Classical homocystinuria is caused by mutations in the cystathionine β-synthase (CBS) gene. Previous experiments in bacterial and yeast cells showed that many mutant CBS enzymes misfold and that chemical chaperones enable proper folding of a number of mutations. In the present study, we tested the extent of misfolding of 27 CBS mutations previously tested in E. coli under the more folding-permissive conditions of mammalian CHO-K1 cells and the ability of chaperones to rescue the conformation of these mutations. Expression of mutations in mammalian cells increased the median activity 16-fold and the amount of tetramers 3.2-fold compared with expression in bacteria. Subsequently, we tested the responses of seven selected mutations to three compounds with chaperone-like activity. Aminooxyacetic acid and 4-phenylbutyric acid exhibited only a weak effect. In contrast, heme arginate substantially increased the formation of mutant CBS protein tetramers (up to sixfold) and rescued catalytic activity (up to ninefold) of five out of seven mutations (p.A114V, p.K102N, p.R125Q, p.R266K, and p.R369C). The greatest effect of heme arginate was observed for the mutation p.R125Q, which is non-responsive to in vivo treatment with vitamin B(6). Moreover, the heme responsiveness of the p.R125Q mutation was confirmed in fibroblasts derived from a patient homozygous for this genetic variant. Based on these data, we propose that a distinct group of heme-responsive CBS mutations may exist and that the heme pocket of CBS may become an important target for designing novel therapies for homocystinuria.

• MeSH
• arginin farmakologie   MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• cystathionin-beta-synthasa genetika   metabolismus   MeSH
• fenotyp MeSH
• fibroblasty účinky léků   enzymologie   MeSH
• genetická predispozice k nemoci MeSH
• hem farmakologie   MeSH
• homocystinurie diagnóza   farmakoterapie   enzymologie   genetika   MeSH
• homozygot MeSH
• katalytická doména MeSH
• konformace proteinů MeSH
• lidé MeSH
• molekulární chaperony farmakologie   MeSH
• molekulární modely MeSH
• mutace * MeSH
• poruchy proteostázy diagnóza   farmakoterapie   enzymologie   genetika   MeSH
• sbalování proteinů MeSH
• substrátová specifita MeSH
• transfekce MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• práce podpořená grantem MeSH

Cystathionine β-synthase (CBS) deficiency is usually confirmed by assaying the enzyme activity in cultured skin fibroblasts. We investigated whether CBS is present in human plasma and whether determination of its activity in plasma could be used for diagnostic purposes. We developed an assay to measure CBS activity in 20 μL of plasma using a stable isotope substrate - 2,3,3-(2)H serine. The activity was determined by measurement of the product of enzyme reaction, 3,3-(2)H-cystathionine, using LC-MS/MS. The median enzyme activity in control plasma samples was 404 nmol/h/L (range 66-1,066; n = 57). In pyridoxine nonresponsive CBS deficient patients, the median plasma activity was 0 nmol/ho/L (range 0-9; n = 26), while in pyridoxine responsive patients the median activity was 16 nmol/hour/L (range 0-358; n = 28); this overlapped with the enzyme activity from control subject. The presence of CBS in human plasma was confirmed by an in silico search of the proteome database, and was further evidenced by the activation of CBS by S-adenosyl-L-methionine and pyridoxal 5'-phosphate, and by configuration of the detected reaction product, 3,3-(2)H-cystathionine, which was in agreement with the previously observed CBS reaction mechanism. We hypothesize that the CBS enzyme in plasma originates from liver cells, as the plasma CBS activities in patients with elevated liver aminotransferase activities were more than 30-fold increased. In this study, we have demonstrated that CBS is present in human plasma and that its catalytic activity is detectable by LC-MS/MS. CBS assay in human plasma brings new possibilities in the diagnosis of pyridoxine nonresponsive CBS deficiency.

• MeSH
• biochemická analýza krve metody   normy   MeSH
• chromatografie kapalinová MeSH
• cystathionin-beta-synthasa nedostatek   metabolismus   MeSH
• homocystinurie krev   diagnóza   enzymologie   MeSH
• imunoenzymatické techniky metody   normy   MeSH
• kalibrace MeSH
• krevní plazma chemie   enzymologie   metabolismus   MeSH
• lidé MeSH
• pyridoxalfosfát farmakologie   MeSH
• S-adenosylmethionin farmakologie   MeSH
• stabilita enzymů MeSH
• studie případů a kontrol MeSH
• tandemová hmotnostní spektrometrie metody   normy   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• validační studie MeSH

• MeSH
• homocystinurie diagnóza   genetika   MeSH
• incidence MeSH
• mutace MeSH
• plošný screening MeSH
• Publikační typ
• kongresy MeSH
• Geografické názvy
• Česká republika MeSH

• MeSH
• cystathionin-beta-synthasa genetika   nedostatek   MeSH
• finanční podpora výzkumu jako téma MeSH
• homocystinurie enzymologie   genetika   MeSH
• lidé MeSH
• mutace MeSH
• mutační analýza DNA MeSH
• Check Tag
• lidé MeSH
• Geografické názvy
• Polsko MeSH

Cystathionine beta-synthase [CBS; l-serine hydro-lyase (adding homocysteine), EC 4.2.1.22] catalyzes the first committed step of transsulfuration and is the enzyme deficient in classical homocystinuria. In this report, we describe the molecular cloning and the complete nucleotide sequence of the human CBS gene. We report a total of 28,046 nucleotides of sequence, which, in addition to the CBS gene, contains approximately 5 kb of the 5' flanking region. The human CBS gene contains 23 exons ranging from 42 to 209 bp. The 5' UTR is formed by 1 of 5 alternatively used exons and 1 invariably present exon, while the 3' UTR is encoded by exons 16 and 17. We also describe the identification of two alternatively used promoter regions that are GC rich (approximately 80%) and contain numerous putative binding sites for Sp1, Ap1, Ap2, and c-myb, but lack the classical TATA box. The CBS locus contains an unusually high number of Alu repeats, which may predispose this gene to deleterious rearrangements. Additionally, we report on a number of DNA sequence repeats that are polymorphic in North American and European Caucasians. Copyright 1998 Academic Press.

• MeSH
• alternativní sestřih * genetika   MeSH
• běloši MeSH
• cystathionin-beta-synthasa * genetika   MeSH
• elementy Alu genetika   MeSH
• exony genetika   MeSH
• klonování DNA MeSH
• lidé MeSH
• minisatelitní repetice genetika   MeSH
• molekulární sekvence - údaje MeSH
• polymorfismus genetický genetika   MeSH
• promotorové oblasti (genetika) genetika   MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA MeSH
• sekvenční homologie nukleových kyselin MeSH
• transkripční faktory genetika   MeSH
• vazebná místa genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH

• Publikační typ
• abstrakt z konference MeSH

CBSs (cystathionine β-synthases) are eukaryotic PLP (pyridoxal 5 *-phosphate)-dependent proteins that maintain cellular homocysteine homoeostasis and produce cystathionine and hydrogen sulfide. In the present study, we describe a novel structural arrangement of the CBS enzyme encoded by the cbs-1 gene of the nematode Caenorhabditis elegans. The CBS-1 protein contains a unique tandem repeat of two evolutionarily conserved catalytic regions in a single polypeptide chain. These repeats include a catalytically active C-terminal module containing a PLP-binding site and a less conserved N-terminal module that is unable to bind the PLP cofactor and cannot catalyse CBS reactions, as demonstrated by analysis of truncated variants and active-site mutant proteins. In contrast with other metazoan enzymes, CBS-1 lacks the haem and regulatory Bateman domain essential for activation by AdoMet (S-adenosylmethionine) and only forms monomers. We determined the tissue and subcellular distribution of CBS-1 and showed that cbs-1 knockdown by RNA interference leads to delayed development and to an approximately 10-fold elevation of homocysteine concentrations in nematode extracts. The present study provides the first insight into the metabolism of sulfur amino acids and hydrogen sulfide in C. elegans and shows that nematode CBSs possess a structural feature that is unique among CBS proteins.

• MeSH
• biokatalýza MeSH
• Caenorhabditis elegans enzymologie   MeSH
• cystathionin-beta-synthasa chemie   genetika   metabolismus   MeSH
• cytoplazma enzymologie   MeSH
• homeostáza MeSH
• homocystein metabolismus   MeSH
• konzervovaná sekvence MeSH
• kvarterní struktura proteinů MeSH
• lidé MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• orgánová specificita MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• terciární struktura proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• alely MeSH
• cystathionin-beta-synthasa genetika   nedostatek   MeSH
• dítě MeSH
• homocystinurie genetika   MeSH
• lidé MeSH
• mutace MeSH
• novorozenec MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mužské pohlaví MeSH
• novorozenec MeSH
• ženské pohlaví MeSH
• Geografické názvy
• Česká republika MeSH
• Slovenská republika MeSH

Recent studies suggest CNNM2 (cyclin M2) to be part of the long-sought basolateral Mg2+ extruder at the renal distal convoluted tubule, or its regulator. In the present study, we explore structural features and ligand-binding capacities of the Bateman module of CNNM2 (residues 429-584), an intracellular domain structurally equivalent to the region involved in Mg2+ handling by the bacterial Mg2+ transporter MgtE, and AMP binding by the Mg2+ efflux protein CorC. Additionally, we studied the structural impact of the pathogenic mutation T568I located in this region. Our crystal structures reveal that nucleotides such as AMP, ADP or ATP bind at only one of the two cavities present in CNNM2429-584. Mg2+ favours ATP binding by alleviating the otherwise negative charge repulsion existing between acidic residues and the polyphosphate group of ATP. In crystals CNNM2429-584 forms parallel dimers, commonly referred to as CBS (cystathionine β-synthase) modules. Interestingly, nucleotide binding triggers a conformational change in the CBS module from a twisted towards a flat disc-like structure that mostly affects the structural elements connecting the Bateman module with the transmembrane region. We furthermore show that the T568I mutation, which causes dominant hypomagnesaemia, mimics the structural effect induced by nucleotide binding. The results of the present study suggest that the T568I mutation exerts its pathogenic effect in humans by constraining the conformational equilibrium of the CBS module of CNNM2, which becomes 'locked' in its flat form.

• MeSH
• cykliny chemie   genetika   metabolismus   MeSH
• cystathionin-beta-synthasa chemie   genetika   metabolismus   MeSH
• konformace proteinů MeSH
• krystalizace MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• mutace genetika   MeSH
• nukleotidy chemie   metabolismus   MeSH
• sekundární struktura proteinů MeSH
• sekvence aminokyselin MeSH
• vazebná místa fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH