In all organisms methionine is an important amino acid in proteins as well as the key component of S-adenosyl methionine (SAM), the main donor of methyl groups in the biosynthesis of biomolecules like choline, creatine, and adrenaline, as well as in DNA methylation. While bacteria possess biosynthetic pathways for methionine, this amino acid is essential for humans. Following SAM demethylation, homocysteine (HCys) is formed. HCys accumulates when its recycling pathways become impaired, leading to hyperhomocysteinemia, that is typical in cardiovascular diseases, neurological/psychiatric disorders and cancer. Whether excessive HCys is causing the pathological condition or is only a biomarker of a metabolic aberration still remains to be established.
In the present project we will evaluate the biological properties of HCys-derived molecules.
First of all we propose a bio-based route for the synthesis of homohypotaurine, the decarboxylation product of homocystein sulfinic acid (HCSA). Both compounds are not available from commercial suppliers. Thus a chemical synthesis and an enzyme-mediated synthesis will be employed to synthesize HCSA and homohypotaurine, respectively. The biological effects of homohypotaurine, both in bacteria and in mammals, are less known than those of homotaurine, its oxidation product,in widespread pharmaceutical and laboratory use. With this project we therefore aim to fill this gap of knowledge.
Furthermore, we plan to investigate the metabolic role of biomolecules related to taurine. Particular emphasis will be devoted to thiotaurine, a biomolecule releasing hydrogen sulfide (H2S), and to the role of these molecules in controlling inflammation. The specific signaling pathways involved will be dissected and proteomic profiling of human neutrophils carried out. The aim of this study is to identify the proteins that change their expression level or undergo post-translational modifications, including nitrosylation/nitration, persulfidation.
While a great deal of work is carried out on taurine and homotaurine (the latter, free or derivatized, is in widespread pharmaceutical and laboratory use), an extensive search of the literature retrieved more limited information on the properties of homohypotaurine, the decarboxylation product of HCSA. The properties of this latter molecule and of HCSA, structurally the closest homologues of GABA and L-Glutamate, the major inhibitory and excitatory neurotransmitters in the CNS, respectively, need a more in-depth investigation. To date it is known that HCSA acts as an agonist of the metabotropic glutamate receptors (mGluR) [16], and that it increases glucose uptake in skeletal muscle via stimulation of AMP-activated protein kinase [17]. These findings point to a role of HCSA in diabetes.
With this work De Biase's group will also set out the basis for a deeper investigation of the possible biological activity of homohypotaurine as compared to homotaurine. Depending on the outcome of the work planned in this project, we will seek for collaborations to assay the biological activity of homohypotaurine on eukaryotic cells and laboratory animals. In fact, even though this work will begin with testing bacterial GAD (with HCSA and HCA) and GABA transaminase (with homohypotaurine and homotaurine), human GAD (available in De Biase¿s group) will eventually be considered for activity on both HCSA and HCA.
Daniela De Biase and Carlotta Zamparelli (both in this project) have already worked in collaboration (and with other groups) to evaluate the oligomeric and conformational state of GAD at different pHs and in the presence/absence of the cofactor PLP [18,19]. Indeed both human and bacterial GAD have much lower affinity for the aminated form of PLP, i.e. PMP (pyridoxamine 5'-phosphate), which is produced as a product of the so-called ¿abortive decarboxylation-transamination¿ reaction. Thus, PMP is lost and the enzymes converts into the apo-inactive form. This is a likely side reaction when using substrates like HCSA or HCA, different from L-glutamate, which remains the preferred substrate of the enzyme. We will employ analytical ultracentrifugation (AUC) and several other spectroscopic techniques to monitor the extent of the overall conformational and/or oligomeric changes when GAD decarboxylates HCSA and HCA. This will be carried out on bacterial GAD and, as a perspective continuation of this project, eventually on human GAD. This is a rather interesting aspect if one considers that one of the major autoantigens in type I diabetes is GAD65 (one of the two isoforms of human GAD) that most of the time is intracellularly in the apo-inactive form.
Non-steroidal anti-inflammatory drugs are among the most commonly used drugs. Despite efforts to produce non-steroidal anti-inflammatory drugs that do not cause gastrointestinal ulceration and bleeding, these adverse effects remain major limitations to their use. On the contrary it has been reported that H2S donors can increase the resistance of the gastric mucosa to injury and accelerate damaged tissue repair [20]. These observations suggest that anti-inflammatory molecules that are chemically modified to release H2S will exhibit improved efficacy and reduced toxicity. In this context thiotaurine biomolecule structurally related to taurine and to hypotaurine, particularly abundant in neutrophil granulocytes, has a thiosulfonate (RSO2SH) group which can potentially release H2S. This biocompound could mimic the role of H2S in inflammation, but also play a protective role on the gastric mucosa when administered with an anti-inflammatory drug.
The mechanisms involved in the interaction of the reactive oxygen (ROS) and nitrogen species (RNS) with sulfinates have only recently been investigated [21]. Interestingly, the oxidation of sulfinates by radicals is accompanied by the generation of highly reactive sulfonyl radicals which can promote oxidative reactions. In light of this, establishing the pathophysiological role of sulfonyl radicals will be an outcome of this work.
References
16. Shi Q. et al. (2003) J. Pharmacol. Exp. Ther. 305, 131
17. Kim J.H. et al. (2011) J. Biol. Chem. 286, 7567
18. Kass I. et al. (2014) Proc. Natl. Acad. Sci. USA 111, E2524
19. Giovannercole F. et al. (2017) Protein Eng. Des. Sel. 30, 237
20. Wallace JL (2007) Trends Pharmacol Sci, 28, 501
21. Baseggio Conrado A. et al. (2014) Free Radic. Res. 48, 1300.