Cystathionine beta-synthase (CBS)-deficient homocystinuria (HCU) is the most common inborn error of sulfur amino acid metabolism. The pyridoxine non-responsive form of the disease manifests itself by massively increasing plasma and tissue concentrations of homocysteine, a toxic intermediate of methionine metabolism that is thought to be the major cause of clinical complications including skeletal deformities, connective tissue defects, thromboembolism and cognitive impairment. The current standard of care involves significant dietary interventions that, despite being effective, often adversely affect quality of life of HCU patients, leading to poor adherence to therapy and inadequate biochemical control with clinical complications. In recent years, the unmet need for better therapeutic options has resulted in development of novel enzyme and gene therapies and exploration of pharmacological approaches to rescue CBS folding defects caused by missense pathogenic mutations. Here, we review scientific evidence and current state of affairs in development of recent approaches to treat HCU.

• Klíčová slova
• chaperones, cystathionine beta-synthase, enzyme therapy, gene therapy, homocystinuria, pegtibatinase, proteasome inhibitors,
• MeSH
• cystathionin-beta-synthasa genetika   metabolismus   MeSH
• homocystinurie * farmakoterapie   genetika   metabolismus   MeSH
• kvalita života MeSH
• lidé MeSH
• missense mutace MeSH
• tromboembolie * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• cystathionin-beta-synthasa MeSH

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
• Názvy látek
• arginin MeSH
• cystathionin-beta-synthasa MeSH
• hem MeSH
• heme arginate MeSH Prohlížeč
• molekulární chaperony MeSH

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.

• MeSH
• cirkulární dichroismus MeSH
• cystathionin-beta-synthasa nedostatek   genetika   metabolismus   MeSH
• Escherichia coli metabolismus   MeSH
• konformace proteinů MeSH
• lidé MeSH
• missense mutace MeSH
• molekulární modely MeSH
• proteolýza MeSH
• S-adenosylmethionin MeSH
• sbalování proteinů * MeSH
• spektrofotometrie ultrafialová MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• cystathionin-beta-synthasa MeSH
• S-adenosylmethionin MeSH

Protein misfolding has been proposed to be a common pathogenic mechanism in many inborn errors of metabolism including cystathionine β-synthase (CBS) deficiency. In this work, we describe the structural properties of nine CBS mutants that represent a common molecular pathology in the CBS gene. Using thermolysin in two proteolytic techniques, we examined conformation of these mutants directly in crude cell extracts after expression in E. coli. Proteolysis with thermolysin under native conditions appeared to be a useful technique even for very unstable mutant proteins, whereas pulse proteolysis in a urea gradient had limited values for the study of the majority of CBS mutants due to their instability. Mutants in the active core had either slightly increased unfolding (p.A114V, p.E302K and p.G307S) or extensive unfolding with decreased stability (p.H65R, p.T191M, p.I278T and p.R369C). The extent of the unfolding inversely correlated with the previously determined degree of tetrameric assembly and with the catalytic activity. In contrast, mutants bearing aminoacid substitutions in the C-terminal regulatory domain (p.R439Q and p.D444N) had increased global stability with decreased flexibility. This study shows that proteolytic techniques can reveal conformational abnormalities even for CBS mutants that have activity and/or a degree of assembly similar to the wild-type enzyme. We present here a methodological strategy that may be used in cell lysates to evaluate properties of proteins that tend to misfold and aggregate and that may be important for conformational studies of disease-causing mutations in the field of inborn errors of metabolism.

• MeSH
• časové faktory MeSH
• cystathionin-beta-synthasa genetika   MeSH
• denaturace proteinů MeSH
• dimerizace MeSH
• Escherichia coli metabolismus   MeSH
• kinetika MeSH
• konformace proteinů MeSH
• lidé MeSH
• močovina chemie   MeSH
• mutace * MeSH
• rozpouštědla MeSH
• sbalování proteinů MeSH
• terciární struktura proteinů MeSH
• thermolysin chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cystathionin-beta-synthasa MeSH
• močovina MeSH
• rozpouštědla MeSH
• thermolysin MeSH

Cystathionine β-synthase (CBS) is a modular enzyme which catalyzes condensation of serine with homocysteine. Cross-talk between the catalytic core and the C-terminal regulatory domain modulates the enzyme activity. The regulatory domain imposes an autoinhibition action that is alleviated by S-adenosyl-l-methionine (AdoMet) binding, by deletion of the C-terminal regulatory module, or by thermal activation. The atomic mechanisms of the CBS allostery have not yet been sufficiently explained. Using pulse proteolysis in urea gradient and proteolytic kinetics with thermolysin under native conditions, we demonstrated that autoinhibition is associated with changes in conformational stability and with sterical hindrance of the catalytic core. To determine the contact area between the catalytic core and the autoinhibitory module of the CBS protein, we compared side-chain reactivity of the truncated CBS lacking the regulatory domain (45CBS) and of the full-length enzyme (wtCBS) using covalent labeling by six different modification agents and subsequent mass spectrometry. Fifty modification sites were identified in 45CBS, and four of them were not labeled in wtCBS. One differentially reactive site (cluster W408/W409/W410) is a part of the linker between the domains. The other three residues (K172 and/or K177, R336, and K384) are located in the same region of the 45CBS crystal structure; computational modeling showed that these amino acid side chains potentially form a regulatory interface in CBS protein. Subtle differences at CBS surface indicate that enzyme activity is not regulated by conformational conversions but more likely by different allosteric mechanisms.

• MeSH
• alosterické místo MeSH
• cystathionin-beta-synthasa antagonisté a inhibitory   chemie   metabolismus   MeSH
• hydrofobní a hydrofilní interakce MeSH
• interakce mezi receptory a ligandy fyziologie   MeSH
• katalytická doména fyziologie   MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• terciární struktura proteinů fyziologie   MeSH
• výpočetní biologie metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• srovnávací studie MeSH
• Názvy látek
• cystathionin-beta-synthasa MeSH