Browsing by Subject "Steroid Hydroxylases"
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Item Genetic Determinants of Human Serum Sterol Levels(2012-12-14) Stiles, Ashlee Renee 1986-; DeBerardinis, Ralph J.; Horton, Jay D.; Cohen, Jonathan C.; Russell, David W.Dozens of different cholesterol metabolites are synthesized by mammalian cells and are known to play important physiological roles in the liver, brain, and immune system. These metabolites are ultimately inactivated by conversion into bile acids in the liver and are thereafter excreted from the body. Mutations in several genes encoding enzymes that metabolize cholesterol have been identified and the clinical consequences of these mutations range from progressive central nervous system neuropathy to spastic paraplegia to liver failure in children. There are several genes in the cholesterol metabolic pathway in which human mutations and their clinical consequences have not yet been identified. For example, the metabolite 24-hydroxycholesterol is produced in the brain by cholesterol 24-hydroxylase (encoded by the CYP46A1 gene). Disruption of this gene in the mouse causes severe learning defects, but it is not known whether mutations in the human gene cause the same phenotype. To overcome the uncertainty inherent in guessing what the phenotype arising from mutations in the human CYP46A1 gene might be and to detect mutations in other genes specifying cholesterol metabolic enzymes, we developed mass-spectrometry based methods to measure >60 different sterol metabolites in small volumes (200 µl) of human serum. We applied these methods to quantify sterols in 3,230 serum samples derived from clinically well-characterized subjects participating in the Dallas Heart Study. Large intra-individual variation was detected in the serum levels of approximately 22 of the >60 sterols assessed. To identify the genes underlying the observed variation, we took both targeted and untargeted approaches. In the targeted approach, advantage was taken of the fact that the substrates and products of many sterol metabolizing enzymes are known and we thus sequenced the corresponding genes in subjects with very high or low levels of the sterol in question. In the untargeted approach, the 3,230 individuals whose sterol levels were measured were genotyped for 9,229 non-synonymous (amino acid-changing) variations (SNPs) in multiple human genes. For 24-hydroxycholesterol, the metabolite mentioned above, both approaches yielded concordant results. Direct sequencing of the oxysterol 7α-hydroxylase gene (CYP39A1), which uses 24-hydroxycholesterol as a substrate, identified several mutations that decrease enzyme activity and thus lead to increased serum levels of this sterol substrate. In the genetic linkage analysis, a SNP in CYP39A1 was strongly (P=2.1 x 10-27) correlated with serum 24-hydroxycholesterol levels. Expression analysis indicated that this SNP also decreased CYP39A1 enzyme activity. Together, these results illustrate that our approach has the ability to identify genetic determinants of serum sterol levels. Additional correlations were identified between a SNP in EPHX2 and levels of 24,25-epoxycholesterol (P=9.6 x 10-25) and a SNP in SDR42E1 and levels of 8-dehydrocholesterol levels (P=1.5 x 10-15). Biochemical assays were performed to characterize these previously unidentified genetic associations and to reveal their effect on oxysterol and sterol metabolism. Simultaneously, SNP genotypes and sterol levels were correlated with specific clinical phenotypes in an effort to shed light on the role of non-cholesterol sterols in disease. The next step is to apply these methods to the other 19 sterols that are routinely detected in serum and to correlate the analytical and genetic findings with the >100 clinical phenotypes of the 3,230 subjects whose sterol levels we have measured.Item Identification of an Interferon Signaling Pathway Linking Membrane Cholesterol Accessibility to Host Defense Against Pathogens(2020-08-01T05:00:00.000Z) Abrams, Michael Edward; Shiloh, Michael; Alto, Neal; Radhakrishnan, Arun; Schoggins, John W.Interferon-γ (IFN-γ) is a multipotent cytokine that is critical to the host innate immune defense against bacterial infection, and functions through transcriptional induction of hundreds of IFN-γ stimulated genes (γ-ISGs). However, the antibacterial roles of many γ-ISGs remain poorly defined. Here, I describe my efforts to characterize mechanisms by which specific γ-ISGs confer cell-intrinsic immunity against bacterial pathogens. Unexpectedly, I found that IFN-γ-activated macrophages secreted a soluble product that potently inhibited infection of the Gram-positive intracellular pathogen Listeria monocytogenes. To identify this factor, I first created a cDNA lentiviral library of more than 400 highly representative γ-ISGs and carried out a gain-of-function flow cytometry screen to determine the effect of each gene on infection. The results of this screen identified eight genes that potently reduce infection. Next, I determined whether these γ-ISGs produced a soluble molecule that could suppress L. monocytogenes infection in trans. Notably, conditioned media from Cholesterol 25-Hydroxylase (CH25H)-expressing cells potently enhanced bacterial resistance of naive cells, suggesting it may be responsible for the previously observed activity in macrophages. Indeed, Ch25h-/- macrophages failed to produce a soluble antibacterial metabolite, while the enzymatic product of CH25H, 25-Hydroxycholesterol (25HC), potently inhibited L. monocytogenes infection. I demonstrated that 25HC inhibits infection of non-phagocytic cells from diverse tissue lineages, and that administration of 25HC to mice significantly reduced bacterial burden in an oral gavage model. Furthermore, I found that 25HC blocks L. monocytogenes cell-to-cell spread by attenuating the formation of plasma membrane protrusions. In collaboration with the post-doctoral fellow Kristen Johnson, I have used toxin-based biosensors to determine that activation of acyl-CoA: cholesterol acyltransferase (ACAT) rapidly mobilized a specific pool of cholesterol termed “accessible cholesterol” from the plasma membrane (PM) to the ER. Importantly, the antibacterial function of 25HC was dependent on this ability to deplete PM accessible cholesterol. Together, these studies have uncovered a heretofore unknown mechanism by which IFN-mediated reorganization of the PM restricts dissemination of intracellular pathogens.Item Role of Cholesterol 24-Hydroxylase in Hippocampal Long-Term Potentiation(2009-06-18) Ramirez, Denise Marie O'Donnell; Russell, David W.The mammalian brain contains a disproportionately large percentage of the body's cholesterol, steady-state levels of which are maintained within a narrow range to preserve membrane function. The brain is denied access to circulating lipoproteins by the blood-brain barrier and therefore relies on de novo cholesterol synthesis through the mevalonate pathway to meet the tissue's requirement for this essential lipid. A small amount of brain cholesterol is turned over daily in select neurons by cholesterol 24-hydroxylase, which catalyzes the production of the membrane-permeable oxysterol 24(S)-hydroxycholesterol and represents the major pathway of cholesterol catabolism in this organ. Mice lacking 24-hydroxylase have a decreased rate of brain cholesterol synthesis and exhibit deficiencies in spatial, associative, and motor learning. Hippocampal slices prepared from these mice are unable to support the induction of long-term potentiation, a type of synaptic strengthening thought to underlie learning and memory. The ability of 24-hydroxylase knockout slices to exhibit long-term potentiation can be restored by treatment with geranylgeraniol, an isoprenoid end-product of the mevalonate pathway. Mechanistic insight into the role of geranylgeraniol in long-term potentiation has been revealed by calcium imaging studies in neurons cultured from wild-type and 24-hydroxylase knockout embryos. Neurons from mice lacking 24-hydroxylase have specific defects in N-methyl-D-aspartate (NMDA) receptor function, a subtype of ionotropic glutamate receptor essential for long-term potentiation. The subunit composition of NMDA receptors located in various functional pools is normal in 24-hydroxylase knockout hippocampus, suggesting that geranylgeraniol does not affect expression of NMDA receptors. Localization studies of 24-hydroxylase show the enzyme is predominantly expressed in the endoplasmic reticulum throughout the soma and dendrites of selected hippocampal, cerebellar, and cortical neurons, consistent with a postsynaptic need for cholesterol turnover in neurons of brain regions important for learning and memory. These findings reveal that cholesterol turnover is important to produce a constant supply of geranylgeraniol, which in turn is necessary for the induction of long-term potentiation and presumably learning in mice.