Catalysis of 5-methyltetrahydrofolate to MeFox facilitates folate biofortification in crops

Folate deficiency is a global health problem. Biofortification has been considered a cost-effective means to tackle this problem. Here, we describe the genetic cloning and functional identification of a previously uncharacterised plant protein, designated as CTM, which functions as an enzyme in folate metabolism. Plant CTMs are capable of catalysing 5-methyl-tetrahydrofolate to MeFox, a pyrazino-s-triazine derivative of 4α-hydroxy-5-methyl-tetrahydrofolate. The natural asparagine-to-glycine substitution caused by an A-to-G single nucleotide variation in maize CTM enhances its enzymatic activity, as demonstrated by in vitro enzymatic assays and in silico analyses using a maize CTM structure model based on a monomeric sorghum CTM crystal. Loss of the CTM function led to accumulation of 5-methyl-tetrahydrofolate, and overexpression of the maize CTM carrying the G-allele boosted the metabolic flow towards MeFox, and showed no negative impacts on plant growth. Our results suggest that CTM, which has evolved 5-methyl-tetrahydrofolate-to-MeFox converting activity in plants, could be valuable for developing folate-biofortified crops to provide an alternative to the challenge presented by the global folate deficiency.

D614G substitution enhances the stability of trimeric SARS-CoV-2 spike protein

SARS-CoV-2 spike protein with D614G substitution has become the dominant variant in the ongoing COVID-19 pandemic. Several studies to characterize the new virus expressing G614 variant show that it exhibits increased infectivity compared to the ancestral virus having D614 spike protein. Here, using in-silico mutagenesis and energy calculations, we analyzed inter-residue interaction energies and thermodynamic stability of the dominant (G614) and the ancestral (D614) variants of spike protein trimer in ‘closed’ and ‘partially open’ conformations. We find that the local interactions mediated by aspartate at the 614th position are energetically frustrated and create unfavourable environment. Whereas, glycine at the same position confers energetically favourable environment and strengthens intra-as well as inter-protomer association. Such changes in the local interaction energies enhance the thermodynamic stability of the spike protein trimer as free energy difference (ΔΔG) upon glycine substitution is −2.6 kcal/mol for closed conformation and −2.0 kcal/mol for open conformation. Our results on the structural and energetic basis of enhanced stability hint that G614 may confer increased availability of functional form of spike protein trimer and consequent in higher infectivity than the D614 variant.

MicroRNA regulation postbleomycin due to the R213G extracellular superoxide dismutase variant is predicted to suppress inflammatory and immune pathways

Oxidative stress is a key contributor to the development of dysregulated inflammation in acute lung injury (ALI). A naturally occurring single nucleotide polymorphism in the key extracellular antioxidant enzyme, extracellular superoxide dismutase (EC-SOD), results in an arginine to glycine substitution (R213G) that promotes resolution of inflammation and protection against bleomycin-induced ALI. Previously we found that mice harboring the R213G mutation in EC-SOD exhibit a transcriptomic profile consistent with a striking suppression of inflammatory and immune pathways 7 days postbleomycin. However, the alterations in noncoding regulatory RNAs in wild-type (WT) and R213G EC-SOD lungs have not been examined. Therefore, we used next-generation microRNA (miR) Sequencing of lung tissue to identify dysregulated miRs 7 days after bleomycin in WT and R213G mice. Differential expression analysis identified 92 WT and 235 R213G miRs uniquely dysregulated in their respective genotypes. Subsequent pathway analysis identified that these miRs were predicted to regulate approximately half of the differentially expressed genes previously identified. The gene targets of these altered miRs indicate suppression of immune and inflammatory pathways in the R213G mice versus activation of these pathways in WT mice. Triggering receptor expressed on myeloid cells 1 (TREM1) signaling was identified as the inflammatory pathway with the most striking difference between WT and R213G lungs. miR-486b-3p was identified as the most dysregulated miR predicted to regulate the TREM1 pathway. We validated the increase in TREM1 signaling using miR-486b-3p antagomir transfection. These findings indicate that differential miR regulation is predicted to regulate the inflammatory gene profile, contributing to the protection against ALI in R213G mice.