共查询到20条相似文献,搜索用时 15 毫秒
1.
Hnízda A Majtan T Liu L Pey AL Carpenter JF Kodíček M Kožich V Kraus JP 《Biochemistry》2012,51(23):4755-4763
Protein misfolding due to missense mutations is a common pathogenic mechanism in cystathionine β-synthase (CBS) deficiency. In our previous studies, we successfully expressed, purified, and characterized nine CBS mutant enzymes containing the following patient mutations: P49L, P78R, A114V, R125Q, E176K, R266K, P422L, I435T, and S466L. These purified mutants exhibited full heme saturation, normal tetrameric assembly, and high catalytic activity. In this work, we used several spectroscopic and proteolytic techniques to provide a more thorough insight into the conformation of these mutant enzymes. Far-UV circular dichroism, fluorescence, and second-derivative UV spectroscopy revealed that the spatial arrangement of these CBS mutants is similar to that of the wild type, although the microenvironment of the chromophores may be slightly altered. Using proteolysis with thermolysin under native conditions, we found that the majority of the studied mutants is more susceptible to cleavage, suggesting their increased local flexibility or propensity for local unfolding. Interestingly, the presence of the CBS allosteric activator, S-adenosylmethionine (AdoMet), increased the rate of cleavage of the wild type and the AdoMet-responsive mutants, while the proteolytic rate of the AdoMet-unresponsive mutants was not significantly changed. Pulse proteolysis analysis suggested that the protein structure of the R125Q and E176K mutants is significantly less stable than that of the wild type and the other mutants. Taken together, the proteolytic data shows that the conformation of the pathogenic mutants is altered despite retained catalytic activity and normal tetrameric assembly. This study demonstrates that the proteolytic techniques are useful tools for the assessment of the biochemical penalty of missense mutations in CBS. 相似文献
2.
Régnier V Billard JM Gupta S Potier B Woerner S Paly E Ledru A David S Luilier S Bizot JC Vacano G Kraus JP Patterson D Kruger WD Delabar JM London J 《PloS one》2012,7(1):e29056
Background
The cystathionine β-synthase (CBS) gene, located on human chromosome 21q22.3, is a good candidate for playing a role in the Down Syndrome (DS) cognitive profile: it is overexpressed in the brain of individuals with DS, and it encodes a key enzyme of sulfur-containing amino acid (SAA) metabolism, a pathway important for several brain physiological processes.Methodology/Principal Findings
Here, we have studied the neural consequences of CBS overexpression in a transgenic mouse line (60.4P102D1) expressing the human CBS gene under the control of its endogenous regulatory regions. These mice displayed a ∼2-fold increase in total CBS proteins in different brain areas and a ∼1.3-fold increase in CBS activity in the cerebellum and the hippocampus. No major disturbance of SAA metabolism was observed, and the transgenic mice showed normal behavior in the rotarod and passive avoidance tests. However, we found that hippocampal synaptic plasticity is facilitated in the 60.4P102D1 line.Conclusion/Significance
We demonstrate that CBS overexpression has functional consequences on hippocampal neuronal networks. These results shed new light on the function of the CBS gene, and raise the interesting possibility that CBS overexpression might have an advantageous effect on some cognitive functions in DS. 相似文献3.
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Majtan T Freeman KM Smith AT Burstyn JN Kraus JP 《Archives of biochemistry and biophysics》2011,508(1):4417-30
Human cystathionine β-synthase (CBS), a pivotal enzyme in the metabolism of homocysteine, is a pyridoxal-5′-phosphate-dependent enzyme that also contains heme, a second cofactor whose function is still unclear. One strategy for elucidation of heme function is its replacement with different metalloporphyrins or with porphyrins containing different substituent groups. This paper describes a novel expression approach and purification of cobalt CBS (CoCBS), which results in a high yield of fully active, high purity enzyme, in which heme is substituted by Co-protoporphyrin IX (CoPPIX). Metal content analysis showed that the enzyme contained 92% cobalt and 8% iron. CoCBS was indistinguishable from wild-type FeCBS in its activity, tetrameric oligomerization, PLP saturation and responsiveness to the allosteric activator, S-adenosyl-l-methionine. The observed biochemical and spectral characteristics of CoCBS provide further support for the suggestion that heme is involved in structural integrity and folding of this unusual enzyme. 相似文献
5.
Senemar S Doroudchi M Pezeshki AM Bazrgar M Torab-Jahromi A Ghaderi A 《Molecular biology reports》2009,36(2):353-356
Iranian population with an Indo-European origin is one of the oldest populations in the world. Historical evidence suggests
the close similarity in the origin of Iranian, European and north Indian population. However, there are few anthropological
and genetic evidences on this subject. This study, which is the first report from Iran, was performed to investigate the genetic
origin of Iranian population using a polymorphism in Cystathionine beta synthase (CBS) gene known as 844INS68bp in this respect,
genomic DNA was extracted from the whole blood of 480 healthy normal blood donors referred to Fars Blood Transfusion Center,
using a salting out method. The fragment containing 844INS68bp was amplified, the normal fragment was 174 bp and the fragment
containing the insertion was 242 bp in length. Results indicated that 418 (87.08%) out of 480 individuals had a normal (N/N)
genotype, 59 (12.29%) individuals were heterozygote (N/I) and 3 (0.63%) had homozygote a mutated genotype (I/I). The total
frequency of 844INS68bp allele was found 6.8% which is similar to with the reported in White Caucasians. Comparison of the
genotype of this study with the polymorphism in other populations revealed that Southern Iranian population has a great similarity
with other Caucasians populations’ especially South Italy and North America while differed from East Asian and African populations.
These results are in agreement with the result of other studied polymorphisms. Therefore, despite the great admixture of Iranian
population with the neighboring non-Caucasian populations during the time, Iranian population still share a genetic background
with other Caucasian populations. 相似文献
6.
Background
Human uterus undergoes distinct molecular and functional changes during pregnancy and parturition. Hydrogen sulfide (H2S) has recently been shown to play a key role in the control of smooth muscle tension. The role of endogenous H2S produced locally in the control of uterine contractility during labour is unknown.Methodology/Principal Findings
Human myometrium biopsies were obtained from pregnant women undergoing cesarean section at term. Immunohistochemistry analysis showed that cystathionine-γ-lyase (CSE) and cystathionine-β-synthetase (CBS), the principle enzymes responsible for H2S generation, were mainly localized to smooth muscle cells of human pregnant myometrium. The mRNA and protein expression of CBS as well as H2S production rate were down-regulated in labouring tissues compared to nonlabouring tissues. Cumulative administration of L-cysteine (10−7–10−2 mol/L), a precursor of H2S, caused a dose-dependent decrease in the amplitude of spontaneous contractions in nonlabouring and labouring myometrium strips. L-cysteine at high concentration (10−3 mol/L) increased the frequency of spontaneous contractions and induced tonic contraction. These effects of L-cysteine were blocked by the inhibitors of CBS and CSE. Pre-treatment of myometrium strips with glibenclamide, an inhibitor of ATP-sensitive potassium (KATP) channels, abolished the inhibitory effect of L-cysteine on spontaneous contraction amplitude. The effects of L-cysteine on the amplitude of spontaneous contractions and baseline muscle tone were less potent in labouring tissues than that in nonlabouring strips.Conclusion/Significance
H2S generated by CSE and CBS locally exerts dual effects on the contractility of pregnant myometrium. Expression of H2S synthetic enzymes is down-regulated during labour, suggesting that H2S is one of the factors involved in the transition of pregnant uterus from quiescence to contractile state after onset of parturition. 相似文献7.
Kabil O Weeks CL Carballal S Gherasim C Alvarez B Spiro TG Banerjee R 《Biochemistry》2011,50(39):8261-8263
Human CBS is a PLP-dependent enzyme that clears homocysteine, gates the flow of sulfur into glutathione, and contributes to the biogenesis of H(2)S. The presence of a heme cofactor in CBS is enigmatic, and its conversion from the ferric- to ferrous-CO state inhibits enzyme activity. The low heme redox potential (-350 mV) has raised questions about the feasibility of the ferrous-CO state forming under physiological conditions. Herein, we provide the first evidence of reversible inhibition of CBS by CO in the presence of a human flavoprotein and NADPH. These data provide a mechanism for cross talk between two gas-signaling systems, CO and H(2)S, via heme-mediated allosteric regulation of CBS. 相似文献
8.
Byung-Cheon Jeong Si Hoon Park Kyoung Shin Yoo Jeong Sheop Shin Hyun Kyu Song 《Biochemical and biophysical research communications》2013,430(1):265-271
The single cystathionine β-synthase (CBS) pair proteins from Arabidopsis thaliana have been identified as being a redox regulator of the thioredoxin (Trx) system. CBSX1 and CBSX2, which are two of the six Arabidopsis cystathione β-synthase domain-containing proteins that contain only a single CBS pair, have close sequence similarity. Recently, the crystal structure of CBSX2 was determined and a significant portion of the internal region was disordered. In this study, crystal structures of full-length CBSX1 and the internal loop deleted (Δloop) form are reported at resolutions of 2.4 and 2.2 Å, respectively. The structures of CBSX1 show that they form anti-parallel dimers along their central twofold axis and have a unique ~155° bend along the side. This is different from the angle of CBSX2, which is suggestive of the flexible nature of the relative angle between the monomers. The biochemical data that were obtained using the deletion as well as point mutants of CBSX1 confirmed the importance of AMP-ligand binding in terms of enhancing Trx activity. 相似文献
9.
Summary Among several established mouse, rat, and Chinese hamster cell lines that were screened for cystathionine -synthase (CBS) activity, mouse 3T3 and Chinese hamster Don fibroblasts were found to contain no detectable activity. Somatic cell hybrids between human fibroblasts KG-7 with normal CBS activity and Don/a23TK- cells (series XXI) were examined for CBS activity and for human chromosome content. Only chromosome 21 cosegregated with CBS activity. Because the activities measured could represent either Chinese hamster or human gene products, we have prepared a new series of hybrids between Don/a23TK- cells and mutant human fibroblasts from a patient with homocystinuria due to deficiency of functional CBS mRNA. None of these (series XXV) hybrids contained detectable CBS activity, although collectively all human chromosomes were represented. Our results suggest that the human gene for CBS, called CBS, and thus for the most common form of homocystinuria, is located on chromosome 21. 相似文献
10.
Leo A. J. Kluijtmans Henk J. Blom Godfried H. J. Boers Bernard A. van Oost Frans J. M. Trijbels Lambert P. W. J. van den Heuvel 《Human genetics》1995,96(2):249-250
Direct sequencing of the coding region of the cystathionine -synthase (CBS) gene in two homocystinuric patients revealed the presence of two novel missense mutations. The first mutation, a 1111G A transition, resulted in the substitution of the evolutionary conserved valine-371 by a methionine residue (V371M) and created a new NlaIII restriction site. The second mutation, a GA transition at base-pair 494, resulted in an amino acid change from cysteine to tyrosine (C165Y) and abolished a BsoFI restriction site. Both mutations were found in a compound heterozygous state with the previously described 833T C transition. 相似文献
11.
To determine the l-methionine (l-Met) concentration in an extract from dried blood spots (DBSs) for newborn mass screening for homocystinuria (HCU) due to cystathionine β-synthase (CBS) deficiency, a new fluorometric microplate assay using a methionine-specific dehydrogenase (MetDH) and the diaphorase/reazusrin system was established. We created by directed mutagenesis an NAD(+)-dependent MetDH from phenylalanine dehydrogenase (PheDH) showing higher substrate specificity toward l-Met than l-phenylalanine (l-Phe). However, it also exhibited notable activity for branched-chain amino acids (BCAAs). BCAAs in blood clearly interfered with the determination of l-Met in the DBS specimens using a single application of MetDH. To measure l-Met selectively, we used a branched-chain amino acid transaminase (BCAT) to eliminate the BCAAs in the specimens and screened for a BCAT with low activity toward l-Met. In microplate assays using MetDH, pretreatment of specimens with the BCAT from Lactobacillus delbrueckii subsp. bulgaricus coupled with l-glutamate oxidase minimized the effects of BCAAs, and l-Met concentrations were determined with high accuracy even at elevated BCAA concentrations. This enzymatic end-point assay is suitable for determining l-Met concentrations in DBSs for neonatal screening for HCU due to CBS deficiency. 相似文献
12.
Using immunohistochemistry, we examined the distribution of the hydrogen sulfide (H2S) synthesizing enzymes cystathionine β-synthase (CBS) and cystathionine Γ-lyase (CSE) in tissues of the nemertean Cerebratulus marginatus Renier, 1804 (Heteronemertea: Lineidae). The expression of both CBS and CSE was found in the body wall and in the receptor cells of the canals of the cerebral organs; the expression of CBS was found in the foregut and intestine; and CSE was found in the brain, lateral nerve cords, and cerebral organs. The peculiarities of the distribution and the possible functional role of H2S-synthesizing elements in nemerteans are discussed. 相似文献
13.
Yoo KS Ok SH Jeong BC Jung KW Cui MH Hyoung S Lee MR Song HK Shin JS 《The Plant cell》2011,23(10):3577-3594
Plant thioredoxins (Trxs) participate in two redox systems found in different cellular compartments: the NADP-Trx system (NTS) in the cytosol and mitochondria and the ferredoxin-Trx system (FTS) in the chloroplast, where they function as redox regulators by regulating the activity of various target enzymes. The identities of the master regulators that maintain cellular homeostasis and modulate timed development through redox regulating systems have remained completely unknown. Here, we show that proteins consisting of a single cystathionine β-synthase (CBS) domain pair stabilize cellular redox homeostasis and modulate plant development via regulation of Trx systems by sensing changes in adenosine-containing ligands. We identified two CBS domain-containing proteins in Arabidopsis thaliana, CBSX1 and CBSX2, which are localized to the chloroplast, where they activate all four Trxs in the FTS. CBSX3 was found to regulate mitochondrial Trx members in the NTS. CBSX1 directly regulates Trxs and thereby controls H(2)O(2) levels and regulates lignin polymerization in the anther endothecium. It also affects plant growth by regulating photosynthesis-related [corrected] enzymes, such as malate dehydrogenase, via homeostatic regulation of Trxs. Based on our findings, we suggest that the CBSX proteins (or a CBS pair) are ubiquitous redox regulators that regulate Trxs in the FTS and NTS to modulate development and maintain homeostasis under conditions that are threatening to the cell. 相似文献
14.
Jean François Chassé Véronique Paul Raphael Escañez Pierre Kamoun Jacqueline London 《Mammalian genome》1997,8(12):917-921
The human cystathionine β-synthase (CBS) gene spans in excess of 30 kb and consists of 19 exons, with three different 5′ untranslated regions including three different exons 1 (exons 1 a, b, and c). Exon la and 1b are 390 bp apart from each other and are linked to exon 2 in cDNA « a » and cDNA « b ». Exon 1c, which linked to exon 5 in cDNA « c », is 7 kb apart from exon 1b. All splice sites conform to the GT/AG rule, including those from exon la or 1b to exon 2 and from exon 1c to exon 5. Upstream of exons la and 1b, we found two putative promoter sequences with high C + G nucleotide content, one CAAT box at —70 nucleotides (for exon lb), no TATA box, several Sp1 binding regulatory consensus sequences, and some other regulatory sequences. Human adult and fetal Northern blots hybridized with total cDNA containing exon 1b, or specific probes from exons 1 (b and c) showed mRNAs of 2.5 kb, 2.7 kb, and 3.7 kb. These results suggest that the mRNAs containing the different exons 1 are under the control of different promoters. 相似文献
15.
Beard RS Bearden SE 《American journal of physiology. Heart and circulatory physiology》2011,300(1):H13-H26
Homocysteine (Hcy), a cardiovascular and neurovascular disease risk factor, is converted to hydrogen sulfide (H(2)S) through the transsulfuration pathway. H(2)S has attracted considerable attention in recent years for many positive effects on vascular health and homeostasis. Cystathionine β-synthase (CBS) is the first, and rate-limiting, enzyme in the transsulfuration pathway. Mutations in the CBS gene decrease enzymatic activity, which increases the plasma Hcy concentration, a condition called hyperhomocysteinemia (HHcy). Animal models of CBS deficiency have provided invaluable insights into the pathological effects of transsulfuration impairment and of both mild and severe HHcy. However, studies have also highlighted the complexity of HHcy and the need to explore the specific details of Hcy metabolism in addition to Hcy levels per se. There has been a relative paucity of work addressing the dysfunctional H(2)S production in CBS deficiency that may contribute to, or even create, HHcy-associated pathologies. Experiments using CBS knockout mice, both homozygous (-/-) and heterozygous (+/-), have provided 15 years of new knowledge and are the focus of this review. These murine models present the opportunity to study a specific mechanism for HHcy that matches one of the etiologies in many human patients. Therefore, the goal of this review was to integrate and highlight the critical information gained thus far from models of CBS deficiency and draw attention to critical gaps in knowledge, with particular emphasis on the modulation of H(2)S metabolism. We include findings from human and animal studies to identify important opportunities for future investigation that should be aimed at generating new basic and clinical understanding of the role of CBS and transsulfuration in cardiovascular and neurovascular disease. 相似文献
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Microtubule dynamics play essential roles in intracellular organization and cell division. They result from structural and biochemical properties of αβ-tubulin heterodimers and how these polymerizing subunits interact with themselves and with regulatory proteins. A broad understanding of the underlying mechanisms has been established, but fundamental questions remain unresolved. The lack of routine access to recombinant αβ-tubulin represents an obstacle to deeper insight into αβ-tubulin structure, biochemistry, and recognition. Indeed, the widespread reliance on animal brain αβ-tubulin means that very few in vitro studies have taken advantage of powerful and ordinarily routine techniques like site-directed mutagenesis. Here we report new methods for purifying wild-type or mutant yeast αβ-tubulin from inducibly overexpressing strains of Saccharomyces cerevisiae. Inducible overexpression is an improvement over existing approaches that rely on constitutive expression: it provides higher yields while also allowing otherwise lethal mutants to be purified. We also designed and purified polymerization-blocked αβ-tubulin mutants. These "blocked" forms of αβ-tubulin give a dominant lethal phenotype when expressed in cells; they cannot form microtubules in vitro and when present in mixtures inhibit the polymerization of wild-type αβ-tubulin. The effects of blocking mutations are very specific, because purified mutants exhibit normal hydrodynamic properties, bind GTP, and interact with a tubulin-binding domain. The ability to overexpress and purify wild-type αβ-tubulin, or mutants like the ones we report here, creates new opportunities for structural studies of αβ-tubulin and its complexes with regulatory proteins, and for biochemical and functional studies of microtubule dynamics and its regulation. 相似文献
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