apolipoprotein scaffold, in addition to interchangeability of the lipid and scaffold components, makes ND a versatile delivery platform.