Blood Lipids, Lipoproteins, and Apolipoproteins With Risk of Coronary Heart Disease: A Prospective Study Among Racially Diverse Populations.

BACKGROUND: Comprehensive blood lipoprotein profiles and their association with incident coronary heart disease (CHD) among racially and geographically diverse populations remain understudied. METHODS AND RESULTS: We conducted nested case-control studies of CHD among 3438 individuals (1719 pairs), including 1084 White Americans (542 pairs), 1244 Black Americans (622 pairs), and 1110 Chinese adults (555 pairs). We examined 36 plasma lipids, lipoproteins, and apolipoproteins, measured by nuclear magnetic resonance spectroscopy, with incident CHD among all participants and subgroups by demographics, lifestyle, and metabolic health status using conditional or unconditional logistic regression adjusted for potential confounders. Conventionally measured blood lipids, that is, total cholesterol, triglycerides, low-density lipoprotein-cholesterol, and high-density lipoprotein-cholesterol, were each associated with incident CHD, with odds ratios (ORs) being 1.33, 1.32, 1.24, and 0.79 per 1-SD increase among all participants. Seventeen lipoprotein biomarkers showed numerically stronger associations than conventional lipids, with ORs per 1-SD among all participants ranging from 1.35 to 1.57 and a negative OR of 0.78 (all false discovery rate <0.05), including apolipoprotein B100 to apolipoprotein A1 ratio (OR, 1.57 [95% CI, 1.45-1.7]), low-density lipoprotein-triglycerides (OR, 1.55 [95% CI, 1.43-1.69]), and apolipoprotein B (OR, 1.49 [95% CI, 1.37-1.62]). All these associations were significant and consistent across racial groups and other subgroups defined by age, sex, smoking, obesity, and metabolic health status, including individuals with normal levels of conventionally measured lipids. CONCLUSIONS: Our study highlighted several lipoprotein biomarkers, including apolipoprotein B/ apolipoprotein A1 ratio, apolipoprotein B, and low-density lipoprotein-triglycerides, strongly and consistently associated with incident CHD. Our results suggest that comprehensive lipoprotein measures may complement the standard lipid panel to inform CHD risk among diverse populations.

Verteporfin is a substrate-selective γ-secretase inhibitor that binds the amyloid precursor protein transmembrane domain.

This work reports substrate-selective inhibition of a protease with broad substrate specificity based on direct binding of a small-molecule inhibitor to the substrate. The target for these studies was γ-secretase protease, which cleaves dozens of different single-span membrane protein substrates, including both the C99 domain of the human amyloid precursor protein and the Notch receptor. Substrate-specific inhibition of C99 cleavage is desirable to reduce production of the amyloid-ß polypeptide without inhibiting Notch cleavage, a major source of toxicity associated with broad specificity γ-secretase inhibitors. In order to identify a C99-selective inhibitors of the human γ-secretase, we conducted an NMR-based screen of FDA-approved drugs against C99 in model membranes. From this screen, we identified the small-molecule verteporfin with these properties. We observed that verteporfin formed a direct 1:1 complex with C99, with a KD of 15-47 µM (depending on the membrane mimetic used), and that it did not bind the transmembrane domain of the Notch-1 receptor. Biochemical assays showed that direct binding of verteporfin to C99 inhibits γ-secretase cleavage of C99 with IC50 values in the range of 15-164 µM, while Notch-1 cleavage was inhibited only at higher concentrations, and likely via a mechanism that does not involve binding to Notch-1. This work documents a robust NMR-based approach to discovery of small-molecule binders to single-span membrane proteins and confirmed that it is possible to inhibit γ-secretase in a substrate-specific manner.

DNA Sequence Modulates the Efficiency of NEIL1-Catalyzed Excision of the Aflatoxin B1-Induced Formamidopyrimidine Guanine Adduct.

Dietary exposure to aflatoxins is a significant risk factor in the development of hepatocellular carcinomas. Following bioactivation by microsomal P450s, the reaction of aflatoxin B1 (AFB1) with guanine (Gua) in DNA leads to the formation of stable, imidazole ring-opened 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxyaflatoxin B1 (AFB1-FapyGua) adducts. In contrast to most base modifications that result in destabilization of the DNA duplex, the AFB1-FapyGua adduct increases the thermal stability of DNA via 5'-interface intercalation and base-stacking interactions. Although it was anticipated that this stabilization might make these lesions difficult to repair relative to helix distorting modifications, prior studies have shown that both the nucleotide and base excision repair pathways participate in the removal of the AFB1-FapyGua adduct. Specifically for base excision repair, we previously showed that the DNA glycosylase NEIL1 excises AFB1-FapyGua and catalyzes strand scission in both synthetic oligodeoxynucleotides and liver DNA of exposed mice. Since it is anticipated that error-prone replication bypass of unrepaired AFB1-FapyGua adducts contributes to cellular transformation and carcinogenesis, the structural and thermodynamic parameters that modulate the efficiencies of these repair pathways are of considerable interest. We hypothesized that the DNA sequence context in which the AFB1-FapyGua adduct is formed might modulate duplex stability and consequently alter the efficiencies of NEIL1-initiated repair. To address this hypothesis, site-specific AFB1-FapyGua adducts were synthesized in three sequence contexts, with the 5' neighbor nucleotide being varied. DNA structural stability analyses were conducted using UV absorbance- and NMR-based melting experiments. These data revealed differentials in thermal stabilities associated with the 5'-neighbor base pair. Single turnover kinetic analyses using the NEIL1 glycosylase demonstrated corresponding sequence-dependent differences in the repair of this adduct, such that there was an inverse correlation between the stabilization of the duplex and the efficiency of NEIL1-mediated catalysis.

Analysis of DNA binding by human factor xeroderma pigmentosum complementation group A (XPA) provides insight into its interactions with nucleotide excision repair substrates.

Xeroderma pigmentosum (XP) complementation group A (XPA) is an essential scaffolding protein in the multiprotein nucleotide excision repair (NER) machinery. The interaction of XPA with DNA is a core function of this protein; a number of mutations in the DNA-binding domain (DBD) are associated with XP disease. Although structures of the central globular domain of human XPA and data on binding of DNA substrates have been reported, the structural basis for XPA's DNA-binding activity remains unknown. X-ray crystal structures of the central globular domain of yeast XPA (Rad14) with lesion-containing DNA duplexes have provided valuable insights, but the DNA substrates used for this study do not correspond to the substrates of XPA as it functions within the NER machinery. To better understand the DNA-binding activity of human XPA in NER, we used NMR to investigate the interaction of its DBD with a range of DNA substrates. We found that XPA binds different single-stranded/double-stranded junction DNA substrates with a common surface. Comparisons of our NMR-based mapping of binding residues with the previously reported Rad14-DNA crystal structures revealed similarities and differences in substrate binding between XPA and Rad14. This includes direct evidence for DNA contacts to the residues extending C-terminally from the globular core, which are lacking in the Rad14 construct. Moreover, mutation of the XPA residue corresponding to Phe-262 in Rad14, previously reported as being critical for DNA binding, had only a moderate effect on the DNA-binding activity of XPA. The DNA-binding properties of several disease-associated mutations in the DBD were investigated. These results suggest that for XPA mutants exhibiting altered DNA-binding properties, a correlation exists between the extent of reduction in DNA-binding affinity and the severity of symptoms in XP patients.

Observation of two modes of inhibition of human microsomal prostaglandin E synthase 1 by the cyclopentenone 15-deoxy-Δ(12,14)-prostaglandin J(2).

Microsomal prostaglandin E synthase 1 (MPGES1) is an enzyme that produces the pro-inflammatory molecule prostaglandin E(2) (PGE(2)). Effective inhibitors of MPGES1 are of considerable pharmacological interest for the selective control of pain, fever, and inflammation. The isoprostane, 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)), a naturally occurring degradation product of prostaglandin D(2), is known to have anti-inflammatory properties. In this paper, we demonstrate that 15d-PGJ(2) can inhibit MPGES1 by covalent modification of residue C59 and by noncovalent inhibition through binding at the substrate (PGH(2)) binding site. The mechanism of inhibition is dissected by analysis of the native enzyme and the MPGES1 C59A mutant in the presence of glutathione (GSH) and glutathione sulfonate. The location of inhibitor adduction and noncovalent binding was determined by triple mass spectrometry sequencing and with backbone amide H/D exchange mass spectrometry. The kinetics, regiochemistry, and stereochemistry of the spontaneous reaction of GSH with 15d-PGJ(2) were determined. The question of whether the anti-inflammatory properties of 15d-PGJ(2) are due to inhibition of MPGES1 is discussed.

γ-Hydroxy-1,N2-propano-2'-deoxyguanosine DNA adduct conjugates the N-terminal amine of the KWKK peptide via a carbinolamine linkage.

The γ-hydroxy-1,N(2)-propano-2'-deoxyguanosine adduct (γ-OH-PdG) was introduced into 5'-d(GCTAGCXAGTCC)-3'·5'-d(GGACTCGCTAGC)-3' (X = γ-OH-PdG). In the presence of excess peptide KWKK, (13)C isotope-edited NMR revealed the formation of two spectroscopically distinct DNA-KWKK conjugates. These involved the reaction of the KWKK N-terminal amino group with the N(2)-dG propylaldehyde tautomer of the γ-OH-PdG lesion. The guanine N1 base imino resonance at the site of conjugation was observed in isotope-edited (15)N NMR experiments, suggesting that the conjugated guanine was inserted into the duplex and that the guanine imino proton was protected from exchange with water. The conjugates could be reduced in the presence of NaCNBH(3), suggesting that they existed, in part, as imine (Schiff base) linkages. However, (13)C isotope-edited NMR failed to detect the imine linkages, suggesting that these KWKK conjugates existed predominantly as diastereomeric carbinolamines, in equilibrium with trace amounts of the imines. The structures of the diastereomeric DNA-KWKK conjugates were predicted from potential energy minimization of model structures derived from the refined structure of the fully reduced cross-link [ Huang, H., Kozekov, I. D., Kozekova, A., Rizzo, C. J., McCullough, A., Lloyd, R. S., and Stone, M. P. ( 2010 ) Biochemistry , 49 , 6155 -6164 ]. Molecular dynamics calculations carried out in explicit solvent suggested that the conjugate bearing the S-carbinolamine linkage was the major species due to its potential for intramolecular hydrogen bonding. These carbinolamine DNA-KWKK conjugates thermally stabilized duplex DNA. However, the DNA-KWKK conjugates were chemically reversible and dissociated when the DNA was denatured. In this 5'-CpX-3' sequence, the DNA-KWKK conjugates slowly converted to interstrand N(2)-dG:N(2)-dG DNA cross-links and ring-opened γ-OH-PdG derivatives over a period of weeks.