Journal: Clinical Ophthalmology (Auckland, N.Z.)

Article Title: TRAP1 Improves Diabetic Retinopathy by Preserving Mitochondrial Function

doi: 10.2147/OPTH.S521660

Figure Lengend Snippet: In the retina of STZ-induced diabetic retinopathy (DR) rat models, the level of TRAP1 decreases. (A) Changes in body weight during the modeling period of diabetic and control rats. (n=8) ( B ) FBG levels during the modeling period of diabetic and control rats. (n=8) ( C ) Representative micrographs of retinal sections stained with H&E from control, diabetic for 4 weeks, diabetic for 8 weeks, and diabetic for 12 weeks groups. Scale bar=50μm. (n=3) ( D ) Western blot analysis of total TRAP1 in retinal tissues from control, diabetic for 4 weeks, diabetic for 8 weeks, and diabetic for 12 weeks groups. Quantitative data are shown in the right panel. (n=3) ( E ) Total RNA was extracted from retinal tissues of control, diabetic for 4 weeks, diabetic for 8 weeks, and diabetic for 12 weeks groups, and TRAP1 mRNA expression was detected by qRT-PCR. (n=3) ( F ) Immunofluorescence staining of retinal tissue sections from each group of rats using anti-TRAP1 antibody (yellow) and DAPI (blue) for nuclear staining. The error bars in the above histograms represent the mean±SD of independent experiments.*P<0.05, **P<0.01, ***P<0.001,****P<0.0001.

Article Snippet: Additionally, TRAP1 Polyclonal antibody (10325-1-AP), Beta Actin Polyclonal antibody (20536-1-AP), COXIV Polyclonal antibody (11242-1-AP), Tom20 Polyclonal antibody (11802-1-AP), HRP-conjugated Goat Anti-Rabbit IgG(H+L) (SA00001-2), CoraLite594-conjugated Goat Anti-Rabbit IgG(H+L) (SA00013-4), and CoraLite488-conjugated Goat Anti-Mouse IgG(H+L) (SA00013-1) are sourced from Proteintech (Wuhan, China).

Techniques: Control, Staining, Western Blot, Expressing, Quantitative RT-PCR, Immunofluorescence

Journal: Clinical Ophthalmology (Auckland, N.Z.)

Article Title: TRAP1 Improves Diabetic Retinopathy by Preserving Mitochondrial Function

doi: 10.2147/OPTH.S521660

Figure Lengend Snippet: HG promotes mitochondrial dysfunction in ARPE-19 cells, accompanied by a decrease in TRAP1 levels. ( A ) Cell viability of ARPE-19 cells cultured with different glucose concentrations for 4 days. (n=4) ( B ) Cell viability of ARPE-19 cells cultured with 50mM glucose for 1–6 days. (n=4) ( C ) Intracellular ROS levels in cells cultured with 50mM glucose for 1–6 days. (n=3) ( D ) Identification of Δψ m through JC-1 staining after culturing ARPE-19 cells with 50mM glucose for 6 days. Images captured under fluorescence microscope show red fluorescence representing polymer form, indicating intact Δψ m, and green fluorescence representing monomer form, indicating decreased Δψ m. Quantified data presented on the right. Scale bar=20μm. (n=3) ( E ) ARPE-19 cells cultured with 50mM glucose for 6 days and treated with Calcein AM (1X) and CoCl2 (1X). Images captured under fluorescence microscope shown on the left. Quantified data presented on the right. Scale bar=20μm. (n=3) ( F ) TEM images of mitochondrial ultrastructure in ARPE-19 cells cultured with 50mM glucose for 6 days and control ARPE-19 cells. Quantified data presented on the right. Scale bar=2μm. ( G ) Western blot analysis of TRAP1 in ARPE-19 cells cultured with 50mM glucose for 1–6 days. (n=3) ( H ) Western blot analysis of intramitochondrial TRAP1 in ARPE-19 cells cultured with 50mM glucose for 1–6 days after mitochondrial extraction. (n=3) ( I ) Quantified data corresponding to Figure ( G ). ( J ) qRT-PCR analysis of TRAP1 mRNA expression in ARPE-19 cells cultured with 50mM glucose for 1–6 days. (n=3) ( K ) Quantified data corresponding to Figure ( H ). The error bars in the above histograms represent the mean±SD of independent experiments. ns P>0.05, *P<0.05, **P<0.01, ***P<0.001,****P<0.0001.

Article Snippet: Additionally, TRAP1 Polyclonal antibody (10325-1-AP), Beta Actin Polyclonal antibody (20536-1-AP), COXIV Polyclonal antibody (11242-1-AP), Tom20 Polyclonal antibody (11802-1-AP), HRP-conjugated Goat Anti-Rabbit IgG(H+L) (SA00001-2), CoraLite594-conjugated Goat Anti-Rabbit IgG(H+L) (SA00013-4), and CoraLite488-conjugated Goat Anti-Mouse IgG(H+L) (SA00013-1) are sourced from Proteintech (Wuhan, China).

Techniques: Cell Culture, Staining, Fluorescence, Microscopy, Polymer, Control, Western Blot, Extraction, Quantitative RT-PCR, Expressing

Journal: Clinical Ophthalmology (Auckland, N.Z.)

Article Title: TRAP1 Improves Diabetic Retinopathy by Preserving Mitochondrial Function

doi: 10.2147/OPTH.S521660

Figure Lengend Snippet: TRAP1 rescues mitochondrial damage under high-glucose stimulation. ( A ) The left panel depicts Western blot analysis of TRAP1 overexpression and knockdown cell lines. The right panel shows the quantified data. (n=3) ( B ) Total RNA was extracted from ARPE-19 cells overexpressing or knockdown for TRAP1, followed by qRT-PCR to assess TRAP1 mRNA expression. (n=3) ( C ) Immunofluorescence staining of TRAP1 (red) and Tom20 (green) demonstrates their colocalization in TRAP1 OE and shTRAP1 cells. Cell nuclei were stained with DAPI (blue). Scale bar = 20μm. (n=3) ( D )The upper panel illustrates cell viability of NC group, cells transduced with overexpression empty virus, and cells transduced with knockdown empty virus cultured for 6 days under 50mM glucose and control conditions. The lower panel represents cell viability of NC, TRAP1 OE , and shTRAP1 cells cultured for 6 days under 50mM glucose and control conditions. (n=4) ( E ) Intracellular ROS levels were measured after 6 days of incubation with 50mM glucose. (n=3) ( F ) Cells cultured with 50mM glucose for 6 days were subjected to JC-1 staining to assess Δψ m. Fluorescence microscopy images are shown. Scale bar = 20μm. (n=3) ( G ) Quantified data from panel ( F ) are presented. ( H ) Cells cultured with 50mM glucose for 6 days were treated with Calcein AM (1X) and CoCl2 (1x). Fluorescence microscopy images are shown. Scale bar = 20μm. (n=3) ( I ) Quantified data from panel ( H ) are presented. ( J ) TEM was performed to evaluate mitochondrial ultrastructure after 6 days of incubation with 50mM glucose. Scale bar = 2μm. ( K ) Quantified data from panel ( J ) are presented. The error bars in the above histograms represent the mean±SD of independent experiments. ns P>0.05, *P<0.05, **P<0.01, ***P<0.001,****P<0.0001.

Article Snippet: Additionally, TRAP1 Polyclonal antibody (10325-1-AP), Beta Actin Polyclonal antibody (20536-1-AP), COXIV Polyclonal antibody (11242-1-AP), Tom20 Polyclonal antibody (11802-1-AP), HRP-conjugated Goat Anti-Rabbit IgG(H+L) (SA00001-2), CoraLite594-conjugated Goat Anti-Rabbit IgG(H+L) (SA00013-4), and CoraLite488-conjugated Goat Anti-Mouse IgG(H+L) (SA00013-1) are sourced from Proteintech (Wuhan, China).

Techniques: Western Blot, Over Expression, Knockdown, Quantitative RT-PCR, Expressing, Immunofluorescence, Staining, Transduction, Virus, Cell Culture, Control, Incubation, Fluorescence, Microscopy

Journal: Clinical Ophthalmology (Auckland, N.Z.)

Article Title: TRAP1 Improves Diabetic Retinopathy by Preserving Mitochondrial Function

doi: 10.2147/OPTH.S521660

Figure Lengend Snippet: Knocking down TRAP1 affects oxidative stress and related mitochondrial functions in ARPE-19 cells. ( A ) Volcano plot of differentially expressed genes (DEGs) between shTRAP1 and NC, with yellow representing significantly upregulated genes and blue representing significantly downregulated genes (|log2 fold change|>1, log10 adjusted p-values<0.05). ( B ) Heatmap of DEGs between shTRAP1 and NC. ( C ) Venn diagram showing a high overlap between DEGs in the shTRAP1 group and oxidative stress-related genes (OS) and diabetic retinopathy-related risk genes (DR). ( D ) Gene Ontology (GO) enrichment analysis of DEGs. ( E ) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs.

Article Snippet: Additionally, TRAP1 Polyclonal antibody (10325-1-AP), Beta Actin Polyclonal antibody (20536-1-AP), COXIV Polyclonal antibody (11242-1-AP), Tom20 Polyclonal antibody (11802-1-AP), HRP-conjugated Goat Anti-Rabbit IgG(H+L) (SA00001-2), CoraLite594-conjugated Goat Anti-Rabbit IgG(H+L) (SA00013-4), and CoraLite488-conjugated Goat Anti-Mouse IgG(H+L) (SA00013-1) are sourced from Proteintech (Wuhan, China).

Techniques:

Journal: Clinical Ophthalmology (Auckland, N.Z.)

Article Title: TRAP1 Improves Diabetic Retinopathy by Preserving Mitochondrial Function

doi: 10.2147/OPTH.S521660

Figure Lengend Snippet: TRAP1 alleviates high glucose-induced cellular damage by inhibiting mitochondrial ferroptosis.( A ) Activity of shTRAP1 cells treated with 50 mM glucose for 6 days or co-treated with Fer-1, Z-VAD, 3-MA, and Nec-1. (n=3) ( B ) Activity of NC, TRAP1 OE , and shTRAP1 cells treated with Erastin for 6 days or co-treated for 6 days. (n=4) ( C ) Intracellular MDA concentration of NC, TRAP1 OE , and shTRAP1 cells treated with Erastin for 6 days or co-treated for 6 days. (n=3) ( D ) Changes in intracellular Fe 2+ concentration of NC, TRAP1 OE , and shTRAP1 cells treated with Erastin for 6 days or co-treated for 6 days. (n=3) ( E ) Ratio of GSH to GSSG in NC, TRAP1 OE , and shTRAP1 cells treated with Erastin for 6 days or co-treated for 6 days. (n=3) ( F ) Left panel: Fluorescence microscopy images showing the content of lipid ROS in NC, TRAP1 OE , and shTRAP1 cells treated with Erastin for 6 days or co-treated for 6 days. R-BODIPY: Reduced BODIPY(red). O-BODIPY: Oxidized BODIPY(green). Right panel: Quantitative data. Scale bar=20μm. (n=3) ( G ) Activity of NC, TRAP1 OE , and shTRAP1 cells treated with 50 mM glucose for 6 days or co-treated with Fer-1. (n=4) ( H ) Intracellular MDA concentration of NC, TRAP1 OE , and shTRAP1 cells treated with 50 mM glucose for 6 days or co-treated with Fer-1. (n=3) ( I ) Changes in intracellular Fe 2+ concentration of NC, TRAP1 OE , and shTRAP1 cells treated with 50 mM glucose for 6 days or co-treated with Fer-1. (n=3) ( J ) Ratio of GSH to GSSG in NC, TRAP1 OE , and shTRAP1 cells treated with 50 mM glucose for 6 days or co-treated with Fer-1. (n=3) ( K ) Left panel: Fluorescence microscopy images showing the content of lipid ROS in NC, TRAP1 OE , and shTRAP1 cells treated with 50 mM glucose for 6 days or co-treated with Fer-1. Right panel: Quantitative data. Scale bar= 20μm. (n=3) ( L ) In Control, OE, and OE+HG groups of ARPE19 cells, immunoprecipitation (IP) was performed using TRAP1 antibody, and the enriched proteins were analyzed by Western Blot with antibodies against ACSL1, ACSL4, and CYB5R1. The error bars in the above histograms represent the mean±SD of independent experiments. ns P>0.05, *P<0.05, **P<0.01, ***P<0.001,****P<0.0001.

Article Snippet: Additionally, TRAP1 Polyclonal antibody (10325-1-AP), Beta Actin Polyclonal antibody (20536-1-AP), COXIV Polyclonal antibody (11242-1-AP), Tom20 Polyclonal antibody (11802-1-AP), HRP-conjugated Goat Anti-Rabbit IgG(H+L) (SA00001-2), CoraLite594-conjugated Goat Anti-Rabbit IgG(H+L) (SA00013-4), and CoraLite488-conjugated Goat Anti-Mouse IgG(H+L) (SA00013-1) are sourced from Proteintech (Wuhan, China).

Techniques: Activity Assay, Concentration Assay, Fluorescence, Microscopy, Control, Immunoprecipitation, Western Blot

Journal: Cell death & disease

Article Title: Point mutations of the mitochondrial chaperone TRAP1 affect its functions and pro-neoplastic activity.

doi: 10.1038/s41419-025-07467-6

Figure Lengend Snippet: 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.

Article Snippet: Primary antibodies were incubated for 16 h at 4 °C (TRAP1 Santa Cruz #sc-73604, Citrate Synthase Abcam #ab96600).

Techniques: High Throughput Screening Assay, Functional Assay, Phospho-proteomics

Journal: Cell death & disease

Article Title: Point mutations of the mitochondrial chaperone TRAP1 affect its functions and pro-neoplastic activity.

doi: 10.1038/s41419-025-07467-6

Figure Lengend Snippet: 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 (P600A, P600B). 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.

Article Snippet: Primary antibodies were incubated for 16 h at 4 °C (TRAP1 Santa Cruz #sc-73604, Citrate Synthase Abcam #ab96600).

Techniques: Sequencing, Residue, Mutagenesis, Western Blot, Expressing

Journal: Cell death & disease

Article Title: Point mutations of the mitochondrial chaperone TRAP1 affect its functions and pro-neoplastic activity.

doi: 10.1038/s41419-025-07467-6

Figure Lengend Snippet: 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 (PmutA, PmutB). 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 (Δηmut). 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.

Article Snippet: Primary antibodies were incubated for 16 h at 4 °C (TRAP1 Santa Cruz #sc-73604, Citrate Synthase Abcam #ab96600).

Techniques: Activity Assay, Recombinant, Mutagenesis, Residue, Labeling, Sequencing

Journal: Cell death & disease

Article Title: Point mutations of the mitochondrial chaperone TRAP1 affect its functions and pro-neoplastic activity.

doi: 10.1038/s41419-025-07467-6

Figure Lengend Snippet: 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).

Article Snippet: Primary antibodies were incubated for 16 h at 4 °C (TRAP1 Santa Cruz #sc-73604, Citrate Synthase Abcam #ab96600).

Techniques: Activity Assay, Expressing, Mutagenesis, Immunoprecipitation, Residue

Journal: Cell death & disease

Article Title: Point mutations of the mitochondrial chaperone TRAP1 affect its functions and pro-neoplastic activity.

doi: 10.1038/s41419-025-07467-6

Figure Lengend Snippet: 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.

Article Snippet: Primary antibodies were incubated for 16 h at 4 °C (TRAP1 Santa Cruz #sc-73604, Citrate Synthase Abcam #ab96600).

Techniques: Activity Assay, Focus Forming Assay, Expressing, Invasion Assay, Mutagenesis, Software