Small aminothiol compounds improve the function of Arg to Cys variant proteins: effect on the human cystathionine ß-synthase p.R336C.

The key regulatory point of L-methionine (Met) and L-homocysteine (Hcy) degradation is catalyzed by cystathionine beta-synthase (CBS). CBS deficiency is caused by mutations in CBS gene, often resulting in protein misfolding. The prevalence of CBS deficiency in Qatar is 1/1800, ∼200-fold higher than the worldwide prevalence of 1/344 000. Almost all patients bear the CBS p.R336C variant. More than 20 years ago, it was shown in vitro that two unrelated protein variants with a substitution of an arginine (Arg) residue by cysteine (Cys) could be rescued by cysteamine (mercaptoethylamine), likely via formation of a disulfide between Cys and cysteamine, functionally mimicking the wild-type (WT) Arg side-chain. Based on these findings, we aimed to study whether cysteamine was able to improve the function of p.R336C CBS variant. Additionally, we tested the effect of mercaptoethylguanidine (MEG), a compound with a guanidino and a thiol function that may resemble Arg structure better than cysteamine. Three purified recombinant CBS proteins (p.R336C, p.R336H and WT) were pre-incubated with cysteamine, MEG or Cys (as negative control), and CBS activity and stability were measured. Pre-incubation with cysteamine and MEG increased the enzymatic activity of the p.R336C protein, which was absent upon pre-incubation with Cys. The WT and the p.R336H variant enzyme activity presented no increase with any of the tested compounds. Our results show that cysteamine and MEG are able to specifically improve the function of the CBS p.R336C variant, suggesting that any Arg-to-Cys substitution accessible to these small molecules may be converted back to a moiety resembling Arg.

Insights into the regulatory domain of cystathionine Beta-synthase: characterization of six variant proteins.

Cystathionine beta-synthase (CBS) catalyzes the formation of cystathionine from homocysteine and serine. CBS is allosterically activated by S-adenosylmethionine (SAM), which binds to its C-terminal regulatory domain. Mutations in this domain lead to variants with high residual activity but lacking SAM activation. We characterized six C-terminal CBS variants (p.P427L, p.D444N, p.V449G, p.S500L, p.K523Sfs*18, and p.L540Q). To understand the effect of C-terminal mutations on the functional/structural properties of CBS, we performed dynamic light scattering, differential scanning fluorimetry, limited proteolysis, enzymatic characterization, and determination of SAM-binding affinity. Kinetic data confirm that the enzymatic function of these variants is not impaired. Although lacking SAM activation, the p.P427L and p.S500L were able to bind SAM at a lower extent than the wild type (WT), confirming that SAM binding and activation can be two independent events. At the structural level, the C-terminal variants presented various effects, either showing catalytic core instability and increased susceptibility toward aggregation or presenting with similar or higher stability than the WT. Our study highlights as the common feature to the C-terminal variants an impaired binding of SAM and no increase in enzymatic activity with physiological concentrations of the activator, suggesting the loss of regulation by SAM as a potential pathogenic mechanism.

NO* binds human cystathionine ß-synthase quickly and tightly.

The hexa-coordinate heme in the H2S-generating human enzyme cystathionine ß-synthase (CBS) acts as a redox-sensitive regulator that impairs CBS activity upon binding of NO(•) or CO at the reduced iron. Despite the proposed physiological relevance of this inhibitory mechanism, unlike CO, NO(•) was reported to bind at the CBS heme with very low affinity (Kd = 30-281 µm). This discrepancy was herein reconciled by investigating the NO(•) reactivity of recombinant human CBS by static and stopped-flow UV-visible absorption spectroscopy. We found that NO(•) binds tightly to the ferrous CBS heme, with an apparent Kd ≤ 0.23 µm. In line with this result, at 25 °C, NO(•) binds quickly to CBS (k on ∼ 8 × 10(3) m(-1) s(-1)) and dissociates slowly from the enzyme (k off ∼ 0.003 s(-1)). The observed rate constants for NO(•) binding were found to be linearly dependent on [NO(•)] up to ∼ 800 µm NO(•), and >100-fold higher than those measured for CO, indicating that the reaction is not limited by the slow dissociation of Cys-52 from the heme iron, as reported for CO. For the first time the heme of human CBS is reported to bind NO(•) quickly and tightly, providing a mechanistic basis for the in vivo regulation of the enzyme by NO(•). The novel findings reported here shed new light on CBS regulation by NO(•) and its possible (patho)physiological relevance, enforcing the growing evidence for an interplay among the gasotransmitters NO(•), CO, and H2S in cell signaling.

Reduced response of Cystathionine Beta-Synthase (CBS) to S-Adenosylmethionine (SAM): Identification and functional analysis of CBS gene mutations in Homocystinuria patients.

A reduced response of cystathionine beta-synthase (CBS) to its allosteric activator S-adenosylmethionine (SAM) has been reported to be a cause of CBS dysfunction in homocystinuria patients. In this work we performed a retrospective analysis of fibroblast data from 62 homocystinuria patients and found that 13 of them presented a disturbed SAM activation. Their genotypic background was identified and the corresponding CBS mutant proteins were produced in E. coli. Nine distinct mutations were detected in 22 independent alleles: the novel mutations p.K269del, p.P427L, p.S500L and p.L540Q; and the previously described mutations p.P49L, p.C165Rfs*2, p.I278T, p.R336H and p.D444N. Expression levels and residual enzyme activities, determined in the soluble fraction of E. coli lysates, strongly correlated with the localization of the affected amino acid residue. C-terminal mutations lead to activities in the range of the wild-type CBS and to oligomeric forms migrating faster than tetramers, suggesting an abnormal conformation that might be responsible for the lack of SAM activation. Mutations in the catalytic core were associated with low protein expression levels, decreased enzyme activities and a higher content of high molecular mass forms. Furthermore, the absence of SAM activation found in the patients' fibroblasts was confirmed for all but one of the characterized recombinant proteins (p.P49L). Our study experimentally supports a deficient regulation of CBS by SAM as a frequently found mechanism in CBS deficiency, which should be considered not only as a valuable diagnostic tool but also as a potential target for the development of new therapeutic approaches in classical homocystinuria.

A liquid chromatography mass spectrometry method for the measurement of cystathionine ß-synthase activity in cell extracts.

BACKGROUND: In order to correctly assess the efficacy of therapy or diet in intervention studies on the activity of cystathionine ß-synthase (CBS) a sensitive analytical method is necessary. METHODS: An electrospray LC-MS/MS method preceded by a solid phase extraction step was developed for the measurement of CBS activity in cell extracts. Nonafluoropentanoic acid was used as an ionpair to provide the underivatized cystathionine the desired retention on a C18 column. RESULTS: A detection limit of 50pmol cystathionine/h/mg protein was achieved. In fibroblasts, intra- and inter-assay CVs for the CBS activity were 5.2% and 14.7%, respectively. A K(m) value of 8µmol/L for homocysteine, and 2.5µmol/L for serine was calculated. In fibroblasts wildtype, heterozygous, and homozygous CBS activity ranges measured were 8.5-27.0, 4.2-13.4, 0.0-0.7nmol/h×mg protein, respectively. The method was applied to a study where rats were fed 2 diets. Increase of dietary methionine (7.7 versus 3.8mg/kg methionine) significantly increased the CBS activity in rat liver lysates from a median of 58.0 to a median of 71.5 (P=0.037)nmol/h×mg protein. In a lymphoblasts cell culture experiment, the addition of Hcy to the culture media increased the activity of CBS 3 fold. CONCLUSION: This LC-MS/MS is able to diagnose CBS deficiency at the enzyme level, and can accurately measure the effect diets or therapy might have on the CBS activity in a variety of cell types.