Absence Of Cyclophilin Research Articles

Cyclophilin D ablation is associated with increased end-ischemic mitochondrial hexokinase activity

Both the absence of cyclophilin D (CypD) and the presence of mitochondrial bound hexokinase II (mtHKII) protect the heart against ischemia/reperfusion (I/R) injury. It is unknown whether CypD determines the amount of mtHKII in the heart. We examined whether CypD affects mtHK in normoxic, ischemic and preconditioned isolated mouse hearts. Wild type (WT) and CypD−/− mouse hearts were perfused with glucose only and subjected to 25 min ischemia and reperfusion. At baseline, cytosolic and mtHK was similar between hearts. CypD ablation protected against I/R injury and increased ischemic preconditioning (IPC) effects, without affecting end-ischemic mtHK. When hearts were perfused with glucose, glutamine, pyruvate and lactate, the preparation was more stable and CypD ablation−resulted in more protection that was associated with increased mtHK activity, leaving little room for additional protection by IPC. In conclusion, in glucose only-perfused hearts, deletion of CypD is not associated with end-ischemic mitochondrial-HK binding. In contrast, in the physiologically more relevant multiple-substrate perfusion model, deletion of CypD is associated with an increased mtHK activity, possibly explaining the increased protection against I/R injury.

Cyclophilin A enhances macrophage differentiation and lipid uptake in high glucose conditions: a cellular mechanism for accelerated macro vascular disease in diabetes mellitus.

BackgroundVascular disease in diabetes is initiated by monocyte adhesion to vascular endothelium, transmigration and formation of foam cells. Increasing clinical evidence supports a role for the secretory protein, cyclophilin A in diabetic vascular disease. The means by which cyclophilin A contributes to vascular lesion development in diabetes is however largely unknown.MethodsIn this study we investigated using THP1 cells and human monocytes whether cyclophilin A under hyperglycemic conditions, functions in the inflammatory cascade as a chemoattractant and increases lipid uptake by formation of foam cells invitro. We developed an invitro model of monocytes cultured in 20 mm glucose (high glucose) equivalent to 360 mg/dL of plasma glucose levels. These monocytes were then differentiated into macrophages using PMA and subsequently transformed to lipid laden foam cells using oxidized low density lipoproteins in the presence and absence of cyclophilin A. This cellular model was used to study monocyte to macrophage differentiation, transmigration and foam cell formation. A similar cellular model using siRNA mediated transient elimination of the cyclophilin A gene as well as chemical inhibitors were used to further confirm the role of cyclophilin A in the differentiation and foam cell formation process.ResultsCyclophilin A effectively increased migration of high glucose treated monocytes to the endothelial cell monolayer (p < 0.0001). In the presence of cyclophilin A, differentiated macrophages, when treated with oxLDL had a 36 percent increase in intracellular lipid accumulation (p = 0.01) when compared to cells treated with oxLDL alone. An increased flux of reactive oxygen species was also observed (p = 0.01). Inflammatory cytokines such as TNF-α, MCP-1 and cyclophilin A were significantly increased. Silencing cyclophilin A in THP-1 cells and human monocytes using siRNA or chemical inhibitor, TMN355 resulted in decrease in lipid uptake by 65–75% even after exposure to oxidized LDL. The expression of scavenger receptors expressed during differentiation process, CD36 and LOX-1 were decreased (p < 0.0001). Levels of extracellular cyclophilin A and other inflammatory cytokines such as TNF-α and MCP-1also significantly reduced.ConclusionsTaken together, we describe here a possible cellular basis by which cyclophilin A may accelerate atherogenesis in diabetes mellitus.Electronic supplementary materialThe online version of this article (doi:10.1186/s12933-016-0467-5) contains supplementary material, which is available to authorized users.

Role of Cyclophilin on Amyloid Formation and Protein Disaggregation

Oligomers and fibrils of protein and peptides are implicated in memory storage, neurotransmission and formation of amyloid diseases. It is believed that partially misfolded/folded states are trapped in a certain conformation and it eventually leads to formation of larger and more stable fibrillar aggregates. Recently, our laboratory demonstrated that AdK-aggregate could be disaggregated by cyclophilin CyP (LdCyP) in an isomerase- independent fashion, resulting in reactivation [Ref. 1]. We also observed the formation of elongated fibrils when lysozyme was co-incubated with cyclophilin [Ref. 2]. Very recently we tested the self aggregation process of insulin in the presence and absence of cyclophilin. The formation of insulin fiber was confirmed by ThT fluorescence assay when insulin was incubated in acidic condition at 65oC. However, co-incubation with cyclophilin, hinder the formation of amyloid fiber. Cyclophilin also showed potential to disaggregate amyloid aggregate of insulin. Cyclophilin itself was not formed amyloid fibril upon incubation for 50 days. TEM images, however, indicated formations of amorphous type of aggregates upon prolong incubation for several days at 65 oC. In our study, possibly, cyclophilin, helped to preserve the insulin native structure intact even at high temperature and did not allow it to partially unfold that may initiate the amyloid formation. Ref. 1. Mukherjee, D ; Patra, H ; Laskar, A ; Dasgupta, A ; Maiti, NC and Datta, AK, Archives Biochem. Biophys. 2013, 537, 82-90 Ref. 2. Das S., Pal U., Das S. and Maiti N.C. , Journal of Proteins and Proteomics, 4(2), 129-137, 2013.

Mitochondrially Mediated Integrin αIIbβ3 Protein Inactivation Limits Thrombus Growth

When platelets are strongly stimulated, a procoagulant platelet subpopulation is formed that is characterized by phosphatidylserine (PS) exposure and epitope modulation of integrin αIIbβ3 or a loss of binding of activation-dependent antibodies. Mitochondrial permeability transition pore (mPTP) formation, which is essential for the formation of procoagulant platelets, is impaired in the absence of cyclophilin D (CypD). Here we investigate the mechanisms responsible for these procoagulant platelet-associated changes in integrin αIIbβ3 and the physiologic role of procoagulant platelet formation in the regulation of platelet aggregation. Among strongly stimulated adherent platelets, integrin αIIbβ3 epitope changes, mPTP formation, PS exposure, and platelet rounding were closely associated. Furthermore, platelet mPTP formation resulted in a decreased ability to recruit additional platelets. In the absence of CypD, integrin αIIbβ3 function was accentuated in both static and flow conditions, and, in vivo, a prothrombotic phenotype occurred in mice with a platelet-specific deficiency of CypD. CypD-dependent proteolytic events, including cleavage of the integrin β3 cytoplasmic domain, coincided closely with integrin αIIbβ3 inactivation. Calpain inhibition blocked integrin β3 cleavage and inactivation but not mPTP formation or PS exposure, indicating that integrin inactivation and PS exposure are mediated by distinct pathways subsequent to mPTP formation. mPTP-dependent alkalinization occurred in procoagulant platelets, suggesting a possible alternative mechanism for enhancement of calpain activity in procoagulant platelets. Together, these results indicate that, in strongly stimulated platelets, mPTP formation initiates the calpain-dependent cleavage of integrin β3 and associated regulatory proteins, resulting in integrin αIIbβ3 inactivation, and demonstrate a novel CypD-dependent negative feedback mechanism that limits platelet aggregation and thrombotic occlusion.

Enhancing Mitochondrial Calcium Buffering Capacity Reduces Aggregation of Misfolded SOD1 and Motor Neuron Cell Death without Extending Survival in Mouse Models of Inherited Amyotrophic Lateral Sclerosis

Mitochondria have been proposed as targets for toxicity in amyotrophic lateral sclerosis (ALS), a progressive, fatal adult-onset neurodegenerative disorder characterized by the selective loss of motor neurons. A decrease in the capacity of spinal cord mitochondria to buffer calcium (Ca(2+)) has been observed in mice expressing ALS-linked mutants of SOD1 that develop motor neuron disease with many of the key pathological hallmarks seen in ALS patients. In mice expressing three different ALS-causing SOD1 mutants, we now test the contribution of the loss of mitochondrial Ca(2+)-buffering capacity to disease mechanism(s) by eliminating ubiquitous expression of cyclophilin D, a critical regulator of Ca(2+)-mediated opening of the mitochondrial permeability transition pore that determines mitochondrial Ca(2+) content. A chronic increase in mitochondrial buffering of Ca(2+) in the absence of cyclophilin D was maintained throughout disease course and was associated with improved mitochondrial ATP synthesis, reduced mitochondrial swelling, and retention of normal morphology. This was accompanied by an attenuation of glial activation, reduction in levels of misfolded SOD1 aggregates in the spinal cord, and a significant suppression of motor neuron death throughout disease. Despite this, muscle denervation, motor axon degeneration, and disease progression and survival were unaffected, thereby eliminating mutant SOD1-mediated loss of mitochondrial Ca(2+) buffering capacity, altered mitochondrial morphology, motor neuron death, and misfolded SOD1 aggregates, as primary contributors to disease mechanism for fatal paralysis in these models of familial ALS.

Modulation of F0F1‐ATP synthase activity by cyclophilin D regulates matrix adenine nucleotide levels

Cyclophilin D was recently shown to bind to and decrease the activity of F(0)F(1)-ATP synthase in submitochondrial particles and permeabilized mitochondria [Giorgio V et al. (2009) J Biol Chem, 284, 33982-33988]. Cyclophilin D binding decreased both ATP synthesis and hydrolysis rates. In the present study, we reaffirm these findings by demonstrating that, in intact mouse liver mitochondria energized by ATP, the absence of cyclophilin D or the presence of cyclosporin A led to a decrease in the extent of uncoupler-induced depolarization. Accordingly, in substrate-energized mitochondria, an increase in F(0)F(1)-ATP synthase activity mediated by a relief of inhibition by cyclophilin D was evident in the form of slightly increased respiration rates during arsenolysis. However, the modulation of F(0)F(1)-ATP synthase by cyclophilin D did not increase the adenine nucleotide translocase (ANT)-mediated ATP efflux rate in energized mitochondria or the ATP influx rate in de-energized mitochondria. The lack of an effect of cyclophilin D on the ANT-mediated adenine nucleotide exchange rate was attributed to the ∼ 2.2-fold lower flux control coefficient of the F(0)F(1)-ATP synthase than that of ANT, as deduced from measurements of adenine nucleotide flux rates in intact mitochondria. These findings were further supported by a recent kinetic model of the mitochondrial phosphorylation system, suggesting that an ∼ 30% change in F(0)F(1)-ATP synthase activity in fully energized or fully de-energized mitochondria affects the ADP-ATP exchange rate mediated by the ANT in the range 1.38-1.7%. We conclude that, in mitochondria exhibiting intact inner membranes, the absence of cyclophilin D or the inhibition of its binding to F(0)F(1)-ATP synthase by cyclosporin A will affect only matrix adenine nucleotides levels.

Critical role for the mitochondrial permeability transition pore and cyclophilin D in platelet activation and thrombosis

AbstractMany of the cellular responses that occur in activated platelets resemble events that take place following activation of cell-death pathways in nucleated cells. We tested the hypothesis that formation of the mitochondrial permeability transition pore (MPTP), a key signaling event during cell death, also plays a critical role in platelet activation. Stimulation of murine platelets with thrombin plus the glycoprotein VI agonist convulxin resulted in a rapid loss of mitochondrial transmembrane potential (Δψm) in a subpopulation of activated platelets. In the absence of cyclophilin D (CypD), an essential regulator of MPTP formation, murine platelet activation responses were altered. CypD-deficient platelets exhibited defects in phosphatidylserine externalization, high-level surface fibrinogen retention, membrane vesiculation, and procoagulant activity. Also, in CypD-deficient platelet-rich plasma, clot retraction was altered. Stimulation with thrombin plus H2O2, a known activator of MPTP formation, also increased high-level surface fibrinogen retention, phosphatidylserine externalization, and platelet procoagulant activity in a CypD-dependent manner. In a model of carotid artery photochemical injury, thrombosis was markedly accelerated in CypD-deficient mice. These results implicate CypD and the MPTP as critical regulators of platelet activation and suggest a novel CypD-dependent negative-feedback mechanism regulating arterial thrombosis.

In VitroAssembly Properties of Wild-Type and Cyclophilin-Binding Defective Human Immunodeficiency Virus Capsid Proteins in the Presence and Absence of Cyclophilin A

The cellular protein cyclophilin A (CypA) binds specifically to the human immunodeficiency virus type 1 (HIV-1) capsid (CA) protein and is incorporated into HIV-1 particles at a molar ratio of 1:10 (CypA/CA). Structural analysis of a CA–CypA complex suggested that CypA may destabilize interactions in the viral capsid and thus promote uncoating. We analyzed the influence of CypA on thein vitroassembly properties of wild-type (WT) CA and derivatives containing substitutions of Gly89 in the Cyp-binding loop. All variant proteins were significantly impaired in CypA binding. In the presence of CypA at a molar ratio of 1:10 (CypA/CA), WT CA assembled into hollow cylinders that were similar to those observed in the absence of CypA but slightly longer. Higher CypA concentrations inhibited cylinder formation. Variant CA proteins G89L and G89F yielded similar cylinders as the WT protein but were significantly more resistant to CypA. Cryoelectron microscopic analysis of WT cylinders assembled in the presence of CypA revealed direct binding of CypA to the outer surface. Electron diffraction patterns generated from these cylinders indicated that CypA causes local disorder. The addition of CypA to preassembled cylinders had little effect, however, and cylinders were only disrupted when incubated with a threefold molar excess of CypA for several hours. These results suggest that CypA does not efficiently destabilize CA interactions at the molar ratio observed in the virion and therefore is unlikely to serve as an uncoating factor.

Gag Protein from Human Immunodeficiency Virus Type 1 Assembles in the Absence of Cyclophilin A

Human immunodeficiency virus type 1 (HIV-1) replication requires coordinated activities of host and viral factors. We reported previously that interactions of the host factor cyclophilin A with HIV-1 Gag polyproteins affected Gag processing and maturation of virus particles (Streblowet al.,1998.Virology245, 197–202). We now usein vitrotranslation and physical analysis of Gag structures to refine our understanding of how cyclophilin A affects HIV-1 replication. Gag assembled into oligomeric structuresin vitroin the presence or absence of cyclophilin A, and proteins synthesized under the two conditions were equally susceptible to cleavage by exogenous HIV-1 protease. These and previous data show that Cyclophilin A is required at a step between Gag assembly and Gag processing/virion morphogenesis. Cyclophilin A may be required for Gag conformational changes subsequent to assembly, that are required for efficient dimerization and activation of the viral protease.

Demonstration of ternary immunophilin-calcineurin complexes with the immunosuppressants cyclosporin and macrolide FK506

The specificity of cyclosporin A (CsA) binding to the major intracellular receptor proteins, cyclophilin A and B, as well as the interaction of CsA with the phosphatase calcineurin were investigated. Binding of photoaffinity-labeled CsA (PL-CS), a photoaffinity probe of CsA, to recombinant human cyclophilin A and B is saturable and specific. Non-specific PL-CS binding to calcineurin is observed in the absence of cyclophilin and calmodulin. In the presence of cyclophilin, cyclosporin-calcineurin binding becomes specific. Ternary complexes containing an equimolar ratio of cyclophilin A or B, PL-CS and calcineurin are resolved using the chemical-crosslinking technique. The formation of these complexes is specific, calcium- but not calmodulin-dependent, and is only inhibitable by cyclosporins, which bind cyclophilin. The drug-immunophilin complex binds to the calcineurin A subunit. The proteolytic 43 kDa product of calcineurin A retains binding properties, suggesting that the C-terminal domains are not necessary for complex formation. A trimeric complex of FKBP-calcineurin is also formed with FK506, but not with rapamycin. As expected, these complexes are only competed with by homologous derivatives. Chemical crosslinking of photolabeled Jurkat T-cells strongly suggests that drug-calcineurin complexes are of biological relevance.

Angiotensin-converting enzyme preferentially hydrolyzes trans isomer of proline-containing substrate

An analysis of the hydrolysis kinetics of the synthetic angiotensin-converting enzyme (ACE) substrate benzoyl-phenylalanyl-alanyl-proline (BPAP) in the intact lung suggested that 12-15% of the BPAP was in a form that could not be hydrolyzed by ACE in the time course of a single pass through the lungs [C. A. Dawson et al. Am. J. Physiol. 257 (Heart Circ. Physiol. 26): H853-H865, 1989]. BPAP has been found to exist as a mixture of cis and trans isomers in a ratio of approximately 14:86 in aqueous solution at equilibrium. Thus, one possible explanation for the incomplete hydrolysis of BPAP on passage through the intact lung is that the trans form is the preferred substrate for ACE. To examine this hypothesis, we measured BPAP hydrolysis by ACE in vitro over a range of ACE concentrations and in the presence and absence of the peptidyl-prolyl cis-trans isomerase cyclophilin. In the presence of a sufficient concentration of ACE and in the absence of cyclophilin, hydrolysis of [3H]BPAP by ACE followed biexponential progress curves, consistent with the hypothesis that the rate of hydrolysis of the majority (approximately 87%) of the substrate is proportional to ACE concentration, whereas the hydrolysis rate of the remaining substrate fraction is independent of enzyme concentration. The addition of cyclophilin resulted in an increase in the ACE-independent rate constant, an effect that was reversed by the cyclophilin inhibitor cyclosporin A. These results suggest that the enzyme-independent rate constant represents the rate of cis-trans isomerization and that the enzyme-dependent rate constant represents the hydrolysis of the trans isomer.(ABSTRACT TRUNCATED AT 250 WORDS)