DNA-dependent protein kinase (DNA-PK), which is involved in DNA double-stranded break repair and V(D)J recombination, comprises a DNA-targeting component called Ku and an approximately 460 kDa catalytic subunit, DNA-PKcs. Here, we describe the cloning of the DNA-PKcs cDNA and show that DNA-PKcs falls into the phosphatidylinositol (PI) 3-kinase family. Biochemical assays, however, indicate that DNA-PK phosphorylates proteins but has no detectable activity toward lipids. Strikingly, DNA-PKcs is most similar to PI kinase family members involved in cell cycle control, DNA repair, and DNA damage responses. These include the FKBP12-rapamycin-binding proteins Tor1p, Tor2p, and FRAP, S. pombe rad3, and the product of the ataxia telangiectasia gene, mutations in which lead to genomic instability and predisposition to cancer. The relationship of these proteins to DNA-PKcs provides important clues to their mechanisms of action.

BACKGROUND: Nuclear magnetic resonance (NMR) spectra of serum obtained under quantitative conditions for lipoprotein particle analyses contain additional signals that could potentially serve as useful clinical biomarkers. One of these signals that we named GlycA originates from a subset of glycan N-acetylglucosamine residues on enzymatically glycosylated acute-phase proteins. We hypothesized that the amplitude of the GlycA signal might provide a unique and convenient measure of systemic inflammation.

Scavenger receptor BI (SR-BI) mediates the selective uptake of high density lipoprotein (HDL) cholesteryl ester (CE), a process by which HDL CE is taken into the cell without internalization and degradation of the HDL particle. The biochemical mechanism by which SR-BI mediates the selective uptake of HDL CE is poorly understood. Given that CE transfer will occur to some extent from HDL to protein-free synthetic membranes, one hypothesis is that the role of SR-BI is primarily to tether HDL close to the cell surface to facilitate CE transfer from the particle to the plasma membrane. In the present study, this hypothesis was tested by comparing the selective uptake of HDL CE mediated by mouse SR-BI (mSR-BI) with that mediated by rat CD36 (rCD36), a closely related class B scavenger receptor. Both mSR-BI and rCD36 bind HDL with high affinity, and both receptors mediate HDL CE selective uptake. However, SR-BI mediates selective uptake of HDL CE with a 7-fold greater efficiency than rCD36. HDL CE selective uptake mediated by rCD36 is dependent on HDL binding to the receptor, since a mutation that blocks HDL binding also blocks HDL CE selective uptake. These data lead us to hypothesize that one component of HDL CE selective uptake is the tethering of HDL particles to the cell surface. To explore the molecular domains responsible for the greater efficiency of selective uptake by mSR-BI, we compared binding and selective uptake among mSR-BI, scavenger receptor BII, and various chimeric receptors formed from mSR-BI and rCD36. The results show that the extracellular domain of mSR-BI is essential for efficient HDL CE uptake, but the C-terminal cytoplasmic tail also has a major influence on the selective uptake process.The risk for developing atherosclerosis and cardiovascular disease is inversely related to plasma concentrations of high density lipoprotein (HDL) 1 cholesterol (1). Although the mechanism of this protective effect remains uncertain, it has been known for some time that HDL plays a pivotal role in the transport of free cholesterol and cholesteryl esters (CE) through the plasma. HDL participates in reverse cholesterol transport (2), a process involving the uptake of free cholesterol from peripheral tissue and its subsequent delivery (as free cholesterol or CE) to steroidogenic (for hormone synthesis) and hepatic (for bile acid synthesis) tissues. HDL provides CE to cells via the selective uptake pathway in which HDL CE is taken into the cell without the internalization and lysosomal degradation of the HDL particle (3-8). Recent studies identified a cell surface receptor, scavenger receptor BI (SR-BI), which binds HDL particles and mediates the selective uptake of HDL CE in transfected cells (9). Immunochemical analysis of SR-BI in rodents indicates that it is expressed most abundantly in the liver and in steroidogenic cells of the adrenal gland and ovary (9 -11), where the selective uptake of HDL CE is greatest (4, 7). SR-BI expression is regulated by gonadotropins and adrenocorticotropic hormone coordinately w...

, we showed that SR-BI-mediated cholesterol efflux was highly correlated (r 2 ‫؍‬ 0.985) with HDL phosphatidylcholine content. The effects of varying HDL phospholipid composition on SR-BI-mediated free cholesterol flux were not correlated with changes in either the K d or B max values for high affinity binding to SR-BI. We conclude that SR-BI-mediated free cholesterol flux is highly sensitive to HDL phospholipid composition. Thus, factors that regulate cellular SR-BI expression and the local modification of HDL phospholipid composition will have a large impact on reverse cholesterol transport.The deposition of cholesterol in peripheral cells is opposed by the process of reverse cholesterol transport (RCT) 1 where high density lipoproteins (HDL) remove free cholesterol (FC) from cells and deliver it back to the liver for excretion (1-3). The flux of FC between cells and HDL is bi-directional. Depending on the direction of the FC concentration gradient between cells and lipoproteins, either net efflux or net influx of cholesterol can occur (4, 5). The creation of a cholesterol gradient depends upon many properties of the acceptors and the cell plasma membrane. Such factors include the cholesterol and phospholipid content of the acceptors and plasma membrane (4, 5), the existence of cholesterol domains within the plasma membrane (6 -10), and the size, number, and composition of acceptor particles (11-13).Recent studies have shown that, when cells express scavenger receptor BI (SR-BI), the bi-directional flux of FC between cells and HDL is accelerated (8,14,15). The mechanism by which SR-BI mediates FC flux is uncertain. However, recent studies from our laboratory demonstrated that binding of the acceptor particles to SR-BI is not a requirement for SR-BImediated cholesterol efflux (7,8). Rather SR-BI induces a reorganization of the plasma membrane cholesterol, and this reorganization is linked to enhanced FC flux (7,8,16). Regardless of the mechanism, evidence is accumulating to support the importance of SR-BI-mediated FC flux in RCT. Recent studies of Ji and colleagues (17) showed that either attenuation or overexpression of hepatic SR-BI in mice led to significantly decreased or increased delivery of HDL FC into bile. In addition, the expression of SR-BI in peripheral cells and in foam cells of the arterial wall suggests a role for SR-BI in the removal of FC from the periphery (15, 18, 19).SR-BI-mediated FC flux requires phospholipid in the acceptor (15), and studies have shown that cholesterol efflux from cells is highly correlated with the concentration of HDL phospholipid in serum (20,21). Also, the stimulation of cholesterol efflux upon phospholipid supplementation of serum is closely linked to the levels of SR-BI among cell types (14). These observations are consistent with epidemiological data demonstrating that humans with low HDL phospholipid levels have a high incidence of coronary artery disease (22). These findings suggest that changes in HDL phospholipid content may alter SR-BI-mediated FC flux. The cu...

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LP-IR scores had strong associations with multiple measures, HOMA-IR, and GDR, the former being more reflective of hepatic and the latter of peripheral insulin sensitivity, and may represent a simple means to identify individuals with IR.

BackgroundGlycA is a novel spectroscopic marker of systemic inflammation with low intra-individual variability and other attributes favoring its clinical use in patients with chronic inflammatory and autoimmune diseases. GlycA is unique in its composite nature, reflecting both increased glycan complexity and circulating acute phase protein levels during local and systemic inflammation. Recent studies of GlycA from cross-sectional, observational and interventional studies have been highly informative, demonstrating that GlycA is elevated in acute and chronic inflammation, predicts death in healthy individuals and is associated with disease severity in patients with chronic inflammatory diseases such as rheumatoid arthritis, psoriasis and lupus. Moreover, following treatment with biological therapy in psoriasis, reduction in skin disease severity was accompanied by a decrease in GlycA levels and improvement in vascular inflammation.ConclusionsCollectively, these findings suggest GlycA is a marker that tracks systemic inflammation and subclinical vascular inflammation. However, larger prospective studies and randomized trials are necessary in order to assess the impact of novel therapies on GlycA in patients with chronic inflammatory conditions, which may be concomitant with cardiovascular benefits.

Exposure of mammalian cells to UV radiation alters gene expression and cell cycle progression; some of these responses may ensure survival or serve as mutation-avoidance mechanisms, lessening the consequences of UV-induced DNA damage. We showed previously that UV irradiation increases phosphorylation of the p34 subunit of human replication protein A (RPA) and that this hyperphosphorylation correlated with loss of activity of the DNA replication complex. To characterize further the role of RPA hyperphosphorylation in the cellular response to UV irradiation and to determine which protein kinases might be involved, we identified by phosphopeptide analysis the sites phosphorylated in the p34 subunit of RPA (RPA-p34) from HeLa cells before and after exposure to 30 J/m 2 UV light. In unirradiated HeLa cells, RPA-p34 is phosphorylated primarily at Ser-23 and Ser-29. At least four of the eight serines and one threonine in the N-terminal 33 residues of RPA-p34 can become phosphorylated after UV irradiation. Two of these sites (Ser-23 and Ser-29) are known to be sites phosphorylated by Cdc2 kinase; two others (Thr-21 and Ser-33) are consensus sites for the DNA-dependent protein kinase (DNA-PK); the fifth site (Ser-11, -12, or -13) does not correspond to the (Ser/Thr)-Gln DNA-PK consensus. All five can be phosphorylated in vitro by incubating purified RPA with purified DNA-PK. Two additional sites, probably Ser-4 and Ser-8, are phosphorylated in vivo after UV irradiation and in vitro by purified DNA-PK. The capacity of purified DNA-PK to phosphorylate many of these same sites on RPA-p34 in vitro implicates DNA-PK or a kinase with similar specificity in the UV-induced hyperphosphorylation of RPA in vivo.Replication protein A (RPA 1 ; also known as human SSB) is a trimeric single-stranded DNA-binding protein necessary for DNA replication (1-3), recombination (4), and repair (5, 6).RPA binds to DNA through its 70-kDa subunit (7). In vitro, the DNA binding activity of RPA is essential for DNA unwinding at the origin of replication. The p70 subunit alone, or SSB proteins from other species (8 -12), also can cause unwinding, but specific protein-protein interactions between RPA and polymerase ␣-primase and SV40 large tumor antigen are necessary for assembly of the initiation complex at the SV40 origin of replication and for DNA replication. Neither RPA-p70 alone nor heterologous SSBs substitute for RPA in these interactions (13). Thus, the RPA-p34 and RPA-p13 subunits are primarily responsible for the specificity of protein-protein interactions in DNA replication and possibly also in DNA repair. The RPA protein was shown to interact with XPA (XP group A) (14 -16), XPF-ERCC1 (XPF-excision repair cross-complementing rodent repair deficiency 1 (XP group F)) (17) and XPG (XP group G) (15, 17) proteins in excision repair. RPA-p34 is phosphorylated in vitro by DNA-PK, a DNA-activated protein kinase that participates in double-strand break repair (18 -24). RPA also interacts with several transcription factors, including VP16, Gal4, and...