Genetic,structural, and functional analysis of pathogenic variations causing methylmalonyl-CoA epimerase deficiency

Human methylmalonyl-CoA epimerase (MCEE) catalyzes the interconversion of d-methylmalonyl-CoA and l-methylmalonyl-CoA in propionate catabolism. Autosomal recessive pathogenic variations in MCEE reportedly cause methylmalonic aciduria (MMAuria) in eleven patients. We investigated a cohort of 150 individuals suffering from MMAuria of unknown origin, identifying ten new patients with pathogenic variations in MCEE. Nine patients were homozygous for the known nonsense variation p.Arg47* (c.139C > T), and one for the novel missense variation p.Ile53Arg (c.158T > G). To understand better the molecular basis of MCEE deficiency, we mapped p.Ile53Arg, and two previously described pathogenic variations p.Lys60Gln and p.Arg143Cys, onto our 1.8 Å structure of wild-type (wt) human MCEE. This revealed potential dimeric assembly disruption by p.Ile53Arg, but no clear defects from p.Lys60Gln or p.Arg143Cys. We solved the structure of MCEE-Arg143Cys to 1.9 Å and found significant disruption of two important loop structures, potentially impacting surface features as well as the active-site pocket. Functional analysis of MCEE-Ile53Arg expressed in a bacterial recombinant system as well as patient-derived fibroblasts revealed nearly undetectable soluble protein levels, defective globular protein behavior, and using a newly developed assay, lack of enzymatic activity - consistent with misfolded protein. By contrast, soluble protein levels, unfolding characteristics and activity of MCEE-Lys60Gln were comparable to wt, leaving unclear how this variation may cause disease. MCEE-Arg143Cys was detectable at comparable levels to wt MCEE, but had slightly altered unfolding kinetics and greatly reduced activity. These studies reveal ten new patients with MCEE deficiency and rationalize misfolding and loss of activity as molecular defects in MCEE-type MMAuria.     

Structural and functional analysis of missense mutations in fumarylacetoacetate hydrolase, the gene deficient in hereditary tyrosinemia type 1

Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disease caused by a deficiency of the enzyme involved in the last step of tyrosine degradation, fumarylacetoacetate hydrolase (FAH). Thus far, 34 mutations in the FAH gene have been reported in various HT1 patients. Site-directed mutagenesis of the FAH cDNA was used to investigate the effects of eight missense mutations found in HTI patients on the structure and activity of FAH. Mutated FAH proteins were expressed in Escherichia coli and in mammalian CV-1 cells. Mutations N16I, F62C, A134D, C193R, D233V, and W234G lead to enzymatically inactive FAH proteins. Two mutations (R341W, associated with the pseudo-deficiency phenotype, and Q279R) produced proteins with a level of activity comparable to the wild-type enzyme. The N16I, F62C, C193R, and W234G variants were enriched in an insoluble cellular fraction, suggesting that these amino acid substitutions interfere with the proper folding of the enzyme. Based on the tertiary structure of FAH, on circular dichroism data, and on solubility measurements, we propose that the studied missense mutations cause three types of structural effects on the enzyme: 1) gross structural perturbations, 2) limited conformational changes in the active site, and 3) conformational modifications with no significant effect on enzymatic activity.     

Structural and functional analyses of disease-causing missense mutations in Bloom syndrome protein

Bloom syndrome (BS) is an autosomal recessive disorder characterized by genomic instability and the early development of many types of cancer. Missense mutations have been identified in the BLM gene (encoding a RecQ helicase) in affected individuals, but the molecular mechanism and the structural basis of the effects of these mutations remain to be elucidated. We analysed five disease-causing missense mutations that are localized in the BLM helicase core region: Q672R, I841T, C878R, G891E and C901Y. The disease-causing mutants had low ATPase and helicase activities but their ATP binding abilities were normal, except for Q672, whose ATP binding activity was lower than that of the intact BLM helicase. Mutants C878R, mapping near motif IV, and G891E and C901Y, mapping in motif IV, displayed severe DNA-binding defects. We used molecular modelling to analyse these mutations. Our work provides insights into the molecular basis of BLM pathology, and reveals structural elements implicated in coupling DNA binding to ATP hydrolysis and DNA unwinding. Our findings will help to explain the mechanism underlying BLM catalysis and interpreting new BLM causing mutations identified in the future.     

Three-dimensional structural location and molecular functional effects of missense SNPs in the T cell receptor Vbeta domain

The mechanisms by which human single nucleotide polymorphisms (SNPs) influence susceptibility to disease are not yet well understood. In a previous study, we developed a structure-based model that may be used to identify which missense SNPs are neutral and which are deleterious to protein function and so potentially involved in disease (Wang and Moult, Hum Mutat 2001;263-270). The model has now been applied to a set of 54 missense cSNPs in the 46 functional T-cell receptor Vbeta-genes. Most of these missense cSNPs are found to be neutral, but 10 are identified as likely deleterious to protein function. Only one was previously associated with disease. We suggest that the others may be disease related but that redundancy in the T-cell response prevents any simple, monogenic effect. Therefore, these SNPs are the most likely contributors to complex, polygenic disease traits. It has been noted that there is a surprisingly high (74%) fraction of nonsynonymous SNPs in these genes. Contrary to expectation, the analysis shows that these are not associated with an unusually high fraction of deleterious SNPs, nor do they significantly contribute to a larger range of antigen recognition or a reduced superantigen-binding repertoire.     

Mutational analysis of SRY: nonsense and missense mutations in XY sex reversal

Summary XY females (n=17) were analysed for mutations in SRY (sex-determining region Y gene), a gene that has recently been equated with the testis determining factor (TDF). SRY sequences were amplified by the polymerase chain reaction (PCR) and analysed by both the single strand conformational polymorphism assay (SSCP) and DNA sequencing. The DNA from two individuals gave altered SSCP patterns; only these two individuals showed any DNA sequence variation. In both cases, a single base change was found, one altering a tryptophan codon to a stop codon, the other causing a glycine to arginine amino acid substitution. These substitutions lie in the high mobility group (HMG)-related box of the SRY protein, a potential DNA-binding domain. The corresponding regions of DNA from the father of one individual and the paternal uncle of the other, were sequenced and found to be normal. Thus, in both cases, sex reversal is associated with de novo mutations in SRY. Combining this data with two previously published reports, a total of 40 XY females have now been analysed for mutations in SRY. The number of de novo mutations in SRY is now doubled to four, adding further strength to the argument that SRY is TDF.     

Metalloproteomics: forward and reverse approaches in metalloprotein structural and functional characterization

About one-third of all proteins are associated with a metal. Metalloproteomics is defined as the structural and functional characterization of metalloproteins on a genome-wide scale. The methodologies utilized in metalloproteomics, including both forward (bottom-up) and reverse (top-down) technologies, to provide information on the identity, quantity, and function of metalloproteins are discussed. Important techniques frequently employed in metalloproteomics include classical proteomic tools such as mass spectrometry and 2D gels, immobilized-metal affinity chromatography, bioinformatic sequence analysis and homology modeling, X-ray absorption spectroscopy and other synchrotron radiation based tools. Combinative applications of these techniques provide a powerful approach to understand the function of metalloproteins.     

Bioinformatics approaches for structural and functional analysis of proteins in secondary metabolism in Withania somnifera

Withania somnifera (Ashwagandha) is an affluent storehouse of large number of pharmacologically active secondary metabolites known as withanolides. These secondary metabolites are produced by withanolide biosynthetic pathway. Very less information is available on structural and functional aspects of enzymes involved in withanolides biosynthetic pathways of Withiana somnifera. We therefore performed a bioinformatics analysis to look at functional and structural properties of these important enzymes. The pathway enzymes taken for this study were 3-Hydroxy-3-methylglutaryl coenzyme A reductase, 1-Deoxy-d-xylulose-5-phosphate synthase, 1-Deoxy-d-xylulose-5-phosphate reductase, farnesyl pyrophosphate synthase, squalene synthase, squalene epoxidase, and cycloartenol synthase. The prediction of secondary structure was performed for basic structural information. Three-dimensional structures for these enzymes were predicted. The physico-chemical properties such as pI, AI, GRAVY and instability index were also studied. The current information will provide a platform to know the structural attributes responsible for the function of these protein until experimental structures become available.     

Mapping missense and nonsense mutations in genecI of bacteriophage lambda: Marker effects

Summary Amber and missense mutations in genecI of bacteriophage lambda were mapped by reciprocal four-factor crosses, selecting recombinants between the outside markers (N amber andO amber). Distances betweencI missense mutations were additive. SeveralcIamber mutants recombined with othercI mutations with a higher-frequency than expected from the map location. Multiple exchanges in theN-O region occurred at a frequency greater than expected by chance. This high negative interference was especially marked in crosses with thecIamber mutations that were strong recombiners.A newind mutation,ind2, was found neartsU51, to the left of the previously-knownindl mutation, which is located almost in the center of genecI. The mutationc50 maps to the right oftsU50 andc71. Mutationsc60, andts71, which differ in phenotype, are apparently at the same site.     

Computational approaches for predicting the biological effect of p53 missense mutations: a comparison of three sequence analysis based methods

Prediction of the biological effect of missense substitutions has become important because they are often observed in known or candidate disease susceptibility genes. In this paper, we carried out a 3-step analysis of 1514 missense substitutions in the DNA-binding domain (DBD) of TP53, the most frequently mutated gene in human cancers. First, we calculated two types of conservation scores based on a TP53 multiple sequence alignment (MSA) for each substitution: (i) Grantham Variation (GV), which measures the degree of biochemical variation among amino acids found at a given position in the MSA; (ii) Grantham Deviation (GD), which reflects the 'biochemical distance' of the mutant amino acid from the observed amino acid at a particular position (given by GV). Second, we used a method that combines GV and GD scores, Align-GVGD, to predict the transactivation activity of each missense substitution. We compared our predictions against experimentally measured transactivation activity (yeast assays) to evaluate their accuracy. Finally, the prediction results were compared with those obtained by the program Sorting Intolerant from Tolerant (SIFT) and Dayhoff's classification. Our predictions yielded high prediction accuracy for mutants showing a loss of transactivation ( approximately 88% specificity) with lower prediction accuracy for mutants with transactivation similar to that of the wild-type (67.9 to 71.2% sensitivity). Align-GVGD results were comparable to SIFT (88.3 to 90.6% and 67.4 to 70.3% specificity and sensitivity, respectively) and outperformed Dayhoff's classification (80 and 40.9% specificity and sensitivity, respectively). These results further demonstrate the utility of the Align-GVGD method, which was previously applied to BRCA1. Align-GVGD is available online at http://agvgd.iarc.fr.     

Structural and functional consequences of missense mutations in exon 5 of the lipoprotein lipase gene

Missense mutations in exon 5 of the LPL gene are the most common reported cause of LPL deficiency. Exon 5 is also the region with the strongest homology to pancreatic and hepatic lipase, and is conserved in LPL from different species. Mutant LPL proteins from post-heparin plasma from patients homozygous for missense mutations at amino acid positions 176, 188, 194, 205, and 207, and from COS cells transiently transfected with the corresponding cDNAs were quantified and characterized, in an attempt to determine which aspect of enzyme function was affected by each specific mutation. All but one of the mutant proteins were present, mainly as partially denatured LPL monomer, rendering further detailed assessment of their catalytic activity, affinity to heparin, and binding to lipoprotein particles difficult. However, the fresh unstable Gly(188)-->Glu LPL and the stable Ile(194)-->Thr LPL, although in native conformation, did not express lipase activity. It is proposed that many of the exon 5 mutant proteins are unable to achieve or maintain native dimer conformation, and that the Ile(194)-->Thr substitution interferes with access of lipid substrate to the catalytic pocket. These results stress the importance of conformational evaluation of mutant LPL. Absence of catalytic activity does not necessarily imply that the substituted amino acid plays a specific direct role in catalysis.     

Structural and functional in silico analysis of LRRK2 missense substitutions

The LRRK2 gene (Leucine-Rich Repeat Kinase 2, PARK8) is mutated in a significant number of cases of autosomal dominant Parkinson’s disease (PD) and in some sporadic cases of late-onset PD. LRRK2 is a large, complex protein that comprises several interaction domains: armadillo, ankyrin, leucine-rich repeats and WD40 domains; two catalytic domains: ROC-GTPase and serine/threonine kinase; and a COR domain (unknown function). Pathogenic mutations are scattered all over the domains of LRRK2, although the prevalence of mutations in some domains is higher (ROC-GTPase, COR and kinase). In this work, we model the structure of each domain to predict and explore the effects of described missense mutations and polymorphisms. The results allow us to postulate the possible effects of pathogenic mutations in the function of the protein, and hypothesize the importance of some polymorphisms that have not been linked directly to PD, but act as risk factors for the disease. In our analysis, we also study the effects of PD-related mutations in the kinase domain structure and in the phosphorylation of the activation loop to determine effects on kinase activity.     

Hypoxanthine-guanine phosphoribosyltransferase deficiency: analysis of HPRT mutations by direct sequencing and allele-specific amplification

Summary The Lesch-Nyhan syndrome is a severe X chromosome-linked human disease caused by a virtual absence of hypoxanthine-guanine phosphoribosyltransferase (HPRT) activity. A partial deficiency in the activity of this enzyme can result in gouty arthritis. To determine the genetic basis for reduction or loss of enzyme activity, we have amplified and sequenced the coding region of HPRT cDNA from four patients: one with LeschNyhan syndrome (HPRT_Perth) and three with partial deficiencies of HPRT activity, which have been designated HPRT_Urangan, HPRT_Swan and HPRT_Toowong. In all four patients, the only mutation identified was a single base substitution in exons 2 or 3 of the coding region, which in each case predicts a single amino acid substitution in the translated protein. Each base change was confirmed by allele-specific amplification of the patient's genomic DNA. It is interesting to note that the mutation found for HPRT_Perth is identical to that reported for HPRT_Flint. It appears that the two mutations are de novo events.     

Evolutionary,structural and functional relationships revealed by comparative analysis of syntenic genes in Rhizobiales

Background  

Comparative genomics has provided valuable insights into the nature of gene sequence variation and chromosomal organization of closely related bacterial species. However, questions about the biological significance of gene order conservation, or synteny, remain open. Moreover, few comprehensive studies have been reported for rhizobial genomes.     

Camptosemin, a tetrameric lectin of Camptosema ellipticum: structural and functional analysis

Lectins have been classified into a structurally diverse group of proteins that bind carbohydrates and glycoconjugates with high specificity. They are extremely useful molecules in the characterization of saccharides, as drug delivery mediators, and even as cellular surface makers. In this study, we present camptosemin, a new lectin from Camptosema ellipticum. It was characterized as an N-acetyl-d-galactosamine-binding homo-tetrameric lectin, with a molecular weight around 26 kDa/monomers. The monomers were stable over a wide range of pH values and exhibited pH-dependent oligomerization. Camptosemin promoted adhesion of breast cancer cells and hemagglutination, and both activities were inhibited by its binding of sugar. The stability and unfolding/folding behavior of this lectin was characterized using fluorescence and far-UV circular dichroism spectroscopies. The results indicate that chemical unfolding of camptosemin proceeds as a two-state monomer-tetramer process. In addition, small-angle X-ray scattering shows that camptosemin behaves as a soluble and stable homo-tetramer molecule in solution.     

Expression and molecular analysis of mutations in prolidase deficiency.

Prolidase (E.C.3.4.13.9) cleaves iminodipeptides. Prolidase deficiency (PD; McKusick 170100) is an autosomal recessive disorder with highly variable penetrance. We have identified two novel alleles in the prolidase gene (PEPD) by direct sequencing of PCR-amplified cDNA from a PD individual asymptomatic at age 11 years: a 551G-->A transition in exon 8 (R184Q) and a 833G-->A transition in exon 12 (G278D). To assess the biochemical phenotypes of these and two previously identified PEPD mutations (G448R and delE452), we have designed a transient-expression system for prolidase in COS-1 cells. The enzyme was expressed as a fusion protein carrying an N-terminal tag, the HA1 epitope of influenza hemagglutinin, allowing its immunological discrimination from the endogenous enzyme with a monoclonal antibody. Expression of the R184Q mutation produced 7.4% of control enzymatic activity whereas the expression of the G278D, G448R, and delE452 mutations produced inactive enzymes. Western analysis of the R184Q, G278D, and G448R prolidases revealed stable immunoreactive material whereas the delE452 prolidase was not detectable. Pulse-chase metabolic labeling of cells followed by immunoprecipitation revealed that the delE452 mutant protein was synthesized but had an increased rate of degradation.     

Dihydropyrimidine dehydrogenase (DPD) deficiency: identification and expression of missense mutations C29R, R886H and R235W

Dihydropyrimidine dehydrogenase (DPD) deficiency (McKusick 274270) is an autosomal recessive disease characterized by thymine-uraciluria in homozygous-deficient patients and associated with a variable clinical phenotype. Cancer patients with this defect should not be treated with the usual dose of 5-fluorouracil because of the expected lethal toxicity. In addition, heterozygosity for mutations in the DPD gene increases the risk of toxicity in cancer patients treated with this drug. Sequence analysis in a patient with complete DPD deficiency, previously shown to be heterozygous for the ΔC1897 frameshift mutation, revealed the presence of a novel missense mutation, R235W. Expression of this novel mutation and previously identified missense mutations C29R and R886H in Escherichia coli showed that both C29R and R235W lead to a mutant DPD protein without significant residual enzymatic activity. The R886H mutation, however, resulted in about 25% residual enzymatic activity and is unlikely to be responsible for the DPD-deficient phenotype. We show that the E. coli expression system is a valuable tool for examining DPD enzymatic variants. In addition, two new patients who were both heterozygous for the C29R mutation and the common splice donor site mutation were identified. Only one of these patients showed convulsive disorders during childhood, whereas the other showed no clinical phenotype, further illustrating the lack of correlation between genotype and phenotype in DPD deficiency. Received: 20 June 1997 / Accepted: 26 August 1997