A combined LDL receptor/LDL receptor adaptor protein 1 mutation as the cause for severe familial hypercholesterolemia

Familial hypercholesterolemia (FH) results from impaired catabolism of plasma low density lipoproteins (LDL), thus leading to high cholesterol, atherosclerosis, and a high risk of premature myocardial infarction. FH is commonly caused by defects of the LDL receptor or its main ligand apoB, together mediating cellular uptake and clearance of plasma LDL. In some cases FH is inherited by mutations in the genes of PCSK9 and LDLRAP1 (ARH) in a dominant or recessive trait. The encoded proteins are required for LDL receptor stability and internalization within the LDLR pathway. To detect the underlying genetic defect in a family of Turkish descent showing unregular inheritance of severe FH, we screened the four candidate genes by denaturing gradient gel electrophoresis (DGGE) mutation analysis. We identified different combinatory mixtures of LDLR- and LDLRAP1-gene defects as the cause for severe familial hypercholesterolemia in this family. We also show for the first time that a heterozygous LDLR mutation combined with a homozygous LDLRAP1 mutation produces a more severe hypercholesterolemia phenotype in the same family than a homozygous LDLR mutation alone.     

Association of the low-density lipoprotein receptor with caveolae in hamster and rat liver

The association of the low-density lipoprotein (LDL) receptor with detergent resistant hepatic membranes was investigated using discontinuous sucrose gradients. In liver homogenates from both hamsters and rats, the fractions with the highest concentrations of LDL receptor coincided with the location of caveolin-1, a marker of the cholesterol-rich caveolae. Feeding the animals diets enriched in cholesterol slightly shifted both LDL receptor and caveolin-1 to positions of lower density. The cholesterol content of the caveolae fractions was increased 2-fold in animals fed cholesterol-supplemented diets. In homogenates of CHO cells, fractionated in the same manner, the LDL receptor was absent from the caveolae fractions but was present in denser fractions near the bottom of the gradient. Addition of caveolin-1 antibody to solubilized caveolae from liver coimmunoprecipitated the LDL receptor. These observations suggest that in liver, the LDL receptor is mainly located in caveolae. This location contrasts with the clathrin-coated pit location observed in fibroblasts and CHO cells.     

Crystal Structure of Dinitrogenase Reductase-activating Glycohydrolase (DRAG) Reveals Conservation in the ADP-Ribosylhydrolase Fold and Specific Features in the ADP-Ribose-binding Pocket

Protein-reversible ADP-ribosylation is emerging as an important post-translational modification used to control enzymatic and protein activity in different biological systems. This modification regulates nitrogenase activity in several nitrogen-fixing bacterial species. ADP-ribosylation is catalyzed by ADP-ribosyltransferases and is reversed by ADP-ribosylhydrolases. The structure of the ADP-ribosylhydrolase that acts on Azospirillum brasilense nitrogenase (dinitrogenase reductase-activating glycohydrolase, DraG) has been solved at a resolution of 2.5 Å. This bacterial member of the ADP-ribosylhydrolase family acts specifically towards a mono-ADP-ribosylated substrate. The protein shows an all-α-helix structure with two magnesium ions located in the active site. Comparison of the DraG structure with orthologues deposited in the Protein Data Bank from Archaea and mammals indicates that the ADP-ribosylhydrolase fold is conserved in all domains of life. Modeling of the binding of the substrate ADP-ribosyl moiety to DraG is in excellent agreement with biochemical data.     

Specificity of reversible ADP-ribosylation and regulation of cellular processes

Proper and timely regulation of cellular processes is fundamental to the overall health and viability of organisms across all kingdoms of life. Thus, organisms have evolved multiple highly dynamic and complex biochemical signaling cascades in order to adapt and survive diverse challenges. One such method of conferring rapid adaptation is the addition or removal of reversible modifications of different chemical groups onto macromolecules which in turn induce the appropriate downstream outcome. ADP-ribosylation, the addition of ADP-ribose (ADPr) groups, represents one of these highly conserved signaling chemicals. Herein we outline the writers, erasers and readers of ADP-ribosylation and dip into the multitude of cellular processes they have been implicated in. We also review what we currently know on how specificity of activity is ensured for this important modification.     

多发性骨髓瘤细胞株ARH-77L1逆转录酶基因的克隆及体外表达研究

通过菌落原位分子杂交,从多发性骨髓瘤细胞株ARH-77 cDNA文库获得L1逆转录酶基因5’端序列．随后使用3’RACE技术,获得L1逆转录酶基因3’端序列及poly(A)尾．生物信息学分析表明：该L1逆转录酶DNA序列有一长552bp开放性阅读框,编码184个氨基酸残基的多肽链,其相对分子质量约为21kDa．同时将编码L1逆转录酶保守区的开放性阅读框DNA片段与原核表达载体pQE30连接,得到重组原核表达质粒,利用大肠杆菌表达并获得L1逆转录酶融合蛋白．     

Unrestrained poly-ADP-ribosylation provides insights into chromatin regulation and human disease

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Molecular cloning and polymorphism analysis of the prion protein gene in Tan sheep of Ningxia, China

The resistance or susceptibility of sheep to scrapie is associated with polymorphisms of the prion protein gene (PRNP), particularly, single nucleotide polymorphisms (SNPs) in amino acid positions 136, 154 and 171. The prion protein (PrP) gene sequence and the deduced amino acid alignment of prion protein in Tan sheep, a local Chinese sheep breed traditionally raised in Ningxia, northwestern China, were determined and variability of the PrP amino acids sequence was analyzed in this study. The PrP nucleic acids and amino acids sequences of 112 Tan sheep were highly homogenous, although polymorphism of the PrP gene was detected at several sites, particularly codons 106, 154, and 171. The analysis of both sequences revealed that the most predominant allele at codons 136, 154 and 171 in Tan sheep was ARQ, which was known to be associated with high susceptibility to scrapie in sheep. The result suggests that Tan sheep is potentially susceptible to scrapie. Our findings provide valuable information for future breeding projects to scrapie resistance in Tan sheep.     

AMN Directs Endocytosis of the Intrinsic Factor‐Vitamin B12 Receptor Cubam by Engaging ARH or Dab2

Cubam is a multi‐ligand receptor involved in dietary uptake of intrinsic factor‐vitamin B₁₂ in the small intestine and reabsorption of various low‐molecular‐weight proteins (such as albumin, transferrin, apolipoprotein A‐I and vitamin D‐binding protein) in the kidney. Cubam is composed of two proteins: cubilin and amnionless. Cubilin harbors ligand binding capabilities, while amnionless provides membrane anchorage and potential endocytic capacity via two FXNPXF signals within the cytosolic domain. These signals are similar to the FXNPXY signals found in members of the low‐density lipoprotein receptor superfamily, which associate with clathrin‐associated sorting proteins, including Disabled‐2 (Dab2) and autosomal recessive hypercholesterolemia (ARH), during endocytosis. We therefore investigated the functionality of each amnionless FXNPXF signal and their respective interaction with sorting proteins. By sequential mutation and expression of a panel of amnionless mutants combined with yeast two‐hybrid analyses, we demonstrate that the signals are functionally redundant and both are able to mediate endocytosis of cubam through interaction with Dab2 and ARH.     

Serum 11-deoxycorticosterone levels in adrenal-regeneration hypertension under conditions of quiescence and stress.

A time course study to measure adrenal cortical function was undertaken for the period prior to the development of hypertension until the onset of hypertension in the adrenal-regeneration hypertension (ARH) model. Quiescent rat kills were used so that all adrenal cortical parameters investigated would reflect basal or resting levels for controls. Thus a more accurate determination of the differences between control and experimental animals could be made. A radioimmunoassay procedure for deoxycorticosterone was developed to measure this steroid in individual rat serum samples. Elevated serum deoxycorticosterone levels were observed in rats with regenerating adrenals when they were killed under quiescent conditions. This agreed with our recently reported in vitro finding of restoration of cholesterol side chain cleavage activity while 11beta-hydroxylase activity remained imparied 25 days after adrenal enucleation. When rats were killed after ether stress, deoxycorticosterone levels were elevated in both control rats and in rats with regenerating adrenals but the difference was not significant. In contrast, after ether stress serum corticosterone levels were lower in rats with regenerating adrenals than in controls. These studies, in conjunction with our previous in vitro findings, point to the importance of deoxycorticosterone in the pathogenesis of adrenal regeneration hypertension and help to explain the anomalous corticosteroid secretion rate data found in this experimental hypertension model.     

Structure and function of the ARH family of ADP-ribosyl-acceptor hydrolases

ADP-ribosylation is a post-translational protein modification, in which ADP-ribose is transferred from nicotinamide adenine dinucleotide (NAD⁺) to specific acceptors, thereby altering their activities. The ADP-ribose transfer reactions are divided into mono- and poly-(ADP-ribosyl)ation. Cellular ADP-ribosylation levels are tightly regulated by enzymes that transfer ADP-ribose to acceptor proteins (e.g., ADP-ribosyltransferases, poly-(ADP-ribose) polymerases (PARP)) and those that cleave the linkage between ADP-ribose and acceptor (e.g., ADP-ribosyl-acceptor hydrolases (ARH), poly-(ADP-ribose) glycohydrolases (PARG)), thereby constituting an ADP-ribosylation cycle. This review summarizes current findings related to the ARH family of proteins. This family comprises three members (ARH1-3) with similar size (39 kDa) and amino acid sequence. ARH1 catalyzes the hydrolysis of the N-glycosidic bond of mono-(ADP-ribosyl)ated arginine. ARH3 hydrolyzes poly-(ADP-ribose) (PAR) and O-acetyl-ADP-ribose. The different substrate specificities of ARH1 and ARH3 contribute to their unique roles in the cell. Based on a phenotype analysis of ARH1^−/− and ARH3^−/− mice, ARH1 is involved in the action by bacterial toxins as well as in tumorigenesis. ARH3 participates in the degradation of PAR that is synthesized by PARP1 in response to oxidative stress-induced DNA damage; this hydrolytic reaction suppresses PAR-mediated cell death, a pathway termed parthanatos.