Novel missense MTTP gene mutations causing abetalipoproteinemia

Objective

The microsomal triglyceride transfer protein (MTTP) plays a critical role in the formation of hepatic very low density lipoprotein. Abetalipoproteinemia (ABL) is a rare, naturally occurring extreme form of MTTP inhibition, which is characterized by the virtual absence of apolipoprotein (apo) B-containing lipoproteins in blood. The goal of this study was to examine the effect that four novel MTTP missense mutations had on protein interactions, expression and lipid-transfer activity, and to determine which mutations were responsible for the ABL phenotype observed in two patients.

Approach and results

In two patients with ABL, we identified in MTTP a novel frameshift mutation (K35Ffs*37), and four novel missense mutations, namely, G264R, Y528H, R540C, and N649S. When transiently expressed in COS-7 cells, all missense MTTP mutations interacted with apoB17, apoB48, and protein disulfide isomerase. Mutations Y528H and R540C, however, displayed negligible levels of MTTP activity and N649S displayed a partial reduction relative to the wild-type MTTP. In contrast, G264R retained full lipid-transfer activity.

Conclusions

These studies indicate that missense mutations Y528H, R540C, and N649S appear to cause ABL by reducing MTTP activity rather than by reducing binding of MTTP with protein disulfide isomerase or apoB. The region of MTTP containing amino acids 528 and 540 constitutes a critical domain for its lipid-transfer activity.     

Refolding of the catalytic and hinge domains of human MT1-mMP expressed in Escherichia coli and its characterization

The catalytic and hinge domain (Tyr112-Ile318) of the human membrane type-1 matrix metalloproteinase (MT1-MMP; MMP-14), containing hexa-histidines at the C-terminus (chMT1-MMP), was overexpressed in Escherichia coli. The expressed polypeptide was almost exclusively found in the inclusion body, and then purified by a single Ni2+-NTA agarose column chromatography after solubilization with 6 M urea. During refolding, the 26.9 kDa chMT1-MMP was processed to a 24.3 kDa intermediate form and then to a 22.2 kDa mature form. By Western blot analysis and mass spectrometry combined with N-terminal sequencing, the intermediate form was identified as a mixture of the Tyr112-Thr299 with a translation-initiating methionine and Ile114-Thr299, and that the mature form corresponds to Ile114-Pro290. These results demonstrate that the mature form was generated by successive autoproteolysis of the N- and C-terminal sites between Thr299-Thr300, Ala113-Ile114, and Pro290-Thr291 during refolding. Catalytic activity of the mature chMT1-MMP was demonstrated by a peptide cleavage assay. In addition, it has gelatinolytic activity and is able to activate proMMP-2 to the mature MMP-2. These results indicate that the refolded chMT1-MMP retains characteristics of MT1-MMP.     

Generation of a stable activated thrombin activable fibrinolysis inhibitor variant

Activated thrombin activable fibrinolysis inhibitor (TAFIa), generated upon activation of TAFI, exerts an antifibrinolytic effect. TAFIa is a thermolabile enzyme, inactivated through a conformational change. The objective of the current study was to generate a stable variant of human TAFIa. Using a site-directed as well as a random mutagenesis approach to generate a library of TAFI mutants, we identified two mutations that increase TAFIa stability, i.e. a Ser305 to Cys and a Thr329 to Ile mutation, respectively. Combining these mutations in TAFI-Ala147-Ile325, the most stable isoform of TAFIa (half-life of 9.4 +/- 0.4 min), revealed a TAFIa half-life of 70 +/- 3.1 min (i.e. an 11-fold increase versus 6.3 +/- 0.3 min for TAFIa-Ala147-Thr325, the most frequently occurring isoform of TAFI in humans) at 37 degrees C. Moreover, clot lysis (induced by tissue plasminogen activator) experiments in which TAFI-Ala147-Cys305-Ile325-Ile329 was added to TAFI-depleted plasma revealed a 50% clot lysis time of 313 +/- 77 min (i.e. a 3.0-fold increase versus 117 +/- 10 min for TAFI-Ala147-Thr325). The availability of a more stable TAFIa variant will facilitate the search for inhibitors and allow further structural analysis to elucidate the mechanisms of the instability of TAFIa.     

Amino acid pairing preferences in parallel beta-sheets in proteins

Statistical approaches have been applied to examine amino acid pairing preferences within parallel beta-sheets. The main chain hydrogen bonding pattern in parallel beta-sheets means that, for each residue pair, only one of the residues is involved in main chain hydrogen bonding with the strand containing the partner residue. We call this the hydrogen bonded (HB) residue and the partner residue the non-hydrogen bonded (nHB) residue, and differentiate between the favorability of a pair and that of its reverse pair, e.g. Asn(HB)-Thr(nHB)versus Thr(HB)-Asn(nHB). Significantly (p < or = 0.000001) favoured pairings were rationalised using stereochemical arguments. For instance, Asn(HB)-Thr(nHB) and Arg(HB)-Thr(nHB) were favoured pairs, where the residues adopted favoured chi1 rotamer positions that allowed side-chain interactions to occur. In contrast, Thr(HB)-Asn(nHB) and Thr(HB)-Arg(nHB) were not significantly favoured, and could only form side-chain interactions if the residues involved adopted less favourable chi1 conformations. The favourability of hydrophobic pairs e.g. Ile(HB)-Ile(nHB), Val(HB)-Val(nHB) and Leu(HB)-Ile(nHB) was explained by the residues adopting their most preferred chi1 and chi2 conformations, which enabled them to form nested arrangements. Cysteine-cysteine pairs are significantly favoured, although these do not form intrasheet disulphide bridges. Interactions between positively and negatively charged residues were asymmetrically preferred: those with the negatively charged residue at the HB position were more favoured. This trend was accounted for by the presence of general electrostatic interactions, which, based on analysis of distances between charged atoms, were likely to be stronger when the negatively charged residue is the HB partner. The Arg(HB)-Asp(nHB) interaction was an exception to this trend and its favorability was rationalised by the formation of specific side-chain interactions. This research provides rules that could be applied to protein structure prediction, comparative modelling and protein engineering and design. The methods used to analyse the pairing preferences are automated and detailed results are available (http://www.rubic.rdg.ac.uk/betapairprefsparallel/).     

Structural and ICAM-1-docking properties of a cyclic peptide from the I-domain of LFA-1: an inhibitor of ICAM-1/LFA- 1-mediated T-cell adhesion

The purpose of this work was to study the conformation of cyclic peptide 1, cyclo(1,12)-Pen1-Ile2-Thr3-Asp4-Gly5-Glu6-Ala7- Thr8-Asp9-Ser10-Gly11-Cys12-OH, derived from the I-domain of the LFA-1 alpha-subunit. We found that cyclic peptide 1 can bind to the D1-domain of ICAM-1 and inhibit ICAM-1/LFA-1-mediated homotypic and heterotypic T-cell adhesion. To understand the bioactive conformation and binding requirements for cyclic peptide 1, its solution structure was studied using NMR, CD, and molecular dynamics simulations. Furthermore, possible binding properties between the cyclic peptide and the D1-domain of ICAM-1 were evaluated using docking experiments. This cyclic peptide has a stable betaII -turn at Asp4- Gly5-Glu6-Ala7 and a betaI-turn at Pen1-Ile2-Thr3-Asp4; a less stable betaV-turn is found at the C-terminal region. The beta-turn at Asp4- Gly5-Glu6-Ala7 was also found in the X-ray structure of the I-domain of LFA-1. Our CD studies showed that the peptide binds to calcium/magnesium and forms a 1:1 (peptide:calcium/magnesium) complex with low cation concentrations and multiple types of complexes with higher cation concentrations. Binding to divalent cations causes a conformational change in peptide 1; this is consistent with our previous study that binding of peptide 1 to ICAM-1 was influenced by divalent cations. Docking studies show the interaction between cyclic peptide 1 and the D1-domain of ICAM-1; it indicates that the Ile2-Thr3-Asp4-Gly4-Glu6-Ala7-Thr8 sequence interacts with the F and C strands of the D1-domain. Finally, these studies will help us design a new generation of selective peptides that may bind better to the D1-domain of ICAM-1.     

ApoB100-LDL Acts as a Metabolic Signal from Liver to Peripheral Fat Causing Inhibition of Lipolysis in Adipocytes

Background

Free fatty acids released from adipose tissue affect the synthesis of apolipoprotein B-containing lipoproteins and glucose metabolism in the liver. Whether there also exists a reciprocal metabolic arm affecting energy metabolism in white adipose tissue is unknown.

Methods and Findings

We investigated the effects of apoB-containing lipoproteins on catecholamine-induced lipolysis in adipocytes from subcutaneous fat cells of obese but otherwise healthy men, fat pads from mice with plasma lipoproteins containing high or intermediate levels of apoB100 or no apoB100, primary cultured adipocytes, and 3T3-L1 cells. In subcutaneous fat cells, the rate of lipolysis was inversely related to plasma apoB levels. In human primary adipocytes, LDL inhibited lipolysis in a concentration-dependent fashion. In contrast, VLDL had no effect. Lipolysis was increased in fat pads from mice lacking plasma apoB100, reduced in apoB100-only mice, and intermediate in wild-type mice. Mice lacking apoB100 also had higher oxygen consumption and lipid oxidation. In 3T3-L1 cells, apoB100-containing lipoproteins inhibited lipolysis in a dose-dependent fashion, but lipoproteins containing apoB48 had no effect. ApoB100-LDL mediated inhibition of lipolysis was abolished in fat pads of mice deficient in the LDL receptor (Ldlr^−/−Apob ^100/100).

Conclusions

Our results show that the binding of apoB100-LDL to adipocytes via the LDL receptor inhibits intracellular noradrenaline-induced lipolysis in adipocytes. Thus, apoB100-LDL is a novel signaling molecule from the liver to peripheral fat deposits that may be an important link between atherogenic dyslipidemias and facets of the metabolic syndrome.     

The apolipoprotein B Q3405E polymorphism has no effect on its low-density-lipoprotein receptor binding affinity

A better understanding of the apolipoprotein B100 (apoB100) sequences involved in binding to the low-density lipoprotein (LDL) receptor will be achieved by studying the effects of polymorphisms and rare mutations of apoB100. Upon re-examination of apoB100 DNA sequencing discrepancies, a charge-change polymorphism, Q3405E, was found in the putative LDL receptor binding domain of the protein. Positively charged lysine and arginine side chains of the protein have been demonstrated to participate in the ligand. This led us to propose that the presence of an additional negative charge in close proximity could have an impact on the binding affinity. The polymorphism is the result of a C-to-G transition at nucleotide 10422. Population screening revealed 20 of the less common glutamate alleles at an allele frequency of 0.9%. The effect of the presence of one glutamate allele on the binding affinity of LDL for the LDL receptor was investigated in seven heterozygous individuals by a competitive dual-label fibroblast binding assay. One individual who was homozygous for the glutamate allele was discovered and her LDL examined in a competitive displacement binding assay. The additional negative charge at residue 3405 had no detectable affect on the binding affinity. Received: 8 May 1996 / Revised: 9 July 1996     

Primary structure comparison of the proposed low density lipoprotein (LDL) receptor binding domain of human and pig apolipoprotein B: implications for LDL-receptor interactions

Apolipoprotein B (apoB) is the predominant protein in low density lipoprotein (LDL) and is responsible for LDL binding to the LDL receptor. Although the primary amino acid sequence of human apoB has been determined, little is known about the structural domains involved in mediating apoB binding to the LDL receptor. Amino acid sequence comparisons across species lines provide a means of defining structures that are essential for function. We have sequenced a l.l kb fragment of pig apoB genomic DNA, corresponding to a 363 amino acid segment proposed to mediate human apoB binding to the LDL receptor. In human apoB this domain contains two regions enriched in positively charged amino acids flanking two disulfide-linked cysteine residues. The pig amino acid sequence shared 72% identity with the human sequence. However, there were differences that have significant structural and functional implications. Human apoB arginine-3,359 corresponds to a critical arginine (position 142) residue in the apoE LDL receptor binding domain. In the pig, this arginine residue was not conserved. Also, the two disulfide-linked cysteine residues found near the proposed apoB binding domain were not conserved in the pig. Despite these differences, pig LDL had a higher affinity than human LDL for both the pig and human LDL receptor. Thus, these features are not required for high affinity binding of pig LDL to the LDL receptor, and may not be necessary for the binding of human LDL to the LDL receptor.     

Study of highly constitutively active mutants suggests how cAMP activates cAMP receptor protein

The cAMP receptor protein (CRP) of Escherichia coli undergoes a conformational change in response to cAMP binding that allows it to bind specific DNA sequences. Using an in vivo screening method following the simultaneous randomization of the codons at positions 127 and 128 (two C-helix residues of the protein interacting with cAMP), we have isolated a series of novel constitutively active CRP variants. Sequence analysis showed that this group of variants commonly possesses leucine or methionine at position 127 with a beta-branched amino acid at position 128. One specific variant, T127L/S128I CRP, showed extremely high cAMP-independent DNA binding affinity comparable with that of cAMP-bound wild-type CRP. Further biochemical analysis of this variant and others revealed that Leu(127) and Ile(128) have different roles in stabilizing the active conformation of CRP in the absence of cAMP. Leu(127) contributes to an improved leucine zipper at the dimer interface, leading to an altered intersubunit interaction in the C-helix region. In contrast, Ile(128) stabilizes the proper position of the beta4/beta5 loop by functionally communicating with Leu(61). By analogy, the results suggest two direct local effects of cAMP binding in the course of activating wild-type CRP: (i) C-helix repositioning through direct interaction with Thr(127) and Ser(128) and (ii) the concomitant reorientation of the beta4/beta5 loop. Finally, we also report that elevated expression of T127L/S128I CRP markedly perturbed E. coli growth even in the absence of cAMP, which suggests why comparably active variants have not been described previously.     

Two polymorphic forms of human histamine methyltransferase: structural, thermal, and kinetic comparisons

BACKGROUND: Histamine plays important biological roles in cell-to-cell communication; it is a mediator in allergic responses, a regulator of gastric acid secretion, a messenger in bronchial asthma, and a neurotransmitter in the central nervous system. Histamine acts by binding to histamine receptors, and its local action is terminated primarily by methylation. Human histamine N-methyltransferase (HNMT) has a common polymorphism at residue 105 that correlates with the high- (Thr) and low- (Ile) activity phenotypes. RESULTS: Two ternary structures of human HNMT have been determined: the Thr105 variant complexed with its substrate histamine and reaction product AdoHcy and the Ile105 variant complexed with an inhibitor (quinacrine) and AdoHcy. Our steady-state kinetic data indicate that the recombinant Ile105 variant shows 1.8- and 1.3-fold increases in the apparent K(M) for AdoMet and histamine, respectively, and slightly (16%) but consistently lower specific activity as compared to that of the Thr105 variant. These differences hold over a temperature range of 25 degrees C-45 degrees C in vitro. Only at a temperature of 50 degrees C or higher is the Ile105 variant more thermolabile than the Thr105 enzyme. CONCLUSIONS: HNMT has a 2 domain structure including a consensus AdoMet binding domain, where the residue 105 is located on the surface, consistent with the kinetic data that the polymorphism does not affect overall protein stability at physiological temperatures but lowers K(M) values for AdoMet and histamine. The interactions between HNMT and quinacrine provide the first structural insights into a large group of pharmacologic HNMT inhibitors and their mechanisms of inhibition.     

Theoretical 3D model of histamine N-methyltransferase: insights into the effects of a genetic polymorphism on enzymatic activity and thermal stability

Histamine N-methyltransferase (HNMT) catalyzes the N-methylation of histamine in mammals. The experimentally determined HNMT three-dimensional (3D) structure is not available. However, there is a common genetic polymorphism for human HNMT (Thr105Ile) that reduces enzymatic activity and is a risk factor for asthma. To obtain insights into mechanisms responsible for the effects of that polymorphism on enzymatic activity and thermal stability, we predicted the 3D structure of HNMT using the threading method and molecular dynamics simulations in water. Herein, we report a theoretical 3D model of human HNMT which reveals that polymorphic residue Thr105Ile is located in the turn between a beta strand and an alpha helix on the protein surface away from the active site of HNMT. Ile105 energetically destabilizes folded HNMT because of its low Chou-Fasman score for forming a turn conformation and the exposure of its hydrophobic side chain to aqueous solution. It thus promotes the formation of misfolded proteins that are prone to the clearance by proteasomes. This information explains, for the first time, how genetic polymorphisms can cause enhanced protein degradation and why the thermal stability of allozyme Ile105 is lower than that of Thr105. It also supports the hypothesis that the experimental observation of a significantly lower level of HNMT enzymatic activity for allozyme Ile105 than that with Thr105 is due to a decreased concentration of allozyme Ile105, but not an alternation of the active-site topology of HNMT caused by the difference at residue 105.     

Conformations of the central transforming region (Ile 55-Met 67) of the p21 protein and their relationship to activation of the protein

The GTP-binding p21 protein, encoded by the ras-oncogene, becomes transforming if amino acid substitutions are made at critical positions in the polypeptide chain, e.g., at Gly 12, Gly 13, Ala 59, Gln 61 and Glu 63. Most of these substitutions occur in two phosphate-binding loop regions, Tyr 4-Thr 20, herein designated as segment 1, and Ile 55-Met 67, herein designated, as segment 2. These two segments are homologous to two corresponding regions in the two purine nucleotide binding proteins, bacterial elongation factor (EF-tu) (Val 12-Thr 28 corresponds to segment 1; His 78-Ile 92 corresponds to segment 2) and adenylate kinase (ADK) (Lys 9-Cys 25 corresponds to segment 1 and Tyr 95-Arg 107 corresponds to segment 2). We find that the conformations of the segment 1 region in the p21 protein, EF-tu and ADK are similar to one another and that the conformation of the segment 2 region of EF-tu is superimposable on that of segment 2 of ADK. Furthermore, the relative position of the two segments in EF-tu is strikingly similar to that of the two segments in ADK. In the originally proposed X-ray structure for the p21 protein, the conformation of segment 2 in the p21 protein is not similar to that found for the other two proteins, and its disposition relative to segment 1 and the remainder of the protein is also different from that observed for the other two proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhanced clearance from plasma of low density lipoproteins containing a truncated apolipoprotein, apoB-89

Previously we have reported on a kindred with hypobetalipoproteinemia in which three sisters were found to be compound heterozygotes for two newly described truncated forms of apoB. apoB-40 and apoB-89. ApoB-89-containing low density lipoproteins (LDL) bound with increased affinity to cultured normal human fibroblasts and were internalized and degraded at increased rates, suggesting that the low plasma concentrations of apoB-89-LDL of the patients could be due to enhanced rates of clearance through LDL-receptors. To examine this hypothesis, apoB-89-LDL was isolated from the three study subjects and apoB-100-LDL from two control subjects. LDL was conjugated to the radioiodinated residualizing label, dilactitol tyramine (*I-DLT, containing either 125I or 131I). *I-DLT-apoB-89-LDL and *I-DLT-apoB-100-LDL were simultaneously injected into ear veins of rabbits. The clearance from plasma and hepatic accumulations of both radiolabeled LDL fractions were followed over 24 h. Fractional catabolic rates (FCR) of apoB-89-LDL were 0.105 +/- 0.012 h-1 compared to 0.054 +/- 0.007 h-1 for apoB-100-LDL. In agreement with the enhanced clearance from plasma, 1.72 to 1.87 times more *I-DLT-apoB-89-LDL than *I-DLT-apoB-100-LDL accumulated in the livers 24 h after injection. There was no significant difference in splenic accumulation, suggesting that LDL-receptors rather than scavenger receptors mediated the enhanced clearance of apoB-89-LDL. To assess further the importance of LDL-receptors, *I-DLT-apoB-89-LDL and *I-DLT-apoB-100-LDL were reductively methylated to inhibit their interactions with LDL-receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of amino acid residues responsible for the alpha5 subunit binding selectivity of L-655,708, a benzodiazepine binding site ligand at the GABA(A) receptor

L-655,708 is a ligand for the benzodiazepine site of the gamma-aminobutyric acid type A (GABA(A)) receptor that exhibits a 100-fold higher affinity for alpha5-containing receptors compared with alpha1-containing receptors. Molecular biology approaches have been used to determine which residues in the alpha5 subunit are responsible for this selectivity. Two amino acids have been identified, alpha5Thr208 and alpha5Ile215, each of which individually confer approximately 10-fold binding selectivity for the ligand and which together account for the 100-fold higher affinity of this ligand at alpha5-containing receptors. L-655,708 is a partial inverse agonist at the GABA(A) receptor which exhibited no functional selectivity between alpha1- and alpha5-containing receptors and showed no change in efficacy at receptors containing alpha1 subunits where amino acids at both of the sites had been altered to their alpha5 counterparts (alpha1Ser205-Thr,Val212-Ile). In addition to determining the binding selectivity of L-655,708, these amino acid residues also influence the binding affinities of a number of other benzodiazepine (BZ) site ligands. They are thus important elements of the BZ site of the GABA(A) receptor, and further delineate a region just N-terminal to the first transmembrane domain of the receptor alpha subunit that contributes to this binding site.     

Oxidized phospholipids, linked to apolipoprotein B of oxidized LDL, are ligands for macrophage scavenger receptors

Previous studies have shown that macrophage receptors for oxidized LDL (OxLDL) recognize both the lipid and protein moieties, and that a monoclonal antibody against OxLDL, EO6, also recognizes both species. The present studies show directly that during LDL oxidation phospholipids become covalently attached to apolipoprotein B (apoB). After exhaustive extraction of lipids, apoB of native LDL contained 4 +/- 3 moles of phosphorus/mole protein. In contrast, apoB of OxLDL contained approximately 75 moles of phosphorus/mole protein. Saponification of this apoB released phosphorus, choline, and saturated fatty acids in a molar ratio of 1.0:0.98:0.84. When LDL was reductively methylated prior to oxidation, the amount of phospholipid covalently bound was reduced by about 80%, indicating that the phospholipids attach at lysine epsilon amino groups. Progressive decreases in the phospholipid associated with apoB of OxLDL decreased the ability of the protein to compete for binding to macrophage scavenger receptors and decreased its reactivity with antibody EO6.We postulate that some oxidized phospholipids containing fatty acid aldehydes at the sn-2 position bind to lysine residues of apoB while others remain unreacted within the lipid phase. This would account for the interchangeability of lipid and apolipoprotein of OxLDL with respect to receptor binding and antibody recognition.     

Variants of the microsomal triglyceride transfer protein gene are associated with plasma cholesterol levels and body mass index.

The microsomal triglyceride transfer protein (MTP) is required for the assembly and secretion of apolipoprotein B (apoB)-containing lipoproteins from liver and intestine. We set out to study the phenotypic modulation of all common genetic variants in the MTP gene. In addition, we aimed at characterizing the association between the various polymorphisms. A total of 564 healthy men were genotyped for the MTP -493 G/T, -400 A/T, and -164 T/C promoter polymorphisms, as well as the Q/H 95, I/T 128, Q/E 244, and H/Q 297 missense polymorphisms. The -493 G/T, -164 T/C, and I/T 128 polymorphisms showed to be in almost complete linkage disequilibrium. Subjects homozygous for the less common -493 T, -164 C, and T 128 alleles showed significantly lower plasma total and LDL cholesterol levels and plasma LDL apoB levels, and also significantly higher body mass index (BMI) and plasma insulin levels compared with carriers of the common alleles. The associations between plasma total cholesterol and MTP -493 genotype was verified in a cohort consisting of 1,117 disease-free control subjects of the West of Scotland Coronary Prevention Study (WOSCOPS). None of the other polymorphisms showed any significant change in either lipid and lipoprotein levels or anthropometric variables.In summary, two promoter polymorphisms and one missense polymorphism in the MTP gene alter plasma total and LDL cholesterol levels, plasma LDL apoB levels, BMI, and insulin levels. This may, in turn, have implications for genetic regulation of cardiovascular risk factors.     

Association of Histamine N-Methyltransferase Thr105Ile Polymorphism with Parkinson’s Disease and Schizophrenia in Han Chinese: A Case-Control Study

Parkinson’s disease (PD) and schizophrenia (SCZ) are frequent central nervous disorders that have unclear etiologies but that show similarities in their pathogenesis. Since elevated histamine levels in the brain have been associated with PD and SCZ, we wanted to explore whether the Thr105Ile substitution in the histamine N-methyltransferase gene (HNMT-Thr105Ile), which impairs histamine degradation, is associated with either disease. We used the ligase detection reaction to genotype a case-control cohort of Han Chinese patients with PD or SCZ and healthy controls at the HNMT-Thr105Ile locus. The Ile allele was associated with reduced risk of PD (OR 0.516, 95%CI 0.318 to 0.838, p = 0.007) and of SCZ (OR 0.499, 95%CI 0.288 to 0.865, p = 0.011). Genotype frequencies and minor allele frequencies were similar between patients and controls when we compared males with females or early-onset patients with late-onset ones. Genotype and allele frequencies were not significantly different between PD patients with dyskinesia and PD patients without dyskinesia. Our results suggest that the heterozygous Thr/Ile genotype at the HNMT-Thr105Ile locus and the minor Ile105 allele protect against PD and SCZ in Han Chinese.     

Association of polymorphisms in glutamate-cysteine ligase catalytic subunit and microsomal triglyceride transfer protein genes with nonalcoholic fatty liver disease

Genetic and environmental factors are important for the development of nonalcoholic fatty liver disease (NAFLD). The aim of the present study was to examine the single nucleotide polymorphism (SNP) -129C/T (rs17883901) in glutamate-cysteine ligase catalytic subunit (GCLC) and SNPs I128T (rs3816873) and Q95H (rs61733139) in microsomal triglyceride transfer protein (MTTP) in NAFLD. Eighty-three patients with a diagnosis of NAFLD and 93 healthy subjects were included in the study. Tetra amplification refractory mutation system-polymerase chain reaction was designed to detect the SNPs. There were no significant differences in the polymorphism of -129C/T (rs17883901) of the GCLC gene among NAFLD and control groups (p?>?0.05). A significant difference was observed between NAFLD and control group regarding the SNP I128T (rs3816873) in the coding region of the MTTP gene (p?相似文献   

An ApaLI restriction site polymorphism is associated with the MB19 polymorphism in apolipoprotein B

An apolipoprotein (apo) B-specific monoclonal antibody, MB19, detects a commonly occurring two-allele genetic polymorphism in human apoB (Young, S. G., S. J. Bertics, L. K. Curtiss, D. C. Casal, and J. L. Witztum. 1986. Proc. Natl. Acad. Sci. USA. 83: 1101-1105). Antibody MB19 binds to two different allotypes of apoB, MB19(1) and MB19(2), with high and low affinity, respectively. The epitope for antibody MB19 is located within apoB-100 thrombolytic fragment T4 (apoB-100 amino acid residues 1-1297). In this study, we examined the relationship of the MB19 polymorphism to a C----T nucleotide substitution at apoB cDNA nucleotide 421. This nucleotide substitution results in a Thr----Ile substitution at apoB-100 amino acid 71, and it changes an ApaLI restriction endonuclease site in the apoB gene. The nucleotide substitution was easily detectable by ApaLI digestion of a 141-base pair fragment of the apoB gene obtained by enzymatic amplification of genomic DNA. In 62 subjects, the MB19 phenotype, as determined by radioimmunoassays, correlated perfectly with the ApaLI restriction site polymorphism in the amplified DNA. The apoB allotype MB19(1) is associated with an Ile at residue 71 and the absence of the ApaLI site, whereas the apoB allotype MB19(2) is associated with a Thr at residue 71 and the presence of the ApaLI site. We conclude that the amino acid substitution at residue 71 probably accounts for the MB19 polymorphism in apoB.     

Interaction between shrimp and white spot syndrome virus through PmRab7-VP28 complex: an insight using simulation and docking studies

White spot disease is a devastating disease of shrimp Penaeus monodon in which the shrimp receptor protein PmRab7 interacts with viral envelop protein VP28 to form PmRab7–VP28 complex, which causes initiation of the disease. The molecular mechanism implicated in the disease, the dynamic behavior of proteins as well as interaction between both the biological counterparts that crafts a micro-environment feasible for entry of virus into the shrimp is still unknown. In the present study, we applied molecular modeling (MM), molecular dynamics (MD) and docking to compute surface mapping of infective amino acid residues between interacting proteins. Our result showed that α-helix of PmRab7 (encompassing Ser74, Ile143, Thr184, Arg53, Asn144, Thr184, Arg53, Arg79) interacts with β-sheets of VP28 (containing Ser74, Ile143, Thr184, Arg53, Asn144, Thr184, Arg53, Arg79) and Arg69-Ser74, Val75-Ile143, Leu73-Ile143, Arg79-Asn144, Ala198-Ala182 bonds contributed in the formation of PmRab7–VP28 complex. Further studies on the amino acid residues and bonds may open new possibilities for preventing PmRab7–VP28 complex formation, thus reducing chances of WSD. The quantitative predictions provide a scope for experimental testing in future as well as endow with a straightforward evidence to comprehend cellular mechanisms underlying the disease.