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. 2024 Jan 26;9(5):5485-5495.
doi: 10.1021/acsomega.3c07195. eCollection 2024 Feb 6.

Analyzing the Effects of Single Nucleotide Polymorphisms on hnRNPA2/B1 Protein Stability and Function: Insights for Anticancer Therapeutic Design

Kunal Dutta  1 , Viacheslav Kravtsov  1 , Katerina Oleynikova  2 , Alexey Ruzov  2 , Ekaterina V Skorb  1 , Sergey Shityakov  1
Affiliations

Analyzing the Effects of Single Nucleotide Polymorphisms on hnRNPA2/B1 Protein Stability and Function: Insights for Anticancer Therapeutic Design

Kunal Dutta et al. ACS Omega. .

Abstract

Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/B1) is a pivotal player in m6A recognition, RNA metabolism, and antiviral responses. In the context of cancer, overexpression of hnRNPA2/B1, abnormal RNA levels, and m6A depositions are evident. This study focuses on two significant nonsynonymous single nucleotide polymorphisms (nsSNPs) within hnRNPA2/B1, namely, F66L and E92K. Our structural analyses reveal decreased stability in these mutants, with E92K being predicted to undergo destabilizing post-translational methylation. Furthermore, our extensive analysis of 44,239 tumor samples from the COSMIC database uncovers that amino acid position 92 exhibits the second-highest mutation frequency within hnRNPA2/B1, particularly associated with breast and lung cancers. This experimental data aligns with our theoretical studies, highlighting the substantial impact of the nsSNP at position 92 on hnRNPA2/B1's stability and functionality. Given the critical role of pre-mRNA splicing, transcription, and translation regulation in cellular function, it is important to assess the impact of these nsSNPs on the stability and function of the hnRNPA2/B1 protein to design more efficient anticancer therapeutics.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Biophysical impacts of nsSNPs on hnRNPA2B1.
Figure 2
Figure 2
Structural stability of the wild-type and mutant-type hnRNPA2/B1. Scatter plots of RMSD versus Rosetta total score for the protein (A) wild-type, (B) F66L, (C) E92K, and (D) exponential curve fitting; the wild-type hnRNPA2B1 is depicted by a black line, and F66L and E92K are depicted by a different pattern of dotted lines. RMSD, root-mean-square deviation; REU, Rosetta energy units.
Figure 3
Figure 3
Two histograms displaying the positions of mutations (A) and single base substitutions (B) across the hnRNPA2/B1 (ENST00000618183) gene from COSMIC (the Catalogue of Somatic Mutations in Cancer). These mutations are shown at the amino acid level at two different resolutions. Amino acid position 92 is highlighted with a red arrow in both diagrams. The substitutions are color-coded by residue according to the scheme used in Ensembl. The visualization was performed by using COSMIC online tools.
Figure 4
Figure 4
Rosetta energy decomposition analyses for substitution mutants, i.e., F66L and E92K, of hnRNPA2/B1. fa_atr, Lennard-Jones attractive between atoms in different residues; fa_rep, Lennard-Jones repulsive between atoms in different residues; fa_sol, Lazaridis-Karplus solvation energy; fa_intra_sol_xover4, intraresidue Lazaridis-Karplus solvation, counted for the atom pairs beyond the torsion relationship; lk_ball_wtd, a weighted sum of anisotropic contribution to the solvation; fa_elec, Coulombic electrostatic potential with a distance-dependent dielectric; fa_dun, internal energy of side chain rotamers as derived from Dunbrack’s statistics; p_aa_pp, the probability of amino acid at Φ/Ψ angles; ref, reference energy for each amino acid, balancing internal energy of amino acid terms; rama_prepro, backbone torsion preference term that takes into account of whether the preceding amino acid is proline or not; REU, Rosetta energy units.
Figure 5
Figure 5
Evolutionary conservation profiles of hnRNPA2/B1. (A) Color protein molecule according to the Eisenberg hydrophobicity scale. (B) Wild-type, (C) F66L, (D) E92K, and their respective conservation scores (right); F66, E92, and substitutions are highlighted by black rectangles. Conservation profile scale depicted by a color-coded map (bottom) according to the NACCESS algorithm.
Figure 6
Figure 6
Structural stability analyses before and after post-translational modification E92K of hnRNPA2/B1. (A) Cartoon model of hnRNPA2/B1, E92K methylated (cyan), and unmethylated (green). Cαdist, a difference of distance between the α carbon of E92Kmet and E92Kunmet. REUs of E92K methylated (cyan) and unmethylated (green) are shown next protein structures, (B) scatter plots of RMSD versus Rosetta total score, (C) SASA versus amino acid residue number, (D) RMSD versus amino acid residue number, (E) Rg versus amino acid residue number of methylated, unmethylated E92K of hnRNPA2/B1. REU, Rosetta energy units; RMSD, root-mean-square deviation; Rg, radius of gyration. A threshold is depicted by horizontal dotted lines; E92Kmet and E92Kunmet are marked by vertical dotted lines.
Figure 7
Figure 7
Molecular interaction network of human hnRNPA2/B1. The black node at the center, hnRNPA2/B1, physical interactions (bold line) and coexpression (line), and degree of interactions are depicted proportionally by the size of the nodes.
Figure 8
Figure 8
Binding site analyses of hnRNPA2/B1. (A) hnRNPA2/B1 docked with RNA; the drug binding site is depicted by protein surface presentation. (B) Superimposed protein structure of hnRNPA2/B1 docked with CPT wild-type (blue), F66L (pale green), E92K (pale cyan), and 2D ligand interactions; (C) wild-type, (D) F66L, and (E) E92K.
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