Structural analysis of human SEPHS2 protein, a selenocysteine machinery component, over-expressed in triple negative breast cancer

Abstract

Selenophosphate synthetase 2 (SEPHS2) synthesizes selenide and ATP into selenophosphate, the selenium donor for selenocysteine (Sec), which is cotranslationally incorporated into selenoproteins. The action and regulatory mechanisms of SEPHS2 as well as its role in carcinogenesis (especially breast cancer) remain ambiguous and need further clarification. Therefore, lacking an experimentally determined structure for SEPHS2, we first analyzed the physicochemical properties of its sequence, modeled its three-dimensional structure and studied its conformational behavior to identify the key residues (named HUB nodes) responsible for protein stability and to clarify the molecular mechanisms by which it induced its function. Bioinformatics analysis evidenced higher amplification frequencies of SEPHS2 in breast cancer than in other cancer types. Therefore, because triple negative breast cancer (TNBC) is biologically the most aggressive breast cancer subtype and its treatment represents a challenge due to the absence of well-defined molecular targets, we evaluated SEPHS2 expression in two TNBC cell lines and patient samples. We demonstrated mRNA and protein overexpression to be correlated with aggressiveness and malignant tumor grade, suggesting that this protein could potentially be considered a prognostic marker and/or therapeutic target for TNBC.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1

Sequence analysis of human SEPHS2. The negatively and positively charged residues are shown in red and cyan, respectively. We report predictions for secondary structure (Jnet), disorder propensity (Meta_d and IUPred), globularity propensity (GlobPL), predicted and experimentally determined phosphorylation sites (NetPhos and Phosphosite), sulfination sites (Sulfinator), glycosylation sites (NetNGlyc and NetOGlyc), and molecular recognition features (MorfPred and ANCHOR).

Figure 2

BLAST alignment between human SEPHS2 and human SEPHS1. The 310-helices, α-helices and β-strands are shown in cyan, red and yellow, respectively.

Figure 3

Complete SEPHS2 model obtained by the molecular modeling approach. In detail, 310-helices and α-helices are reported in red, β-strands in yellow and loops in green.

Figure 4

(A) RMSD, (B) gyration radius, (C) H-bond, (D) RMSF, and (E) solvent accessible surface plots and (F) partial densities for SEPHS2 during MD simulations at neutral (in red) and acidic (in black) pH.

Figure 5

Projection plot and cluster analysis results for SEPHS2 during MD simulations at neutral (A,B) and acidic pH (C,D), respectively.

Figure 6

Residue interaction network for SEPHS2 at the end of the neutral pH simulation. HUB nodes are in pink, and the other residues are in light blue.

Figure 7

Residue interaction network for SEPHS2 at the end of the acidic pH simulation. HUB nodes are in pink, and the other residues are in light blue.

Figure 8

SEPHS2 levels in MDA-MB468 and MDA-MB231 cells compared to MCF10A cells and in TNBC tissues compared to their normal counterparts based on RT-qPCR analysis. The p-values

Figure 9

(A) Immunohistochemical observation of human SEPHS2 expression at 200x magnification in normal mammary tissues and grade 2 (G2) and 3 (G3) TNBC patients. In the panel related to a tissue section from a G2 patient, only the neoplastic lesion is visible. In the panel related to a tissue section from a G3 patient, two black arrows indicate a lobular ductule of a normal mammary gland immersed in the adipose stroma; moreover, clusters of neoplastic cells are found in the same stroma. (B) Kaplan–Meier curves of the overall survival of TNBC patients. Blue line: score = 2 and green line: score = 3.