Mutation-Structure-Function Relationship Based Integrated Strategy Reveals the Potential Impact of Deleterious Missense Mutations in Autophagy Related Proteins on Hepatocellular Carcinoma (HCC): A Comprehensive Informatics Approach

Abstract

Autophagy, an evolutionary conserved multifaceted lysosome-mediated bulk degradation system, plays a vital role in liver pathologies including hepatocellular carcinoma (HCC). Post-translational modifications (PTMs) and genetic variations in autophagy components have emerged as significant determinants of autophagy related proteins. Identification of a comprehensive spectrum of genetic variations and PTMs of autophagy related proteins and their impact at molecular level will greatly expand our understanding of autophagy based regulation. In this study, we attempted to identify high risk missense mutations that are highly damaging to the structure as well as function of autophagy related proteins including LC3A, LC3B, BECN1 and SCD1. Number of putative structural and functional residues, including several sites that undergo PTMs were also identified. In total, 16 high-risk SNPs in LC3A, 18 in LC3B, 40 in BECN1 and 43 in SCD1 were prioritized. Out of these, 2 in LC3A (K49A, K51A), 1 in LC3B (S92C), 6 in BECN1 (S113R, R292C, R292H, Y338C, S346Y, Y352H) and 6 in SCD1 (Y41C, Y55D, R131W, R135Q, R135W, Y151C) coincide with potential PTM sites. Our integrated analysis found LC3B Y113C, BECN1 I403T, SCD1 R126S and SCD1 Y218C as highly deleterious HCC-associated mutations. This study is the first extensive in silico mutational analysis of the LC3A, LC3B, BECN1 and SCD1 proteins. We hope that the observed results will be a valuable resource for in-depth mechanistic insight into future investigations of pathological missense SNPs using an integrated computational platform.

Keywords: autophagy related proteins; computational analysis; hepatocellular carcinoma; missense single nucleotide polymorphisms; post-translational modifications.

Figure 1

Schematic representation of prioritization pipeline. Identification of the most deleterious mutations in LC3A, LC3B, BECN1 and SCD1 based on various in silico tools. Abbreviations used: DG, free energy change; DDG, the predicted free energy change value.

Figure 2

Conservation analysis of potential modified residues in LC3A protein. Sequences of LC3A proteins (human, mouse, rat, cattle and pig) were aligned using Multalin version 5.4.1 (http://multalin.toulouse.inra.fr/multalin/). High risk deleterious missense SNPs are mentioned in red color at respective positions while overlap with various types of posttranslational modifications are given in blue color.

Figure 3

Conservation analysis of potential modified residues in LC3B protein. Sequences of LC3B proteins (human, mouse, rat and cattle) were aligned using Multalin version 5.4.1 (http://multalin.toulouse.inra.fr/multalin/). High risk deleterious missense SNPs are mentioned in red color at respective positions while overlap with various types of posttranslational modifications are given in blue color.

Figure 4

Conservation analysis of potential modified residues in BECN1 protein. Sequences of BECN1 proteins (human, mouse, rat, cattle and pig) were aligned using Multalin version 5.4.1 (http://multalin.toulouse.inra.fr/multalin/). High risk deleterious missense SNPs are mentioned in red color at respective positions while overlap with various types of posttranslational modifications are given in blue color.

Figure 5

Conservation analysis of potential modified residues in SCD1 protein. Sequences of SCD1 proteins (human, mouse, rat, cattle and pig) were aligned using Multalin version 5.4.1 (http://multalin.toulouse.inra.fr/multalin/). High risk deleterious missense SNPs are mentioned in red color at respective positions while overlap with various types of posttranslational modifications are given in blue color.

Figure 6

Representation of WALTZ and DDG scores for the mutations associated with HCC. (A) Difference in WALTZ scores with respect to its frequency of occurrence (red dot represents WALTZ score for LC3B Y113C); (B) Difference in stability scores with respect to its frequency of occurrence (red dot represents DDG scores for BECN1 I403T , SCD1 R126S and SCD1 Y218C).

Figure 7

Human LC3A (PDB ID: 4ZDV) (A), LC3B (PDB ID: 2ZJD) (B), BECN1 (C) and SCD1 (D) in cartoon and a ball-and-stick representations. Ligand binding pockets were annotated by FTsite and COACH servers. Ligand binding sites coinciding with highly deleterious missense mutations are shown as ball-and-sticks in red color. Illustration was created using PYMOL.