published_or_final_version / abstract / Biological Sciences / Doctoral / Doctor of Philosophy

In order to support rampant cell growth, tumor cells must reprogram metabolism to simultaneously drive macromolecular biosynthesis and energy production. Mitochondrial uncoupling proteins (UCPs) oppose this phenotype by inducing futile mitochondrial respiration that is disengaged from ATP synthesis. We found that uncoupling protein 3 (UCP3) was normally expressed in follicular and epidermal keratinocytes and that its levels were augmented by calcium-induced differentiation in vitro. Over-expression of a UCP3 transgene targeted to the basal epidermis by the keratin-5 promoter (K5-UCP3) led to increased differentiation of both epidermal and bulge stem cells, the progenitors of most squamous carcinomas. Consistent with this phenotype, K5-UCP3 mice were completely protected from chemically induced skin carcinogenesis. To define the mechanisms by which UCP3 conferred such strong tumor resistance, we interbred K5-UCP3 mice with a “pre-initiated” mouse model, and found that UCP3 over-expression blocked tumor promotion. Uncoupled epidermis displayed reduced proliferation after treatment with tumor promoter, along with diminished activation of Akt signaling. This effect corresponded to decreased Akt activation by epidermal growth factor (EGF) in K5-UCP3 cells, along with UCP3 overexpressing primary human keratinocytes. Mechanistic studies revealed that uncoupling drove global lipid catabolism, along with impaired recruitment of Akt to the plasma membrane. Over-expression of wild type Akt rescued tumor promoter-induced proliferation and two-stage chemical carcinogenesis in bi-transgenic mice. Collectively, these findings demonstrate that mitochondrial uncoupling is an effective strategy to limit cell proliferation and tumorigenesis through inhibition of Akt, and suggest a novel mechanism of crosstalk between mitochondrial metabolism and growth signaling. / text

<p>There are many types of membrane lipids throughout Nature. Still little is known about synthesizing pathways and how different lipids affect the embedded membrane proteins. The most common lipids are glycolipids since they dominate plant green tissue. Glycolipids also exist in mammal cells as well as in most Gram-positive bacteria. Glycosyltransferases (GTs) catalyze the final enzymatic steps for these glycolipids. In the bacteria <i>Acholeplasma laidlawii</i> and <i>Streptococcus pneumonie</i> and in the plant <i>Arabidopsis thaliana</i>, GTs for mono-/di-glycosyl-diacylglycerol (-DAG) are suggested to be regulated to keep a certain membrane curvature close to a bilayer/nonbilayer phase transition. The monoglycosylDAGs are nonbilayer-prone with small headgroups, hence by themselves they will not form bilayer structures.</p><p>Here we have determined the genes encoding the main glycolipids of <i>A. laidlawii</i> and <i>S. pneumonie</i>. We have also shown that these GTs belong to a large enzyme group widely spread in Nature, and that all four enzymes are differently regulated by membrane lipids. The importance of different lipid properties were traced in a lipid mutant of <i>Escherichia coli</i> lacking the major (75 %), nonbilayer-prone/zwitterionic, lipid phosphatidylethanolamine. Introducing the genes for the GTs of <i>A. laidlawii</i> and two analogous genes from <i>A. thaliana</i> yielded new strains containing 50 percent of glyco-DAG lipids. The monoglyco-DAG strains contain significant amounts of nonbilayer-prone lipids while the diglyco-DAG strains contain no such lipids. Comparing these new strains for viability and the state of membrane-associated functions made it possible to connect different functions to certain lipid properties. In summary, a low surface charge density of anionic lipids is important in <i>E.coli</i> membranes, but this fails to be supportive if the diluting species have a too large headgroup. This indicates that a certain magnitude of the curvature stress is crucial for the membrane bilayer <i>in vivo</i>.</p>

The effects of different dietary lipids on the growth, nutrition and tissue lipid profiles of rainbow trout raised to market size on a commercially available ration were examined. Rainbow trout of 80 g mean initial weight were fed pellets prepared according to Oregon Moist Pellet specifications for 20 weeks. Salmon oil (0MP-1), soybean oil (OMP-2), linseed oil (OMP-3), chicken fat (OMP-4), pork lard (OMP-5) and beef tallow (OMP-6) were used for the 6% lipid component of the diets. No differences in feed conversion or growth rate were observed across diet groups. Trout nutrition was monitored by three blood parameters -- red blood cell (RBC) fragility (hemolysis), packed cell volume (PCV) (hematocrit), and percent hemoglobin. The OMP-1 diet appeared to have caused oxidative stress in trout as measured by hemolysis, while hematocrit and percent hemoglobin values were uniform across diet groups. Tissue levels of total n-3 fatty acids were highest in the OMP-3 group and decreased in the order OMP-1 > OMP-2 = OMP-4 = OMP-5 = OMP-6. However, unaltered linolenic acid (18:3 n-3) comprised almost 50% of the n-3 fatty acid content of OMP-3 trout tissue lipid. The level of total 20:5 n-3 plus 22:6 n-3 (18%), was 33% higher in tissue from OMP-1 trout than tissue from all other diet groups which held fairly constant at 12% across OMP groups 3-6. The lowest tissue level of total 20:5 n-3 plus 22:6 n-3 was found in the OMP-2 trout, 10%. Trout raised on the OMP-1 diet also retained higher tissue stores of 20 and 22 carbon monoenes than trout fed OMP diets 2-6, but less than dietary levels. In contrast, trout fed OMP diets 2-6 retained higher tissue levels of these same fatty acids than dietary levels. The diet ratio of polyunsaturated fatty acids (PUFA)/22:1 appeared to influence whether 22:1 was conserved or oxidized. The most favorable balance of trout tissue monoenes, n-6 fatty acids and total 20:5 n-3 plus 22:6 n-6 for human health was found in trout fed the OMP-6 diet. / Graduation date: 1988

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Suspended lipid bilayers, or black lipid membranes (BLMs), have been used to study the electrophysiological properties of ion channels (ICs); however, BLMs assembled from natural, non-polymerizable lipids are inherently unstable due to the non-covalent associations on which they are based. Lifetimes of several hours are commonly observed in BLMs until rupture due to mechanical, thermal, or chemical insults. One potential improvement is the use of polymerizable phospholipids (poly(lipids)). BLMs prepared using dienoyl functionalized poly(lipids) and binary mixtures of fluid, non-polymerizable lipids with poly(lipids) were investigated for IC recordings.poly(BLMs) exhibited enhanced lifetimes from several hours to upwards of 4 weeks while maintaining IC functionality for one week. Activity of ICs that require membrane fluidity was retained using binary phospholipid mixtures of fluid and polymeric phospholipids. IC activity was retained by inducing domain formation, wherein ICs incorporated into the fluid domains. The binary membranes exhibited marked enhancement in stability resulting from fractional poly(lipids) polymerization. Additionally, ICs can be reconstituted into the fluid domains following photopolymerization and subsequent domain formation, a key requirement when UV-sensitive ICs are utilized. Here, the electrical properties, stability, and incorporation of pore-forming ICs, including hemolysin, alamethicin, and gramicidin, into poly(lipid) membranes are reported. Potential applications developing ligand-gated IC based sensors for high throughput screening are being investigated.In parallel to the characterization of poly(lipids) for potential long-term IC membranes, a model ligand-gated IC was expressed, characterized, and reconstituted into non-polymerizable lipids. Mutant K_ATP channels were expressed in mammalian and yeast systems. The orientations of mutant K_ATP channels were studied using electrophysiological and immunohistochemical techniques. Large quantities were expressed and purified from <italic>Pichia pastoris</italic> and functionally reconstituted into BLMs. ATP and long-chaing coenzyme A ester sensitivity was maintained in reconstituted in BLMs. K_ATP channels will serve as a model system for testing the effect of poly(lipid) BLMs on IC function. Future utilization of poly(lipid) BLMs in combination with ligand-gated ICs offer major advancements to potential increased throughput for IC screening.

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