Structural and functional characterization of disease-associated NOTCH4: a potential modulator of PI3K/AKT-mediated insulin signaling pathway

Type 2 diabetes mellitus (T2DM) is one of the top five leading causes of global morbidity and mortality attributed to non-communicable diseases. Several genes are involved in disease susceptibility to T2DM. Genetic variants falling in regulatory areas are likely to have an impact on gene function. The aim of the present study was to identify an SNP and its related protein that may have possible interactions with PI3K/AKT-dependent insulin signaling and was not previously reported to be associated with T2DM in the context of glucose homeostasis. Apropos, the genetic variants and their association with T2DM were explored. The single nucleotide polymorphisms (SNPs) associated with T2DM were identified using genome-wide association (GWAS) studies. 120 proxy SNPs were selected for further analysis on the basis of linkage disequilibrium score, regulome score and lack of pre-existing evidence of association with the canonical PI3K/AKT pathway affected in T2DM. We found that rs415929 SNP is directly associated with NOTCH4, which likely interacts with the signaling proteins of the PI3K/AKT pathway. Structural and functional characterization of NOTCH4 was therefore performed using in silico approaches. Characterization of NOTCH4 revealed that it is an acidic protein with greater half-life, is thermally unstable and has hydrophilic propensity. It is an extracellular, secretory protein acting as signaling peptide. The RAM and ANK domains of NOTCH4 are involved in interactions with AKT. The important interacting residues of RAM and AKT1 are RAM: Arg1489 with AKT: Glu397, while ANK: Glu1695 and AKT: Lys112 are the important interactions between ANK domain of NOTCH4 and AKT1. The MD simulation of NOTCH4 and its cytoplasmic domain giving 0.11-5.875 angstrom and 0.029-7.744 angstrom RMSF values in 10 ns interval validates the structural stability and flexibility of protein residues in a cellular environment. Thus, in this study, the plausible role of NOTCH4 in T2DM via modulation of PI3K/AKT-dependent insulin signaling was identified, which may be further validated through in vitro and in vivo analysis.