Multifactor Dimensionality Reduction Analysis Identifies Specific Nucleotide Patterns Promoting Genetic Polymorphisms

The fidelity of DNA replication serves as the nidus for both genetic evolution and genomic instability fostering disease. Single nucleotide polymorphisms (SNPs) constitute greater than 80% of the genetic variation between individuals. A new theory regarding DNA replication fidelity has emerged in which selectivity is governed by base-pair geometry through interactions between the selected nucleotide, the complementary strand, and the polymerase active site. We hypothesize that specific nucleotide combinations in the flanking regions of SNP fragments are associated with mutation

Dioxin Alters Human Low-Density and Very Low-Density Lipoprotein Structure with Evidence for Specific Quenching of TRP-48 in Apolipoprotein C-H

Low-density lipoproteins (LDL) and very low-density lipoproteins (VLDL) are essential for transporting cholesterol and triglycerides in the blood. Intimate to this process is the interaction between the lipoprotein molecule and its cellular receptor. This pathway involves the LDL receptor (LDLR) interacting with apolipoprotein Apo B-100 in both LDLs and VLDLs and additionally lipoprotein lipase (LPL) with Apo C-I1 in the case of VLDL. Recent investigations link 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure in humans to heart disease. Due to TCDD\u27s non-polar nature, it is reasonable to expect this compound to bind serum lipoproteins in vivo. Here we show TCDD to tightly bind human LDL and VLDL in vitro inducing a change in the lipoprotein conformation using fluorescence spectroscopy. We also show TCDD\u27s ability to bind Trp-48 within the lipase binding region of apolipoprotein C-11. In order to understand TCDD\u27s affinity for the protein components of lipoproteins, computational studies show TCDD\u27s ability to form hydrogen bonds with certain amino acid residues, specifically tryptophan. These results indicate TCDD has a far greater ability to bind protein components within lipoprotein molecules compared with polychlorinated biphenyls (PCBs) controls. Circular dichroism spectroscopy (CD) shows that TCDD binding LDL and apolipoprotein C-I1 denatures the protein\u27s secondary structure. In the case of apolipoprotein C-11, the inner core composed of a four-a-helix bundle is lost. CD also shows denaturing of LDL when treated with TCDD with a loss of both a-helix and Psheet secondary structure. These studies show that at low concentrations of TCDD (3: 1 TCDD to protein), TCDD binds and diminishes overall protein structure. Furthermore, this work shows that when TCDD binds the apolipoprotein C-11, apoC-I1 is unable to bind lipoprotein lipase. This interaction may lead to the inactivation of LDL and VLDL receptor recognition contributing to a buildup of serum lipoproteins and lipids thus hastening the development of atherosclerosis

Conserved Rhodopsin Intradiscal Structural Motifs Mediate Stabilization: Effects of Zinc

Retinitis pigmentosa (RP), a neurodegenerative disorder, can arise from single point mutations in rhodopsin, leading to a cascade of protein instability, misfolding, aggregation, rod cell death, retinal degeneration, and ultimately blindness. Divalent cations, such as zinc and copper, have allosteric effects on misfolded aggregates of comparable neurodegenerative disorders including Alzheimer disease, prion diseases, and ALS. We report that two structurally conserved low-affinity zinc coordination motifs, located among a cluster of RP mutations in the intradiscal loop region, mediate dose-dependent rhodopsin destabilization. Disruption of native interactions involving histidines 100 and 195, through site-directed mutagenesis or exogenous zinc coordination, results in significant loss of receptor stability. Furthermore, chelation with EDTA stabilizes the structure of both wild-type rhodopsin and the most prevalent rhodopsin RP mutation, P23H. These interactions suggest that homeostatic regulation of trace metal concentrations in the rod outer segment of the retina may be important both physiologically and for an important cluster of RP mutations. Furthermore, with a growing awareness of allosteric zinc binding domains on a diverse range of GPCRs, such principles may apply to many other receptors and their associated diseases

Comprehensive Biochemical Analysis of Rare Prostacyclin Receptor Variants: STUDY OF ASSOCIATION OF SIGNALING WITH CORONARY ARTERY OBSTRUCTION*

Currently, pharmacogenetic studies are at an impasse as the low prevalence (<2%) of most variants hinder their pharmacogenetic analysis with population sizes often inadequate for sufficiently powered studies. Grouping rare mutations by functional phenotype rather than mutation site can potentially increase sample size. Using human population-based studies (n = 1,761) to search for dysfunctional human prostacyclin receptor (hIP) variants, we recently discovered 18 non-synonymous mutations, all with frequencies less than 2% in our study cohort. Eight of the 18 had defects in binding, activation, and/or protein stability/folding. Mutations (M113T, L104R, and R279C) in three highly conserved positions demonstrated severe misfolding manifested by impaired binding and activation of cell surface receptors. To assess for association with coronary artery disease, we performed a case-control study comparing coronary angiographic results from patients with reduced cAMP production arising from the non-synonymous mutations (n = 23) with patients with non-synonymous mutations that had no reduction in cAMP (n = 17). Major coronary artery obstruction was significantly increased in the dysfunctional mutation group in comparison with the silent mutations. We then compared the 23 dysfunctional receptor patients with 69 age- and risk factor-matched controls (1:3). This verified the significantly increased coronary disease in the non-synonymous dysfunctional variant cohort. This study demonstrates the potential utility of in vitro functional characterization in predicting clinical phenotypes and represents the most comprehensive characterization of human prostacyclin receptor genetic variants to date

Acceleration of cardiovascular disease by a dysfunctional prostacyclin receptor mutation - Potential implications for cyclooxygenase-2 inhibition

Recent increased adverse cardiovascular events observed with selective cyclooxygenase-2 inhibition led to the withdrawal of rofecoxib ( Vioxx) and valdecoxib ( Bextra), but the mechanisms underlying these atherothrombotic events remain unclear. Prostacyclin is the major end product of cyclooxygenase-2 in vascular endothelium. Using a naturally occurring mutation in the prostacyclin receptor, we report for the first time that a deficiency in prostacyclin signaling through its G protein-coupled receptor contributes to atherothrombosis in human patients. We report that a prostacyclin receptor variant ( R212C) is defective in adenylyl cyclase activation in both patient blood and in an in vitro COS-1 overexpression system. This promotes increased platelet aggregation, a hallmark of atherothrombosis. Our analysis of patients in 3 separate white cohorts reveals that this dysfunctional receptor is not likely an initiating factor in cardiovascular disease but that it accelerates the course of disease in those patients with the greatest risk factors. R212C was associated with cardiovascular disease only in the high cardiovascular risk cohort ( n = 980), with no association in the low-risk cohort ( n = 2293). In those at highest cardiovascular risk, both disease severity and adverse cardiovascular events were significantly increased with R212C when compared with age- and risk factor-matched normal allele patients. We conclude that for haploinsufficient mutants, such as the R212C, the enhanced atherothrombotic phenotype is likely dependent on the presence of existing atherosclerosis or injury ( high risk factors), analogous to what has been observed in the cyclooxygenase-2 inhibition studies or prostacyclin receptor knockout mice studies. Combining both biochemical and clinical approaches, we conclude that diminished prostacyclin receptor signaling may contribute, in part, to the underlying adverse cardiovascular outcomes observed with cyclooxygenase-2 inhibition

Acceleration of Cardiovascular Disease by a Dysfunctional Prostacyclin Receptor Mutation: Potential Implications for Cyclooxygenase-2 Inhibition

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