Studies on the reactions of formaldehyde with protein N-termini and related biomolecules

Formaldehyde (HCHO) is a reactive electrophile that is both a human metabolite and a pollutant. Above threshold levels HCHO is carcinogenic, likely as a consequence of its reactions with biomolecules. NMR studies have provided insight into the reactions of HCHO with amino acids and nucleobases, the latter of which may underpin HCHO-driven toxicity. HCHO is produced by the actions of demethylases in the regulation of transcription, likely causing relatively high local nuclear concentrations of HCHO. Endogenous HCHO is sufficient to cause fatal malignancies in mice deficient of HCHO protection mechanisms. The work described in this thesis explores the possibility that HCHO reacts with N-terminal residues in proteins and its biochemical consequences. The N-terminus offers the unique scenario where two nucleophiles, i.e. the N-terminal free amine and an adjacent reactive group (either the side chain of the first amino acid or the adjacent peptide bond) are in close proximity, so enabling stable ring formation upon reaction with HCHO.
Introduction. In Chapter 1, an overview of the chemistry of HCHO and its historical and modern uses are provided. The cellular detoxification mechanisms and known reactions of HCHO with biomolecules are discussed, along with biochemical reactions that release HCHO. This is followed by a description of the role of aldehydes in metabolism in prokaryotes and eukaryotes.
N-Terminal prolines. In Chapter 2, studies testing the hypothesis that histone tails might act as a HCHO sink are reported. Among the four isolated canonical histone tails, H2B shows exceptional susceptibility to reactions with HCHO. The product of the reaction of H2B with HCHO was characterized by MS and NMR, revealing formation of a stable fused 5,5-bicyclic adduct. These findings were then validated in a biological context; the results show that in situ generated HCHO results in the same adduct. Studies on the inhibition of 4-oxalocrotonate tautomerase (4-OT), which bears an N-terminal catalytic proline, show that HCHO can inhibit enzyme activity both in vitro and in cellulo.
N-Terminal cysteines. Competition experiments of N-terminal prolines with free cysteine reveal that cysteine reacts even more efficiently than N-terminal prolines with HCHO. Chapter 3 describes the reactions of N-terminal cysteines with HCHO. Initial studies on model peptides showed that N-terminal cysteines react with HCHO to form a stable thiazolidine; N-terminal threonine or serine residues form the analogous oxazolidine, but those adducts are less stable. Asparagine Synthetase (ASNS), which bears an N-terminal catalytic cysteine was developed as a model to investigate reactions with HCHO with N-terminal cysteine residues in vitro and in cellulo. ASNS uses its N-terminal cysteine to deamidate glutamine, forming ammonia, which travels through an intramolecular tunnel to react with β-aspartyl-AMP, forming asparagine. HCHO inhibits the N-terminal glutaminase reaction, while the asparagine synthesis activity is retained, as shown by studies with NH4Cl rather than glutamine. Addition of HCHO leads to an increase in ASNS levels in cellulo and depletes intracellular asparagine.
Metampicillin. The reactions of HCHO with nucleophilic drugs are important in medicine. In Chapter 4, NMR was used to investigate the structure of metampicillin, which is produced by reaction of ampicillin with HCHO. Metampicillin, which has been used to treat biliary infections in humans and animals, was proposed to have HCHO-derived imine or hemiaminal structures. In collaboration with Raphael Reinbold, it is shown that metampicillin is a cyclic aminal.
CV19. As a consequence of the CV-19 pandemic, collaborative work was carried out on nucleophilic cysteine proteases. In Chapter 5, mass spectrometry based assays were developed to study inhibition of two proteases of SARS-CoV-2, namely Mpro and PLPro.

Advisor: Schofield, CJ