Tumor Necrosis Factor Receptor-associated Protein Research Articles

Development of a Fluorescence Probe for High-Throughput Screening of Allosteric Inhibitors Targeting TRAP1.

Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a molecular chaperone implicated in pro-tumorigenic pathways by regulating the folding of substrate proteins (clients) within cancer cells. Recent research has pinpointed a potentially druggable allosteric site within the client binding site (CBS) of TRAP1, suggesting this site might offer a more effective strategy for developing potent and selective TRAP1 inhibitors. However, the absence of reliable assay systems has hindered quantitative evaluation of inhibitors. In this study, we have developed a fluorescent probe, Rho6TPP, designed to target the CBS. Utilizing fluorescence polarization-based high-throughput screening assays, Rho6TPP exhibits excellent signal-to-noise ratio (>20), Z factor (>0.6), and Z' factor (>0.6). Additionally, it facilitates comparative analysis of existing small molecules and discovery of novel binders. MitoTam, a mitochondria-targeted tamoxifen, emerges as a potent CBS-targeting TRAP1 inhibitor. Our findings highlight the potential of Rho6TPP as a crucial tool for advancing the development of CBS-targeting TRAP1 inhibitors.

The role of TRAP1 in regulating mitochondrial dynamics during acute hypoxia-induced brain injury

Brain damage caused by acute hypoxia is associated with the physiological activities of mitochondria. Although mitochondria being dynamically regulated, our comprehensive understanding of the response of specific brain cell types to acute hypoxia remains ambiguous. Tumor necrosis factor receptor-associated protein 1 (TRAP1), a mitochondrial-based molecular chaperone, plays a role in controlling mitochondrial movements. Herein, we demonstrated that acute hypoxia significantly alters mitochondria morphology and functionality in both in vivo and in vitro brain injury experiments. Summary-data-based Mendelian Randomization (SMR) analyses revealed possible causative links between mitochondria-related genes and hypoxia injury. Advancing the protein-protein interaction network and molecular docking further elucidated the associations between TRAP1 and mitochondrial dynamics. Furthermore, it was shown that TRAP1 knockdown levels variably affected the expression of key mitochondrial dynamics proteins (DRP1, FIS1, and MFN1/2) in primary hippocampal neurons, astrocytes, and BV-2 cell, leading to changes in mitochondrial structure and function. Understanding the function of TRAP1 in altering mitochondrial physiological activity during hypoxia-induced acute brain injury could help serve as a potential therapeutic target to mitigate neurological damage.

Genome-wide identification of the TRAF gene family in humpback grouper (Cromileptes altivelis) and analysis of their expression in response to Vibrio harveyi challenge

TRAF (Tumor necrosis factor receptor-associated factor) proteins are key mediators of signal transduction in cell signaling and immune regulation within the toll-like receptor (TLR) and tumor necrosis factor (TNFR) superfamily. Despite the importance of TRAF genes in teleost innate immunity, study on their functions in C. altivelis is limited. This study utilized bioinformatics methods to identify and named eight TRAF genes (CaTRAF2a, CaTRAF2a-like, CaTRAF2b, CaTRAF3, CaTRAF4a, CaTRAF5, CaTRAF6 and CaTRAF7) in C. altivelis. Phylogenetic, syntenic and molecular evolution revealed that all CaTRAF members were evolutionarily conserved in teleost. Domain analysis indicated the presence of a conserved N-terminal RING finger domain in all CaTRAF proteins. Most CaTRAF proteins also featured a MATH domain at the C-terminal, with the exception of CaTRAF7 which contained seven repeat WD40 domains. In addition, qRT-PCR was used to detect the expression patterns of nine different tissues and eight different embryonic development stages of healthy fish, and it was found that there were spatial and tissue specificities among the members. HE staining revealed evident pathological lesions in the tissues post V. harveyi infection. Atrophy and significant bending of the gill lamellae were observed in the gills, while irregular cell shapes, increased fat vacuoles, and enlarged cell volume were noted in the liver. Intestinal tissues displayed thickening of the muscle layer, elongation of intestinal villi, and increased folds. Moreover, the expression of TRAF gene changed significantly after V. harveyi infection. These results would help to clarify the molecular role of CaTRAF gene in the regulation of immune and inflammatory responses in C. altivelis.

TRAP1 drives smooth muscle cell senescence and promotes atherosclerosis via HDAC3-primed histone H4 lysine 12 lactylation.

Vascular smooth muscle cell (VSMC) senescence is crucial for the development of atherosclerosis, characterized by metabolic abnormalities. Tumour necrosis factor receptor-associated protein 1 (TRAP1), a metabolic regulator associated with ageing, might be implicated in atherosclerosis. As the role of TRAP1 in atherosclerosis remains elusive, this study aimed to examine the function of TRAP1 in VSMC senescence and atherosclerosis. TRAP1 expression was measured in the aortic tissues of patients and mice with atherosclerosis using western blot and RT-qPCR. Senescent VSMC models were established by oncogenic Ras, and cellular senescence was evaluated by measuring senescence-associated β-galactosidase expression and other senescence markers. Chromatin immunoprecipitation (ChIP) analysis was performed to explore the potential role of TRAP1 in atherosclerosis. VSMC-specific TRAP1 deficiency mitigated VSMC senescence and atherosclerosis via metabolic reprogramming. Mechanistically, TRAP1 significantly increased aerobic glycolysis, leading to elevated lactate production. Accumulated lactate promoted histone H4 lysine 12 lactylation (H4K12la) by down-regulating the unique histone lysine delactylase HDAC3. H4K12la was enriched in the senescence-associated secretory phenotype (SASP) promoter, activating SASP transcription and exacerbating VSMC senescence. In VSMC-specific Trap1 knockout ApoeKO mice (ApoeKOTrap1SMCKO), the plaque area, senescence markers, H4K12la, and SASP were reduced. Additionally, pharmacological inhibition and proteolysis-targeting chimera (PROTAC)-mediated TRAP1 degradation effectively attenuated atherosclerosis in vivo. This study reveals a novel mechanism by which mitonuclear communication orchestrates gene expression in VSMC senescence and atherosclerosis. TRAP1-mediated metabolic reprogramming increases lactate-dependent H4K12la via HDAC3, promoting SASP expression and offering a new therapeutic direction for VSMC senescence and atherosclerosis.

Glutamine deprivation in glioblastoma stem cells triggers autophagic SIRT3 degradation to epigenetically restrict CD133 expression and stemness.

Glioblastoma multiforme (GBM) is a highly malignant brain tumor, and glioblastoma stem cells (GSCs) are the primary cause of GBM heterogeneity, invasiveness, and resistance to therapy. Sirtuin 3 (SIRT3) is mainly localized in the mitochondrial matrix and plays an important role in maintaining GSC stemness through cooperative interaction with the chaperone protein tumor necrosis factor receptor-associated protein 1 (TRAP1) to modulate mitochondrial respiration and oxidative stress. The present study aimed to further elucidate the specific mechanisms by which SIRT3 influences GSC stemness, including whether SIRT3 serves as an autophagy substrate and the mechanism of SIRT3 degradation. We first found that SIRT3 is enriched in CD133+ GSCs. Further experiments revealed that in addition to promoting mitochondrial respiration and reducing oxidative stress, SIRT3 maintains GSC stemness by epigenetically regulating CD133 expression via succinate. More importantly, we found that SIRT3 is degraded through the autophagy-lysosome pathway during GSC differentiation into GBM bulk tumor cells. GSCs are highly dependent on glutamine for survival, and in these cells, we found that glutamine deprivation triggers autophagic SIRT3 degradation to restrict CD133 expression, thereby disrupting the stemness of GSCs. Together our results reveal a novel mechanism by which SIRT3 regulates GSC stemness. We propose that glutamine restriction to trigger autophagic SIRT3 degradation offers a strategy to eliminate GSCs, which combined with other treatment methods may overcome GBM resistance to therapy as well as relapse.

Identification of Six Pathogenic Genes for Tibetan Familial Ventricular Septal Defect by Whole Exome Sequencing

IntroductionVentricular septal defect (VSD) is the most common congenital heart malformation in children. This study aimed to investigate potential pathogenic genes associated with Tibetan familial VSD. MethodsWhole genomic DNA was extracted from eight Tibetan children with VSD and their healthy parents (a total of 16 individuals). Whole-exome sequencing was performed using the Illumina HiSeq platform. After filtration, detection, and annotation, single nucleotide variations and insertion-deletion markers were examined. Comparative evaluations using the Sorting Intolerant from Tolerant, PolyPhen V2, Mutation Taster, and Combined Annotation Dependent Depletion databases were conducted to predict harmful mutant genes associated with the etiology of Tibetan familial VSD. ResultsA total of six missense mutations in genetic disease-causing genes associated with the development of Tibetan familial VSD were identified: activin A receptor type II-like 1 (c.652 C > T: p.R218 W), ATPase cation transporting 13A2 (c.1363 C > T: p.R455 W), endoplasmic reticulum aminopeptidase 1 (c.481 G > A: p.G161 R), MRI1 (c.629 G > A: p.R210Q), tumor necrosis factor receptor-associated protein 1 (c.224 G > A: p.R75H), and FBN2 (c.2260 G > A: p.G754S). The Human Gene Mutation Database confirmed activin A receptor type II-like 1, MRI1, and tumor necrosis factor receptor-associated protein 1 as pathogenic mutations, while FBN2 was classified as a probable pathogenic mutation. ConclusionsThis novel study directly screens genetic variations associated with Tibetan familial VSD using whole-exome sequencing, providing new insights into the pathogenesis of VSD.

Tumour Necrosis Factor Receptor-Associated Protein 1 (TRAP1): A Pivotal Regulator of Smooth Muscle Cell Senescence and Atherosclerosis through Metabolic Reprogramming

Tumour Necrosis Factor Receptor-Associated Protein 1 (TRAP1): A Pivotal Regulator of Smooth Muscle Cell Senescence and Atherosclerosis through Metabolic Reprogramming

The development of cancers research based on mitochondrial heat shock protein 90

Mitochondrial heat shock protein 90 (mtHsp90), including Tumor necrosis factor receptor-associated protein 1 (TRAP1) and Hsp90 translocated from cytoplasm, modulating cellular metabolism and signaling pathways by altering the conformation, activity, and stability of numerous client proteins, and is highly expressed in tumors. mtHsp90 inhibition results in the destabilization and eventual degradation of its client proteins, leading to interference with various tumor-related pathways and efficient control of cancer cell development. Among these compounds, gamitrinib, a specific mtHsp90 inhibitor, has demonstrated its safety and efficacy in several preclinical investigations and is currently undergoing evaluation in clinical trials. This review aims to provide a comprehensive overview of the present knowledge pertaining to mtHsp90, encompassing its structure and function. Moreover, our main emphasis is on the development of mtHsp90 inhibitors for various cancer therapies, to present a thorough overview of the recent pre-clinical and clinical advancements in this field.

Targeting the Mitochondrial Chaperone TRAP1 Alleviates Vascular Pathologies in Ischemic Retinopathy.

Activation of hypoxia-inducible factor 1α (HIF1α) contributes to blood-retinal barrier (BRB) breakdown and pathological neovascularization responsible for vision loss in ischemic retinal diseases. During disease progression, mitochondrial biology is altered to adapt to the ischemic environment created by initial vascular dysfunction, but the mitochondrial adaptive mechanisms, which ultimately contribute to the pathogenesis of ischemic retinopathy, remain incompletely understood. In the present study, it is identified that expression of mitochondrial chaperone tumor necrosis factor receptor-associated protein 1 (TRAP1) is essential for BRB breakdown and pathologic retinal neovascularization in mouse models mimicking ischemic retinopathies. Genetic Trap1 ablation or treatment with small molecule TRAP1 inhibitors, such as mitoquinone (MitoQ) and SB-U015, alleviate retinal pathologies via proteolytic HIF1α degradation, which is mediated by opening of the mitochondrial permeability transition pore and activation of calcium-dependent protease calpain-1. These findings suggest that TRAP1 can be a promising target for the development of new treatments against ischemic retinopathy, such as retinopathy of prematurity and proliferative diabetic retinopathy.

Inhibition of TRAP1 Accelerates the DNA Damage Response, Activation of the Heat Shock Response and Metabolic Reprogramming in Colon Cancer Cells.

Colorectal cancer (CRC) is one of the major causes of cancer-related mortality worldwide. The tumor microenvironment plays a significant role in CRC development, progression and metastasis. Oxidative stress in the colon is a major etiological factor impacting tumor progression. Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a mitochondrial member of the heat shock protein 90 (HSP90) family that is involved in modulating apoptosis in colon cancer cells under oxidative stress. We undertook this study to provide mechanistic insight into the role of TRAP1 under oxidative stress in colon cells. We first assessed the The Cancer Genome Atlas (TCGA) CRC gene expression dataset to evaluate the expression of TRAP1 and its association with oxidative stress and disease progression. We then treated colon HCT116 cells with hydrogen peroxide to induce oxidative stress and with the TRAP1 inhibitor gamitrinib-triphenylphosphonium (GTPP) to inhibit TRAP1. We examined the cellular proteomic landscape using liquid chromatography tandem mass spectrometry (LC-MS/MS) in this context compared to controls. We further examined the impact of treatment on DNA damage and cell survival. TRAP1 expression under oxidative stress is associated with the disease outcomes of colorectal cancer. TRAP1 inhibition under oxidative stress induced metabolic reprogramming and heat shock factor 1 (HSF1)-dependent transactivation. In addition, we also observed enhanced induction of DNA damage and cell death in the cells under oxidative stress and TRAP1 inhibition in comparison to single treatments and the nontreatment control. These findings provide new insights into TRAP1-driven metabolic reprogramming in response to oxidative stress.

Head or tail? A molecular dynamics approach to the complex structure of TNF-associated factor TRAF2.

Tumor necrosis factor receptor-associated factor proteins (TRAFs) are trimeric proteins that play a fundamental role in signaling, acting as intermediaries between the tumor necrosis factor (TNF) receptors and the proteins that transmit the downstream signal. The monomeric subunits of all the TRAF family members share a common tridimensional structure: a C-terminal globular domain and a long coiled-coil tail characterizing the N-terminal section. In this study, the dependence of the TRAF2 dynamics on the length of its tail was analyzed in silico. In particular, we used the available crystallographic structure of a C-terminal fragment of TRAF2 (168 out of 501 a.a.), TRAF2-C, and that of a longer construct, addressed as TRAF2-plus, that we have re-constructed using the AlphaFold2 code. The results indicate that the longer N-terminal tail of TRAF2-plus has a strong influence on the dynamics of the globular regions in the protein C-terminal head. In fact, the quaternary interactions among the TRAF2-C subunits change asymmetrically in time, while the movements of TRAF2-plus monomers are rather limited and more ordered than those of the shorter construct. Such findings shed a new light on the dynamics of TRAF subunits and on the protein mechanism in vivo, since TRAF monomer-trimer equilibrium is crucial for several reasons (receptor recognition, membrane binding, hetero-oligomerization).

The mitochondrial chaperone TRAP-1 regulates the glutamine metabolism in tumor cells

Understanding cancer cell metabolism always provides information on hidden dimensions of tumor adaptations. Warburg's theory that cancer cells opt for aerobic glycolysis over the mitochondrial oxidative phosphorylation (OXPHOS) system is widely accepted. However, the hypothesis does not explain the mitochondrion's role in these cells. Here, we demonstrate that intact mitochondria are used for anaplerotic functions and ATP production by utilizing glutamine with the help of mitochondrial chaperone TRAP-1 (Tumor Necrosis Factor Receptor-associated Protein 1). TRAP-1 otherwise promotes aerobic glycolysis by lowering the mitochondrial OXPHOS in the presence of glucose. Here, we show that TRAP-1 maintains mitochondrial integrity and augments glutamine metabolism upon glucose deprivation to meet the cellular energy demand. The enhanced PER and ECAR correlating with increased ATP production suggest that glutamine fuels mitochondria in the presence of TRAP-1. We also found that TRAP1-dependent glutamine utilization involves the HIF2α-SLC1A5-GLS axis and is independent of hypoxia. Subsequently, we show that the metastatic potential of tumor cells is linked with glucose utilization, whereas the proliferative potential is linked with both glucose and glutamine utilization. Our findings establish that TRAP-1 contributes to enhanced glutamine utilization through the HIF2α-SLC1A5-GLS axis. Our results endow that TRAP-1 inhibitors can be potential drug candidates to combat tumor metabolism. Therefore, their use, either alone or in combination with existing chemotherapeutic agents, may target tumor metabolism and improve anticancer treatment response.

TRAP1 Is Expressed in Human Retinal Pigment Epithelial Cells and Is Required to Maintain their Energetic Status.

Age-related macular degeneration (AMD) is the leading cause of severe vision loss and blindness in elderly people worldwide. The damage to the retinal pigment epithelium (RPE) triggered by oxidative stress plays a central role in the onset and progression of AMD and results from the excessive accumulation of reactive oxygen species (ROS) produced mainly by mitochondria. Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a mitochondrial molecular chaperone that contributes to the maintenance of mitochondrial integrity by decreasing the production and accumulation of ROS. The present study aimed to evaluate the presence and the role of TRAP1 in the RPE. Here, we report that TRAP1 is expressed in human adult retinal pigment epithelial cells and is located mainly in the mitochondria. Exposure of RPE cells to hydrogen peroxide decreases the levels of TRAP1. Furthermore, TRAP1 silencing increases intracellular ROS production and decreases mitochondrial respiratory capacity without affecting cell proliferation. Together, these findings offer novel insights into TRAP1 functions in RPE cells, opening possibilities to develop new treatment options for AMD.

The value of serum TRAP1 in diagnosing small cell lung cancer and tumor immunity.

This study set out to investigate the clinical significance of serum tumor necrosis factor receptor-associated protein 1 (TRAP1) in diagnosing small cell lung cancer (SCLC) with different clinical stages, and to compare the diagnostic efficiency with neuron-specific enolase (NSE), carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9). Besides, to analyze the role of serum TRAP1 in tumor immunity. A total of 91 patients with SCLC, 99 patients with non-small cell lung cancer (NSCLC), 102 patients with pulmonary nodules (PN), and 75 healthy people were included. The concentrations of serum TRAP1 was detected by enzyme-linked immunosorbent assay (ELISA). NSE, CEA, and CA19-9 were detected by chemiluminescence. The results showed that level of TRAP1 in Group SCLC was lower than other three groups (P < 0.01), whereas NSE in SCLC was significantly higher than the others (P < 0.01), and the levels of CEA and CA19-9 were higher than healthy people and PN patients (P < 0.01). There was a significant difference in TRAP1 levels between patients with limited-stage disease SCLC (LD-SCLC) and extensive-stage disease SCLC (ED-SCLC) (P < 0.0001). The sensitivity and specificity of TRAP1 in diagnosing LD-SCLC were 0.964 and 0.560, respectively, and the area under the curve (AUC) was 0.819. The sensitivity and specificity in diagnosing ED-SCLC were 0.810 and 0.868, respectively, and the AUC was 0.933, which showed high diagnostic value. The AUC of these two groups can be increased to 0.946 and 0.947 in combination of four biomarkers, effectively improving the diagnosis rate of SCLC. Our findings have revealed that serum TRAP1 has high diagnostic value for SCLC and high diagnostic sensitivity for LD-SCLC. It is a potential biomarker for SCLC. Combined detection can effectively improve the diagnosis rate of SCLC. TRAP1 may be secreted into the circulation by mature immune cells and participates in tumor immunity as a carrier of tumor antigens.

The association of TRAP1 gene and TNFSF4 gene polymorphisms with susceptibility of rheumatoid arthritis in a sample of Egyptian patients

Background/aim The disclosure of new gene polymorphisms and their association with rheumatoid arthritis (RA) susceptibility open new windows for better clarification of disease pathogenesis, leading to discovering new therapeutic targets. The present study aimed to explore the association of tumor necrosis factor receptor-associated protein 1 (TRAP1) gene rs8055172 and tumor necrosis factor superfamily number 4 (TNFSF4) gene rs1234315 with susceptibility of RA in a sample of Egyptian patients. Patients and methods This study included 200 RA patients from the Rheumatology Department Outpatients’ Clinic of Kasr El Ainy Teaching Hospital and Centre of Medical Excellence of National Research Centre, Cairo, Egypt. The study also included 200 healthy participants with no family history of autoimmunity serving as a control group. Genotyping of the studied polymorphisms was done using real-time PCR technique. Results The control group showed no significant deviations from Hardy–Weinberg equilibrium regarding rs8055172 and rs1234315 (P=0.6 and 0.2, respectively). Regarding genotypes of rs8055172, the CC homozygous genotype was more observed among patients. Therefore, the frequency of C allele is higher among RA patients compared with healthy controls (P=0.001). Logistic regression analysis of rs8055172 genotypes with susceptibility of RA was only significant under the recessive model, where patients carrying CC allele have higher susceptibility to develop RA (P=0.001, odds ratio=3.1) compared with patients carrying TT and CT allele. On the other hand, distribution of TNFSF4 (rs1234315) genotypes showed no significant difference between controls and RA group (P=0.7). Conclusions Our results indicate that the TRAP1 gene rs8055172 associates with RA in a population of Egyptians from Cairo, while TNFSF4 gene rs1234315 plays no role in disease susceptibility. A large-scale study to assess the association between TRAP1 gene polymorphism, TRAP1 mRNA expression, and TRAP1 protein level, is needed to clarify the role of TRAP1 gene polymorphism in RA pathogenesis.

Structure, Function, and Inhibitors of the Mitochondrial Chaperone TRAP1.

Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a mitochondrial molecular chaperone modulating cellular metabolism and signaling pathways by altering the conformation, activity, and stability of numerous substrate proteins called clients. It exerts its chaperone function as an adaptive response to counter cellular stresses instead of maintaining housekeeping protein homeostasis. However, the stress-adaptive machinery becomes dysregulated to support the progression and maintenance of human diseases, such as cancers; therefore, TRAP1 has been proposed as a promising target protein for anticancer drug development. In this review, by collating recent reports on high-resolution TRAP1 structures and structure-activity relationships of inhibitors, we aimed to provide better insights into the chaperoning mechanism of the emerging drug target and to suggest an efficient strategy for the development of potent TRAP1 inhibitors.

An association study on the risk, glucocorticoids effectiveness, and prognosis of systemic lupus erythematosus: insight from mitochondrial DNA copy number.

This study aimed to explore the role of mitochondrial DNA copy number (mtDNAcn) in the risk, glucocorticoid (GC) effectiveness, and prognosis of systemic lupus erythematosus (SLE) and its interactions with environmental factors and tumor necrosis factor receptor-associated protein 1 (TRAP1) genetic polymorphisms. We first conducted a case-control study of 1198 subjects (595 SLE patients and 603 healthy controls). Subsequently, we followed up with patients to assess the effectiveness of GC treatment and the prognosis of SLE. Real-time fluorescent quantitative PCR (qPCR) was used to quantify mtDNAcn. Associations were estimated using logistic regression, and prognosis analysis was performed using Kaplan-Meier analysis and Cox proportional hazards models. Interactions on multiplicative and additive scales were also evaluated. Individuals with low mtDNAcn had an increased risk of SLE (P &lt; 0.001). Low mtDNAcn was associated with poor GC effectiveness in patients with spicy food consumption or with arthritis (P &lt; 0.05). mtDNAcn was significantly related to the prognosis of SLE in the drinking subgroup (P = 0.018). Furthermore, we found significant interactions between mtDNAcn and environmental factors/TRAP1 genetic polymorphisms on the risk, GC effectiveness, and prognosis of SLE. Our data suggest that low mtDNAcn is associated with an increased risk of SLE. Alteration of mtDNAcn may be associated with GC effectiveness and prognosis in certain subgroups of SLE. The interactions between mtDNAcn, environmental factors, and TRAP1 gene polymorphisms may jointly affect the risk, GC effectiveness, and prognosis of SLE.

Metabolic targeting of NRF2 potentiates the efficacy of the TRAP1 inhibitor G-TPP through reduction of ROS detoxification in colorectal cancer

Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a mitochondrial homolog of HSP90 chaperones. It plays an important role in protection against oxidative stress and apoptosis by regulating reactive oxidative species (ROS). To further elucidate the mechanistic role of TRAP1 in regulating tumor cell survival, we used gamitrinib-triphenylphosphonium (G-TPP) to inhibit TRAP1 signaling pathways in colon cancer. Inhibition of TRAP1 by G-TPP disrupted redox homeostasis and induced cell death. However, colon cancers show a wide range of responses to G-TPP treatment through the induction of variable ER stress responses and ROS accumulation. Interestingly, a strong inverse correlation was observed between the expression of TRAP1 and antioxidant genes in colon tumor tissues using the GSE106582 database. Using a luciferase reporter assay, we detected increased transcriptional activation of antioxidant response elements (AREs) in G-TPP-treated DLD1 and RKO cells but not in SW48 cells. We found that G-TPP induced upregulation of GRP78, CHOP and PARP cleavage in G-TPP-sensitive cells (SW48). In contrast, G-TPP treatment of G-TPP-resistant cells (DLD1 and RKO) resulted in excessive activation of the antioxidant gene NRF2, leading to ROS detoxification and improved cell survival. The NRF2 target genes HO1 and NQO1 were upregulated in G-TPP-treated DLD1 cells, making the cells more resistant to G-TPP treatment. Furthermore, treatment with both a NRF2 inhibitor and a TRAP1 inhibitor led to excessive ROS production and exacerbated G-TPP-induced cell death in G-TPP-resistant cells. Taken together, dual targeting of TRAP1 and NRF2 may potentially overcome colon cancer resistance by raising cellular ROS levels above the cytotoxic threshold.

Activation of mitochondrial TRAP1 stimulates mitochondria-lysosome crosstalk and correction of lysosomal dysfunction

SummaryNumerous studies have established the involvement of lysosomal and mitochondrial dysfunction in the pathogenesis of neurodegenerative disorders such as Alzheimer’s and Parkinson diseases. Building on our previous studies of the neurodegenerative lysosomal lipidosis Niemann–Pick C1 (NPC1), we have unexpectedly discovered that activation of the mitochondrial chaperone tumor necrosis factor receptor-associated protein 1 (TRAP1) leads to the correction of the lysosomal storage phenotype in patient cells from multiple lysosomal storage disorders including NPC1. Using small compound activators specific for TRAP1, we find that activation of this chaperone leads to a generalized restoration of lysosomal and mitochondrial health. Mechanistically, we show that this process includes inhibition of oxidative phosphorylation and reduction of oxidative stress, which results in activation of AMPK and ultimately stimulates lysosome recycling. Thus, TRAP1 participates in lysosomal-mitochondrial crosstalk to maintain cellular homeostasis and could represent a potential therapeutic target for multiple disorders.

Hyperacetylation of Toll‐like receptor 4 (TLR4): a potential factor contributing to non‐alcoholic liver disease (NAFLD), a silent killer in obesity

BackgroundObesity frequently leads to non‐alcoholic fatty liver disease (NAFLD), the most common liver problem affecting the American population. In fact, the prevalence of NAFLD in adult Americans ranges from 25‐46%. To date, there is no specific treatment for NAFLD. In severe cases, NAFLD can lead to fibrosis and cirrhosis which evolves into nonalcoholic steatohepatitis (NASH). Most patients with NAFLD experience no symptoms, making it a silent killer. Evidence has suggested that Toll‐like receptor 4 (TLR4) signaling, a key component of innate immunity, is implicated in the pathogenesis of NAFLD; however, the mechanisms of TLR4 activation remain unclear. We hypothesized that hyperacetylation of TLR4 leads to its overactivation in obesity, contributing to NAFLD development.MethodsEight‐week‐old male C57BL/6 mice were randomized into two experimental groups: the control group (n=4) received a regular chow diet (5% fat, 48.7% carbohydrates [3.2% sucrose], 24.1% protein) and the Western diet (WD) group (n=4) received a WD (21% fat, 50% carbohydrates [34% sucrose], and 20% protein) for 36 weeks. Body weight was obtained and intraperitoneal glucose tolerance test (IPGTT) was performed over the course of the dietary protocol at weekly and monthly intervals, respectively. During terminal experiments, liver tissue was isolated and processed for cross‐section histology. Staining with H&amp;E and Oil‐Red‐O were used for grading of liver injury with the NAFLD Score, which involves summation of steatosis (0–3), lobular inflammation (0–3), and hepatocellular ballooning (0–2) scores. A final score of 0–2 indicates no NASH, 3–4 indicates borderline NASH, and 5–8 indicates NASH. TLR4 and its downstream protein expression was detected by western blot and immunoprecipitation was utilized to determine the acetylation levels of TLR4.ResultsThe WD group exhibited increased body weight (41.22 ± 7.71 g vs 29.94 ± 1.16 g controls, p&lt;0.05) and liver weight (1.27 ± 0.0.07 g vs 1.27 ± 0.07 g controls, p&lt;0.05). Results from IPGTT demonstrated that male mice develop early intolerance to glucose, within 8 weeks on WD. Hepatic histology showed ballooned hepatocytes, lobular inflammation, and steatosis, confirming presence of NAFLD with borderline NASH. Livers from the WD group exhibited increased TLR4 expression (1.4‐fold increase, p&lt;0.05). Of note, this was accompanied by an increase in TLR4 signaling in WD livers as confirmed by elevated expression of the downstream signaling protein TNF receptor associated factor 6 (TRAF6) (0.8‐fold increase, p&lt;0.05). Strikingly, immunoprecipitation assay revealed that TLR4 is hyperacetylated in the livers from the WD group.ConclusionsOur results demonstrate for the first time that hepatic TLR4 is hyperacetylated in obesity‐related NAFLD. This hyperacetylation may serve as a trigger for the TLR4/TRAF6 signaling activation and may prove to be crucial to the development of NAFLD and NASH in obesity.