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.

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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.

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.