Cholesterol and fatty acyl-coenzymeA thioesters are signalling molecules with role in regulation of genes involved in lipid and glucose transport and metabolism. The studies described herein focused on three proteins that bind lipids and have different cellular functions: steroidogenic acute regulatory protein (StAR), hepatocyte nuclear factor-4a (HNF-4a) and acyl-CoA binding protein (ACBP). First, StAR mediates delivery of cholesterol to inner mitochondrial membrane in steroidogenesis by a poorly understood mechanism. In our studies, fluorescent NBD-cholesterol binding assays demonstrate that StAR binds cholesterol at two binding sites with 32 nM Kds and circular dichroism spectra show that cholesterol binding results in changes of StAR secondary structure. Fluorescent sterol exchange assays between donor and acceptor mitochondrial membranes indicate that StAR significantly increased the formation of rapidly transferable cholesterol domains. Second, HNF-4a, a nuclear receptor, had been shown to bind fatty acyl-CoAs as natural ligands with apparent low affinities obtained with radiolabeled ligand binding assays. Our fluorescence spectroscopy studies demonstrate that HNF-4a ligand binding domain (HNF-4aLBD) binds acyl-CoAs at a single binding site with Kds of 1.6-4 nM. Fluorescence resonance energy transfer (FRET) between HNF-4aLBD tryptophan residues and cis-parinaroyl-CoA yielded an intermolecular distance of 42 Â thus pointing to direct molecular interaction. Third, although ACBP has been detected in the nucleus, it is not known whether ACBP may directly and/or functionally interact with a nuclear acyl-CoA binding protein such as HNF-4a to regulate transcription. Our present studies in vitro and in intact cultured cells, including circular dichroism of HNF-4a in the presence of ACBP, coimmunoprecipitation of HNF-4a/ACBP complexes, ACBP and HNF-4a colocalization in nuclei of cells by confocal microscopy demonstrate a physical association of ACBP and HNF-4a. FRET microscopy data indicated an intermolecular distance of 53 Â between ACBP and HNF-4a in rat hepatoma cells. Functional assays (transactivation of an HNF4a-dependent reporter gene) showed significant increase in the presence of ACBP in two different cell lines. Expression of ACBP anti-sense RNA decreased HNF-4a-mediated transactivation, pointing to a role of ACBP in co-regulating HNF-4a-dependent transcription.

Cholesterol and fatty acyl-coenzymeA thioesters are signalling molecules with role in regulation of genes involved in lipid and glucose transport and metabolism. The studies described herein focused on three proteins that bind lipids and have different cellular functions: steroidogenic acute regulatory protein (StAR), hepatocyte nuclear factor-4a (HNF-4a) and acyl-CoA binding protein (ACBP). First, StAR mediates delivery of cholesterol to inner mitochondrial membrane in steroidogenesis by a poorly understood mechanism. In our studies, fluorescent NBD-cholesterol binding assays demonstrate that StAR binds cholesterol at two binding sites with 32 nM Kds and circular dichroism spectra show that cholesterol binding results in changes of StAR secondary structure. Fluorescent sterol exchange assays between donor and acceptor mitochondrial membranes indicate that StAR significantly increased the formation of rapidly transferable cholesterol domains. Second, HNF-4a, a nuclear receptor, had been shown to bind fatty acyl-CoAs as natural ligands with apparent low affinities obtained with radiolabeled ligand binding assays. Our fluorescence spectroscopy studies demonstrate that HNF-4a ligand binding domain (HNF-4aLBD) binds acyl-CoAs at a single binding site with Kds of 1.6-4 nM. Fluorescence resonance energy transfer (FRET) between HNF-4aLBD tryptophan residues and cis-parinaroyl-CoA yielded an intermolecular distance of 42 Â thus pointing to direct molecular interaction. Third, although ACBP has been detected in the nucleus, it is not known whether ACBP may directly and/or functionally interact with a nuclear acyl-CoA binding protein such as HNF-4a to regulate transcription. Our present studies in vitro and in intact cultured cells, including circular dichroism of HNF-4a in the presence of ACBP, coimmunoprecipitation of HNF-4a/ACBP complexes, ACBP and HNF-4a colocalization in nuclei of cells by confocal microscopy demonstrate a physical association of ACBP and HNF-4a. FRET microscopy data indicated an intermolecular distance of 53 Â between ACBP and HNF-4a in rat hepatoma cells. Functional assays (transactivation of an HNF4a-dependent reporter gene) showed significant increase in the presence of ACBP in two different cell lines. Expression of ACBP anti-sense RNA decreased HNF-4a-mediated transactivation, pointing to a role of ACBP in co-regulating HNF-4a-dependent transcription.

The kinetics of the reaction catalyzed by acyl-CoA: sn-glycerol-3-phosphate O-acyltransferase solubilized from Euglena gracilis microsomes were examined. For myristoyl-, palmitoyl-, stearoyl-, and oleoyl-CoAs, the initial reaction rates rose with increasing substrate concentration up to an optimal concentration that varied from 18.5 to 25 μ M, well above the respective critical micelle concentrations. At higher substrate concentrations, reaction was progressively inhibited. Arachidoyl-CoA was a relatively poor substrate for the acyltransferase, and substrate inhibition was not seen with it. Km values for acyl-CoAs ranged from 13 to 20 μ M while the corresponding V values varied almost 40-fold. Bovine serum albumin, among other effects, caused a change in the kinetic pattern of the reaction acyl-CoA dependency. Both acyl-CoA micelles and albumin-bound acyl-CoA were substrates. The binding of palmitoyl- and oleoyl-CoA was 2.7 and 1.5 mol, respectively, per mol of albumin. The critical micelle concentration of palmitoyl-CoA under the reaction conditions was shown by low angle light scattering photometry to be 7.1 p.M. The sn-glycerol 3-phosphate concentration dependency of the acyltransferase initial velocity exhibited Michaelis-Menten kinetics with Km values of 1.3 and 2.9 mM in the presence of 12.5 and 25 μM palmitoyl-CoA, respectively. The substrate analogues sn-glyceraldehyde 3-phosphate and dihydroxyacetone phosphate inhibited the reaction.

Bioluminescence is the chemical production of light that results when a luciferase enzyme catalyzes the luminogenic oxidation of a small-molecule luciferin substrate. The numerous luciferases and luciferins nature has evolved can be used to illuminate biological processes, from in vitro assays to imaging processes in live animals. However, we can improve the utility of bioluminescence through modification of these enzymes and substrates. My thesis work focuses on developing reporters that expand the bioluminescent toolkit and improving our understanding of how bioluminescence works on a molecular level. The first part of my thesis focuses on characterizing luciferases and luciferins that improve bioluminescence imaging in vivo. Some of our luciferins can outperform the natural D-luciferin substrate in live mouse imaging, while others are selectively utilized by mutant luciferases in live mouse brain. We also engineered luciferins that can selectively report on endogenous enzymatic activity in live mice. The second part of my thesis focuses on determining the molecular details of how enzymes related to firefly luciferase, long-chain fatty acyl-CoA synthetases (ACSLs), can function as latent luciferases. I have determined the structure for one of these enzymes and improved its bioluminescent activity with synthetic luciferins enough to image in live mouse brain. I also characterized the selectivity in chimerized enzymes that combine firefly luciferase and ACSLs. In summary, my work improves the utility of bioluminescence for in vivo use and informs us about how evolutionarily-related enzymes function as luciferases on a molecular level.

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