Justina Šileikytė

Regulation of the Mitochondrial Permeability Transition Pore by the Outer Membrane Does Not Involve the Peripheral Benzodiazepine Receptor (Translocator Protein of 18 kDa (TSPO))

Background: TSPO has been proposed to be a critical regulator of the permeability transition pore (PTP). Results: TSPO-null mitochondria and cardiac tissue show no difference from controls in pore function, response to ligands, or response to ischemia/reperfusion injury. Conclusion: Regulation of the PTP by the outer membrane must rely on unknown proteins. Significance: Our results call into question studies implicating TSPO in pathological processes through the PTP. Translocator protein of 18 kDa (TSPO) is a highly conserved, ubiquitous protein localized in the outer mitochondrial membrane, where it is thought to play a key role in the mitochondrial transport of cholesterol, a key step in the generation of steroid hormones. However, it was first characterized as the peripheral benzodiazepine receptor because it appears to be responsible for high affinity binding of a number of benzodiazepines to non-neuronal tissues. Ensuing studies have employed natural and synthetic ligands to assess the role of TSPO function in a number of natural and pathological circumstances. Largely through the use of these compounds and biochemical associations, TSPO has been proposed to play a role in the mitochondrial permeability transition pore (PTP), which has been associated with cell death in many human pathological conditions. Here, we critically assess the role of TSPO in the function of the PTP through the generation of mice in which the Tspo gene has been conditionally eliminated. Our results show that 1) TSPO plays no role in the regulation or structure of the PTP, 2) endogenous and synthetic ligands of TSPO do not regulate PTP activity through TSPO, 3) outer mitochondrial membrane regulation of PTP activity occurs though a mechanism that does not require TSPO, and 4) hearts lacking TSPO are as sensitive to ischemia-reperfusion injury as hearts from control mice. These results call into question a wide variety of studies implicating TSPO in a number of pathological processes through its actions on the PTP.

Channel Formation by Yeast F-ATP Synthase and the Role of Dimerization in the Mitochondrial Permeability Transition

Background: Whether channel formation is a general feature of F-ATP synthase dimers across species is unknown. Results: Yeast F-ATP synthase dimers form Ca2+-dependent channels, and the e and g subunits facilitate pore formation in situ through dimerization. Conclusion: F-ATP synthase dimers form the permeability transition pore of yeast. Significance: Ca2+-dependent channel formation is a conserved feature of F-ATP synthases. Purified F-ATP synthase dimers of yeast mitochondria display Ca2+-dependent channel activity with properties resembling those of the permeability transition pore (PTP) of mammals. After treatment with the Ca2+ ionophore ETH129, which allows electrophoretic Ca2+ uptake, isolated yeast mitochondria undergo inner membrane permeabilization due to PTP opening. Yeast mutant strains ΔTIM11 and ΔATP20 (lacking the e and g F-ATP synthase subunits, respectively, which are necessary for dimer formation) display a striking resistance to PTP opening. These results show that the yeast PTP originates from F-ATP synthase and indicate that dimerization is required for pore formation in situ.

Regulation of the Inner Membrane Mitochondrial Permeability Transition by the Outer Membrane Translocator Protein (Peripheral Benzodiazepine Receptor)

We studied the properties of the permeability transition pore (PTP) in rat liver mitochondria and in mitoplasts retaining inner membrane ultrastructure and energy-linked functions. Like mitochondria, mitoplasts readily underwent a permeability transition following Ca2+ uptake in a process that maintained sensitivity to cyclosporin A. On the other hand, major differences between mitochondria and mitoplasts emerged in PTP regulation by ligands of the outer membrane translocator protein of 18 kDa, TSPO, formerly known as the peripheral benzodiazepine receptor. Indeed, (i) in mitoplasts, the PTP could not be activated by photo-oxidation after treatment with dicarboxylic porphyrins endowed with protoporphyrin IX configuration, which bind TSPO in intact mitochondria; and (ii) mitoplasts became resistant to the PTP-inducing effects of N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide and of other selective ligands of TSPO. Thus, the permeability transition is an inner membrane event that is regulated by the outer membrane through specific interactions with TSPO.

Shedding light on the mitochondrial permeability transition

The mitochondrial permeability transition is an increase of permeability of the inner mitochondrial membrane to ions and solutes with an exclusion size of about 1500Da. It is generally accepted that the permeability transition is due to opening of a high-conductance channel, the permeability transition pore. Although the molecular nature of the permeability transition pore remains undefined, a great deal is known about its regulation and role in pathophysiology. This review specifically covers the characterization of the permeability transition pore by chemical modification of specific residues through photoirradiation of mitochondria after treatment with porphyrins. The review also illustrates the basic principles of the photodynamic effect and the mechanisms of phototoxicity and discusses the unique properties of singlet oxygen generated by specific porphyrins in discrete mitochondrial domains. These experiments provided remarkable information on the role, interactions and topology of His and Cys residues in permeability transition pore modulation and defined an important role for the outer membrane 18kDa translocator protein (formerly known as the peripheral benzodiazepine receptor) in regulation of the permeability transition.

Switch from inhibition to activation of the mitochondrial permeability transition during hematoporphyrin-mediated photooxidative stress. Unmasking pore-regulating external thiols.

We have studied the mitochondrial permeability transition pore (PTP) under oxidizing conditions with mitochondria-bound hematoporphyrin, which generates reactive oxygen species (mainly singlet oxygen, (1)O(2)) upon UV/visible light-irradiation and promotes the photooxidative modification of vicinal targets. We have characterized the PTP-modulating properties of two major critical sites endowed with different degrees of photosensitivity: (i) the most photovulnerable site comprises critical histidines, whose photomodification by vicinal hematoporphyrin causes a drop in reactivity of matrix-exposed (internal), PTP-regulating cysteines thus stabilizing the pore in a closed conformation; (ii) the most photoresistant site coincides with the binding domains of (external) cysteines sensitive to membrane-impermeant reagents, which are easily unmasked when oxidation of internal cysteines is prevented. Photooxidation of external cysteines promoted by vicinal hematoporphyrin reactivates the PTP after the block caused by histidine photodegradation. Thus, hematoporphyrin-mediated photooxidative stress can either inhibit or activate the mitochondrial permeability transition depending on the site of hematoporphyrin localization and on the nature of the substrate; and selective photomodification of different hematoporphyrin-containing pore domains can be achieved by fine regulation of the sensitizer/light doses. These findings shed new light on PTP modulation by oxidative stress.

Discovery, Synthesis, and Optimization of Diarylisoxazole-3-carboxamides as Potent Inhibitors of the Mitochondrial Permeability Transition Pore

The mitochondrial permeability transition pore (mtPTP) is a Ca2+‐requiring mega‐channel which, under pathological conditions, leads to the deregulated release of Ca2+ and mitochondrial dysfunction, ultimately resulting in cell death. Although the mtPTP is a potential therapeutic target for many human pathologies, its potential as a drug target is currently unrealized. Herein we describe an optimization effort initiated around hit 1, 5‐(3‐hydroxyphenyl)‐N‐(3,4,5‐trimethoxyphenyl)isoxazole‐3‐carboxamide, which was found to possess promising inhibitory activity against mitochondrial swelling (EC50<0.39 μM) and showed no interference on the inner mitochondrial membrane potential (rhodamine 123 uptake EC50>100 μM). This enabled the construction of a series of picomolar mtPTP inhibitors that also potently increase the calcium retention capacity of the mitochondria. Finally, the therapeutic potential and in vivo efficacy of one of the most potent analogues, N‐(3‐chloro‐2‐methylphenyl)‐5‐(4‐fluoro‐3‐hydroxyphenyl)isoxazole‐3‐carboxamide (60), was validated in a biologically relevant zebrafish model of collagen VI congenital muscular dystrophies.

N‐Phenylbenzamides as Potent Inhibitors of the Mitochondrial Permeability Transition Pore

Persistent opening of the mitochondrial permeability transition pore (PTP), an inner membrane channel, leads to mitochondrial dysfunction and renders the PTP a therapeutic target for a host of life‐threatening diseases. Herein, we report our effort toward identifying small‐molecule inhibitors of this target through structure–activity relationship optimization studies, which led to the identification of several potent analogues around the N‐phenylbenzamide compound series identified by high‐throughput screening. In particular, compound 4 (3‐(benzyloxy)‐5‐chloro‐N‐(4‐(piperidin‐1‐ylmethyl)phenyl)benzamide) displayed noteworthy inhibitory activity in the mitochondrial swelling assay (EC50=280 nm), poor‐to‐very‐good physicochemical as well as in vitro pharmacokinetic properties, and conferred very high calcium retention capacity to mitochondria. From the data, we believe compound 4 in this series represents a promising lead for the development of PTP inhibitors of pharmacological relevance.

The translocator protein (peripheral benzodiazepine receptor) mediates rat-selective activation of the mitochondrial permeability transition by norbormide

We have investigated the mechanism of rat-selective induction of the mitochondrial permeability transition (PT) by norbormide (NRB). We show that the inducing effect of NRB on the PT (i) is inhibited by the selective ligands of the 18kDa outer membrane (OMM) translocator protein (TSPO, formerly peripheral benzodiazepine receptor) protoporphyrin IX, N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide and 7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one; and (ii) is lost in digitonin mitoplasts, which lack an intact OMM. In mitoplasts the PT can still be induced by the NRB cationic derivative OL14, which contrary to NRB is also effective in intact mitochondria from mouse and guinea pig. We conclude that selective NRB transport into rat mitochondria occurs via TSPO in the OMM, which allows its translocation to PT-regulating sites in the inner membrane. Thus, species-specificity of NRB toward the rat PT depends on subtle differences in the structure of TSPO or of TSPO-associated proteins affecting its substrate specificity.