Investigation on MMACHC pathological mutants involved in cblC disease, a rare inborn metabolic disorder of vitamin B12

The cblC disease is a rare inborn disorder of the vitamin B12 (cobalamin, Cbl) metabolism characterized by combined methylmalonic aciduria and homocystinuria. The clinical consequences are heterogenic and severe, and even when early treated with current therapies, the affected children manifest neurocognitive, ophtalmological and cardiovasculardysfunctions. The molecular genetic cause of the disease was found in the mutations the MMACHC gene coding for MMACHC, a 282 amino acid protein responsible for the intracellular trafficking and processing of the various forms of Cbl. Although the crystal structure of the wild-type protein (WT) has been solved, many molecular features of MMACHC physiopathology remain to be understood and a study on the effect of each specific pathogenic mutation on the resulting protein is still lacking. In this PhD thesis we present a comprehensive methodological approach for the biophysical characterization of MMACHC-WT and the MMACHC-R161Q variant, resulting from the mostfrequent missense pathological mutation found in cblC patients. The recombinant MMACHC-WT protein produced in Escherichia coli was first characterized using circular dichroism, UV-visible absorbance, fluorescence spectroscopy, molecular dynamics, size exclusion chromatography and native gel electrophoresis. These analyses provided a detailed description of the protein’s secondary and tertiary structure, stability, oligomerization propensity, and ability to correctly process Cbl. The same experimental pipeline was applied to the MMACHC-R161Q variant, revealing a different stability and an impaired dimerization, that directly correlate to its reduced enzymatic activity. These results were further enriched by calorimetric studies: differential scanning calorimetry unveiled acomplex thermal unfolding pathway that is significantly different among the two variants, while isothermal titration calorimetry directly quantified the binding of the two proteins to the natural ligands, confirming the weaker interactions established by MMACHC-R161Q, and provided the thermodynamic parameters governing these interactions. Moreover, we demonstrated as a proof-of-principle that the use of non-specific stabilizers (osmolytes) can partially restore functionality in MMACHC-R161Q, establishing the basis for a therapeutic treatment based on protein stabilization. In this framework, we also performed an in silico screening of drug-like small molecules to identify potential pharmacological chaperones, followed by an in vitro evaluation of their effects on the isolated protein. Taken together, these results provide a molecular interpretation of the MMACHC-R161Q pathogenic mechanism and establish a biophysical platform suitable for investigating disease causing variants. This comparative approach also provided the opportunity to investigate previously unexplored biophysical aspects of the wild-type enzyme, thereby deepening our understanding of the molecular mechanisms responsible for MMACHC function and dysfunction. Our results not only advance the current knowledge of the alterations underlying the disease but also represents the first step toward the rational design of personalized therapeutic strategies. Furthermore, the biophysics-based methodological toolbox presented here may be extended to the study of other rare disorders sharing similar molecular mechanisms driving the pathogenic events.