p53-binding protein-1 (53BP1) is phosphorylated in response to DNA damage and rapidly relocalizes to presumptive sites of DNA damage along with Mre11 and the phosphorylated histone 2A variant, ␥-H2AX. 53BP1 associates with the BRCA1 tumor suppressor, and knockdown experiments with small interfering RNA have revealed a role for the protein in the checkpoint response to DNA damage. By generating mice defective in m53BP1 (m53BP1 tr/tr ), we have created an animal model to further explore its biochemical and genetic roles in vivo. We find that m53BP1 tr/tr animals are growth-retarded and show various immune deficiencies including a specific reduction in thymus size and T cell count. Consistent with a role in responding to DNA damage, we find that m53BP1 tr/tr mice are sensitive to ionizing radiation (␥-IR), and cells from these animals exhibit chromosomal abnormalities consistent with defects in DNA repair. Thus, 53BP1 is a critical element in the DNA damage response and plays an integral role in maintaining genomic stability.

It is expected that the attendant structural heterogeneity of human high density lipoprotein (HDL) complexes is a determinant of its varied metabolic functions. To determine structural heterogeneity of HDL, major apolipoprotein stoichiometry profiles in human HDL were determined. First, HDL was separated into two main populations, with and without apolipoprotein (apo) A-II, LpA-I and LpA-I/A-II respectively. Each main population was further separated into six individual subfractions using size exclusion chromatography (SEC). Protein proximity profiles (PPP) of major apolipoproteins in each individual subfraction was determined by optimally cross-linking apolipoproteins within individual particles with bis(sulfosuccinimidyl)suberate (BS3), a bifunctional cross linker, followed by molecular weight determination by MALDI-MS. The PPPs of LpA-I subfractions indicated that the number of apoA-I molecules increased from two to three to four upon increase in the LpA-I particle size. On the other hand, the entire population of LpA-I/A-II demonstrated the presence of only two proximal apoA-I molecules per particle, while the number of apoA-II molecules varied from one dimeric apoA-II to two and then to three. For most of the above PPP profiles, an additional population that contained a single molecule of apoC-III in addition to apoA-I and/or apoA-II was detected. Upon composition analyses of individual subpopulations, LpA-I/A-II displayed comparable proportions for total protein (~58%), phospholipids (~21%) total cholesterol (~16%), triglycerides (~5%) and free cholesterol (~4%) across subfractions. LpA-I components, on the other hand, showed significant variability. This novel information on HDL subfractions will form a basis for better understanding particle specific functions of HDL.

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Objective Reverse cholesterol transport (RCT) comprises cholesterol efflux from ABCA1-expressing macrophages to apo AI giving nascent high density lipoprotein (nHDL), esterification of nHDL-free cholesterol (FC), selective hepatic extraction of HDL-lipids, and hepatic conversion of HDL-cholesterol to bile salts, which are excreted. We tested this model by identifying the fates of nHDL-[3H]FC, [14C]PL, and [125I]apo AI in serum in vitro and in vivo. Approach and Results During in vitro incubation of human serum, nHDL-[3H]FC and [14C]PL rapidly transfer to HDL and low density lipoproteins (LDL; t1/2 = 2–7 min) while nHDL-[125I]apo AI transfers solely to HDL (t1/2<10 min) and to the lipid-free form (t1/2 >480 min). Following injection into mice nHDL-[3H]FC and [14C]PL rapidly transfer to liver (t1/2 ~ 2–3 min) whereas apo AI clears with t1/2 = ~460 min. The plasma nHDL-[3H]FC esterification rate is slow (0.46%/h) compared to hepatic uptake. Phospholipid transfer protein enhances nHDL-[14C]PL but not nHDL-[3H]FC transfer to cultured Huh7 hepatocytes. Conclusions nHDL-FC, PL, and apo AI enter different pathways in vivo. Most nHDL-[3H]FC and [14C]PL are rapidly extracted by the liver via scavenger receptor class B member 1 and spontaneous transfer; hepatic PL uptake is promoted by phospholipid transfer protein. nHDL-[125I]apo AI transfers to HDL and to the lipid-free form that can be recycled to nHDL formation. Cholesterol esterification by LCAT is a minor process in nHDL metabolism. These findings could guide the design of therapies that better mobilize peripheral tissue-FC to hepatic disposal.

Serum opacity factor (SOF), a virulence determinant of Streptococcus pyogenes, converts plasma high density lipoproteins (HDL) to three distinct species: lipid-free apolipoprotein (apo) A-I, neo HDL, a small discoidal HDL-like particle, and a large cholesteryl ester-rich microemulsion (CERM), that contains the cholesterol esters (CE) of up to ~400,000 HDL particles and apo E as its major protein. Similar SOF reaction products are obtained with HDL, total plasma lipoproteins and whole plasma. We hypothesized that hepatic uptake of CERM-CE via multiple apo E dependent receptors would be faster than that of HDL-CE. We tested our hypothesis using human hepatoma cells and lipoprotein receptor-specific Chinese hamster ovary (CHO) cells. [ 3 H]CE uptake by HepG2 and Huh7 cells from HDL after SOF treatment, which transfers >90% of HDL-CE to CERM, was respectively 2.4 and 4.5 times faster than from control HDL. CERM-[ 3 H]CE uptake was inhibited by LDL and HDL, suggestive of uptake by both the LDL receptor (LDL-R) and scavenger receptor class B type I (SR-BI). Studies in CHO cells specifically expressing LDL-R and SR-BI confirmed CERM-[ 3 H]CE uptake by both receptors. RAP and heparin inhibit CERM-[ 3 H]CE but not HDL-[ 3 H]CE uptake thereby implicating LRP-1 and cell surface proteoglycans in this process. These data demonstrate that SOF treatment of HDL increases CE uptake via multiple hepatic apo E receptors. In so doing, SOF might increase hepatic disposal of plasma cholesterol in a way that is therapeutically useful. KeywordsHDL function; cholesteryl ester uptake; Huh7; HepG2; apo E; LDL-R; LRP; SR-BI; RAP; heparin Serum opacity factor (SOF), a virulence determinant of Streptococcus pyogenes, converts HDL to lipid-free (LF) apo A-I, neo HDL, which is a small HDL-like particle, and a large cholesteryl ester-rich microemulsion (CERM) that contains the cholesteryl esters (CE) of 400,000 HDL particles and monomeric apo E and its heterodimer with apo A-II as its sole apos (1-6). Recombinant SOF (rSOF) is potent and catalytic; rSOF (1 μg/mL) quantitatively 1 This work was supported by grants-in-aid from the National Institutes of Health (HL-30914 and HL-56865 to H.J.P.) and the converts HDL to CERM, neo HDL, and LF apo A-I with a halftime of ~30 min (4;5). Based on the reaction products and kinetics, we proposed a model for the rSOF reaction in which rSOF is a heterodivalent fusogenic protein that uses a docking site to displace apo A-I and bind to exposed CE surfaces on HDL (4). The initial rSOF-HDL complex recruits additional HDL with its binding-delipidation site and through multiple fusion steps forms large CERM and releases neo HDL and LF apo A-I (4). Importantly, CERM contain apo E. We hypothesized that with its high apo E and CE contents, CERM could transfer large amounts of cholesterol to the liver for disposal via LDL receptor (LDL-R) or other apo E receptors (4). Department of Veterans Affairs (HSCCERM and chylomicron remnants (CR) share some properties suggesting that they might be cleared via similar pa...

Objective To identify the role of triglyceride-rich lipoproteins (TGRLs) and apoE, a major apolipoprotein in TGRLs, in adipose tissue inflammation with high-fat diet (HFD)–induced obesity. Methods Male apoE−/− and C57BL/6J wild-type (WT) mice fed HFD for 12 weeks were assessed for metabolic and inflammatory parameters. ApoE−/− and WT mice were orally gavaged with [3H]palmitic acid to examine the role of apoE in fat delivery to adipose tissue. VLDL from obese apoE−/− mice were intravenously injected into lean WT or apoE−/− mice to test potential contribution of TGRLs-derived fat delivery to inflammation in adipose tissue and the role of apoE. Results ApoE−/− mice gained less body weight, and had less fat mass and lower triglyceride levels in skeletal muscle than WT. ApoE−/− mice on HFD had better insulin sensitivity than WT even when comparing body weight–matched mice. Compared to WT mice, apoE−/− mice on HFD had lower levels of inflammatory cytokines/chemokines and CD11c in adipose tissue, and lower levels of inflammatory markers in skeletal muscle. At 6 hours after oral gavage with [3H]palmitic acid, incorporation of [3H]palmitic acid into adipose tissue and skeletal muscle was lower in apoE−/− mice. After repeated daily injection for 3 days, VLDL from obese apoE−/− mice induced inflammation in adipose tissue of recipient WT but not apoE−/− mice. Conclusion In HFD-induced obesity, apoE plays an important role in inflammation in adipose tissue and skeletal muscle, likely by mediating TGRL-derived fat delivery to these tissues.

Objective Recombinant streptococcal serum opacity factor (rSOF) mediates the in vitro disassembly of human plasma HDL into lipid-free (LF) apolipoprotein (apo) A-I, a neo HDL that is cholesterol-poor, and a cholesteryl ester-rich microemulsion (CERM) containing apolipoprotein E. Given the occurrence of apolipoprotein E on the CERM, we tested the hypothesis that rSOF injection into mice would reduce total plasma cholesterol clearance via apo E-dependent hepatic LDL receptors (LDLR). Methods and Results rSOF (4 μg) injection into wild type C57BL/6J mice forms neo HDL, CERM, and LF apo A-I, as observed in vitro, and reduced plasma total cholesterol (−43%, t1/2 = 44 ± 18 min) whereas control saline injections had a negligible effect. Similar experiments with apo E−/− and LDLR−/− mice respectively reduced plasma total cholesterol ~0 and 20%. rSOF is potent; injection of 0.18 μg rSOF produces 50% of maximum reduction of plasma cholesterol 3 h post injection, corresponding to a ~0.5 mg human dose. Most cholesterol is cleared hepatically (>99%), with rSOF treatment increasing clearance by 65%. Conclusion rSOF injection into mice forms a CERM that is cleared via hepatic LDLR that recognize apo E. This reaction could provide an alternative mechanism for reverse cholesterol transport.

Human plasma high density lipoproteins (HDL), the primary vehicle for reverse cholesterol transport, are the target of serum opacity factor (SOF), a virulence determinant of Streptococcus pyogenes that turns serum opaque. HDL comprise a core of neutral lipids-cholesteryl esters and some triglyceridesurrounded by a surface monolayer of cholesterol, phospholipids, and specialized proteinsapolipoproteins (apos) A-I and A-II. HDL is an unstable particle residing in a kinetic trap from which it can escape via chaotropic, detergent or thermal perturbation. Recombinant (r) SOF catalyzes the transfer of nearly all neutral lipids of ~100,000 HDL particles (D ~ 8.5 nm) into a single, large cholesteryl ester-rich microemulsion (CERM; D >100 nm) leaving a new HDL-like particle-neo HDL (D ~5.8 nm) while releasing lipid-free (LF) apo A-I. CERM formation and apo A-I release have similar kinetics suggesting parallel or rapid consecutive steps. By using complementary physico-chemical methods, we have refined the mechanistic model for HDL opacification. According to size exclusion chromatography, HDL containing non-labile apo A-I resists rSOFmediated opacification. Based on kinetic cryo electron microscopy, rSOF (10 nM) catalyzes the conversion of HDL (4 μM) to neo HDL via a step-wise mechanism in which intermediate-size particles are seen. Kinetic turbidimetry revealed opacification as a rising exponential reaction with a rate constant k = (4.400 ± 0.004) × 10 −2 min −1 . Analysis of the kinetic data using transition state theory gave an enthalpy, entropy and free energy of activation of ΔH ‡ = 73.9 kJ/mol, ΔS ‡ = −66.87 J/°K, and ΔG ‡ = 94.6 kJ/mol respectively. The free energy of activation for opacification is nearly identical to that for the displacement of apo A-I from HDL by guanidine hydrochloride. We conclude that apo A-I lability is required for HDL opacification, LF apo A-I desorption is the rate-limiting step, and nearly all HDL particles contain at least one labile copy of apo A-I. KeywordsSerum opacity factor; electron cryomicroscopy; HDL remodeling; lipid fusion; apolipoprotein A-I; Streptococcus pyogenes Cardiovascular disease (CVD) is a major source of mortality and morbidity and identification of therapies that address its underlying causes is an important public health priority.