No description available.

No description available.

No description available.

Diacylglycerol kinases (DGK’s) tightly regulate the intracellular levels of diacylglycerol (DAG) and phosphatidic acid (PA). DAG is an important intermediate in lipid biosynthetic pathways and acts as a lipid second messenger in a number of signaling pathways. Similarly, since PA serves as a potent signaling lipid and is a precursor for lipid biosynthesis, intracellular PA levels must be tightly regulated. There are ten isoforms of DGK in mammals, but we have decided to focus solely on the epsilon form (DGKε) in this work. DGKε is the only isoform that shows specificity for the acyl chains of its DAG substrate; as a consequence, it contributes to the dramatic enrichment of cellular lipids with sn-1 stearoyl and sn-2 arachidonoyl. The most notable example is the highly enriched bioactive lipid 1-stearoyl-2-arachidonoyl phosphatidylinositol. We have purified active human DGKε to near homogeneity and thoroughly characterized its stability as well as examined its secondary structure with CD. We also purified a truncated form (DGKε Δ40) that shows increased stability compared to the full-length protein. Our purified fractions are well suited for a wide range of exciting applications and studies. We have begun incorporating DGKε into liposomes in order to develop a liposome-based assay, which would be a dramatic improvement over the presently used micelle-based assay. This purification also allows for high throughput screens of chemical compounds to test for a specific inhibitor. These studies will reveal valuable information about the structural and functional properties of DGKε and will aid in the development of therapies for DGKε-related diseases. / Thesis / Master of Science (MSc)

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The basic unit of eukaryotic chromatin is the nucleosome core, which contains 147 base pairs of DNA wrapped around an octamer of core histone proteins. Linker histones bind through their globular domain at the nucleosome dyad and to internucleosomal DNA through their C-terminal basic tail. The Saccharomyces cerevisiae linker histone homologue, Hho1p, contains two domains, GI and GII, that have sequence similarity to the globular domain of the canonical linker histone H1. The individual domains of Hho1p differ in their structural and functional properties, for example in 10 mM sodium phosphate GI is folded while GII exists as two species: folded and 'unfolded'. In Chapter 2 the structure of the second globular domain of Hho1p, GII, is further investigated. NMR studies indicate residual structure in the 'unfolded' form of GII, especially at the start of helices I and III. Chapter 3 considers the structural roles of Hho1p within chromatin. Semi-quantitative Western blotting is used to measure the abundance of Hho1p relative to nucleosomes in yeast. Analysis of reconstituted nucleosome arrays containing NGIL (Hho1p with the second globular domain removed) are indistinguishable from those containing full-length Hho1p, in gel-based assays and by analytical ultracentrifugation, suggesting the GII domain may not have a major role in chromatin compaction. Chapter 4 focuses on the interaction of Hho1p with chromatin proteins. Chemical cross-linking and gel filtration indicate that Hho1p does not interact significantly with the putative HMGB1 homologues Hmo1p and Nhp6ap in vitro. Hho1p and Htz1p, the yeast histone H2A.Z subtype, do not appear to interact directly in co-immunoprecipitation and chemical cross-linking assays, while chromatin immunoprecipitation studies show no evidence of colocalisation across the ADH2 and PHO5 genes. Hho1p and Sir2p cross-link in solution, but purification difficulties precluded further investigation. The effect of phosphorylation on the interaction of Hho1p and related truncation proteins with DNA and chromatin are investigated in Chapter 5. Phosphorylation reduces their affinity for linear DNA, but has different effects on the binding to four-way junction DNA for Hho1p and NGIL, compared with LGII (the linker region and GII domain of Hho1p). Phosphorylation has no obvious effect on the affinity of these proteins for chromatin in sucrose gradient centrifugation assays. NMR spectroscopy studies show that the linker region is mostly unstructured, with a short region showing some α-helical character. Phosphorylation of the linker domain changes its structural character.

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The homologous p10 fusion-associated small transmembrane (FAST) proteins of the fusogenic avian (ARV) and Nelson Bay (NBV) reoviruses are the smallest known proteins capable of mediating syncytiogenesis. Their extremely small size precludes them from following the paradigmatic membrane fusion pathway proposed for enveloped viral fusion proteins. I exploited the sequence conservation/divergence and differential syncytiogenic rates between ARV and NBV to define functional motifs in the p10 ectodomains. Using chimeric p10 constructs, I determined the 40-residue ectodomain (sizes refer to ARV) comprises two distinct functional motifs essential for syncytiogenesis. Cellular syncytiogenic and surface biotinylation assays identified an indivisible, 25- residue, N-terminal ectodomain motif required for cystine loop fusion peptide formation. I further determined the roles of this cystine loop in promoting lipid binding and cholesterol-dependent lipid destabilization. Immunofluorescence staining, FRET analysis and cholesterol depletion/repletion studies identified a second motif comprising the 13 membrane-proximal ectodomain residues (MPER). This motif governs the reversible, cholesterol-dependent assembly of p10 multimers in the plasma membrane. I demonstrate that ARV and NBV homomultimers segregate to separate foci in the plasma membrane, and the four juxtamembrane residues present in the multimerization motif dictate species- specific homomultimerization. I also discovered the novel codependency of p10 multimerization and cholesterol-dependent microdomain localization. The majority of enveloped virus membrane fusion proteins function as stable multimers, which nonetheless must undergo dramatic, irreversible, tertiary structure rearrangements to mediate membrane fusion. Cholesterol-rich membrane microdomains have also been implicated in the function of several enveloped virus fusion proteins, and a limited number of studies have investigated the role of cholesterol in multimerization. My results reveal cholesterol-dependent p10 homomultimerization is an essential aspect of p10- mediated syncytium formation, and I identify the motifs responsible for this process. The reversible nature of p10 cholesterol-dependent multimerization at the plasma membrane is in line with several other studies suggesting that the dynamic clustering and dispersion of cholesterol microdomains, as well as protein transitioning from multimeric to monomeric intermediates, are essential phenomena of protein mediated membrane fusion.

Proteins perform an incredible array of functions facilitated by a diverse set of biochemical properties. Changing these properties is an essential molecular mechanism of evolutionary change, with major questions in protein evolution surrounding this topic. How do new functional biochemical features evolve? How do proteins change following gene duplication events? I used the S100 protein family as a model to probe these aspects of protein evolution. The S100s are signaling proteins that play a diverse range of biological roles binding Calcium ions, transition metal ions, and other proteins. Calcium drives a conformational change allowing S100s to bind to diverse peptide regions of target proteins. I used a phylogenetic approach to understand the evolution of these diverse biochemical features. Chapter I comprises an introduction to the disseration. Chapter II is a co-authored literature review assessing available evidence for global trends in protein evolution. Chapter III describes mapping of transition metal binding onto a maximum likelihood S100 phylogeny. Transition metal binding sites and metal-driven structural changes are a conserved, ancestral features of the S100s. However, they are highly labile at the amino acid level. Chapter IV further characterizes the biophysics of metal binding in the S100A5 lineage, revealing that the oft–cited Ca2+/Cu2+ antagonism of S100A5 is likely due to an experimental artifact of previous studies. Chapter V uses the S100 family to investigate the evolution of binding specificity. Binding specificity for a small set of peptides in the duplicate S100A5 and S100A6 clades. Ancestral sequence reconstruction reveals a pattern of clade-level conservation and apparent subfunctionalization along both lineages. In chapter VI, peptide phage display, deep-sequencing, and machine-learning are combined to quantitatively reconstruct the evolution of specificity in S100A5 and S100A6. S100A5 has subfunctionalized from the ancestor, while S100A6 specificity has shifted. The importance of unbiased approaches to measure specificity are discussed. This work highlights the lability of conserved functions at the biochemical level, and measures changes in specificity following gene duplication. Chapter VII summarizes the results of the dissertation, considers the implications of these results, and discusses limitations and future directions. This dissertation includes both previously published/unpublished and co- authored material.

Cells transduce signals from the external environment to the inside through phosphatidylinositol-3,4,5-phosphate (PIP3), a major signaling lipid on the plasma membrane. PIP3 is generated by the action of a family of lipid kinases called Class I phosphoinositide 3-kinases (PI3Ks) and controls an array of essential cellular functions including growth, proliferation, survival, metabolism and cytoskeletal architecture. PI3Ks are large heterodimeric complexes composed of a catalytic p110 subunit and a regulatory subunit. Crucial to healthy PIP3 production is the interpretation of diverse activating inputs arising from signaling proteins on the membrane by these subunits. A member of the PI3K family, PI3Kγ is a master regulator of immune functions with therapeutic implications in cancer immunity and inflammatory disease. PI3Kγ is distinct from other well studied PI3Ks due to the presence of unique regulatory mechanisms that control its ability to integrate signals from G-protein coupled receptors, small GTPases, immunoglobulin receptors and toll-like receptors. However, unlike the other well characterized PI3Ks, there are significant gaps in understanding of the molecular details of these mechanisms and how regulatory processes are translated into functions elicited by PI3Kγ in its unique milieu within the immune system. To understand PI3Kγ regulation, I utilized a synergy of cutting-edge approaches including protein biochemistry, X-ray crystallography, cryo-electron microscopy and hydrogen-deuterium exchange mass spectrometry to elucidate the unique regulatory features within its catalytic and regulatory subunits and how these features are disrupted in disease. These studies significantly advanced our understanding of how this enzyme functions and provided novel avenues for potentially targeting the enzyme better in therapy. This dissertation will consist of an introduction chapter summarizing PI3Kγ regulation and its role in disease, followed by three data chapters investigating previously uncharacterized regulatory mechanisms that control its function and how these mechanisms are implicated in disease. These data chapters are followed by a final chapter describing conclusions and future directions. In summary, the work presented in this thesis provides novel insights into the unique regulatory features in the catalytic and regulatory subunits of PI3Kγ that mediate its stimulation by upstream activating partners and the mechanisms by which these features are disrupted in disease. Further, these studies have facilitated the effective characterization of potent molecules that can specifically target PI3Kγ in disease. Altogether, the findings of this dissertation constitute a major advancement in our understanding of PI3K regulation. / Graduate