Identification of Mortalin as the Main Interactor of Mycalin A, a Poly-Brominated C-15 Acetogenin Sponge Metabolite, by MS-Based Proteomics

Abstract

Mycalin A (MA) is a polybrominated C-15 acetogenin isolated from the marine sponge Mycale rotalis. Since this substance displays a strong antiproliferative bioactivity towards some tumour cells, we have now directed our studies towards the elucidation of the MA interactome through functional proteomic approaches, (DARTS and t-LIP-MS). DARTS experiments were performed on Hela cell lysates with the purpose of identifying MA main target protein(s); t-LiP-MS was then applied for an in-depth investigation of the MA-target protein interaction. Both these techniques exploit limited proteolysis coupled with MS analysis. To corroborate LiP data, molecular docking studies were performed on the complexes. Finally, biological and SPR analysis were conducted to explore the effect of the binding. Mortalin (GRP75) was identified as the MA's main interactor. This protein belongs to the Hsp70 family and has garnered significant attention due to its involvement in certain forms of cancer. Specifically, its overexpression in cancer cells appears to hinder the pro-apoptotic function of p53, one of its client proteins, because it becomes sequestered in the cytoplasm. Our research, therefore, has been focused on the possibility that MA might prevent this sequestration, promoting the re-localization of p53 to the nucleus and facilitating the apoptosis of tumor cells.

Keywords: drug affinity responsive target stability; heat shock proteins; marine antitumoral compound; molecular docking; multiple reaction monitoring; proteomics; targeted-limited proteolysis.

Figure 1

(A) Mycalin structure. (B) Coomassie stained gel showing proteins protection from subtilisin upon MA interaction. Dashed red lines indicate gel slices submitted to in situ tryptic digestion. Molecular weights alongside the ladder are expressed in kDa. (C) Densitometric analysis of the SDS-PAGE in panel (B), performed through ImageJ and reporting the pixel intensity of each gel region vs. its corresponding molecular weight range. Positive control intensities, rated at 100%, are not reported. As shown by the red arrow, the lowest MA concentration induces the highest pixel intensity increase around 75 kDa, where the protection is also dependent on the molecule amounts. (D) Mortalin and HS71A protection extent (%) calculated from the Mascot matches, as follows: [(Matches_MA − Matches_Ctrl)/Matches_Lysate] × 100. The reported values are the results of two DARTS biological replicates. (E) Immunoblotting analysis with anti-mortalin (i.e., anti-GRP75) and anti-HS71A antibodies. GAPDH has been used as a loading normalizer.

Figure 2

(A) MA-protected mortalin (i.e., GRP75) and HS71A fully tryptic peptides, reported with their Q1 and Q3 m/z values, the calculated MA over negative control fold changes and the corresponding p-values. Fold changes were calculated over the mean of peptides areas coming from three replicates. Schematic mortalin (i.e., GRP75, (B)) and HS71A (C) cartoons reporting the proteins NBDs and SBDs. T-LiP-MS protected peptides are represented as light blue bars, whose thickness is directly proportional to their length.

Figure 3

MA best predicted interaction poses with mortalin NBD (A) and SBD (B) and with HS71A NBD (C) and SBD (D) obtained through molecular docking. MA is represented through green sticks in all of the panels, whereas ADP is represented in purple stick in panel (A) (where the third phosphate is also shown as orange/red spheres and Mg²⁺ as a light green sphere) and in light blue stick in panel (C). MA interacting amino acidic residues are depicted in the zoomed schemes, with hydrophobic interactions, hydrogen bonds and halogen bonds shown in gray, blue and light blue lines, or dotted lines, respectively.

Figure 4

Histogram for data shown in Table 1. * p < 0.05; ** p < 0.01.

Figure 5

SPR analysis. Sensorgrams to evaluate (A) the binding affinity between p53 protein and mortalin (mortalin concentrations were 0.22, 0.44, 0.87, 1.75, 3.5, and 7 nM); and the competition between (B) mortalin-MA and (C) mortalin-MKT077. Concentration of MA or MKT077 were 26, 50, 100, 210, 420, and 850 nM while concentration of mortalin remained 13 nM. The equilibrium dissociation constants (K_D) were derived from the ratio between kinetic dissociation (koff) and association (kon) constants. Experiments were repeated independently three times. Reported K_D is the mean ± SD of three independent experiments.