Hot spots in apolipoprotein A-II misfolding and amyloidosis in mice and men

ApoA-II is the second-major protein of high-density lipoproteins. C-terminal extension in human apoA-II or point substitutions in murine apoA-II cause amyloidosis. The molecular mechanism of apolipoprotein misfolding, from the native predominantly α-helical conformation to cross-β-sheet in amyloid, is unknown. We used 12 sequence-based prediction algorithms to identify two ten-residue segments in apoA-II that probably initiate β-aggregation. Previous studies of apoA-II fragments experimentally verify this prediction. Together, experimental and bioinformatics studies explain why the C-terminal extension in human apoA-II causes amyloidosis and why, unlike murine apoA-II, human apoA-II normally does not cause amyloidosis despite its unusually high sequence propensity for β-aggregation.     

Human apolipoprotein A-II: nucleotide sequence of a cloned cDNA, and localization of its structural gene on human chromosome 1

[3H]yohimbine, a potent and selective alpha 2-adrenergic antagonist was used to label alpha-adrenoceptors in intact human lymphocytes. Binding of [3H]yohimbine was rapid (t1/2 1.5 -2.0 min) and readily reversed by 10 microM phentolamine (t1/2 = 5 - 6 min) and of high affinity (Kd = 3.7 +/- 0.86 nM). At saturation, the total number of binding sites was 19.9 +/- 5.3 fmol/10(7) lymphocytes. Adrenergic agonists competed for [3H]yohimbine binding sites with an order of potency: clonidine greater than (-) epinephrine greater than (-) norepinephrine greater than (+) epinephrine much greater than (-) isoproterenol; adrenergic antagonists with a potency order of yohimbine greater than phentolamine greater than prazosin. These results indicate the presence of alpha 2-adrenoceptors in human lymphocytes.     

Effects of apolipoprotein B-100 on the metabolism of a lipid microemulsion model in rats

In previous studies, it was shown that lipid microemulsions resembling LDL (LDE) but not containing protein, acquire apolipoprotein E when injected into the bloodstream and bind to LDL receptors (LDLR) using this protein as ligand. Aiming to evaluate the effects of apolipoprotein (apo) B-100 on the catabolism of these microemulsions, LDE with incorporated apo B-100 (LDE-apoB) and native LDL, all labeled with radioactive lipids were studied after intraarterial injection into Wistar rats. Plasma decay curves of the labels were determined in samples collected over 10 h and tissue uptake was assayed from organs excised from the animals sacrificed 24 h after injection. LDE-apo B had a fractional clearance rate (FCR) similar to native LDL (0.40 and 0.33, respectively) but both had FCR pronouncedly smaller than LDE (0.56, P<0.01). Liver was the main uptake site for LDE, LDE-apoB, and native LDL, but LDE-apoB and native LDL had lower hepatic uptake rates than LDE. Pre-treatment of the rats with 17α-ethinylestradiol, known to upregulate LDLR, accelerated the removal from plasma of both LDE and LDE-apoB, but the effect was greater upon LDE than LDE-apoB. These differences in metabolic behavior documented in vivo can be interpreted by the lower affinity of LDLR for apo B-100 than for apo E, demonstrated in in vitro studies. Therefore, our study shows in vivo that, in comparison with apo E, apo B is a less efficient ligand to remove lipid particles such as microemulsions or lipoproteins from the intravascular compartment.     

Variants of a cloned synthetic lactose operator: I. A palindromic dimer lactose operator derived from one strand of the cloned 40-base pair operator

Starting with one strand of the 40-bp synthetic operator (Sadler et al., 1978), we have constructed and cloned a 66-bp, palindromic DNA segment with the following sequence

where the horizontal arrows indicate the locations of the two 21-bp “core? operator sequences in this segment and the vertical arrow designates the dyad axis of symmetry. Upon denaturation and rapid renaturation, each strand of this fragment forms a hairpin molecule still retaining an EcoRI cohesive end. Two hairpin molecules can be joined with T4 DNA ligase to form a duplex DNA molecule having no ends (dumbbell form A). Denaturation and rapid renaturation of dumbbell A yields a mixture of two dumbbell forms: dumbbell A which is a substrate for EcoKL, and a new form, dumbbell B, which is not a substrate. Each of the conformations of this DNA fragment have been purified and all are active in binding lactose repressor in vitro.     

Cloning and sequence analysis of a rat liver cDNA coding for a phenobarbital-inducible microheterogenous cytochrome P-450 variant: regulation of its messenger level by xenobiotics

A rat liver cDNA library was prepared from total polyribosomal poly(A)+ RNA extracted from phenobarbital-treated animals. A cDNA clone coding for a phenobarbital-inducible cytochrome P-450 (PB P-450) was identified by differential colony hybridization to cDNAs synthesized from liver poly(A)+RNAs isolated from phenobarbital-treated rats for positive selection and cDNAs from either untreated rats or beta-naphthoflavone-treated rats as negative controls, followed by hybrid-selected translation and analysis of the translation products by immunoprecipitation. As the cloning and screening strategies involve no prior enrichment for specific mRNAs, they also permit the identification of sequences coding for phenobarbital-induced proteins other than cytochromes P-450. This relatively straightforward approach is generally applicable to the molecular cloning of sequences coding for other inducible cytochromes P-450. Nucleic acid sequencing data indicated that the cloned PB P-450 cDNA codes for a cytochrome P-450 variant [designated P-450e(U.C.)] that is very similar, but not identical, to P-450e. Sequence analysis of the section of cDNA specifying the 3'-non-coding region of the mRNA revealed that it lacked the usual poly(A) addition site signal sequence but contained three inverted repeat structures. Solution hybridization analysis demonstrated that PB P-450 mRNA is increased 20-fold by phenobarbital treatment and decreased 3-fold by beta-naphthoflavone treatment.     

A new protein from human plasma lipoproteins: isolation and partial characterization of apolipoprotein G]

A new apolipoprotein has been identified in VHDL1 and in HDL. This protein is immunologically distinct from already isolated apoproteins. It was isolated by column chromatography on hydroxylapatite. In polyacrylamide gel electrophoresis, its mobility is very close to that of apo D. The amino acid composition differs from those of the well characterized polypeptides of the human plasma lipoproteins. It contains glucosamine. The apparent molecular weight is 72 000 +/- 2 000 in the presence and absence of reducing agent. According to the ABCDEF nomenclature, this protein can be named apolipoprotein G (apo G). It is present in a lipoprotein distinct from the lipoproteins A and D among the VHDL1 : this new lipoprotein can be named lipoprotein G (LPG).     

Impact of apolipoprotein A1- or lecithin:cholesterol acyltransferase-deficiency on white adipose tissue metabolic activity and glucose homeostasis in mice

High density lipoprotein (HDL) has attracted the attention of biomedical community due to its well-documented role in atheroprotection. HDL has also been recently implicated in the regulation of islets of Langerhans secretory function and in the etiology of peripheral insulin sensitivity. Indeed, data from numerous studies strongly indicate that the functions of pancreatic β-cells, skeletal muscles and adipose tissue could benefit from improved HDL functionality. To better understand how changes in HDL structure may affect diet-induced obesity and type 2 diabetes we aimed at investigating the impact of Apoa1 or Lcat deficiency, two key proteins of peripheral HDL metabolic pathway, on these pathological conditions in mouse models. We report that universal deletion of apoa1 or lcat expression in mice fed western-type diet results in increased sensitivity to body-weight gain compared to control C57BL/6 group. These changes in mouse genome correlate with discrete effects on white adipose tissue (WAT) metabolic activation and plasma glucose homeostasis. Apoa1-deficiency results in reduced WAT mitochondrial non-shivering thermogenesis. Lcat-deficiency causes a concerted reduction in both WAT oxidative phosphorylation and non-shivering thermogenesis, rendering lcat^?/? mice the most sensitive to weight gain out of the three strains tested, followed by apoa1^?/? mice. Nevertheless, only apoa1^?/? mice show disturbed plasma glucose homeostasis due to dysfunctional glucose-stimulated insulin secretion in pancreatic β-islets and insulin resistant skeletal muscles. Our analyses show that both apoa1^?/? and lcat^?/? mice fed high-fat diet have no measurable Apoa1 levels in their plasma, suggesting no direct involvement of Apoa1 in the observed phenotypic differences among groups.     

Characterization of palmitoylation of ATP binding cassette transporter G1: Effect on protein trafficking and function

ATP-binding cassette transporter G1 (ABCG1) mediates cholesterol efflux onto lipidated apolipoprotein A-I and HDL and plays a role in various important physiological functions. However, the mechanism by which ABCG1 mediates cholesterol translocation is unclear. Protein palmitoylation regulates many functions of proteins such as ABCA1. Here we investigated if ABCG1 is palmitoylated and the subsequent effects on ABCG1-mediated cholesterol efflux. We demonstrated that ABCG1 is palmitoylated in both human embryonic kidney 293 cells and in mouse macrophage, J774. Five cysteine residues located at positions 26, 150, 311, 390 and 402 in the NH₂-terminal cytoplasmic region of ABCG1 were palmitoylated. Removal of palmitoylation at Cys311 by mutating the residue to Ala (C311A) or Ser significantly decreased ABCG1-mediated cholesterol efflux. On the other hand, removal of palmitoylation at sites 26, 150, 390 and 402 had no significant effect. We further demonstrated that mutations of Cys311 affected ABCG1 trafficking from the endoplasmic reticulum. Therefore, our data suggest that palmitoylation plays a critical role in ABCG1-mediated cholesterol efflux through the regulation of trafficking.