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Background:The H 2 S-generating human enzyme cystathionine ␤-synthase (CBS) is inhibited by NO ⅐ and CO. Results: NO ⅐ binds to the ferrous heme in human CBS much more quickly than CO and much more tightly than currently thought. Conclusion:Results support the physiological role of NO ⅐ in CBS regulation. Significance: CBS may integrate the cross-talk among NO ⅐ , CO, and H 2 S, major modulators in human (patho)physiology.

Protein folding in the cell is a tightly regulated process, involving a series of proteins, from molecular chaperones to proteases that assist the folding process and monitor the quality of the final product. Despite this control, genetic or sporadic factors may compromise protein folding and the folded state resulting in the formation of non-native misfolded, destabilised, aggregated or fibrillar species. These are hallmarks of the so-called protein conformational disorders, in which the altered protein conformations result in cell toxicity, functional deficiency or lead to dominant negative effects. Examples of such pathologies include neurodegenerative and metabolic disorders. In recent years, it has become clear that several different small chemical compounds such as osmolytes, protein inhibitors, ligands and cofactors exert a chemical chaperoning effect and are able to rescue folding and trafficking defects, minimising or partly overcoming the pathological consequences of protein misfolding. Here we review the different types of chemical chaperones and provide a structural and energetic rationale for their action. Examples of chemical chaperoning are overviewed and discussed on the basis of the reported effects exerted by chemical compounds at different stages of the protein folding process and protein conformational states.

Galactose-1-phosphate uridylyltransferase (GALT) is a key enzyme in galactose metabolism, particularly important in the neonatal period due to ingestion of galactose-containing milk. GALT deficiency results in the genetic disorder classic galactosemia, whose pathophysiology is still not fully elucidated. Whereas classic galactosemia has been hypothesized to result from GALT misfolding, a thorough functional–structural characterization of GALT most prevalent variants was still lacking, hampering the development of alternative therapeutic approaches. The aim of this study was to investigate the structural–functional effects of nine GALT mutations, four of which account for the vast majority of the mutations identified in galactosemic patients. Several methodologies were employed to evaluate the mutations' impact on GALT function, on the protein secondary and tertiary structures, and on the aggregation propensity. The major structural effect concerns disturbed propensity for aggregation, particularly striking for the p.Q188R variant, resulting from the most frequent (∼60%) allele at a worldwide scale. The absence of major effects at the secondary and tertiary structure levels suggests that the disturbed aggregation results from subtle perturbations causing a higher and/or longer exposure of hydrophobic residues in the variants as compared to WT GALT. The results herein described indicate a possible benefit from introducing proteostasis regulators and/or chemical/pharmacological chaperones to prevent the accumulation of protein aggregates, in new avenues of therapeutic research for classic galactosemia.

Cystathionine ␤-synthase (CBS) is a key enzyme in human (patho)physiology with a central role in hydrogen sulfide metabolism. The enzyme is composed of a pyridoxal 5-phosphate-binding catalytic domain, flanked by the following two domains: a heme-binding N-terminal domain and a regulatory C-terminal domain binding S-adenosyl-L-methionine (AdoMet). CO or NO ⅐ binding at the ferrous heme negatively modulates the enzyme activity. Conversely, AdoMet binding stimulates CBS activity. Here, we provide experimental evidence for a functional communication between the two domains. We report that AdoMet binding significantly enhances CBS inhibition by CO. Consistently, we observed increased affinity (ϳ5-fold) and faster association (ϳ10-fold) of CO to the ferrous heme at physiological AdoMet concentrations. NO ⅐ binding to reduced CBS was also enhanced by AdoMet, although to a lesser extent (ϳ2-fold higher affinity) as compared with CO. Importantly, CO and NO ⅐ binding was unchanged by AdoMet in a truncated form of CBS lacking the C-terminal regulatory domain. These unprecedented observations demonstrate that CBS activation by AdoMet puzzlingly sensitizes the enzyme toward inhibition by exogenous ligands, like CO and NO ⅐ . This further supports the notion that CBS regulation is a complex process, involving the concerted action of multiple physiologically relevant effectors.