Point mutations of the mitochondrial chaperone TRAP1 affect its functions and pro-neoplastic activity

Abstract

The mitochondrial chaperone TRAP1 is a key regulator of cellular homeostasis and its activity has important implications in neurodegeneration, ischemia and cancer. Recent evidence has indicated that TRAP1 mutations are involved in several disorders, even though the structural basis for the impact of point mutations on TRAP1 functions has never been studied. By exploiting a modular structure-based framework and molecular dynamics simulations, we investigated the effect of five TRAP1 mutations on its structure and stability. Each mutation differentially impacts long-range interactions, intra and inter-protomer dynamics and ATPase activity. Changes in these parameters influence TRAP1 functions, as revealed by their effects on the activity of the TRAP1 interactor succinate dehydrogenase (SDH). In keeping with this, TRAP1 point mutations affect the growth and migration of aggressive sarcoma cells, and alter sensitivity to a selective TRAP1 inhibitor. Our work provides new insights on the structure-activity relationship of TRAP1, identifying crucial amino acid residues that regulate TRAP1 proteostatic functions and pro-neoplastic activity.

Fig. 1. High throughput screening and classification of TRAP1 point mutations.

a Circular blot indicating the 310 variants identified for TRAP1 with the MAVISp computational framework. Mutations were stratified according to their effect on protein stability in neutral, uncertain and destabilizing ones. The height of each histogram indicates the pathogenicity score associated to each variants. b Most frequent TRAP1 mutations from COSMIC database and relative table indicating the frequency and pathogenicity score of selected variants. c Cartoon representation of the structure of TRAP1 homodimer TRAP170-704 (Alphafold-Multimer model) with spheres highlighting the Cα atoms of the five residues affected by the selected variants. d The dot plot illustrates the results from the MAVISp analyses. In green are reported the results from the prediction of pathogenicity (AlphaMissense and EVE), loss of function (DeMaSk) and possible effects at the functional sites. In blue are reported the predictions for effects related to long-range (AlloSigma2), phosphorylation (PTM effects) and local effects on interactions within the TRAP1 homodimer. In purple are illustrated the effects related to changes in structural stability or stability in relation to the removal of a PTM. The properties highlighted in blue and purple are the ones referring to the predicted mechanisms altered by the selected variants included in the MAVISp framework.

Fig. 2. Effects of point mutations on TRAP1 stability.

a T600 and its surrounding residues are visualized on the 3D structure of the TRAP1 homodimer. b Residues surrounding T600 in both protomer A (light blue) and B (dark yellow) of TRAP1. Numbering is relative to zTrap1 sequence as in PDB code 4IPE. To convert to hTrap1 numbering 15 should be subtracted. In the zoomed views the structure is rotated and centered on the mutated position. c Change in DF score to mutated positions in protomer A or B for each residue in going from WT to T600P (indicated by red circle) projected onto the protein 3D structure (P_600A, P_600B). Color code for DF scores: blue areas (positive values) correspond to lower mechanical coordination in Trap1 mutants with respect to the wild-type protein, whereas orange ones (negative values) indicate a higher coordination in the former. Gray/white areas are those unaffected by the mutation. d Western blot assessing TRAP1 protein levels in sMPNST cells re-expressing the human WT or T600P TRAP1 forms after knocking-out endogenous TRAP1. Where indicated, cells were treated with the proteasomal inhibitor MG132 10 μM for 6 h. Data are reported as average ± SEM of 3 independent experiments with a two-tail unpaired t-test. e Western blot assessing TRAP1 protein levels in sMPNST cells re-expressing the indicated mutant forms of TRAP1 after knocking-out endogenous TRAP1. Where indicated, cells were treated with 10 μM of MG132 for 6 h. Data are reported as average ± SEM of 3 independent experiments.

Fig. 3. Effect of point mutations on the ATPase activity and molecular dynamics of TRAP1.

a, d, g, j ATPase activity of recombinant WT or mutant forms of human TRAP1 was measured as released PO₄^3-. Data are shown as fold change (with respect to the WT protein) and represent the mean ± SEM of n = 4 independent experiments done in triplicate with a Student’s t test analysis (***p < 0.001; n.s, non-significant). b, e, h, k Change in DF score to mutated positions in protomer A or B for each residue from WT to Mut Trap1 (b D260N; e P381S; h V556M; k A571T), projected onto the protein 3D structure (P_mutA, P_mutB). Protein views are rotated to allow a visualization of the mutation position, the identity of the protomer is labeled. Color code for DF scores: blue areas (positive values) correspond to lower mechanical coordination in Trap1 mutants with respect to the WT protein, whereas orange ones (negative values) indicate a higher coordination in the first. Gray/white areas are those unaffected by the mutation. c, f, i, l Variation in the mechanical connectivity index for each mutant along the sequence (Δη_mut). On the x axis numbering of zTrap1 as in PDB code 4IPE is shown together with the corresponding domains of protomer A and protomer B and the residue numbering is from 85 to 719. NTD: N-terminal Domain, residues 85–310; MD: Middle Domain divided in the subdomains Large Middle Domain (LMD), residues 311–470 and Small Middle Domain (SMD) residues 471–586; CTD: C-Terminal Domain, residues 587–719.

Fig. 4. TRAP1 point mutations differentially affect SDH activity and mitochondrial bioenergetics.

a Succinate dehydrogenase (SDH) activity measured in sMPNST cells expressing either human wild-type or mutant forms of TRAP1. Data are reported as mean±SEM of 3 independent experiments with a two-tail unpaired Student’s t-test with each mutant compared to hTRAP1-WT expressing cells (**p value < 0,01; ***p value < 0,001; n.s. non-significant). b TRAP1 immunoprecipitation in murine sMPNST cells re-expressing either the wild-type or the mutant forms of human TRAP1 after knocking-out endogenous TRAP1. c Root mean square fluctuation (RMSF) per residue considering the backbone atoms only. The mutated positions are indicated with an arrow. On the x axis numbering of zTrap1 is as in PDB code 4IPE and is shown together with the corresponding domains of protomer A and protomer B. For each protomer, residue numbering is from 85 to 719. d–f Quantification of basal oxygen consumption rate (OCR; d), mitochondrial ATP production (e) and extracellular acidification (ECAR; f) measured in sMPNST cells expressing either human wild-type or the mutant forms of TRAP1. Data are reported as mean±SD of 3 independent experiments, with each mutant compared to hTRAP1-WT expressing cells; asterisks indicate significant differences (^∗∗p < 0.01, ^∗p < 0.05; Student’s t-test analysis).

Fig. 5. Effect of TRAP1 point mutations on its pro-neoplastic activity.

a Focus-forming assay on sMPNST cells (scramble, scr, i.e. expressing endogenous TRAP1; TRAP1 KO and re-expressing hWT-TRAP1 in a TRAP1 KO background) grown for 10 days with or without the selective TRAP1 inhibitor compound 5 (25 μM). Data are reported as mean of foci area normalized to scr SMPNST cells, and presented as mean ± SEM (n = 3 independent experiments with 3 replicates for each one); ***p < 0.001 with one-way ANOVA with Bonferroni’s test. b–e Focus-forming and invasion assay on sMPNST cells re-expressing human WT or TRAP1 mutants (b D260N, c V556M; d A571T; e P381S) in an endogenous TRAP1 KO background. Where indicated, cells were treated with 25 μM of compound 5. For focus-forming assays, data are reported as mean of foci area normalized on hWT-TRAP1 expressing cells. For invasion assay data are reported as area covered by invading cells normalized on hTRAP1-WT expressing cells. Data are presented as mean ± SEM of at least 3 independent experiments with 3 replicates for each one); ***p < 0.001 with one-way ANOVA with Bonferroni’s test for focus forming and Student’s t-test for invasion assay. f Spheroids formed by sMPNST cells expressing hTRAP1-WT or hTRAP1-P381S mutant. Spheroid area was measured after 10 days of growth. g Branching morphogenesis assay performed on sMPNST spheroids. Matrigel was added after 3 days of spheroid growth. The spreading of spheroids was estimated by measuring the area of branches using ImageJ software after setting a threshold to highlight and isolate the spheroid and its branches from the background. Data are presented as mean ± SEM of at least 3 independent experiments and analyzed with a Student’s t test analysis; ***p < 0.001.