Translocase of outer mitochondrial membrane 34 (TOMM34) orchestrates heat shock protein 70 (HSP70)/HSP90-mediated transport of mitochondrial precursor proteins. Here, using in vitro phosphorylation and refolding assays, analytical size-exclusion chromatography, and hydrogen/deuterium exchange MS, we found that TOMM34 associates with 14-3-3 proteins after its phosphorylation by protein kinase A (PKA). PKA preferentially targeted two serine residues in TOMM34: Ser93 and Ser160, located in the tetratricopeptide repeat 1 (TPR1) domain and the interdomain linker, respectively. Both of these residues were necessary for efficient 14-3-3 protein binding. We determined that phosphorylation-induced structural changes in TOMM34 are further augmented by binding to 14-3-3, leading to destabilization of TOMM34's secondary structure. We also observed that this interaction with 14-3-3 occludes the TOMM34 interaction interface with ATP-bound HSP70 dimers, which leaves them intact and thereby eliminates an inhibitory effect of TOMM34 on HSP70-mediated refolding in vitro In contrast, we noted that TOMM34 in complex with 14-3-3 could bind HSP90. Both TOMM34 and 14-3-3 participated in cytosolic precursor protein transport mediated by the coordinated activities of HSP70 and HSP90. Our results provide important insights into how PKA-mediated phosphorylation and 14-3-3 binding regulate the availability of TOMM34 for its interaction with HSP70.
- MeSH
- DNA vazebné proteiny genetika metabolismus MeSH
- fosforylace fyziologie MeSH
- lidé MeSH
- MFC-7 buňky MeSH
- mitochondriální membrány metabolismus MeSH
- mitochondriální proteiny metabolismus MeSH
- molekulární chaperony metabolismus MeSH
- proteinkinasy závislé na cyklickém AMP metabolismus MeSH
- proteiny 14-3-3 metabolismus MeSH
- proteiny tepelného šoku HSP70 metabolismus MeSH
- proteiny tepelného šoku HSP72 metabolismus MeSH
- proteiny tepelného šoku HSP90 metabolismus MeSH
- signální transdukce MeSH
- transkripční faktory genetika metabolismus MeSH
- transportní proteiny mitochondriální membrány genetika metabolismus MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Studies have demonstrated that heat shock protein 70 (HSP70) plays an important role in the protection of stressed organisms. The development of strategies for enhancing HSPs expression may provide novel means of minimizing inflammatory lung conditions, such as acute lung injury. This study aimed to examine the effect of L-alanyl-L-glutamine (GLN) inhalation in enhancing pulmonary HSP72 (inducible HSP70) expression and attenuating lung damage in a model of acute lung injury induced by lipopolysaccharide (LPS) inhalation. The experimental rats were randomly assigned to one of four experimental groups: (1) NS: saline inhalation; (2) NS-LPS: pretreatment by saline inhalation 12 h before LPS inhalation; (3) GLN: glutamine inhalation; (4) GLN-LPS: pretreatment by glutamine inhalation 12 h before LPS inhalation. The results show that GLN compared with saline administration, led to significant increase in lung HSP72 both in non LPS-treated rats and LPS-treated rats. In LPS-treated rats, pretreatment by GLN inhalation produced less lung injury as evidenced by the decrease in lung injury score and dramatic decrease in lactate dehydrogenase (LDH) activity and polymorphonuclear leukocyte cell differentiation counts (PMN %) in the bronchoalveolar lavage fluid. The study indicates that prophylactic glutamine inhalation associated with the enhancement of HSP72 synthesis attenuates tissue damage in experimental lung injury.
- MeSH
- aplikace inhalační MeSH
- dipeptidy aplikace a dávkování MeSH
- krysa rodu rattus MeSH
- lipopolysacharidy MeSH
- plíce účinky léků metabolismus MeSH
- poškození plic komplikace metabolismus prevence a kontrola MeSH
- potkani Sprague-Dawley MeSH
- proteiny tepelného šoku HSP72 metabolismus MeSH
- výsledek terapie MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We investigated the effects of repeated hyperthermic bouts on the heat shock response of heat shock protein (HSP) 72 in skeletal muscle. Rats were assigned to control and hyperthermia groups which were exposed to heated water at 42 °C. The hyperthermia group was further divided into sub-groups: a single bout (H30) or four bouts of hyperthermia for 30 min (H30x4). There was an increase in HSP72 protein content of the H30 groups in both extensor digitorum longus (EDL) and soleus muscles. Moreover, HSP72 protein expression in H30x4 group was significantly higher than in H30 group in both EDL and soleus muscles. The HSP72 mRNA was markedly increased from control levels in the H30 and H30x4 group in both types of muscles. However, HSP72 mRNA of the H30x4 group was lower than that of the H30 group in soleus muscles. Heat shock response of HSP72 is activated even after repeated bouts of hyperthermia, with a differential regulation between muscle types.
The purpose of this study was to investigate the influence of heat treatment on glucocorticoid (GC)-induced myopathy. Eight-week-old Wistar rats were randomly assigned to the control, Dex, and Dex + Heat groups. Dexamethasone (2 mg/kg) was injected subcutaneously 6 days per week for 2 weeks in the Dex and Dex + Heat group. In the Dex + Heat group, heat treatment was performed by immersing hindlimbs in water at 42 °C for 60 min, once every 3 days for 2 weeks. The extensor digitorum longus muscle was extracted following 2 weeks of experimentation. In the Dex + Heat group, muscle fiber diameter, capillary/muscle fiber ratio, and level of heat shock protein 72 were significantly higher and atrogene expression levels were significantly lower than in the Dex group. Our results suggest that heat treatment inhibits the development of GC-induced myopathy by decreasing atrogene expression and increasing angiogenesis.
- MeSH
- dexamethason škodlivé účinky MeSH
- glukokortikoidy škodlivé účinky MeSH
- kosterní svalová vlákna metabolismus patologie MeSH
- náhodné rozdělení MeSH
- nemoci svalů chemicky indukované komplikace metabolismus prevence a kontrola MeSH
- potkani Wistar MeSH
- proteinligasy komplexu SCF metabolismus MeSH
- proteiny tepelného šoku HSP72 metabolismus MeSH
- svalová atrofie etiologie metabolismus prevence a kontrola MeSH
- svalové proteiny metabolismus MeSH
- synthasa oxidu dusnatého, typ III metabolismus MeSH
- vaskulární endoteliální růstový faktor A metabolismus MeSH
- vysoká teplota terapeutické užití MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
We examined the membrane expression of inducible Hsp70 and HSP receptors like TLR2, TLR4, CD14, CD36, CD40 and CD91 on fibroblast-like synovial cells (SC) derived from synovial tissue in 23 patients with rheumatoid arthritis (RA), who underwent synovectomy by using flow cytometric analysis. For comparison, autologous skin fibroblasts (SF) derived from the operation wound were tested. Significantly higher Hsp70 expression was found on synovial cells than on skin fibroblasts (median SC 21.4% x SF 5.0%, P < 0.001). Both synovial cells and skin fibroblasts expressed high levels of cell surface CD91 (median SC 80.2% x SF 79.2%), however, no or low levels of CD14, CD40, TLR2, TLR4 and CD36. Further, we observed high co-expression of CD91 and Hsp70 on RA synovial cells (median 18.6%), while skin fibroblasts showed only background Hsp70 expression (median 3.9%, P < 0.001). Since we demonstrated the high prevalence of inducible Hsp70 in RA synovial fluids, we speculate that Hsp70 might be captured onto the membrane of synovial cells from the extracellular space via the CD91 receptor. The significance of the Hsp70 interaction with synovial cells via CD91 remains undefined, but may mediate other non-immune purposes.
- MeSH
- CD antigeny metabolismus MeSH
- dospělí MeSH
- fibroblasty metabolismus MeSH
- kohortové studie MeSH
- lidé středního věku MeSH
- lidé MeSH
- proteiny tepelného šoku HSP70 metabolismus MeSH
- proteiny tepelného šoku HSP72 metabolismus MeSH
- revmatoidní artritida metabolismus MeSH
- senioři MeSH
- synoviální membrána metabolismus MeSH
- synoviální tekutina MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- práce podpořená grantem MeSH
- MeSH
- ischemie mozku * metabolismus patofyziologie MeSH
- králíci MeSH
- neurologické manifestace MeSH
- poranění páteře MeSH
- proteiny tepelného šoku HSP70 sekrece škodlivé účinky MeSH
- proteiny tepelného šoku HSP72 * sekrece škodlivé účinky MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- abstrakty MeSH
