Journal: bioRxiv
Article Title: Functional analyses and integrated mechanisms of cellular destruction by L-amino acid oxidase
doi: 10.1101/2024.09.16.613219
Figure Lengend Snippet: A , Monomeric structure of B. atrox LAAO showing the predicted catalytic and substrate binding residues as sticks: Y372, R322, R90, H223, N172. B , Surface of the substrate binding pocket of LAAO ( A. H. pallas venom (PDB 1TDN) showing positively charged amino acid residues (blue), polar residues (cyan), and hydrophobic residues (green). L-leucine substrate is shown as magenta stick. C , L-amino acid oxidation reaction catalysed by LAAO WT with co-factor FAD (E-FAD) generating H 2 O 2 and ammonia as by products. D - E, Activity of purified LAAO WT or LAAO mutants (52.72 nM) compared to wild type (D, arbitrarily set at 100%) or their initial velocity V (E; µM per second, Y axis). The initial velocity V is defined as the change in the concentration of H 2 O 2 produced by LAAO at each concentration of substrate L-leucine (mM, X axis). In the bottom panel, catalytic activities of selected mutants are shown magnified (100X). F , Keratinocyte cell viability was assessed by incubation 14 nM of LAAO WT or mutants. G-H , Cellular reactive oxygen species (ROS) generated by keratinocytes untreated or treated with 280 nM LAAO WT or LAAO R90A and live stained with CellRox Green (green) and fixed before confocal imaging. Representative images (G) and quantification (H) are shown. Bars indicate mean and error bars show standard deviation. The assays were done as multiple technical replicates in three independent biological replicates (thereafter N=3), represented in blue, green, or pink colours. Black asterisks represent comparison within a single treatment group; red asterisk compares across different treatments. **** P ≤ 0.0001, *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05. Number of all images used for the analysis are indicated in File S1. Scale bar: 20 µm.
Article Snippet: Normal human epidermal keratinocytes at 4000 cells/well (passages 3-6) were co-cultured with 3T3-J2 fibroblasts in a 96 well plate (Corning, 3585; cell viability assay) or a CellCarrier-96 ultra microplates (PerkinElmer, 6055300; confocal imaging) in FAD standard medium (FAD medium (Gibco, custom made) as described previously ).
Techniques: Binding Assay, Activity Assay, Purification, Concentration Assay, Produced, Incubation, Generated, Staining, Imaging, Standard Deviation, Comparison
