Cystathionine beta-synthase variants : identification, characterization and modulation by thiol compounds

Tese de doutoramento, Farmácia (Bioquímica), Universidade de Lisboa, Faculdade de Farmácia, 2014

Cystathionine beta-synthase (CBS) is a cytoplasmatic tetrameric protein that catalyzes the condensation of L-homocysteine (Hcy) with L-serine to generate L-cystathionine. CBS catalyzes a key reaction in the methionine cycle by initiating the transsulfuration pathway, the only metabolic route for the elimination of Hcy, a risk factor for cardiovascular disease. Countless studies have been focusing on revealing the function and structure of CBS and accordingly the knowledge about CBS has grown in the last decades. Each CBS monomer comprises three domains, an N-terminal heme binding domain, a catalytic core that binds pyridoxal-5’-phosphate and a C-terminal regulatory domain. S-adenosylmethionine (SAM) is able to bind CBS in the regulatory domain, resulting in an increase in protein activity and stability. CBS deficiency is the most common inborn error of the methionine cycle. The classical form manifests as a combination of neurological, connective tissue and vascular involvement and to date no effective pharmacological treatments are available. More than 160 mutations have been described in the CBS gene, the majority being missense mutations and conferring an impaired enzyme activity. As such sensitive and precise methods for determination of catalytic activity was developed in order to functional characterize CBS variants. The presented liquid chromatography – tandem mass spectrometry (LC-MS/MS) method revealed to be a fast, reliable, sensitive and precise assay to quantify Cth. The identification of a group of CBS deficient patients with a high residual enzyme activity but unresponsive to SAM was also studied in this thesis. We identified the genetic background of SAM unresponsive patients and found that this phenomenon was caused not only by mutations located in the gene sequence coding for the C-terminal domain, but also by mutations encoding amino acid substitutions in the catalytic core. Since the pathological consequence of SAM unresponsive CBS is still unknown we studied six recombinant C-terminal CBS variant proteins. All of them presented high enzyme activity (71 – 170 % of wild-type (WT)), no evident changes in kinetic parameters but no response to physiological concentrations of SAM. Binding assays and thermal stability studies indicate that some variants retained the ability to bind SAM but not to be activated by this compound, showing that these are two independent phenomena. Since CBS, and particularly catalytic core variants, present a high tendency to aggregate only a few of those variants could be studied after protein purification. Therefore, we also purified and studied seven CBS variants carrying amino acid substitutions in the catalytic core. As expected, most of these presented very limited expression levels and low enzyme activities. From these variants kinetic data and thermal inactivation profiles were obtained for three of them. These CBS variants were more prone to thermal inactivation indicating some level of protein misfolding and interestingly one of them showed a lower affinity for the substrate which correlates well with the structural localization of the affected residue in the loop controlling the access of the substrates to the active site. Last, we investigated the possibility of rescuing a CBS variant with an arginine to cysteine substitution (p.R336C) with thiol compounds. These compounds may bind to the variant cysteine residue resulting in a structure that could resemble the WT arginine. Using purified protein we were able to specifically increase the activity of the target variant protein and also to increase its stability. This observation is not only important for CBS deficiency but could also benefit other diseases caused by missense mutations originating an arginine to cysteine replacement. In summary, the studies presented on this thesis contribute for the growing knowledge about CBS and offer a potential novel therapy for a specific type of missense mutation.

Fundação para a Ciência e a Tecnologia (FCT); European Commission