Cyclophilin Research Articles

Cyclophilin A and EMMPRIN (CD147) in cardiovascular diseases

Cyclophilin A (CyPA) is an abundantly expressed intracellular protein. It exerts a variety of functions due to its peptidyl-prolyl cis-trans isomerase (PPIase) activity. When released into the extracellular space, CyPA binds to its extracellular receptor CD147 (EMMPRIN) and thereby initiates a cascade of inflammatory processes. Recent data indicate that both extra- and intracellular CyPA significantly contribute to cardiovascular inflammation, myocardial ischaemia and reperfusion injury, and myocardial remodelling processes. Thus, CyPA appears to represent a novel target to treat vascular and myocardial inflammation.

CD137-CD137L Interaction Regulates Atherosclerosis via Cyclophilin A in Apolipoprotein E-Deficient Mice

BackgroundOur previous studies showed that increased levels of cyclophilin A (CyPA) may be a valuable marker for predicting the severity of acute coronary syndromes and that interruption of CD137-CD137L interactions diminished the formation and progression of atherosclerosis in apolipoprotein E-deficient (ApoE−/−) mice. Here, we sought to determine whether the proinflammatory factor CyPA is involved in atherosclerosis regulated by CD137-CD137L interactions.Methods and ResultsA constrictive collar was placed around the right carotid arteries of ApoE−/− mice that were fed a high-fat diet to induce atherosclerotic plaque formation. After that, the mice were intraperitoneally injected with anti-CD137 or anti-CD137L in the presence or absence of the recombinant lentiviral vectors LVTHM-CyPA or pGC-FU-CyPA, respectively. Interestingly, activation of CD137-CD137L was negatively correlated with CyPA expression in vivo and in vitro. Stimulating CD137-CD137L interaction significantly increased CyPA, which was concurrent with the upregulation of proinflammatory cytokines, chemokines and matrix metalloproteinases and resulted in the promotion of atherosclerosis in ApoE-/- mice. Silencing CyPA could eliminate these effects, and restoration of CyPA effectively and consistently attenuated the atherosclerotic suppression phenotypes elicited by the blockade of CD137-CD137L.ConclusionThese observations suggest that CD137-CD137L interactions mediated via regulation of CyPA contribute to the progression of atherosclerosis.

Cloning and expression of cyclophilin A gene from Echinoccocus granulosus

Objective In order to assess the value of Echinoccocus granulosus cyclophilin A (EgypA) in immune diagnosis, this novel gene was cloned and expressed. Methods By screening the EST library, the coding region of EgCypA was identified, and the PCR primers were designed based on this sequence.Bioinformatic tools were used to deduce the amino acid sequence of EgCypA and analyzed its biological characteristics.EgCypA was amplified from E.granulosus cDNA library by using PCR.Then, it was cloned into the prokaryotic expression vector pET28a and transformed into E.coli BL21.The recombinant protein was expressed in E.coli BL21 after IPTG induction.The immunogenicity of EgCypA was evaluated by Western blot using the Echinoccocus granulosus and other parasites infected animals′ sera. Results Results of SDS-PAGE electrophoresis show that the recombinant BL21 expressed 18 400-25 000 protein, which was identical with the molecular weight calculated by bioinformatic analysis.Western blot shows that the recombinant protein only reacted with its immune serum and E.granulosus cystic fluid immune serum, and EgCypA immune serum could react with the excretion and secretion antigen from E.granulosus protoscolexes, and no cross-reaction between EgCypA and sera from other parasites infected animals. Conclusions Cloning and expression of EgCypA are successful.EgCypA has good immunologic activity and could be a candidate molecular for immune diagnosis of echinococcosis in early stage.（Chin J Lab Med,2014,37:56-59） Key words: Echinococcus granulosus; Cyclophilin A; Sequence analysis; Recombinant proteins; Blotting, western

HAb18G/CD147 Promotes pSTAT3-Mediated Pancreatic Cancer Development via CD44s

Signal transducer and activator of transcription 3 (STAT3) plays a critical role in initiation and progression of pancreatic cancer. However, therapeutically targeting STAT3 has failed clinically. We previously identified HAb18G/CD147 as an effective target for cancer treatment. In this study, we aimed to investigate the potential role of HAb18G/CD147 in STAT3-involved pancreatic tumorigenesis in vitro and in vivo. The expression of HAb18G/CD147, pSTAT3, and CD44s was determined in tissue microarrays. The tumorigenic function and molecular signaling mechanism of HAb18G/CD147 were assessed by in vitro cellular and clonogenic growth, reporter assay, immunoblot assay, immunofluorescence staining, immunoprecipitation, and in vivo tumor formation using loss or gain-of-function strategies. Highly expressed HAb18G/CD147 promoted cellular and clonogenic growth in vitro and tumorigenicity in vivo. Cyclophilin A (CyPA), a ligand of CD147, stimulated STAT3 phosphorylation and its downstream genes cyclin D1/survivin through HAb18G/CD147-dependent mechanisms. HAb18G/CD147 was associated and colocalized with cancer stem cell marker CD44s in lipid rafts. The inhibitors of STAT3 and survivin, as well as CD44s neutralizing antibodies suppressed the HAb18G/CD147-induced cell growth. High HAb18G/CD147 expression in pancreatic cancer was significantly correlated with the poor tumor differentiation, and the high coexpression of HAb18G/CD147-CD44s-STAT3 associated with poor survival of patients with pancreatic cancer. We identified HAb18G/CD147 as a novel upstream activator of STAT3, which interacts with CD44s and plays a critical role in the development of pancreatic cancer. The data suggest that HAb18G/CD147 could be a promising therapeutic target for highly aggressive pancreatic cancer and a surrogate marker in the STAT3-targeted molecular therapies.

Acetylation of cyclophilin A is required for its secretion and vascular cell activation

Cyclophilin A (CyPA) is a pro-inflammatory mediator involved in oxidative stress-related cardiovascular diseases. It is secreted from vascular smooth muscle cell (VSMC) in response to reactive oxygen species (ROS) in a highly regulated manner. Extracellular CyPA activates VSMCs and endothelial cells (ECs) promoting inflammation, cell growth, and cell death. Recently, it was shown that acetylated CyPA (AcK-CyPA) affects its function. We investigated the role of acetylation of CyPA for its secretion and signalling in vascular cells. We used angiotensin II (Ang II) to create sustained ROS and found significantly increased AcK-CyPA in VSMC. Site-directed mutagenesis showed that lysines K82 and K125 were the predominant CyPA residues acetylated in response to Ang II. Importantly, acetylation of K82 and K125 were required for Ang II-mediated CyPA secretion. ROS inhibitors, Tiron, and N-acetylcysteine inhibited Ang II-induced intracellular CyPA acetylation and also AcK-CyPA secretion. Using secreted CyPA from wild type and K82/125R mutants expressed in transduced VSMC or in vitro acetylated recombinant CyPA, we showed that extracellular AcK-CyPA significantly increased pERK1/2, matrix metalloproteinase-2 activation, and ROS production in VSMC compared with non-acetylated CyPA. Moreover, extracellular AcK-CyPA increased adhesion molecule expression (VCAM-1 and ICAM-1) in EC, which promoted monocyte adhesion. ROS-dependent acetylation of CyPA is required for the generation of extracellular CyPA. Acetylated extracellular CyPA regulates VSMC and EC activation, suggesting that inhibition of acetylation of CyPA may prevent the pathogenesis of oxidative stress-related cardiovascular diseases.

Leukocyte Adhesion and Activation in Atherothrombosis

Platelets interact with a variety of cells, including leukocytes and progenitor cells. Upon activation, platelets release chemokines such as CXCL12/SDF-1 that modulate important functional aspects of vascular inflammation and atherogenesis. Platelet-derived SDF-1 and CyPA regulate chemotaxis, migration, and cell activation in the microenvironment of accumulating platelets. Previously we have shown that platelets are the major source of CXCL12/SDF-1. Platelet-derived SDF-1 provides a strong signal for recruitment of circulating progenitor cells and leukocytes towards vascular and myocardial injury. Patients with severe coronary artery disease have enhanced levels of plasma and platelet-associated SDF-1 that correlates with an enhanced number of circulating progenitor cells and the development of cardiovascular diseases. In patients with both acute myocardial infarction platelet-SDF-1 and an increase in circulating platelet/progenitor cell aggregates, the combination is associated with myocardial recovery and improved prognosis, indicating that platelet-SDF-1 is critical for repair and regeneration of vascular or myocardial injury. Administration of a stable recombinant SDF-1 fusion protein reduces infarct size and preserves myocardial infarction in mice. We showed that platelet-derived SDF-1 is an important survival factor both for platelets (in an autocrine manner) and for surrounding cells. SDF-1 released from activated platelets binds to its receptor CXCR4 and induces internalization of the SDF-1/CXCR4 complex. SDF-1-ligation of CXCR4 augments surface expression of CXCR7; a process coupled via ERK1/2 and cyclophilin A. SDF-1α caused downstream phosphorylation of Erk1/2 and cyclophilin A (CyPA). NIM-811 a CyPA-PPIase-activity inhibitor significantly abolished SDF-1α-driven CXCR7 surface exposure. Moreover, Cypa-/-murine platelets failed to show SDF-1α/CXCR4-mediated CXCR7 translocation. SDF-1α-induced ubiquitination of CXCR7 was essential for its surface translocation and was dependent on Erk1/2 and CyPA-PPIase activity, inhibited by U0126 and NIM-811, respectively. In contrast to wild-type, Cypa-/- murine platelets failed to exhibit a dynamic change in CXCR7 ubiquitination status upon SDF-1α exposure and thus failed in subsequent CXCR7 externalization. SDF-1/CXR4-driven CXCR7 translocation inhibits activation-induced apoptosis of platelets and prolongs platelet survival in vitro and in vivo. Similarly, platelet SDF-1 interacts with monocytes, reduces apoptosis and promotes differentiation of monocytes into macrophages. Inhibition of platelet release and adhesion to endothelial cells reduces monocyte migration and differentiation into macrophages in vitro and attenuates vascular inflammation and atheroprogression in apoE-deficient mice. In conclusion, platelets and platelet interaction with leukocytes are critical in regulation of vascular inflammation and atheroprogression and may offer promising targets for modulating inflammation in disease states. Disclosures:No relevant conflicts of interest to declare.

Cyclophilin-A: a novel biomarker for untreated male essential hypertension

Vascular cytokines, total nitrite, and cyclophilin-A (CyP-A) may be related to the pathogenesis of untreated hypertension. Forty males with normotensive and untreated essential hypertension were recruited in this cytokines survey. Body mass index (BMI), hyperlipidemia, and plasma CyP-A were increased in the hypertensive group (p < 0.05). However, only BMI (p = 0.022) and plasma CyP-A (p = 0.020) were found to be significant contributors to hypertension by multiple regression analysis. CyP-A was also positively correlated with systolic blood pressure (p = 0.029) and diastolic blood pressure (p = 0.047). These findings indicated that plasma CyP-A is a critical molecular biomarker in the early pathogenesis of essential hypertension.

Serum cyclophilin A concentrations in renal transplant recipients receiving cyclosporine A: clinical implications for gingival overgrowth

The aim of this study was to investigate the clinical factors in relation to the cyclosporine A (CsA) induced gingival overgrowth (GO). Seventy-three participants were assigned as GO+ and GO-. Factors including demographic, pharmacological, gingival variables and the serum cyclophilin A (CyPA) concentration were analyzed. The occurrence of GO was 39.72%. Papillary bleeding index (PBI) had a significantly higher risk of GO than plaque index (PI), the ratio of CsA to CyPA, and serum CyPA concentration (odds ratio=364.323, 25.791, 1.002, 0.096, respectively). The severity of GO correlated with PI, the ratio of CsA to CyPA, PBI, serum concentrations of CsA and CyPA (r=0.366, 0.355, 0.344, 0.305, and-0.232, respectively). Since a cross-sectional study is not able to explain whether plaque and inflammation are the cause or consequence of GO, the ratio of CsA to CyPA may be a valuable marker for predicting GO.

The role of cyclophilin D in interspecies differences in susceptibility to hepatotoxic drug-induced mitochondrial injury

Test compound A ((5Z)-6-[(2R,3S)-3-({[(4-Chloro-2-methylphenyl)sulfonyl]amino}methyl) bicyclo[2.2.2]oct-2-yl]hex-5-enoic acid) was withdrawn from premarketing clinical trials due to severe liver injury. Intracellular accumulation of lipids (steatosis) has been observed in human-derived cells and may account for the severe hepatotoxicity. Mitochondrial β-oxidation and ketogenesis play a fundamental role in energy homeostasis. Mitochondrial dysfunction can therefore cause severe deficiency in fatty acid oxidation and apoptosis which finally triggers the hepatocellular injury. Some of hepatotoxic drugs (e.g., salicylic acid, diclofenac and troglitazone) are known to induce mitochondrial dysfunction. This study therefore examined the effect of compound A on the mitochondrial permeability transition (MPT) and membrane potential in mitochondria isolated from mouse, rat and monkey livers. The incubation of rat and monkey mitochondria energized by succinate in the presence of Ca2+ (20μM) and compound A (2.5–10μM) resulted in cyclosporin A (CsA)-sensitive MPT pore opening and a decline in mitochondrial membrane potential in a concentration-dependent manner. However, mouse mitochondria showed low susceptibility to compound A-induced dysfunction. Rat mitochondrial expression of cyclophilin D (CyPD) was about twice that of mouse mitochondria, but the expression levels of other MPT pore proteins (adenine nucleotide translocator and voltage-dependent anion channel) were comparable in both species. An assessment of the effect of compound A on CyPD knockdown cells demonstrated that mitochondrial susceptibility to compound A was attenuated in CyPD knockdown cells. These results suggest that an interspecies difference in the susceptibility to mitochondrial dysfunction induced by compound A exists as a result of species-specific discrepancies in CyPD expression.

Defining reference genes for qPCR normalization to study biotic and abiotic stress responses in Vigna mungo

Expression of ACT, EF1A; H2A, EF1A, ACTand 18S, TUB showed stability under MYMIV, salinity and drought stress, respectively; these are recommended as reference genes for qPCR normalization in Vigna mungo. Accurate gene expression profiling through qPCR depends on selection of appropriate reference gene(s) for normalization. Due to lack of unanimous internal standard, suitable constitutively expressed reference genes are selected that exhibit stable expression under diverse experimental conditions. In this communication, a comparative evaluation of stability among seven V. mungo genes encoding actin (ACT), histone H2A (H2A), elongation factor 1-alpha (EF1A), 18S rRNA (18S), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), cyclophilin (CYP) and tubulin (TUB) under biotic (MYMIV) and abiotic (drought and salinity) stress conditions has been attempted. Specificity and amplification efficiency for each primer pair were verified; however, cumulative assessment of their accumulated transcripts revealed no uniformity. Therefore, individual stability and suitability of these seven candidates have been assessed in silico, by two widely used algorithms, geNorm and Normfinder. Based on the computed results, high stability was obtained for ACT and EF1A during MYMIV stress, while H2A, EFIA and ACT were found to be most suitable in salinity stress experiments and TUB and 18S during drought treatments. Combinations of ACT/TUB or ACT/EFIA were recommended for their use in the pooled analysis, while expression of 18S and CYP showed greater variations and therefore considered unsuitable as reference genes. Additionally, precise quantification of the target gene VmPRX under these stresses was shown to be a function of reference genes' stability, which tends to get affected when normalized with the least stable genes. Hence, use of these normalizers will facilitate accurate and reliable analyses of gene expression in V. mungo.

Cyclophilin A Is Required for Angiotensin II–Induced p47phox Translocation to Caveolae in Vascular Smooth Muscle Cells

Angiotensin II (AngII) signal transduction in vascular smooth muscle cells (VSMC) is mediated by reactive oxygen species (ROS). Cyclophilin A (CyPA) is a ubiquitously expressed cytosolic protein that possesses peptidyl-prolyl cis-trans isomerase activity, scaffold function, and significantly enhances AngII-induced ROS production in VSMC. We hypothesized that CyPA regulates AngII-induced ROS generation by promoting translocation of NADPH oxidase cytosolic subunit p47phox to caveolae of the plasma membrane. Overexpression of CyPA in CyPA-deficient VSMC (CyPA(-/-)VSMC) significantly increased AngII-stimulated ROS production. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors (VAS2870 or diphenylene iodonium) significantly attenuated AngII-induced ROS production in CyPA and p47phox-overexpressing CyPA(-/-)VSMC. Cell fractionation and sucrose gradient analyses showed that AngII-induced p47phox plasma membrane translocation, specifically to the caveolae, was reduced in CyPA(-/-)VSMC compared with wild-type-VSMC. Immunofluorescence studies demonstrated that AngII increased p47phox and CyPA colocalization and translocation to the plasma membrane. In addition, immunoprecipitation of CyPA followed by immunoblotting of p47phox and actin showed that AngII increased CyPA and p47phox interaction. AngII-induced p47phox and actin cell cytoskeleton association was attenuated in CyPA(-/-)VSMC. Mechanistically, inhibition of p47phox phosphorylation and phox homology domain deletion attenuated CyPA and p47phox interaction. Finally, cyclosporine A and CyPA-peptidyl-prolyl cis-trans isomerase mutant, R55A, inhibited AngII-stimulated CyPA and p47phox association in VSMC, suggesting that peptidyl-prolyl cis-trans isomerase activity was required for their interaction. These findings provide the mechanism by which CyPA is an important regulator for AngII-induced ROS generation in VSMC through interaction with p47phox and cell cytoskeleton, which enhances the translocation of p47phox to caveolae.

Abstract 281: Cyclophilin A is a Novel Biomarker for Oxidative Stress and Atherosclerotic Diseases

Background We have recently demonstrated that oxidative stress induces secretion of cyclophilin A (CyPA) from vascular smooth muscle cells, which plays a crucial role in the pathogenesis of atherosclerosis and abdominal aortic aneurysms (AAA) in mice. Thus, we tested our hypothesis that plasma CyPA levels could be a biomarker of atherosclerotic diseases such as coronary artery disease (CAD) and AAA in humans. Methods and Results In consecutive 320 patients undergoing coronary angiography, we examined the relationship between the severity of CAD and plasma CyPA levels measured by an immunoassay. Plasma CyPA levels were significantly higher in patients with significant coronary stenosis (&gt;50%, n=189) compared to those without it (n=131) (P&lt;0.001). A significant positive correlation was noted between plasma CyPA levels and significant coronary stenosis even after adjustment for age, sex, hypertension, diabetes, dyslipidemia and smoking. The average number of coronary arteries with significant stenosis and the need for coronary intervention were significantly increased in the quartiles of higher CyPA levels (both P&lt;0.001). In addition, plasma CyPA levels were significantly correlated with the number of coronary arteries with significant stenosis (P&lt;0.001). Indeed, plasma CyPA levels were a strong predictor of CAD (adjusted odds ratio for CAD, 6.20; 95% confidence interval, 3.14-12.27; P&lt;0.001). Interestingly, plasma levels of CyPA increased according to the number of atherosclerotic risk factors that enhance oxidative stress. Furthermore, plasma CyPA levels significantly reduced after medical treatment of those risk factors. Finally, pathological examinations showed that CyPA was strongly expressed in atherosclerotic coronary plaque of patients with myocardial infarction. In a separate study, we examined the plasma CyPA levels in patients with aortic aneurysms. Plasma CyPA levels were significantly higher in patients with aortic aneurysms (n=58) compared to those without it (n=69) (P&lt;0.001). Importantly, plasma CyPA levels were significantly higher in patients coexisting with CAD (n=18) compared to those only with aortic aneurysms (n=40) (P=0.012). Conclusion Plasma CyPA level is a novel biomarker of atherosclerotic diseases.

Decreased Tissue Levels of Cyclophilin A, a Cyclosporine A Target and Phospho-ERK1/2 in Simvastatin Patients with Abdominal Aortic Aneurysm

Cyclophilin A (CyPA), a cyclosporine A-binding protein, influences abdominal aortic aneurysm (AAA) formation and the ERK1/2 signalling pathway in animal and in vitro studies. Statins decrease CyPA in smooth muscle cells although their influence on CyPA in human AAA is unknown. The study was performed on AAA wall-tissue samples obtained from 30 simvastatin-treated and 15 non-statin patients (2:1 case to control). The patients were matched by age, sex and AAA diameter. We investigated the gene expression of CyPA, its receptor extracellular matrix metalloproteinase inducer (EMMPRIN) by real-time RT-PCR. CyPA and EMMPRIN protein level and phosphorylated extracellular signal-regulated kinases 1 and 2 (ERK1/2) were measured by Western blot. The AAA wall tissue from simvastatin-treated patients had significantly lower CyPA gene expression and protein levels (P = 0.0018, P = 0.0083, respectively). Furthermore, phosphorylation of ERK1 and ERK2 was markedly suppressed in the simvastatin group (P = 0.0002, P = 0.0027, respectively). However, simvastatin did not influence EMMPRIN gene and protein expression. Simvastatin-treated patients with AAA exert lower CyPA messenger RNA (mRNA), as well as CyPA intracellular protein levels and a decreased amount of phospho-ERK1/2. Thus, the interference with signalling pathways leading to CyPA formation and ERK1/2 activation reveals a new anti-inflammatory role of statins in AAA.

Calcium dependent signaling in cardiac hypertrophy and cell death

Ca(2 +) plays a pivotal role in excitation-contraction coupling as well as activation of Ca(2 +) -dependent signaling pathways in the myocardium. The two main established Ca(2 +) -dependent pathways during cardiomyocyte hypertrophy are the Ca(2 +) /calmodulin-calcineurin-NFAT and the Ca(2 +) /calmodulin dependent kinases II -HDAC-MEF2. Both pathways are involved in the transcriptional control of many hypertrophic genes. Recently, cyclophilin D, an immunophilin located on mitochondrial inner membrane, has been highlighted as a regulator of calcium-mediated mitochondrial permeability transition in necrotic cardiac cell death.

Inhibitory effect of cyclophilin A from the hard tick Haemaphysalis longicornis on the growth of Babesia bovis and Babesia bigemina

Haemaphysalis longicornis is known as one of the most important ticks transmitting Babesia parasites in East Asian countries, including Babesia ovata and Babesia gibsoni, as well as Theileria parasites. H. longicornis is not the natural vector of Babesia bovis and Babesia bigemina. Vector ticks and transmitted parasites are thought to have established unique host-parasite interaction for their survival, meaning that vector ticks may have defensive molecules for the growth control of parasites in their bodies. However, the precise adaptation mechanism of tick-Babesia parasites is still unknown. Recently, cyclophilin A (CyPA) was reported to be important for the development of Babesia parasites in ticks. To reveal a part of their adaptation mechanism, the current study was conducted. An injection of B. bovis-infected RBCs into adult female H. longicornis ticks was found to upregulate the expression profiles of the gene and protein of CyPA in H. longicornis (HlCyPA). In addition, recombinant HlCyPA (rHlCyPA) purified from Escherichia coli exhibited significant inhibitory growth effects on B. bovis and B. bigemina cultivated in vitro, without any hemolytic effect on bovine RBCs at all concentrations used. In conclusion, our results suggest that HlCyPA might play an important role in the growth regulation of Babesia parasites in H. longicornis ticks, during natural acquisition from an infected host. Furthermore, rHlCyPA may be a potential alternative chemotherapeutic agent against babesiosis.

Dimers of mitochondrial ATP synthase form the permeability transition pore

Here we define the molecular nature of the mitochondrial permeability transition pore (PTP), a key effector of cell death. The PTP is regulated by matrix cyclophilin D (CyPD), which also binds the lateral stalk of the FOF1 ATP synthase. We show that CyPD binds the oligomycin sensitivity-conferring protein subunit of the enzyme at the same site as the ATP synthase inhibitor benzodiazepine 423 (Bz-423), that Bz-423 sensitizes the PTP to Ca(2+) like CyPD itself, and that decreasing oligomycin sensitivity-conferring protein expression by RNAi increases the sensitivity of the PTP to Ca(2+). Purified dimers of the ATP synthase, which did not contain voltage-dependent anion channel or adenine nucleotide translocator, were reconstituted into lipid bilayers. In the presence of Ca(2+), addition of Bz-423 triggered opening of a channel with currents that were typical of the mitochondrial megachannel, which is the PTP electrophysiological equivalent. Channel openings were inhibited by the ATP synthase inhibitor AMP-PNP (γ-imino ATP, a nonhydrolyzable ATP analog) and Mg(2+)/ADP. These results indicate that the PTP forms from dimers of the ATP synthase.

Extracellular cyclophilin A protects against blast-induced neuronal injury

Blast-induced traumatic brain injury (TBI) and subsequent neurobehavioral deficits are major disabilities suffered by the military and civilian population worldwide. Rigorous scientific research is underway to understand the mechanism of blast TBI and thereby develop effective therapies for protection and treatment. By using an in vitro shock tube model of blast TBI with SH-SY5Y human neuroblastoma cells, we have demonstrated that blast exposure leads to neurobiological changes in an overpressure and time dependent manner. Paradoxically, repeated blast exposures resulted in less neuronal injury compared to single blast exposure and suggested a potential neuroprotective mechanism involving released cyclophilin A (CPA). In the present study, we demonstrate accumulation of CPA in the culture medium after repeated blast exposures supporting the notion of extracellular CPA mediated neuroprotection. Post-exposure treatment of the cells with purified recombinant CPA caused significant protection against blast-induced neuronal injury. Furthermore, repeated blast exposure was associated with phosphorylation of the proteins ERK1/2 and Bad suggesting a potential mechanism of neuroprotection by extracellular CPA and may aid in the development of targeted therapies for protection against blast-induced TBI.

Proteomic methods reveal cyclophilin a function as a host restriction factor against rotavirus infection

Rotavirus (RV) infection is the main cause of acute dehydrating diarrhea in infants and young children below 5 years old worldwide. RV infection causes a global shutoff of host proteins as many other viruses do. However, previous studies revealed that RV could selectively upregulated the expression of some host proteins that then played important roles in RV infection. To globally explor such host proteins that were upregulated in early human rotavirus (HRV) infection, proteomic methods were used and a total of ten upregulated host proteins were unambiguously identified. Cyclophilin A (CYPA), a peptidyl-prolyl cis-trans isomerase, was among these upregulated host proteins. Following infection, CYPA was recruited to the viroplasm and interacted with HRV structural protein VP2; CYPA reduced host susceptibility to HRV infection and inhibited replication of HRV by repressing the expression of viral proteins. Furthermore, we found that the increased expression of CYPA in enterocytes of small intestine correlated to the period when BALB/c mice became resistant to RV diarrhea. Together, we identified CYPA as a novel host restriction factor that confered protection against RV infection and might contribute to host susceptibility to RV diarrhea.

Role of the c subunit of the FO ATP synthase in mitochondrial permeability transition

The term “mitochondrial permeability transition” (MPT) refers to an abrupt increase in the permeability of the inner mitochondrial membrane to low molecular weight solutes. Due to osmotic forces, MPT is paralleled by a massive influx of water into the mitochondrial matrix, eventually leading to the structural collapse of the organelle. Thus, MPT can initiate mitochondrial outer membrane permeabilization (MOMP), promoting the activation of the apoptotic caspase cascade as well as of caspase-independent cell death mechanisms. MPT appears to be mediated by the opening of the so-called “permeability transition pore complex” (PTPC), a poorly characterized and versatile supramolecular entity assembled at the junctions between the inner and outer mitochondrial membranes. In spite of considerable experimental efforts, the precise molecular composition of the PTPC remains obscure and only one of its constituents, cyclophilin D (CYPD), has been ascribed with a crucial role in the regulation of cell death. Conversely, the results of genetic experiments indicate that other major components of the PTPC, such as voltage-dependent anion channel (VDAC) and adenine nucleotide translocase (ANT), are dispensable for MPT-driven MOMP. Here, we demonstrate that the c subunit of the FO ATP synthase is required for MPT, mitochondrial fragmentation and cell death as induced by cytosolic calcium overload and oxidative stress in both glycolytic and respiratory cell models. Our results strongly suggest that, similar to CYPD, the c subunit of the FO ATP synthase constitutes a critical component of the PTPC.

1H, 13C, and 15N Backbone and Sidechain Resonance Assignments for Cyclophilin A from Geobacillus Kaustophilus

1H, 13C, and 15N Backbone and Sidechain Resonance Assignments for Cyclophilin A from Geobacillus Kaustophilus