Bovine seminal plasma (BSP) contains four similar proteins secreted by the seminal vesicles, designated BSP-A1, -A2, -A3, and -30 kDa. These proteins bind to choline phospholipids on the surface of the sperm after ejaculation. These BSP proteins also interact with heparin, apolipoprotein A-I (apoA-I) and apoA-I associated with high-density lipoprotein (HDL). The HDL and heparin present in the female reproductive tract have been implicated in sperm capacitation and the acrosome reaction (AR). This study was undertaken to determine whether or not these BSP proteins and HDL could modulate the capacitation of sperm, and to determine the combined effect of HDL and heparin on capacitation. Washed bovine epididymal sperm were preincubated in buffer containing BSP proteins, washed, and incubated with lipoproteins (HDL, and low- and very low-density lipoproteins) or liposomes with or without apoA-I in the presence or absence of heparin. The percentage of capacitated sperm was evaluated after the AR was induced with lysophosphatidylcholine. HDL alone (160 microg/ml) after an 8-h incubation stimulated the AR of epididymal sperm. The percentage of HDL-enhanced AR further increased when sperm were preincubated with BSP proteins. ApoA-I-liposomes stimulated the AR more rapidly (5 h, 160 microg/ml) than HDL. When sperm were preincubated with BSP proteins, the percentage of apoA-I-enhanced AR further increased. In contrast, when liposomes without apoA-I or when low- or very low-density lipoproteins or lipoprotein-depleted serum was used, no significant increase in the AR was detected with or without BSP proteins. When heparin and HDL or apoA-I-liposomes were used together, their combined effects on the AR were not additive. These results indicate that BSP proteins modulate the process of capacitation induced by heparin, HDL, and apoA-I-liposomes.

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Artemis is a DNA repair factor required for V(D)J recombination, repair of DNA damage induced by ionizing radiation (IR) or radiomimetic drugs, and the maintenance of genome integrity. During V(D)J recombination, Artemis participates in the resolution of hairpin-sealed coding ends, a step crucial to the constitution of the gene encoding for the antigen receptor of lymphocytes. The precise role of Artemis in the repair of IR-induced DNA damage remains to be elucidated. Here we show that Artemis is constitutively phosphorylated in cultured cells and undergoes additional phosphorylation events after irradiation. The IR-induced phosphorylation is mainly, although not solely, dependent on Ataxia-telangiectasia-mutated kinase (ATM). The physiological role of these phosphorylation events remains unknown, as in vitro-generated Artemis mutants, which present impaired IR-induced phosphorylation, still display an activity sufficient to complement the V(D)J recombination defect and the increased radiosensibility of Artemis-deficient cells. Thus, Artemis is an effector of DNA repair that can be phosphorylated by ATM, and possibly by DNA-PKcs and ATR depending upon the type of DNA damage.

Objective-A recent genome-wide association study meta-analysis identified an intronic single nucleotide polymorphism in SMAD3, rs56062135C>T, the minor allele (T) which associates with protection from coronary artery disease. Relevant to atherosclerosis, SMAD3 is a key contributor to transforming growth factor-β pathway signaling. Here, we seek to identify ≥1 causal coronary artery disease-associated single nucleotide polymorphisms at the SMAD3 locus and characterize mechanisms whereby the risk allele(s) contribute to coronary artery disease risk. Approach and Results-By genetic and epigenetic fine mapping, we identified a candidate causal single nucleotide polymorphism rs17293632C>T (D′, 0.97; r 2 , 0.94 with rs56062135) in intron 1 of SMAD3 with predicted functional effects. We show that the sequence encompassing rs17293632 acts as a strong enhancer in human arterial smooth muscle cells. The common allele (C) preserves an activator protein (AP)-1 site and enhancer function, whereas the protective (T) allele disrupts the AP-1 site and significantly reduces enhancer activity (P<0.001). Pharmacological inhibition of AP-1 activity upstream demonstrates that this allele-specific enhancer effect is AP-1 dependent (P<0.001). Chromatin immunoprecipitation experiments reveal binding of several AP-1 component proteins with preferential binding to the (C) allele. We show that rs17293632 is an expression quantitative trait locus for SMAD3 in blood and atherosclerotic plaque with reduced expression of SMAD3 in carriers of the protective allele. Finally, siRNA knockdown of SMAD3 in human arterial smooth muscle cells increases cell viability, consistent with an antiproliferative role. Phosphorylated SMAD3 then forms a complex with the common SMAD4 that subsequently translocates to the nucleus and regulates transcription. 6,7 Relevant to a role in atherosclerosis, in systems genetics analysis of multiple GWAS, we identified TGF-β signaling and SMAD transcriptional activities as enriched pathways for CAD association. 8 However, despite extensive data on the functions of TGF-β with respect to atherosclerosis, 9,10 the roles of SMAD proteins particularly SMAD3 and SMAD3 signaling are less well-understood. Conclusions-TheSMAD3 is expressed at low levels in healthy human aorta by immunohistochemistry and quantitative reverse transcription polymerase chain reaction (PCR).11 There is, however, a marked increase in SMAD3 and other SMAD proteins in fibrofatty lesions, with expression mostly limited to CD68-positive macrophages/macrophage-derived foam cells in these samples. Conversely, in fibrous atherosclerotic plaques, there are high levels of SMAD3 in vascular smooth muscle cells (SMCs), suggesting that the role of SMAD3 in atherosclerosis depends on cell type and stage of atherosclerosis.11 Higher SMAD3 expression in SMCs of the fibrous plaque coincides with TGF-β-mediated synthesis of collagen and other extracellular matrix proteins, which contribute to plaque stability.12 Rare SMAD3 mutations cause aneurysms-osteoarthritis syn...

BackgroundThe TRIB1 locus has been linked to hepatic triglyceride metabolism in mice and to plasma triglycerides and coronary artery disease in humans. The lipid‐associated single nucleotide polymorphisms (SNPs), identified by genome‐wide association studies, are located ≈30 kb downstream from TRIB1, suggesting complex regulatory effects on genes or pathways relevant to hepatic triglyceride metabolism. The goal of this study was to investigate the functional relationship between common SNPs at the TRIB1 locus and plasma lipid traits.Methods and ResultsCharacterization of the risk locus reveals that it encompasses a gene, TRIB1‐associated locus (TRIBAL), composed of a well‐conserved promoter region and an alternatively spliced transcript. Bioinformatic analysis and resequencing identified a single SNP, rs2001844, within the promoter region that associates with increased plasma triglycerides and reduced high‐density lipoprotein cholesterol and coronary artery disease risk. Further, correction for triglycerides as a covariate indicated that the genome‐wide association studies association is largely dependent on triglycerides. In addition, we show that rs2001844 is an expression trait locus (eQTL) for TRIB1 expression in blood and alters TRIBAL promoter activity in a reporter assay model. The TRIBAL transcript has features typical of long noncoding RNAs, including poor sequence conservation. Modulation of TRIBAL expression had limited impact on either TRIB1 or lipid regulatory genes mRNA levels in human hepatocyte models. In contrast, TRIB1 knockdown markedly increased TRIBAL expression in HepG2 cells and primary human hepatocytes.ConclusionsThese studies demonstrate an interplay between a novel locus, TRIBAL, and TRIB1. TRIBAL is located in the genome‐wide association studies identified risk locus, responds to altered expression of TRIB1, harbors a risk SNP that is an eQTL for TRIB1 expression, and associates with plasma triglyceride concentrations.

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DNA-dependent protein kinase (DNA-PK) acts through an essential relationship with DNA to participate in the regulation of multiple cellular processes. Yet the role of DNA as a cofactor in kinase activity remains to be completely elucidated. For example, although DNA-PK activity appears to be required for the resolution of hairpin coding ends in variable diversity joining recombination, kinase activity remains to be demonstrated from hairpin ends or other DNA structures. In the present study we report that DNA-PK is strongly activated from hairpin ends and structured singlestranded DNA, but that the phosphorylation of many heterologous substrates is blocked efficiently by inactivation of the kinase through autophosphorylation. However, substrates that bound efficiently to single-stranded DNA such as p53 and replication protein A were efficiently phosphorylated by DNA-PK from structured DNA. DNA-PK also was found to be active toward heterologous substrates from hairpin ends on double-stranded DNA under conditions where autophosphorylation was minimized. These results suggest that the role of DNA-PK in resolving coding end hairpins is likely to be enzymatic rather than structural, expand understanding of how DNA-PK binding to structured DNA relates to enzyme activity, and suggest a mechanism for autoregulatory control of its kinase activity in the cell. D NA-dependent protein kinase (DNA-PK) is a Ser͞Thr kinase required for the resolution of the hairpin coding ends in variable diversity joining [V(D)J] recombination and for correct DNA end joining in nonhomologous DNA (1). Roles for DNA-PK also have been proposed in DNA replication and the regulation of specific gene transcription by RNA polymerases I and II (2-4). Although physiological substrates for DNA-PK remain to be demonstrated, kinase activity appears to be essential to DNA-PK function in recombination, DNA repair, and transcriptional regulation (5).DNA-PK is comprised of two components: a large catalytic subunit (DNA-PK cs ), which binds DNA with low affinity (6), and the Ku antigen (Ku70͞Ku80), which binds specifically to DNA ends, sequences, and structural transitions in B-form DNA with high affinity (3, 7). DNA-PK cs is a member of the large phosphatidylinositol 3-kinase-related kinase family with several other kinases, including the ataxia telangiectasia gene product and ataxia telangectasia and RAD-3-related kinase (8). Ku appears to be essential for DNA-PK cs function in vivo and likely acts by promoting the recruitment of DNA-PK cs to DNA ends and sequences from which the kinase is activated (3, 9, 10). Ku also contains limited DNA helicase activity and can induce structural transitions in DNA flanking sequence-specific DNA-PK binding sites (11,12). Whether Ku helicase activity contributes to the activation of DNA-PK cs from DNA ends is not known.DNA-PK cs is activated at DNA ends in the presence and absence of Ku and from specific Ku DNA binding sites when recruited by Ku (6, 13). Activation of DNA-PK cs from DNA ends is further stimulated by the p...