Khan, R. H. | bioRxiv Science

bioRxiv2025

Changes in biophysical characteristics of YghA from E. coli due to variation in pH

YghA from E. coli has been reported to be involved in conferring tolerance against furan aldehyde inhibitors which are commonly generated in thermo-acidic pretreatment of lignocellulosic biomass. In this study we report the biophysical characterization of YghA from E. coli as a function of variation in pH. Fluorescence intensity of YghA increased by 2.5-fold in acidic pH as compared to circumneutral pH. In presence of the hydrophilic 8-anilinonaphthalene-1-sulfonic acid (ANS) dye, a 68 - 79.4-fold increase in fluorescence intensity at acidic pH was observed as compared to circumneutral pH, while at basic pH the increase was only 1 to 2-fold. Secondary structure analysis by circular dichroism signal at 222 and 208 nm suggests that the secondary structure of YghA is primarily composed of alpha-helix at pH 7 and the secondary structure is abruptly lost at pH 3 and lower. In agreement with these observations, MD simulations predicted greater structural variations at low pH when compared to neutral or high pH. Interface energy calculations using computational docking protocols suggested that YghA forms relatively more stable complex with NADH. The in silico pulling assay results also show that NADH is more preferred compared to NADPH. Molecular dynamics simulations also indicate that YghA is structurally unstable at acidic pH with significant variations in the root mean square deviation values of the tetramer backbone. Residues GLU 84 at pH 7 together with PRO 24 and LEU 236 at pH 1 were identified as flexible residues and are promising target for future mutagenesis studies targeted towards improving structural stability of YghA.

biochemistry↗

bioRxiv2024

Loss of Neurofibromin Induces Inflammatory Macrophage Phenotypic Switch and Retinal Neovascularization via GLUT1 Activation

Persons with neurofibromatosis type 1 (NF1), a tumor predisposition syndrome, are largely protected from diabetes and exhibit evidence of enhanced glucose metabolism, which is replicated in mice harboring Nf1 mutations. A hallmark of NF1-associated neurofibromas and sarcomas is the high density of inflammatory macrophages and targeting macrophages appears efficacious in models of NF1. Inflammatory macrophages rely on glycolysis to rapidly generate ATP; thus, identifying whether neurofibromin, the protein encoded by the NF1 gene, controls glucose uptake and/or glycolysis in macrophages is therapeutically compelling. Using neurofibromin-deficient macrophages and macrophage-specific Nf1 knockout mice, we demonstrate that neurofibromin complexes with glucose transporter 1 (GLUT1) to restrain its activity and that loss of neurofibromin permits Akt2 to facilitate GLUT1 translocation to the membrane in macrophages. In turn, glucose internalization and glycolysis are highly up regulated and provoke putative reparative (M2) macrophages to undergo inflammatory phenotypic switch. Inflammatory M1 macrophages and inflammatory-like M2 macrophages invest the perivascular stroma of tumors and induce pathologic angiogenesis in mice harboring macrophage-specific Nf1 deletion. These studies identify a clear mechanism for the enhanced glycolysis and low risk for diabetes observed in persons with NF1 and provide a novel therapeutic target for manifestations of NF1.

cell biology↗