TRAP1 and the proteasome regulatory particle TBP7/Rpt3 interact in the endoplasmic reticulum and control cellular ubiquitination of specific mitochondrial proteins

Tumor necrosis factor receptor-associated protein-1 (TRAP1) is a mitochondrial (MITO) antiapoptotic heat-shock protein. The information available on the TRAP1 pathway describes just a few well-characterized functions of this protein in mitochondria. However, our group's use of mass-spectrometric analysis identified TBP7, an AAA-ATPase of the 19S proteasomal subunit, as a putative TRAP1-interacting protein. Surprisingly, TRAP1 and TBP7 colocalize in the endoplasmic reticulum (ER), as demonstrated by biochemical and confocal/electron microscopic analyses, and interact directly, as confirmed by fluorescence resonance energy transfer analysis. This is the first demonstration of TRAP1's presence in this cellular compartment. TRAP1 silencing by short-hairpin RNAs, in cells exposed to thapsigargin-induced ER stress, correlates with upregulation of BiP/Grp78, thus suggesting a role of TRAP1 in the refolding of damaged proteins and in ER stress protection. Consistently, TRAP1 and/or TBP7 interference enhanced stress-induced cell death and increased intracellular protein ubiquitination. These experiments led us to hypothesize an involvement of TRAP1 in protein quality control for mistargeted/misfolded mitochondria-destined proteins, through interaction with the regulatory proteasome protein TBP7. Remarkably, expression of specific MITO proteins decreased upon TRAP1 interference as a consequence of increased ubiquitination. The proposed TRAP1 network has an impact in vivo, as it is conserved in human colorectal cancers, is controlled by ER-localized TRAP1 interacting with TBP7 and provides a novel model of the ER-mitochondria crosstalk.     

Deficient heat shock protein 70 response to stress in leukocytes at onset of type 1 diabetes

Type 1 diabetes is caused by the immune-mediated destruction of pancreatic beta cells. Animal models of the disease demonstrate an increased susceptibility of beta cells to immunological attacks due to their defective stress-responsiveness. To investigate the stress-responsiveness in human type 1 diabetes we analyzed the heat-inducibility of the dominant stress protein heat shock protein (Hsp)70 in diabetic patients at different disease stages. At diabetes-manifestation heat-induced Hsp70 levels in peripheral blood mononuclear cells (PBMC) reached only about 25% of the levels expressed by heat-treated PBMC from non-diabetic subjects (p < 0.05). Heat-responsiveness improved with disease duration and was re-established at more than eight months after disease-manifestation. Hyperthermia-induced Hsp70 expression was decreased by the T-helper 1-associated cytokine interferon-γ and increased by the T-helper 2-associated transforming growth factor-β. We conclude that impaired cellular stress-responsiveness, aggravated by the inflammatory milieu at the onset of type 1 diabetes, contributes to disease manifestation.     

Alzheimer's Abeta fused to green fluorescent protein induces growth stress and a heat shock response

The 42 amino acid Alzheimer's Abeta peptide is involved in the progression of Alzheimer's disease. Here we describe the effects of intracellular Abeta, produced through its attachment to either end of a green fluorescent protein, in yeast. Cells producing Abeta exhibited a lower growth yield and a heat shock response, showing that Abeta fusions promote stress in cells and supporting the notion that intracellular Abeta is a toxic molecule. These studies have relevance in understanding the role of Abeta in the death of neuronal cells, and indicate that yeast may be a new tractable model system for the screening for inhibitors of the stress caused by Abeta.     

The unfolded protein response in human corneal endothelial cells following hypothermic storage: implications of a novel stress pathway

Human corneal endothelial cells (HCEC) have become increasingly important for a range of eye disease treatment therapies. Accordingly, a more detailed understanding of the processing and preservation associated stresses experienced by corneal cells might contribute to improved therapeutic outcomes. To this end, the unfolded protein response (UPR) pathway was investigated as a potential mediator of corneal cell death in response to hypothermic storage. Once preservation-induced failure had begun in HCECs stored at 4 °C, it was noted that necrosis accounted for the majority of cell death but with significant apoptotic involvement, peaking at several hours post-storage (4–8 h). Western blot analysis demonstrated changes associated with apoptotic activation (caspase 9, caspase 3, and PARP cleavage). Further, the activation of the UPR pathway was observed through increased and sustained levels of ER folding and chaperone proteins (Bip, PDI, and ERO1-Lα) in samples experiencing significant cell death. Modulation of the UPR pathway using the specific inhibitor, salubrinal, resulted in a 2-fold increase in cell survival in samples experiencing profound cold-induced failure. Furthermore, this increased cell survival was associated with increased membrane integrity, cell attachment, and decreased necrotic cell death populations. Conversely, addition of the UPR inducer, tunicamycin, during cold exposure resulted in a significant decrease in HCEC survival during the recovery period. These data implicate for the first time that this novel cell stress pathway may be activated in HCEC as a result of the complex stresses associated with hypothermic exposure. The data suggest that the targeted control of the UPR pathway during both processing and preservation protocols may improve cell survival and function of HCEC thus improving the clinical utility of these cells as well as whole human corneas.     

A heat shock protein 90 inhibitor that modulates the immunophilins and regulates hormone receptors without inducing the heat shock response

When a cell encounters external stressors, such as lack of nutrients, elevated temperatures, changes in pH or other stressful environments, a key set of evolutionarily conserved proteins, the heat shock proteins (hsps), become overexpressed. Hsps are classified into six major families with the hsp90 family being the best understood; an increase in cell stress leads to increased levels of hsp90, which leads to cellular protection. A hallmark of hsp90 inhibitors is that they induce a cell rescue mechanism, the heat shock response. We define the unique molecular profile of a compound (SM145) that regulates hormone receptor protein levels through hsp90 inhibition without inducing the heat shock response. Modulation of the binding event between heat shock protein 90 and the immunophilins/homologs using SM145, leads to a decrease in hormone receptor protein levels. Unlike N-terminal hsp90 inhibitors, this hsp90 inhibitor does not induce a heat shock response. This work is proof of principle that controlling hormone receptor expression can occur by inhibiting hsp90 without inducing pro-survival protein heat shock protein 70 (hsp70) or other proteins associated with the heat shock response. Innovatively, we show that blocking the heat shock response, in addition to hsp90, is key to regulating hsp90-associated pathways.     

Translational regulation of the synthesis of a major heat shock protein in HeLa cells

The synthesis of a major heat shock protein (HSP 70) was measured in HeLa cells incubated at 42.5 degrees C and then transferred to 37 degrees C or 30 degrees C. After 90 min, synthesis of HSP 70 decreased by 54 and 85%, respectively, whereas HSP 70 mRNA was reduced at most by 20%. Therefore, the reduced synthesis of HSP 70 could not be accounted for by mRNA turnover. HSP 70 was associated with large polyribosomes (6-10 ribosomes) in cells kept at 42.5 degrees C, but with medium or small polyribosomes in cells transferred to 37 degrees C or 30 degrees C (5-6 or 2-3 ribosomes, respectively). Addition of puromycin to these cells resulted in the release of all ribosomes from HSP 70 mRNA, indicating that they were translationally active. The regulation of HSP 70 synthesis was investigated in cell-free systems prepared from heat-shocked or control cells and incubated at 30 degrees C and 42 degrees C. After 5 min at 42 degrees C, the cell-free system from heat-shocked cells synthesized protein at 3 times the rate of the control cell-free system. This difference was in large part due to synthesis of HSP 70. Addition of HSP mRNA to the control cell-free system stimulated protein synthesis at 42 degrees C, but not at 30 degrees C. These findings suggest that translation of HSP 70 mRNA is specifically promoted at high temperature and repressed during recovery from heat shock by regulatory mechanisms active at the level of initiation.     

Regulation of heat shock protein 70 release in astrocytes: role of signaling kinases

The ability to mount a successful stress response in the face of injury is critical to the long-term viability of individual cells and to the organism in general. The stress response, characterized in part by the upregulation of heat shock proteins, is compromised in several neurodegenerative disorders and in some neuronal populations, including motoneurons (MNs). Because astrocytes have a greater capacity than neurons to survive metabolic stress, and because they are intimately associated with the regulation of neuronal function, it is important to understand their stress response, so that we may to better appreciate the impact of stress on neuronal viability during injury or disease. We show that astrocytes subjected to hyperthermia upregulate Hsp/c70 in addition to intracellular signaling components including activated forms of extracellular-signal-regulated kinase (ERK1/2), Akt, and c-jun N-terminal kinase/stress activated protein kinase (JNK/SAPK). Furthermore, astrocytes release increasing amounts of Hsp/c70 into the extracellular environment following stress, an event that is abrogated when signaling through the ERK1/2 and phosphatidylinositol-3 kinase (PI3K) pathways is compromised and enhanced by inhibition of the JNK pathway. Last, we show that the Hsp/c70 is released from astrocytes in exosomes. Together, these data illustrate the diverse regulation of stress-induced Hsp/c70 release in exosomes, and the way in which the balance of activated signal transduction pathways affects this release. These data highlight how stressful insults can alter the microenvironment of an astrocyte, which may ultimately have implications for the survival of neighboring neurons.     

The role of protein quality control in mitochondrial protein homeostasis under oxidative stress

Mitochondria contribute significantly to the cellular production of ROS. The deleterious effects of increased ROS levels have been implicated in a wide variety of pathological reactions. Apart from a direct detoxification of ROS molecules, protein quality control mechanisms are thought to protect protein functions in the presence of elevated ROS levels. The reactivities of molecular chaperones and proteases remove damaged polypeptides, maintaining enzyme activities, thereby contributing to cellular survival both under normal and stress conditions. We characterized the impact of oxidative stress on mitochondrial protein homeostasis by performing a proteomic analysis of isolated yeast mitochondria, determining the changes in protein abundance after ROS treatments. We identified a set of mitochondrial proteins as substrates of ROS‐dependent proteolysis. Enzymes containing oxidation‐sensitive prosthetic groups like iron/sulfur clusters represented major targets of stress‐dependent degradation. We found that several proteins involved in ROS detoxification were also affected. We identified the ATP‐dependent protease Pim1/LON as a major factor in the degradation of ROS‐modified soluble polypeptides localized in the matrix compartment. As Pim1/LON expression was induced significantly under ROS treatment, we propose that this protease system performs a crucial protective function under oxidative stress conditions.     

Overexpression of tumor necrosis factor receptor-associated protein 1 (TRAP1), leads to mitochondrial aberrations in mouse fibroblast NIH/3T3 cells

Cancer cells undergo uncontrolled proliferation, and aberrant mitochondrial alterations. Tumor necrosis factor receptorassociated protein 1 (TRAP1) is a mitochondrial heat shock protein. TRAP1 mRNA is highly expressed in some cancer cell lines and tumor tissues. However, the effects of its overexpression on mitochondria are unclear. In this study, we assessed mitochondrial changes accompanying TRAP1 overexpression, in a mouse cell line, NIH/3T3. We found that overexpression of TRAP1 leads to a series of mitochondrial aberrations, including increase in basal ROS levels, and decrease in mitochondrial biogenesis, together with a decrease in peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) mRNA levels. We also observed increased extracellular signal-regulated kinase (ERK) phosphorylation, and enhanced proliferation of TRAP1 overexpressing cells. This study suggests that overexpression of TRAP1 might be a critical link between mitochondrial disturbances and carcinogenesis. [BMB Reports 2014; 47(5): 280-285]     

Conditions for a heat shock response during oogenesis and embryogenesis of the amphibian Pleurodeles waltl

The optimal conditions capable of inducing an increase in HSP70 neosynthesis during development of the urodele amphibian Pleurodeles waltl were determined in this study. These conditions depend on temperature, heat shock duration and recovery duration. In oocytes, a heat shock response was repeatedly obtained at 37°C for 15 min followed by 1 h recovery. These results provided evidence for heat shock response at every stage considered. An increase in HSP70 synthesis was noted throughout oogenesis, but it did not lead to an increase in the amount of soluble HSP70, except for stage VI oocytes. Such results suggest that from stage II to stage IV oocytes, an equilibrium occurs between the HSP70 used and the HSP70 neosynthesized. In contrast, in stage VI oocytes, heat shock led to overproduction of HSP70. During early development, the heat shock response was repeatedly obtained only from the gastrula stage with a 37°C shock and a 15min duration of treatment. Surprisingly, during cleavage stage, the soluble HSP70 total amount increased after heat shock at a time when no HSP70 neosynthesis occurred.     

Iron chelation study in a normal human hepatocyte cell line suggests that tumor necrosis factor receptor-associated protein 1 (TRAP1) regulates production of reactive oxygen species

Iron is an essential component of many proteins, and has crucial roles in the proper functioning of proteins involved in cellular respiration, proliferation, and differentiation. It has been recently reported that the deferoxamine (DFO), an iron chelator, induces mitochondrial dysfunction, characterized by an attenuation of oxidative phosphorylation, as well as senescence-like cellular morphology. However, the effects of DFO on mitochondrial heat shock proteins (HSPs) remain poorly understood. In this study, we examined the effect of DFO on tumor necrosis factor receptor-associated protein 1 (TRAP1), a representative mitochondrial HSP, in a normal human hepatocyte cell line, Chang cells. DFO specifically decreased TRAP1 levels, increasing reactive oxygen species (ROS) and caveolin-1 (Cav-1), a marker protein of senescence. To examine whether these effects of DFO are reversed, we established TRAP1-overexpressing Chang cells. DFO treatment to TRAP1-overexpressing cells resulted in decreases in levels of ROS, Cav-1, glutathione peroxidase (GPX), and manganese superoxide dismutase (MnSOD) levels as well as senescence-associated beta-galactosidase (SA beta-gal) activity. These results suggest that TRAP1 might play a role in protecting mitochondria against damaging stimuli via decrease of ROS generation.     

The role of the unfolded protein response in cancer progression: From oncogenesis to chemoresistance

Tumour cells endure both oncogenic and environmental stresses during cancer progression. Transformed cells must meet increased demands for protein and lipid production needed for rapid proliferation and must adapt to exist in an oxygen‐ and nutrient‐deprived environment. To overcome such challenges, cancer cells exploit intrinsic adaptive mechanisms such as the unfolded protein response (UPR). The UPR is a pro‐survival mechanism triggered by accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER), a condition referred to as ER stress. IRE1, PERK and ATF6 are three ER anchored transmembrane receptors. Upon induction of ER stress, they signal in a coordinated fashion to re‐establish ER homoeostasis, thus aiding cell survival. Over the past decade, evidence has emerged supporting a role for the UPR in the establishment and progression of several cancers, including breast cancer, prostate cancer and glioblastoma multiforme. This review discusses our current knowledge of the UPR during oncogenesis, tumour growth, metastasis and chemoresistance.     

Characterization of the heat shock response in cultured sugarcane cells : I. Physiology of the heat shock response and heat shock protein synthesis

Effect of heat shock on the growth of cultured sugarcane cells (Saccharum officinarum L.) was measured. Heat shock (HS) treatment at 36 to 38°C (2 hours) induced the development of maximum thermotolerance to otherwise nonpermissive heat stress at 54°C (7 minutes). Optimum thermotolerance was observed 8 hours after heat shock. Development of thermotolerance was initiated by treatments as short as 30 minutes at 36°C. Temperatures below 36°C or above 40°C failed to induce maximum thermotolerance. In vivo labeling revealed that HS at 32 to 34°C induced several high molecular mass heat shock proteins (HSPs). A complex of 18 kilodalton HSPs required at least 36°C treatment for induction. The majority of the HSPs began to accumulate within 10 minutes, whereas the synthesis of low molecular mass peptides in the 18 kilodalton range became evident 30 minutes after initiation of HS. HS above 38°C resulted in progressively decreased HSP synthesis with inhibition first observed for HSPs larger than 50 kilodaltons. Analysis of two-dimensional gels revealed a complex pattern of label incorporation including the synthesis of four major HSPs in the 18 kilodalton range and continued synthesis of constitutive proteins during HS.