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case_images: TRAP1 mutation increased caspase activity in vitro and in vivo and inflammasome activation, resulting in enhanced apoptosis. (A) Sanger sequencing electropherograms of patient genetic variants. (B) Schematic representation of the domain structure of Trap1. Trap1 has three structural domains, an N-terminal domain (NTD) containing mitochondria targeting sequence (MTS) and an ATPase domain, a middle domain (MD) responsible for client binding, and a C-terminal domain (CTD) for dimerization. The arrows indicate the location of the amino acid switch from Glutamate to Glutamine (p.E93Q) and alanine to threonine switch at the position of 64 (p.A64T) in Trap1. (C, D) Western blot of peripheral blood mononuclear cells (PBMCs) from the patient and control was stimulated with LPS and Pam3C or unstimulated (RPMI) for 30 minutes. Cell lysates were run on a 10% SDS gel, transferred to PVDF membrane, and probed for different antibodies. beta-actin was used as a loading control. (E) A549 cells were transfected with empty vector, Trap1 wild-type (WT), Trap1 p.A64T (A64T), Trap1 p.E93Q (E93Q), and Trap1 p.A64T+Trap1 p.E93Q (A64T+E93Q) and were stimulated with LPS and Pam3C and the cell lysates were analyzed by Western blot. All Western blots were repeated three times. (F) PCP infection inhibits cell viability in A549 Trap1 transfected cells. The cell viability was measured using the XTT Assay. Values are the mean 3 +- SEM from nine replicates of three independent experiments (n=3). (G) Apoptosis detection by flow cytometry with dual annexin V-PI cell labeling. PBMCs from control (n=7) and patient (n=3) were collected and apoptosis was induced and uninduced by staurosporine. Data is represented in a scatter plot (+ median). Statistical analysis was performed using Student's t-test for PI- control PBMCs vs PI- patient PBMCs in media, and PI+ control PBMCs vs PI+ patient PBMCs in staurosporine. n=7 for control and n=3 for patient. * p <= 0.05. Upper left quadrant (Q1), necrotic cells; upper right quadrant (Q2), late apoptotic or necrotic cells; lower left quadrant (Q3), intact cells; lower right quadrant (Q4) early apoptotic cells. The number in each quadrant refers to the percentage of cells in each subpopulation. The figure is representative of 10,000 cells analyzed independently in seven volunteers and a triplicate of patient cells. RPMI, Roswell Park Memorial Institute Medium; SDS, Sodium dodecyl Sulfate; LPS, lipopolysaccharide; Pam3c, Pam3CysSerLys4; PVDF, polyvinylidene fluoride.
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case_images: The clinical course of the patient is as described in  Supplementary Figure S1A . The sequencing revealed two heterozygous rare variants of TRAP1 on chromosome 16p13 (GRCh37): one at g.3739109 C>G (exon 3) resulting in amino acid switch from Glutamate-to-Glutamine at protein position 93 (E93Q) ( Figure 1A ) and another at g.3740885 C>T (exon 2) resulting in alanine-to-threonine switch at position 64 (A64T) ( Figure 1A ). Both E93Q and A64T SNPs lie in the N-terminal ATP-binding domain (NTD) of TRAP1 and were predicted to be deleterious in-silico ( Figure 1B ;  Supplementary Table S1 ). TRAP1 expression and transcript ( Figure 1C ,  Supplementary Figure S1B ). TRAP1 had previously been shown to protect against apoptosis. In line with those data, we observed increased cleaved, active caspase-3 and caspase-7 in the patient's cells, which may be associated with an overall decline in TRAP1 expression ( Figure 1C ). Indeed, CypD was accentuated ( Figure 1C ), in line with caspase-3 augmentation in the patient's cells. We observed increased caspase-1 cleavage and mature IL-1beta in the patient's cells treated with TLR1/2 or TLR4 ligands, suggesting that the mutant TRAP1s exacerbate inflammasome activation ( Figures 1C, E ,  Supplementary Figure S1C ). However, the aggravated apoptosis exerted a limited inhibitory effect on the NFkappaB complex through NFkappaB2, NFkappaB1, and RelB ( Figure 1D ). A64T in line with in-silico prediction, and an additive effect was observed with co-expressed E93Q-A64T double mutant ( Figure 1E ). As was observed in patient cells, mutant TRAP1 reduced NFkappaB signaling, with the most prominent inhibitory effect on NFkappaB1, RelB and p65 activity observed in double E93Q/A64T mutant. In particular, dual E93Q/A64T-transfected cells exhibited highest cell loss (>80%) ( Figure 1F ). Following staurosporine, the patient's annexin-V-positive, PI-positive cells marking cell death significantly doubled ( Figure 1G ).
case_images: The dysregulation of TRAP1 resulted in a defective ETC, resulting in electron leakage and ROS production. (A) The mitochondrial and total ROS were assessed by flow cytometry in A549 cells transfected with WT and variants (A64T, E93Q, and A64T+E93Q). Representative histograms of flow cytometric analysis of MitoSOX and H2DCFDA fluorescence in different experimental groups (n=3). The bar graphs show the percentage of cells positive for mitochondrial ROS and total ROS production. The significance of the results was determined by ANOVA. * p <= 0.05. (B) The level of mitochondrial and total ROS in PBMCs from controls and the patient was measured by flow cytometry. Results are expressed as means +- standard error of the mean (SEM). n=3 per group. * p <= 0.05 (C) Oxygen consumption rate (OCR) trace of the Seahorse Mito Stress assay on A549 transfected with different Trap1 variants. (n = 3 repeats, 5 technical replicates each repeat). (D) Maximal glycolytic rate of A549 transfected with different Trap1 variants. (n = 2 repeats, 5 technical replicates each repeat). Results are expressed as means +- standard error of the mean (SEM). The significance of the results was determined by ANOVA (**p <= 0.01, *** p<= 0.001, **** p <= 0.0001). ETC, Electron transport chain; ROS, Reactive Oxygen Species.
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case_images: TRAP1 regulates mitochondrial oxidative phosphorylation (OXPHOS) and suppresses ROS production, we next evaluated the effect of TRAP1 mutation on ROS production. Total ROS production, as measured by 2',7'-dichlorofluorescein, was incrementally elevated in mutant-TRAP1 cells relative to WT ( Figure 2A ; top panel). Specifically, when stained with mito-SOX which probed specifically for mito-ROS, the frequency and median fluorescence intensity of E93Q- and A64T-transfected cells increased relative to WT ( Figure 2A ; bottom panel). Notably, dual-E93Q/A64T cells had 6-fold increased mito-ROS ( Figure 2A ; bottom panel). Similarly, the patient's PBMC demonstrated markedly increased total and mito-ROS ( Figure 2B ). Compared to cells transfected with WT-TRAP1 or empty vector, the double E93Q/A64T mutant expressing cells had the most markedly reduced mitochondrial respiration over the single mutants, as measured by the Mito-Stress assay ( Figure 2C ). Supplementary Figure S2 ). Similar to the observed decrease in mitochondrial respiration, the double mutant displayed a guarded reduction in the ability to increase glycolysis when the electron transport chain was blocked ( Figure 2D ).