The aim of the study was to examine the potential role of mitochondrial permeability transition pore (mPTP) in the cardioprotective effect of chronic continuous hypoxia (CH) against acute myocardial ischemia/reperfusion (I/R) injury. Adult male Wistar rats were adapted to CH for 3 weeks, while their controls were kept under normoxic conditions. Subsequently, they were subjected to I/R insult while being administered with mPTP inhibitor, cyclosporin A (CsA). Infarct size and incidence of ischemic and reperfusion arrhythmias were determined. Our results showed that adaptation to CH as well as CsA administration reduced myocardial infarct size in comparison to the corresponding control groups. However, administration of CsA did not amplify the beneficial effect of CH, suggesting that inhibition of mPTP opening contributes to the protective character of CH.
- MeSH
- chronická nemoc MeSH
- cyklosporin * farmakologie MeSH
- hypoxie * metabolismus MeSH
- infarkt myokardu metabolismus patologie prevence a kontrola MeSH
- krysa rodu rattus MeSH
- potkani Wistar * MeSH
- přechodový pór mitochondriální permeability * metabolismus MeSH
- reperfuzní poškození myokardu * metabolismus prevence a kontrola patologie MeSH
- srdeční mitochondrie metabolismus účinky léků patologie MeSH
- transportní proteiny mitochondriální membrány metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The mitochondrial permeability transition pore (MPTP) is a calcium-dependent, ion non-selective membrane pore with a wide range of functions. Although the MPTP has been studied for more than 50 years, its molecular structure remains unclear. Short-term (reversible) opening of the MPTP protects cells from oxidative damage and enables the efflux of Ca2+ ions from the mitochondrial matrix and cell signaling. However, long-term (irreversible) opening induces processes leading to cell death. Ca2+ ions, reactive oxygen species, and changes in mitochondrial membrane potential regulate pore opening. The sensitivity of the pore to Ca2+ ions changes as an organism ages, and MPTP opening plays a key role in the pathogenesis of many diseases. Most studies of the MPTP have focused on elucidating its molecular structure. However, understanding the mechanisms that will inhibit the MPTP may improve the treatment of diseases associated with its opening. To evaluate the functional state of the MPTP and its inhibitors, it is therefore necessary to use appropriate methods that provide reproducible results across laboratories. This review summarizes our current knowledge of the function and regulation of the MPTP. The latter part of the review introduces two optimized methods for evaluating the functional state of the pore under standardized conditions.
The mitochondrial permeability transition pore (mtPTP) plays a vital role in altering the structure and function of mitochondria. Cyclophilin D (CypD) is a mitochondrial protein that regulates mtPTP function and a known drug target for therapeutic studies involving mitochondria. While the effect of aromatase inhibition on the mtPTP has been studied previously, the effect of anastrozole on the mtPTP has not been completely elucidated. The role of anastrozole in modulating the mtPTP was evaluated by docking, molecular dynamics and network-guided studies using human CypD data. The peripheral blood mononuclear cells (PBMCs) of patients with mitochondrial disorders and healthy controls were treated with anastrozole and evaluated for mitochondrial permeability transition pore (mtPTP) function and apoptosis using a flow cytometer. Spectrophotometry was employed for estimating total ATP levels. The anastrozole-CypD complex is more stable than cyclosporin A (CsA)-CypD. Anastrozole performed better than cyclosporine in inhibiting mtPTP. Additional effects included inducing mitochondrial membrane depolarization and a reduction in mitochondrial swelling and superoxide generation, intrinsic caspase-3 activity and cellular apoptosis, along with an increase in ATP levels. Anastrozole may serve as a potential therapeutic agent for mitochondrial disorders and ameliorate the clinical phenotype by regulating the activity of mtPTP. However, further studies are required to substantiate our preliminary findings.Communicated by Ramaswamy H. Sarma.
- MeSH
- adenosintrifosfát metabolismus MeSH
- anastrozol farmakologie metabolismus MeSH
- cyklofiliny genetika metabolismus MeSH
- leukocyty mononukleární metabolismus MeSH
- lidé MeSH
- mitochondriální nemoci * metabolismus MeSH
- mitochondrie metabolismus MeSH
- peptidylprolylisomerasa F MeSH
- přechodový pór mitochondriální permeability * metabolismus farmakologie MeSH
- transportní proteiny mitochondriální membrány metabolismus farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Mitochondria and mitochondrial proteins represent a group of promising pharmacological target candidates in the search of new molecular targets and drugs to counteract the onset of hypertension and more in general cardiovascular diseases (CVDs). Indeed, several mitochondrial pathways result impaired in CVDs, showing ATP depletion and ROS production as common traits of cardiac tissue degeneration. Thus, targeting mitochondrial dysfunction in cardiomyocytes can represent a successful strategy to prevent heart failure. In this context, the identification of new pharmacological targets among mitochondrial proteins paves the way for the design of new selective drugs. Thanks to the advances in omics approaches, to a greater availability of mitochondrial crystallized protein structures and to the development of new computational approaches for protein 3D-modelling and drug design, it is now possible to investigate in detail impaired mitochondrial pathways in CVDs. Furthermore, it is possible to design new powerful drugs able to hit the selected pharmacological targets in a highly selective way to rescue mitochondrial dysfunction and prevent cardiac tissue degeneration. The role of mitochondrial dysfunction in the onset of CVDs appears increasingly evident, as reflected by the impairment of proteins involved in lipid peroxidation, mitochondrial dynamics, respiratory chain complexes, and membrane polarization maintenance in CVD patients. Conversely, little is known about proteins responsible for the cross-talk between mitochondria and cytoplasm in cardiomyocytes. Mitochondrial transporters of the SLC25A family, in particular, are responsible for the translocation of nucleotides (e.g., ATP), amino acids (e.g., aspartate, glutamate, ornithine), organic acids (e.g. malate and 2-oxoglutarate), and other cofactors (e.g., inorganic phosphate, NAD+, FAD, carnitine, CoA derivatives) between the mitochondrial and cytosolic compartments. Thus, mitochondrial transporters play a key role in the mitochondria-cytosol cross-talk by leading metabolic pathways such as the malate/aspartate shuttle, the carnitine shuttle, the ATP export from mitochondria, and the regulation of permeability transition pore opening. Since all these pathways are crucial for maintaining healthy cardiomyocytes, mitochondrial carriers emerge as an interesting class of new possible pharmacological targets for CVD treatments.
- MeSH
- adenosintrifosfát metabolismus MeSH
- hypertenze * metabolismus MeSH
- kardiovaskulární nemoci * farmakoterapie metabolismus MeSH
- kyselina asparagová metabolismus MeSH
- lidé MeSH
- maláty metabolismus MeSH
- mitochondriální proteiny metabolismus MeSH
- mitochondrie metabolismus MeSH
- reperfuzní poškození * metabolismus MeSH
- transportní proteiny mitochondriální membrány metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Cyclophilin D (CypD) is a key regulator of mitochondrial permeability transition pore (mPTP) opening. This pathophysiological phenomenon is associated with the development of several human diseases, including ischemia-reperfusion injury and neurodegeneration. Blocking mPTP opening through CypD inhibition could be a novel and promising therapeutic approach for these conditions. While numerous CypD inhibitors have been discovered to date, none have been introduced into clinical practice, mostly owing to their high toxicity, unfavorable pharmacokinetics, and low selectivity for CypD over other cyclophilins. This review summarizes current knowledge of CypD inhibitors, with a particular focus on small-molecule compounds with regard to their in vitro activity, their selectivity for CypD, and their binding mode within the enzyme's active site. Finally, approaches for improving the molecular design of CypD inhibitors are discussed.
- MeSH
- lidé MeSH
- mitochondriální nemoci * farmakoterapie MeSH
- mitochondrie metabolismus MeSH
- peptidylprolylisomerasa F * antagonisté a inhibitory MeSH
- přechodový pór mitochondriální permeability MeSH
- transportní proteiny mitochondriální membrány * metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Values of the calcium retention capacity (CRC) of rat liver mitochondria are highly dependent on the experimental conditions used. When increasing amounts of added calcium chloride are used (1.25-10 nmol), the values of the CRC increase 3-fold. When calcium is added in 75 s intervals, the CRC values increase by 30 % compared with 150 s interval additions. CRC values are not dependent on the calcium/protein ratio in the measured sample in our experimental design. We also show that a more detailed evaluation of the fluorescence curves can provide new information about mitochondrial permeability transition pore opening after calcium is added.
- MeSH
- biologický transport MeSH
- jaterní mitochondrie metabolismus MeSH
- játra metabolismus MeSH
- krysa rodu rattus MeSH
- mitochondriální membrány metabolismus MeSH
- permeabilita MeSH
- přechodový pór mitochondriální permeability metabolismus MeSH
- transportní proteiny mitochondriální membrány metabolismus MeSH
- vápník metabolismus MeSH
- výzkumný projekt MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
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
Eukaryotic protein homeostasis (proteostasis) is largely dependent on the action of highly conserved Hsp70 molecular chaperones. Recent evidence indicates that, apart from conserved molecular allostery, Hsp70 proteins have retained and adapted the ability to assemble as functionally relevant ATP-bound dimers throughout evolution. Here, we have compared the ATP-dependent dimerization of DnaK, human stress-inducible Hsp70, Hsc70 and BiP Hsp70 proteins, showing that their dimerization propensities differ, with stress-inducible Hsp70 being predominantly dimeric in the presence of ATP. Structural analyses using hydrogen/deuterium exchange mass spectrometry, native electrospray ionization mass spectrometry and small-angle X-ray scattering revealed that stress-inducible Hsp70 assembles in solution as an antiparallel dimer with the intermolecular interface closely resembling the ATP-bound dimer interfaces captured in DnaK and BiP crystal structures. ATP-dependent dimerization of stress-inducible Hsp70 is necessary for its efficient interaction with Hsp40, as shown by experiments with dimerization-deficient mutants. Moreover, dimerization of ATP-bound Hsp70 is required for its participation in high molecular weight protein complexes detected ex vivo, supporting its functional role in vivo As human cytosolic Hsp70 can interact with tetratricopeptide repeat (TPR) domain containing cochaperones, we tested the interaction of Hsp70 ATP-dependent dimers with Chip and Tomm34 cochaperones. Although Chip associates with intact Hsp70 dimers to form a larger complex, binding of Tomm34 disrupts the Hsp70 dimer and this event plays an important role in Hsp70 activity regulation. In summary, this study provides structural evidence of robust ATP-dependent antiparallel dimerization of human inducible Hsp70 protein and suggests a novel role of TPR domain cochaperones in multichaperone complexes involving Hsp70 ATP-bound dimers.
- MeSH
- adenosintrifosfát metabolismus MeSH
- fyziologický stres MeSH
- HEK293 buňky MeSH
- krystalografie rentgenová MeSH
- lidé MeSH
- maloúhlový rozptyl MeSH
- molekulární modely MeSH
- multimerizace proteinu MeSH
- proteiny tepelného šoku HSP70 chemie metabolismus MeSH
- transportní proteiny mitochondriální membrány metabolismus MeSH
- ubikvitinligasy metabolismus MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In early stages of Alzheimer's disease (AD), amyloid-β (Aβ) accumulates in neuronal mitochondria where it interacts with a number of biomolecules including 17beta-hydroxysteroide dehydrogenase 10 (17β-HSD10) and cyclophilin D (cypD). It has been hypothesized that 17β-HSD10 interacts with cypD preventing it from opening mitochondrial permeability transition pores and that its regulation during AD may be affected by the accumulation of Aβ. In this work, we demonstrate for the first time that 17β-HSD10 and cypD form a stable complex in vitro. Furthermore, we show that factors, such as pH, ionic environment and the presence of Aβ, affect the ability of 17β-HSD10 to bind cypD. We demonstrate that K+ and Mg2+ ions present at low levels may facilitate this binding. We also show that different fragments of Aβ (Aβ1-40 and Aβ1-42) affect the interaction between 17β-HSD10 and cypD differently and that Aβ1-42 (in contrast to Aβ1-40) is capable of simultaneously binding both 17β-HSD10 and cypD in a tri-complex.
- MeSH
- 17-hydroxysteroidní dehydrogenasy metabolismus MeSH
- Alzheimerova nemoc metabolismus patologie MeSH
- amyloidní beta-protein metabolismus MeSH
- lidé MeSH
- mitochondrie metabolismus MeSH
- peptidylprolylisomerasa F metabolismus MeSH
- přechodový pór mitochondriální permeability MeSH
- techniky in vitro MeSH
- transportní proteiny mitochondriální membrány metabolismus MeSH
- vápník metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
By determining the calcium retention capacity (CRC) of rat liver mitochondria, we confirmed and extended previous observations describing the activation of mitochondrial swelling by phosphate and tert-butyl hydroperoxide (t-BHP). Using CRC measurements, we showed that both phosphate and t-BHP decrease the extent of calcium accumulation required for the full mitochondrial permeability transition pore (MPTP) opening to 35 % of control values and to only 15 % when both phosphate and t-BHP are present in the medium. When changes in fluorescence were evaluated at higher resolution, we observed that in the presence of cyclosporine A fluorescence values return after each Ca(2+) addition to basal values obtained before the Ca(2+) addition. This indicates that the MPTP remains closed. However, in the absence of cyclosporine A, the basal fluorescence after each Ca(2+) addition continuously increased. This increase was potentiated both by phosphate and t-BHP until the moment when the concentration of intramitochondrial calcium required for the full opening of the MPTP was reached. We conclude that in the absence of cyclosporine A, the MPTP is slowly opened after each Ca(2+) addition and that this rate of opening can be modified by various factors such as the composition of the media and the experimental protocol used.
- MeSH
- fosfáty farmakologie MeSH
- jaterní mitochondrie účinky léků metabolismus MeSH
- krysa rodu rattus MeSH
- potkani Wistar MeSH
- terc-butylhydroperoxid farmakologie MeSH
- transportní proteiny mitochondriální membrány metabolismus MeSH
- vápník metabolismus 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
