Publications by authors named "Miroslav Malesevic"

38 Publications

Secreted Extracellular Cyclophilin A is a Novel Mediator of Ventilator Induced Lung Injury.

Am J Respir Crit Care Med 2021 Apr 13. Epub 2021 Apr 13.

Imperial College London, Anaesthetics & Intensive Care, London, United Kingdom of Great Britain and Northern Ireland;

Rationale: Mechanical ventilation is a mainstay of intensive care but contributes to the mortality of patients through ventilator induced lung injury. Extracellular Cyclophilin A is an emerging inflammatory mediator and metalloproteinase inducer, and the gene responsible for its expression has recently been linked to COVID-19 infection.

Objectives: Here we explore the involvement of extracellular Cyclophilin A in the pathophysiology of ventilator-induced lung injury.

Methods: Mice were ventilated with low or high tidal volume for up to 3 hours, with or without blockade of extracellular Cyclophilin A signalling, and lung injury and inflammation were evaluated. Human primary alveolar epithelial cells were exposed to in vitro stretch to explore the cellular source of extracellular Cyclophilin A, and Cyclophilin A levels were measured in bronchoalveolar lavage fluid from acute respiratory distress syndrome patients, to evaluate clinical relevance.

Measurements And Main Results: High tidal volume ventilation in mice provoked a rapid increase in soluble Cyclophilin A levels in the alveolar space, but not plasma. In vivo ventilation and in vitro stretch experiments indicated alveolar epithelium as the likely major source. In vivo blockade of extracellular Cyclophilin A signalling substantially attenuated physiological dysfunction, macrophage activation and matrix metalloproteinases. Finally, we found that patients with acute respiratory distress syndrome showed markedly elevated levels of extracellular Cyclophilin A within bronchoalveolar lavage.

Conclusions: Cyclophilin A is upregulated within the lungs of injuriously ventilated mice (and critically ill patients), where it plays a significant role in lung injury. Extracellular Cyclophilin A represents an exciting novel target for pharmacological intervention.
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http://dx.doi.org/10.1164/rccm.202009-3545OCDOI Listing
April 2021

Cyclophilin A inhibition as potential treatment of human aortic valve calcification.

Pharmacol Res 2020 08 17;158:104888. Epub 2020 May 17.

Unità per lo Studio delle Patologie Aortiche, Valvolari e Coronariche, Centro Cardiologico Monzino IRCCS, Milano, Italy. Electronic address:

Aortic valve stenosis (AS) is a pathological condition that affects about 3% of the population, representing the most common valve disease. The main clinical feature of AS is represented by the impaired leaflet motility, due to calcification, which leads to the left ventricular outflow tract obstruction during systole. The formation and accumulation of calcium nodules are driven by valve interstitial cells (VICs). Unfortunately, to date, the in vitro and in vivo studies were not sufficient to fully recapitulate all the pathological pathways involved in AS development, as well as to define a specific and effective pharmacological treatment for AS patients. Cyclophilin A (CyPA), the most important immunophilin and endogenous ligand of cyclosporine A (CsA), is strongly involved in several detrimental cardiovascular processes, such as calcification. To date, there are no data on the CyPA role in VIC-mediated calcification process of AS. Here, we aimed to identify the role of CyPA in AS by studying VIC calcification, in vitro. In this study, we found that (i) CyPA is up-regulated in stenotic valves of AS patients, (ii) pro-calcifying medium promotes CyPA secretion by VICs, (iii) in vitro treatment of VICs with exogenous CyPA strongly stimulates calcium deposition, and (iv) exogenous CyPA inhibition mediated by CsA analogue MM284 abolished in vitro calcium potential. Thus, CyPA represents a biological target that may act as a novel candidate in the detrimental AS development and its inhibition may provide a novel pharmacological approach for AS treatment.
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http://dx.doi.org/10.1016/j.phrs.2020.104888DOI Listing
August 2020

Cyclophilin A/EMMPRIN Axis Is Involved in Pro-Fibrotic Processes Associated with Thoracic Aortic Aneurysm of Marfan Syndrome Patients.

Cells 2020 01 8;9(1). Epub 2020 Jan 8.

Unità di Medicina Rigenerativa e Biologia Vascolare, Centro Cardiologico Monzino-IRCCS, 20138 Milan, Italy.

Background: Marfan syndrome (MFS) is a genetic disease, characterized by thoracic aortic aneurysm (TAA), which treatment is to date purely surgical. Understanding of novel molecular targets is mandatory to unveil effective pharmacological approaches. Cyclophilin A (CyPA) and its receptor EMMPRIN are associated with several cardiovascular diseases, including abdominal aortic aneurysm. Here, we envisioned the contribution of CyPA/EMMPRIN axis in MFS-related TAA.

Methods: We obtained thoracic aortic samples from healthy controls (HC) and MFS patients' aortas and then isolated vascular smooth muscle cells (VSMC) from the aortic wall.

Results: our findings revealed that MFS aortic tissue samples isolated from the dilated zone of aorta showed higher expression levels of EMMPRIN vs. MFS non-dilated aorta and HC. Interestingly, angiotensin II significantly stimulated CyPA secretion in MFS-derived VSMC (MFS-VSMC). CyPA treatment on MFS-VSMC led to increased levels of EMMPRIN and other MFS-associated pro-fibrotic mediators, such as TGF-β1 and collagen I. These molecules were downregulated by in vitro treatment with CyPA inhibitor MM284. Our results suggest that CyPA/EMMPRIN axis is involved in MFS-related TAA development, since EMMPRIN is upregulated in the dilated zone of MFS patients' TAA and the inhibition of its ligand, CyPA, downregulated EMMPRIN and MFS-related markers in MFS-VSMC.

Conclusions: these insights suggest both a novel detrimental role for CyPA/EMMPRIN axis and its inhibition as a potential therapeutic strategy for MFS-related TAA treatment.
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http://dx.doi.org/10.3390/cells9010154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7016677PMC
January 2020

Influences of cyclosporin A and non-immunosuppressive derivatives on cellular cyclophilins and viral nucleocapsid protein during human coronavirus 229E replication.

Antiviral Res 2020 01 18;173:104620. Epub 2019 Oct 18.

Max von Pettenkofer-Institute, Ludwig-Maximilians-University, Munich, Germany; German Center for Infection Research (DZIF), Partner Site Munich, 80336, Munich, Germany. Electronic address:

The well-known immunosuppressive drug cyclosporin A inhibits replication of various viruses including coronaviruses by binding to cellular cyclophilins thus inactivating their cis-trans peptidyl-prolyl isomerase function. Viral nucleocapsid proteins are inevitable for genome encapsidation and replication. Here we demonstrate the interaction between the N protein of HCoV-229E and cyclophilin A, not cyclophilin B. Cyclophilin inhibitors abolish this interaction. Upon infection, cyclophilin A stays evenly distributed throughout the cell, whereas cyclophilin B concentrates at ER-bleb-like structures. We further show the inhibitory potential of non-immunosuppressive CsA derivatives Alisporivir, NIM811, compound 3 on HCoV-229E-GFP and -Luciferase replication in human Huh-7.5 hepatoma cells at 18 and 48 h time points post infection with EC s at low micromolar ranges. Thus, non-immunosuppressive CsA derivatives effectively inhibit HCoV-229E replication suggesting them as possible candidates for the treatment of HCoV infection. The interruption of interaction between CypA and N protein by CsA and its derivatives suggest a mechanism how CypA inhibitors suppress viral replication.
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http://dx.doi.org/10.1016/j.antiviral.2019.104620DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7114175PMC
January 2020

Regulation of the Minichromosome Maintenance Protein 3 (MCM3) Chromatin Binding by the Prolyl Isomerase Pin1.

J Mol Biol 2018 12 11;430(24):5169-5181. Epub 2018 Oct 11.

Department of Enzymology, Institute for Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, Charles Tanford Protein Center, Kurt-Mothes-Str. 3a, D-06120 Halle/Saale, Germany. Electronic address:

Human Pin1 is a peptidyl prolyl cis/trans isomerase with a unique preference for phosphorylated Ser/Thr-Pro substrate motifs. Here we report that MCM3 (minichromosome maintenance complex component 3) is a novel target of Pin1. MCM3 interacts directly with the WW domain of Pin1. Proline-directed phosphorylation of MCM3 at S112 and T722 are crucial for the interaction with Pin1. MCM3 as a subunit of the minichromosome maintenance heterocomplex MCM2-7 is part of the pre-replication complex responsible for replication licensing and is implicated in the formation of the replicative helicase during progression of replication. Our data suggest that Pin1 coordinates phosphorylation-dependently MCM3 loading onto chromatin and its unloading from chromatin, thereby mediating S phase control.
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http://dx.doi.org/10.1016/j.jmb.2018.10.002DOI Listing
December 2018

Synthesis and biochemical evaluation of two novel N-hydroxyalkylated cyclosporin A analogs.

Org Biomol Chem 2018 06;16(23):4338-4349

Max Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, 06120 Halle/Saale, Germany.

The cyclic undecapeptide cyclosporin A (CsA) is a widely used immunosuppressive agent. Its immunosuppressive properties arise from strong binding to cyclophilins (Cyp) followed by inhibition of the protein calcineurin (CaN) by the binary CsA/Cyp complex and subsequent negative regulation of T-cell activation. In the present study we show a novel way to modify the CsA ring by selective N-hydroxyalkylation of the residues Val5 and d-Ala8. Moreover, the influence of these structural CsA modifications on the ability of the CsA analogs to bind Cyp, to inhibit CaN and to penetrate membranes of living cells was investigated. Our results show that the Val5 N-substitution significantly improved compound cell-permeability and markedly diminished CaN inhibition by the binary CsA analog/CypA complex but to a lesser extent Cyp inhibition. In contrast, the N-alkylation of d-Ala8 gave a product with significantly reduced affinity for Cyp but its immunosuppressive effects remained similar to CsA. Possible explanations of the observed experimental data are provided by computational studies.
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http://dx.doi.org/10.1039/c8ob00980eDOI Listing
June 2018

Pharmacological Cyclophilin Inhibitors Prevent Intoxication of Mammalian Cells with Toxin.

Toxins (Basel) 2018 05 1;10(5). Epub 2018 May 1.

Institute of Pharmacology and Toxicology, University of Ulm Medical Center, 89081 Ulm, Germany.

The toxin (PT) is one important virulence factor causing the severe childhood disease whooping cough which still accounted for approximately 63,000 deaths worldwide in children in 2013. PT consists of PTS1, the enzymatically active (A) subunit and a non-covalently linked pentameric binding/transport (B) subunit. After endocytosis, PT takes a retrograde route to the endoplasmic reticulum (ER), where PTS1 is released into the cytosol. In the cytosol, PTS1 ADP-ribosylates inhibitory alpha subunits of trimeric GTP-binding proteins (Giα) leading to increased cAMP levels and disturbed signalling. Here, we show that the cyclophilin (Cyp) isoforms CypA and Cyp40 directly interact with PTS1 in vitro and that Cyp inhibitors cyclosporine A (CsA) and its tailored non-immunosuppressive derivative VK112 both inhibit intoxication of CHO-K1 cells with PT, as analysed in a morphology-based assay. Moreover, in cells treated with PT in the presence of CsA, the amount of ADP-ribosylated Giα was significantly reduced and less PTS1 was detected in the cytosol compared to cells treated with PT only. The results suggest that the uptake of PTS1 into the cytosol requires Cyps. Therefore, CsA/VK112 represent promising candidates for novel therapeutic strategies acting on the toxin level to prevent the severe, life-threatening symptoms caused by PT.
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http://dx.doi.org/10.3390/toxins10050181DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5983237PMC
May 2018

Screening for Selective Protein Inhibitors by Using the IANUS Peptide Array.

Chembiochem 2018 04 22;19(8):789-792. Epub 2018 Mar 22.

Max Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, 06120, Halle/Saale, Germany.

Finding new road blacks: A peptidic inhibitor of calcineurin (CaN)-mediated nuclear factor of activated T cells (NFAT) dephosphorylation, which is developed through a template-assisted IANUS (Induced orgANisation of strUcture by matrix-assisted togethernesS) peptide array, is cell permeable and able to block the translocation of green fluorescent protein-NFAT fusion protein (GFP-NFAT) into the nucleus after stimulation.
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http://dx.doi.org/10.1002/cbic.201700652DOI Listing
April 2018

Targeting Extracellular Cyclophilin A Reduces Neuroinflammation and Extends Survival in a Mouse Model of Amyotrophic Lateral Sclerosis.

J Neurosci 2017 02 23;37(6):1413-1427. Epub 2016 Dec 23.

Department of Molecular Biochemistry and Pharmacology,

Neuroinflammation is a major hallmark of amyotrophic lateral sclerosis (ALS), which is currently untreatable. Several anti-inflammatory compounds have been evaluated in patients and in animal models of ALS, but have been proven disappointing in part because effective targets have not yet been identified. Cyclophilin A, also known as peptidylprolyl cis-/trans-isomerase A (PPIA), as a foldase is beneficial intracellularly, but extracellularly has detrimental functions. We found that extracellular PPIA is a mediator of neuroinflammation in ALS. It is a major inducer of matrix metalloproteinase 9 and is selectively toxic for motor neurons. High levels of PPIA were found in the CSF of SOD1 mice and rats and sporadic ALS patients, suggesting that our findings may be relevant for familial and sporadic cases. A specific inhibitor of extracellular PPIA, MM218, given at symptom onset, rescued motor neurons and extended survival in the SOD1 mouse model of familial ALS by 11 d. The treatment resulted in the polarization of glia toward a prohealing phenotype associated with reduced NF-κB activation, proinflammatory markers, endoplasmic reticulum stress, and insoluble phosphorylated TDP-43. Our results indicates that extracellular PPIA is a promising druggable target for ALS and support further studies to develop a therapy to arrest or slow the progression of the disease in patients. We provide evidence that extracellular cyclophilin A, also known as peptidylprolyl cis-/trans-isomerase A (PPIA), is a mediator of the neuroinflammatory reaction in amyotrophic lateral sclerosis (ALS) and is toxic for motor neurons. Supporting this, a specific extracellular PPIA inhibitor reduced neuroinflammation, rescued motor neurons, and extended survival in the SOD1 mouse model of familial ALS. Our findings suggest selective pharmacological inhibition of extracellular PPIA as a novel therapeutic strategy, not only for SOD1-linked ALS, but possibly also for sporadic ALS. This approach aims to address the neuroinflammatory reaction that is a major hallmark of ALS. However, given the complexity of the disease, a combination of therapeutic approaches may be necessary.
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http://dx.doi.org/10.1523/JNEUROSCI.2462-16.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6705677PMC
February 2017

Identification of low abundance cyclophilins in human plasma.

Proteomics 2016 11 12;16(21):2815-2826. Epub 2016 Oct 12.

Department of Enzymology, Institute of Biochemistry und Biotechnology, Martin-Luther-University Halle-Wittenberg, Halle, Germany.

Cylophilins (Cyps) belong to the ubiquitously distributed enzyme class of peptidyl prolyl cis/trans isomerases (EC5.2.1.8), which are foldases capable of accelerating slow steps in the refolding of denatured proteins. At least 20 different Cyp isoenzymes are broadly distributed among all organs and cellular compartments in humans. Extracellularly localized Cyps came into the scientific focus recently because of their involvement in the control of inflammatory diseases, as well as viral and bacterial infections. However, detailed insights into Cyp functions are often hampered by the lack of sensitive detection methods. We present an improved method for affinity purification and detection of Cyp in biotic samples in this manuscript. The procedure takes advantage of two novel cyclosporine A derivatives. Derivative 1 was used to capture Cyps from the sample while derivative 2 was applied for selective release from the affinity matrix. Using this approach, eight different Cyp (CypA, CypB, CypC, Cyp40 (PPID), CypE, CypD (PPIF), CypH, and CypL1) were unambiguously detected in healthy human blood plasma. Moreover, extracellular CypA was found to be partially modified by N acetylation on residues Lys44, Lys133, Lys155, as well as N  acetylation at the N-terminal Val residue. N acetylation of Ser2 residue was also found for Cyp40.
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http://dx.doi.org/10.1002/pmic.201600221DOI Listing
November 2016

Inhibition of Aβ(1-40) fibril formation by cyclophilins.

Biochem J 2016 05 18;473(10):1355-68. Epub 2016 Mar 18.

Max Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, D-06120 Halle (Saale), Germany Department of Enzymology, Institute for Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, Weinbergweg 22, D-06120 Halle (Saale), Germany

Cyclophilins interact directly with the Alzheimer's disease peptide Aβ (amyloid β-peptide) and are therefore involved in the early stages of Alzheimer's disease. Aβ binding to CypD (cyclophilin D) induces dysfunction of human mitochondria. We found that both CypD and CypA suppress in vitro fibril formation of Aβ(1-40) at substoichiometric concentrations when present early in the aggregation process. The prototypic inhibitor CsA (cyclosporin A) of both cyclophilins as well as the new water-soluble MM258 derivative prevented this suppression. A SPOT peptide array approach and NMR titration experiments confirmed binding of Aβ(1-40) to the catalytic site of CypD mainly via residues Lys(16)-Glu(22) The peptide Aβ(16-20) representing this section showed submicromolar IC50 values for the peptidyl prolyl cis-trans isomerase activity of CypD and CypA and low-micromolar KD values in ITC experiments. Chemical cross-linking and NMR-detected hydrogen-deuterium exchange experiments revealed a shift in the populations of small Aβ(1-40) oligomers towards the monomeric species, which we investigated in the present study as being the main process of prevention of Aβ fibril formation by cyclophilins.
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http://dx.doi.org/10.1042/BCJ20160098DOI Listing
May 2016

Targeting Extracellular Cyclophilins Ameliorates Disease Progression in Experimental Biliary Atresia.

Mol Med 2015 Oct 24;21(1):657-664. Epub 2015 Jul 24.

Division of Pediatric Surgery, Children's National Medical Center, Washington, District of Columbia, United States of America.

Biliary atresia (BA) is a devastating liver disease of unknown etiology affecting children generally within the first 3 months of life. The disease is manifested by inflammation and subsequent obstruction of the extrahepatic bile ducts, fibrosis and liver failure. The mechanisms responsible for disease pathogenesis are not fully understood, but a number of factors controlled by the SMAD signaling pathway have been implicated. In this study, we investigated the role of a known proinflammatory factor, extracellular cyclophilin A (CypA), in the pathogenesis of biliary atresia using the rhesus rotavirus (RRV) murine model. We used a unique cyclosporine A derivative, MM284, which does not enter cells and therefore inactivates exclusively extracellular cyclophilins, as a potential treatment. We demonstrated that levels of CypA in plasma of RRV-infected mice were increased significantly, and that treatment of mice with MM284 prior to or one day after disease initiation by RRV infection significantly improved the status of mice with experimental BA: weight gain was restored, bilirubinuria was abrogated, liver infiltration by inflammatory cells was reduced and activation of the SMAD pathway and SMAD-controlled fibrosis mediators and tissue inhibitor of metalloproteinases (TIMP)-4 and matrix metalloproteinase (MMP)-7 was alleviated. Furthermore, treatment of human hepatic stellate cells with recombinant cyclophilin recapitulated SMAD2/3 activation, which was also suppressed by MM284 treatment. Our data provide the first evidence that extracellular cyclophilins activate the SMAD pathway and promote inflammation in experimental BA, and suggest that MM284 may be a promising therapeutic agent for treating BA and possibly other intrahepatic chronic disorders.
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http://dx.doi.org/10.2119/molmed.2015.00076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4749494PMC
October 2015

A fluorescence-based array screen for transglutaminase substrates.

Chembiochem 2015 May 4;16(8):1169-74. Epub 2015 May 4.

Department of Pharmaceutical Technology and Biopharmacy, Institute of Pharmacy, Faculty of Sciences I, Biosciences, Martin Luther University Halle-Wittenberg, Weinbergweg 22, 06120 Halle/Saale (Germany).

Transglutaminases (EC 2.3.2.13) form an enzyme family that catalyzes the formation of isopeptide bonds between the γ-carboxamide group of glutamine and the ε-amine group of lysine residues of peptides and proteins. Other primary amines can be accepted in place of lysine. Because of their important physiological and pathophysiological functions, transglutaminases have been studied for 60 years. However, the substrate preferences of this enzyme class remain largely elusive. In this study, we used focused combinatorial libraries of 400 peptides to investigate the influence of the amino acids adjacent to the glutamine and lysine residues on the catalysis of isopeptide bond formation by microbial transglutaminase. Using the peptide microarray technology we found a strong positive influence of hydrophobic and basic amino acids, especially arginine, tyrosine, and leucine. Several tripeptide substrates were synthesized, and enzymatic kinetic parameters were determined both by microarray analysis and in solution.
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http://dx.doi.org/10.1002/cbic.201402709DOI Listing
May 2015

The Novel Extracellular Cyclophilin A (CyPA) - Inhibitor MM284 Reduces Myocardial Inflammation and Remodeling in a Mouse Model of Troponin I -Induced Myocarditis.

PLoS One 2015 20;10(4):e0124606. Epub 2015 Apr 20.

Medizinische Klinik III, Kardiologie und Kreislauferkrankungen, Eberhard Karls Universität Tübingen, Tübingen, Germany.

Cyclophilins are a group of highly conserved cytosolic enzymes that have a peptidylprolyl cis/trans isomerase activity. Cyclophilin A (CyPA) can be secreted in the extracellular space by inflammatory cells and upon cell death. The presence of CyPA in patients with non-ischemic cardiomyopathy is associated with poor clinical prognosis. Here, we investigated the inhibition of extracellular CyPA in a mouse model of troponin I-induced autoimmune myocarditis using the strictly extracellular CyPA-inhibitor MM284. Since A/J mice develop severe inflammation and fibrosis after immunization with murine cardiac troponin I (mcTn I), we used this model to analyze the effects of an extracellular CyPA inhibition. As extracellular CyPA-inhibitor we used the recently described CsA-derivate MM284. In vitro studies confirmed that MM284 inhibits CyPA-induced monocytic migration and adhesion. A/J mice immunized with mcTnI were treated with MM284 or vehicle every second day. After 28 days, we found a considerable reduction of myocardial injury and fibrosis. Further analysis revealed a reduced myocardial presence of T-cells and macrophages compared to control treated animals. Whereas MMP-9 expression was reduced significantly by MM284, we observed no significant reduction of inflammatory cytokines such as IL-6 or TNFα. Extracellular CyPA plays an important role in autoimmune myocarditis for myocardial damage and fibrosis. Our data suggest a new pharmacological approach for the treatment of myocardial inflammation and reduction of cardiac fibrosis by inhibition of extracellular CyPA.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0124606PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4404136PMC
January 2016

Inhibition of extracellular cyclophilins with cyclosporine analog and development of atherosclerosis in apolipoprotein E-deficient mice.

J Pharmacol Exp Ther 2015 Jun 18;353(3):490-5. Epub 2015 Mar 18.

Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia (M.D., V.N.L.V.N., H.L.C., D.S.); Department of Biochemistry, Martin Luther University of Halle-Wittenberg, Halle (Saale), Germany (M.M.); Max Planck Institute for Biophysical Chemistry, Gottingen, Germany (G.F.); and Department of Microbiology, and Immunology and Tropical Medicine, George Washington University, Washington, DC (M.B.)

Cyclophilins exert both intracellular and extracellular activities related to immune responses and inflammation, which have been implicated in pathogenesis of atherosclerosis. Pan-inhibition of cyclophilins has both pro- and antiatherosclerotic properties, but specific contributions of extracellular and intracellular cyclophilins to these effects have not been characterized. Here, using selective inhibitor of extracellular cyclophilins, we investigated the role of these molecules in atherosclerosis. Apolipoprotein E-null mice fed a high-fat diet received intraperitoneal injections every second day of either vehicle or two analogs of cyclosporine A (CsA): [Melle](4)-CsA (NIM811), a nonimmunosupressive cell-permeable inhibitor of both intracellular and extracellular cyclophilins; and [(4R)-4-[(6-carboxy-1H-benzo[d]imidazol-2-yl)-methyl]-4-methyl-l-threonine](1)-CsA (MM284), cell-impermeable analog only inhibiting extracellular cyclophilins. Development of atherosclerosis and composition of plaques in aorta and innominate artery were studied. Both analogs increased abundance and cross-sectional size of the atherosclerotic plaques in aorta but did not affect development of atherosclerosis in innominate artery. Neither compound affected abundance of macrophages and amount of vascular cell adhesion molecule-1 or nitrotyrosine in the plaques of both arteries. Both compounds reduced the amount of collagen in innominate artery without affecting abundance of collagen in aortic sinus. MM284, but not NIM811, significantly reduced plasma concentration of tumor necrosis factor-α (TNFα); neither compound affected plasma concentrations of interleukin (IL)-6, IL-10 or monocyte chemoattractant protein-1. Ratio between different populations of immune cells in blood or isolated from lymph nodes and spleen as well as plasma lipoprotein profile were unaffected by both compounds. In conclusion, selective inhibition of extracellular cyclophilins reduced TNFα levels in plasma but increased atherosclerosis.
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http://dx.doi.org/10.1124/jpet.115.223420DOI Listing
June 2015

Extracellular cyclophilin A activates platelets via EMMPRIN (CD147) and PI3K/Akt signaling, which promotes platelet adhesion and thrombus formation in vitro and in vivo.

Arterioscler Thromb Vasc Biol 2015 Mar 30;35(3):655-63. Epub 2014 Dec 30.

From the Medizinische Klinik III, Kardiologie und Kreislauferkrankungen (P.S., S.N.I.v.U.-S., T.S., O.B., D.H., M.C., H.L., M.G., A.E.M.), Institute of Physiology (P.M., E.-M.S., F.L.), and Institute of Anatomy (A.F.M.), Eberhard Karls-University Tübingen, Tübingen, Germany; Institute of Biochemistry, Abteilung Enzymology, Martin-Luther-University Halle-Wittenberg, Halle, Germany (M.M.); and Max-Planck-Institute für Biophysikalische Chemie Göttingen, BO Halle (Saale), Germany (G.F.).

Objective: Cyclophilin A (CyPA) is secreted under inflammatory conditions by various cell types. Whereas the important role of intracellular CyPA for platelet function has been reported, the effect of extracellular CyPA on platelet function has not been investigated yet.

Approach And Results: Inhibition of extracellular CyPA through a novel specific inhibitor MM284 reduced thrombus after ferric chloride-induced injury in vivo. In vitro extracellular CyPA enhanced thrombus formation even in CyPA(-/-) platelets. Treatment of isolated platelets with recombinant CyPA resulted in platelet degranulation in a time- and dose-dependent manner. Inhibition of the platelet surface receptor extracellular matrix metalloproteinase inducer (cluster of differentiation 147) by an anticluster of differentiation 147 monoclonal antibody significantly reduced CyPA-dependent platelet degranulation. Pretreatment of platelets with CyPA enhanced their recruitment to mouse carotid arteries after arterial injury, which could be inhibited by an anticluster of differentiation 147 monoclonal antibody (intravital microscopy). The role of extracellular CyPA in adhesion could be confirmed by infusing CyPA(-/-) platelets in CyPA(+/+) mice and by infusing CyPA(+/+) platelets in CyPA(-/-) mice. Stimulation of platelets with CyPA induced phosphorylation of Akt, which could in turn be inhibited in the presence of phosphoinositid-3-kinase inhibitors. Akt-1(-/-) platelets revealed a markedly decreased degranulation on CyPA stimulation. Finally, ADP-induced platelet aggregation was attenuated by MM284, as well as by inhibiting paracrine-secreted CyPA without directly affecting Ca(2+)-signaling.

Conclusions: Extracellular CyPA activates platelets via cluster of differentiation 147-mediated phosphoinositid-3-kinase/Akt-signaling, leading to enhanced adhesion and thrombus formation independently of intracellular CyPA. Targeting extracellular CyPA via a specific inhibitor may be a promising strategy for platelet inhibition without affecting critical functions of intracellular CyPA.
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http://dx.doi.org/10.1161/ATVBAHA.114.305112DOI Listing
March 2015

Cyclophilin-facilitated membrane translocation as pharmacological target to prevent intoxication of mammalian cells by binary clostridial actin ADP-ribosylated toxins.

J Mol Biol 2015 Mar 21;427(6 Pt A):1224-38. Epub 2014 Jul 21.

Institute of Pharmacology and Toxicology, University of Ulm Medical Center, 89081 Ulm, Germany. Electronic address:

Clostridium botulinum C2 toxin, Clostridium perfringens iota toxin and Clostridium difficile CDT belong to the family of binary actin ADP-ribosylating toxins and are composed of a binding/translocation component and a separate enzyme component. The enzyme components ADP-ribosylate G-actin in the cytosol of target cells resulting in depolymerization of F-actin, cell rounding and cell death. The binding/translocation components bind to their cell receptors and form complexes with the respective enzyme components. After receptor-mediated endocytosis, the binding/translocation components form pores in membranes of acidified endosomes and the enzyme components translocate through these pores into the cytosol. This step is facilitated by the host cell chaperone heat shock protein 90 and peptidyl-prolyl cis/trans isomerases including cyclophilin A. Here, we demonstrate that a large isoform of cyclophilin A, the multi-domain enzyme cyclophilin 40 (Cyp40), binds to the enzyme components C2I, Ia and CDTa in vitro. Isothermal titration calorimetry revealed a direct binding to C2I with a calculated affinity of 101 nM and to Ia with an affinity of 1.01 μM. Closer investigation for the prototypic C2I revealed that binding to Cyp40 did not depend on its ADP-ribosyltransferase activity but was stronger for unfolded C2I. The interaction of C2I with Cyp40 was also demonstrated in lysates from C2-treated cells by pull-down. Treatment of cells with a non-immunosuppressive cyclosporine A derivative, which still binds to and inhibits the peptidyl-prolyl cis/trans isomerase activity of cyclophilins, protected cells from intoxication with C2, iota and CDT toxins, offering an attractive approach for development of novel therapeutic strategies against binary actin ADP-ribosylating toxins.
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http://dx.doi.org/10.1016/j.jmb.2014.07.013DOI Listing
March 2015

Human coronavirus NL63 replication is cyclophilin A-dependent and inhibited by non-immunosuppressive cyclosporine A-derivatives including Alisporivir.

Virus Res 2014 May 22;184:44-53. Epub 2014 Feb 22.

Max-von-Pettenkofer Institut, Ludwig-Maximilians-Universität, München, Germany. Electronic address:

Until recently, there were no effective drugs available blocking coronavirus (CoV) infection in humans and animals. We have shown before that CsA and FK506 inhibit coronavirus replication (Carbajo-Lozoya, J., Müller, M.A., Kallies, S., Thiel, V., Drosten, C., von Brunn, A. Replication of human coronaviruses SARS-CoV, HCoV-NL63 and HCoV-229E is inhibited by the drug FK506. Virus Res. 2012; Pfefferle, S., Schöpf, J., Kögl, M., Friedel, C., Müller, M.A., Stellberger, T., von Dall'Armi, E., Herzog, P., Kallies, S., Niemeyer, D., Ditt, V., Kuri, T., Züst, R., Schwarz, F., Zimmer, R., Steffen, I., Weber, F., Thiel, V., Herrler, G., Thiel, H.-J., Schwegmann-Weßels, C., Pöhlmann, S., Haas, J., Drosten, C. and von Brunn, A. The SARS-Coronavirus-host interactome: identification of cyclophilins as target for pan-Coronavirus inhibitors. PLoS Pathog., 2011). Here we demonstrate that CsD Alisporivir, NIM811 as well as novel non-immunosuppressive derivatives of CsA and FK506 strongly inhibit the growth of human coronavirus HCoV-NL63 at low micromolar, non-cytotoxic concentrations in cell culture. We show by qPCR analysis that virus replication is diminished up to four orders of magnitude to background levels. Knockdown of the cellular Cyclophilin A (CypA/PPIA) gene in Caco-2 cells prevents replication of HCoV-NL63, suggesting that CypA is required for virus replication. Collectively, our results uncover Cyclophilin A as a host target for CoV infection and provide new strategies for urgently needed therapeutic approaches.
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http://dx.doi.org/10.1016/j.virusres.2014.02.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7114444PMC
May 2014

Anti-inflammatory effects of extracellular cyclosporins are exclusively mediated by CD147.

J Med Chem 2013 Sep 12;56(18):7302-11. Epub 2013 Sep 12.

Max-Planck Research Unit for Enzymology of Protein Folding , Weinbergweg 22, 06120 Halle (Saale), Germany.

Leukocyte trafficking and recruitment is a critical process in host immune surveillance and in inflammatory diseases. Extracellular cyclophilins (eCyps) have been identified as a novel class of chemotactic mediators. The impact of eCyp/CD147 interactions for the recruitment of leukocytes during inflammation was analyzed using a structurally simplified cell-impermeable eCyp inhibitor. This compound was highly effective at inhibiting leukocyte migration toward CypA in vitro as well as in the recruitment of leukocytes during inflammation in a mouse model of experimentally induced peritonitis and delayed-type hypersensitivity reaction. By using CD147-/- mice in combination with the cell-impermeable eCyp inhibitor, we were able to show that the action of eCyps in inflammation is exclusively mediated by interaction with CD147. Our findings suggest that blocking eCyps may be an effective therapeutic target for reducing inflammatory diseases associated with leukocyte recruitment.
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http://dx.doi.org/10.1021/jm4007577DOI Listing
September 2013

Identification of prolyl oligopeptidase as a cyclosporine-sensitive protease by screening of mouse liver extracts.

Biol Chem 2013 Aug;394(8):1057-67

Max Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, D-06120 Halle, Germany.

Cyclosporine A (CsA) is a cyclic undecapeptide well known for its ability to prevent rejection episodes after organ transplantation via gain-of-function. Therefore, biomedical studies on CsA have been focused on both immunosuppressive properties and binding to the biocatalytically-active immune receptors, the cyclophilins. Much less attention has been spent on effects of cyclosporines on the biological function of other proteins. We used a 9-mer fluorescence-quenched peptide library with defined sequences to identify cyclosporine-sensitive proteolysis in mouse liver extracts. A highly soluble [d-Ser]8-CsA derivative was utilized to avoid drug precipitation at extended incubation times. Analysis of the time courses of proteolysis revealed 15 out of 360 peptide sequences where proteolysis exhibited marked sensitivity to the cyclosporine derivative. As a first example, a collagen-derived substrate was selected from those hits to identify the targeted proteolytic pathway. After purification from mouse liver extracts, prolyl oligopeptidase (EC 3.4.21.26) could be identified as a protease sensitive to submicromolar concentrations of cyclosporines. Surprisingly, in a series of cyclosporine derivatives an inverse relationship was found between the inhibition of prolyl oligopeptidase and inhibition of cyclophilin A.
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http://dx.doi.org/10.1515/hsz-2013-0125DOI Listing
August 2013

Fine tuning the inhibition profile of cyclosporine A by derivatization of the MeBmt residue.

Chembiochem 2013 Jan 7;14(1):63-5. Epub 2012 Dec 7.

Max Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, 06120 Halle/Saale, Germany.

Unique respect: The biological properties of four CsA derivatives were fine-tuned by tractable modifications of the MeBmt residue. The new CsA derivatives share strong inhibitory activity toward cyclophilins (Cyps), but each is unique with respect to immunosuppressive action and cellular localization. These CsA analogues can be used to study the physiological roles of extracellular Cyps.
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http://dx.doi.org/10.1002/cbic.201200621DOI Listing
January 2013

Design of cyclic peptides featuring proline predominantly in the cis conformation under physiological conditions.

Chembiochem 2012 Sep 11;13(14):2122-7. Epub 2012 Sep 11.

Max Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, 06120 Halle/Saale, Germany.

Turns are secondary-structure elements that are omnipresent in natively folded polypeptide chains. A large variety of four-residue β-turns exist, which differ mainly in the backbone dihedral angle values of the two central residues i+1 and i+2. The βVI-type turns are of particular biological interest because the i+2 residue is always a proline in the cis conformation and might thus serve as target of peptidyl prolyl cis/trans isomerases (PPIases). We have designed cyclic hexapeptides containing two proline residues that predominantly adopt the cis conformation in aqueous solution. NMR data and MD calculations indicated that the cyclic peptide sequences c-(-DXaa-Ser-Pro-DXaa-Lys-Pro-) result in highly symmetric backbone structures when both prolines are in the cis conformation and the D-amino acids are either alanine or phenylalanine residues. Replacement of the serine residue either by phosphoserine or by tyrosine compromises this symmetry, but further increases the cis conformation content of both prolines. As a result, we obtained a cyclic hexapeptide that exists almost exclusively as the cis-Pro/cis-Pro conformer but shows no cis/trans interconversion even in the presence of the PPIase Pin1, apparently due to an energetically quite favorable but highly restricted conformational space.
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http://dx.doi.org/10.1002/cbic.201200366DOI Listing
September 2012

Oxygen glucose deprivation causes mitochondrial dysfunction in cultivated rat hippocampal slices: protective effects of CsA, its immunosuppressive congener [D-Ser](8)CsA, the novel non-immunosuppressive cyclosporin derivative Cs9, and the NMDA receptor antagonist MK 801.

Mitochondrion 2013 Sep 21;13(5):539-47. Epub 2012 Jul 21.

Institute of Neurosciences, Lithuanian University of Health Sciences, Eiveniu Str. 4, 50009 Kaunas, Lithuania.

We have introduced a sensitive method for studying oxygen/glucose deprivation (OGD)-induced mitochondrial alterations in homogenates of organotypic hippocampal slice cultures (slices) by high-resolution respirometry. Using this approach, we tested the neuroprotective potential of the novel non-immunosuppressive cyclosporin (CsA) derivative Cs9 in comparison with CsA, the immunosuppressive CsA analog [D-Ser](8)CsA, and MK 801, a N-methyl-d-aspartate (NMDA) receptor antagonist. OGD/reperfusion reduced the glutamate/malate dependent (and protein-related) state 3 respiration to 30% of its value under control conditions. All of the above drugs reversed this effect, with an increase to >88% of the value for control slices not exposed to OGD. We conclude that Cs9, [D-Ser](8)CsA, and MK 801, despite their different modes of action, protect mitochondria from OGD-induced damage.
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http://dx.doi.org/10.1016/j.mito.2012.07.110DOI Listing
September 2013

Molecular basis of β-amyloid oligomer recognition with a conformational antibody fragment.

Proc Natl Acad Sci U S A 2012 Jul 18;109(31):12503-8. Epub 2012 Jul 18.

Institute for Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, 06120 Halle, Saale, Germany.

Oligomers are intermediates of the β-amyloid (Aβ) peptide fibrillogenic pathway and are putative pathogenic culprits in Alzheimer's disease (AD). Here we report the biotechnological generation and biochemical characterization of an oligomer-specific antibody fragment, KW1. KW1 not only discriminates between oligomers and other Aβ conformations, such as fibrils or disaggregated peptide; it also differentiates between different types of Aβ oligomers, such as those formed by Aβ (1-40) and Aβ (1-42) peptide. This high selectivity of binding contrasts sharply with many other conformational antibodies that interact with a large number of structurally analogous but sequentially different antigens. X-ray crystallography, NMR spectroscopy, and peptide array measurements imply that KW1 recognizes oligomers through a hydrophobic and significantly aromatic surface motif that includes Aβ residues 18-20. KW1-positive oligomers occur in human AD brain samples and induce synaptic dysfunctions in living brain tissues. Bivalent KW1 potently neutralizes this effect and interferes with Aβ assembly. By altering a specific step of the fibrillogenic cascade, it prevents the formation of mature Aβ fibrils and induces the accumulation of nonfibrillar aggregates. Our data illuminate significant mechanistic differences in oligomeric and fibril recognition and suggest the considerable potential of KW1 in future studies to detect or inhibit specific types of Aβ conformers.
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http://dx.doi.org/10.1073/pnas.1206433109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3412029PMC
July 2012

The FKBP38 catalytic domain binds to Bcl-2 via a charge-sensitive loop.

J Biol Chem 2012 Jun 20;287(23):19665-73. Epub 2012 Apr 20.

Max Planck Research Unit for Enzymology of Protein Folding, 06120 Halle, Saale, Germany.

FKBP38 is a regulator of the prosurvival protein Bcl-2, but in the absence of detailed structural insights, the molecular mechanism of the underlying interaction has remained unknown. Here, we report the contact regions between Bcl-2 and the catalytic domain of FKBP38 derived by heteronuclear NMR spectroscopy. The data reveal that a previously identified charge-sensitive loop near the putative active site of FKBP38 is mainly responsible for Bcl-2 binding. The corresponding binding epitope of Bcl-2 could be identified via a peptide library-based membrane assay. Site-directed mutagenesis of the key residues verified the contact sites of this electrostatic protein/protein interaction. The derived structure model of the complex between Bcl-2 and the FKBP38 catalytic domain features both electrostatic and hydrophobic intermolecular contacts and provides a rationale for the regulation of the FKBP38/Bcl-2 interaction by Ca(2+).
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http://dx.doi.org/10.1074/jbc.M111.317214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3366001PMC
June 2012

Negatively charged phospholipids trigger the interaction of a bacterial Tat substrate precursor protein with lipid monolayers.

Langmuir 2012 Feb 9;28(7):3534-41. Epub 2012 Feb 9.

Institute of Chemistry-Physical Chemistry, Martin-Luther-University Halle-Wittenberg, Von-Danckelmann-Platz 4, 06120 Halle, Germany.

Folded proteins can be translocated across biological membranes via the Tat machinery. It has been shown in vitro that these Tat substrates can interact with membranes prior to translocation. Here we report a monolayer and infrared reflection-absorption spectroscopic (IRRAS) study of the initial states of this membrane interaction, the binding to a lipid monolayer at the air/water interface serving as a model for half of a biological membrane. Using the model Tat substrate HiPIP (high potential iron-sulfur protein) from Allochromatium vinosum, we found that the precursor preferentially interacts with monolayers of negatively charged phospholipids. The signal peptide is essential for the interaction of the precursor protein with the monolayer because the mature HiPIP protein showed no interaction with the lipid monolayer. However, the individual signal peptide interacted differently with the monolayer compared to the complete precursor protein. IRRA spectroscopy indicated that the individual signal peptide forms mainly aggregated β-sheet structures. This β-sheet formation did not occur for the signal peptide when being part of the full length precursor. In this case it adopted an α-helical structure upon membrane insertion. The importance of the signal peptide and the mature domain for the membrane interaction is discussed in terms of current ideas of Tat substrate-membrane interactions.
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http://dx.doi.org/10.1021/la204473tDOI Listing
February 2012

Effects of cyclosporine A and its immunosuppressive or non-immunosuppressive derivatives [D-Ser]8-CsA and Cs9 on mitochondria from different brain regions.

Mitochondrion 2011 May 16;11(3):421-9. Epub 2010 Dec 16.

Leibniz Institute for Neurobiology, Department Behavioral Neurology, Brenneckestr. 6, D-39118 Magdeburg, Germany.

We studied the functional properties of isolated brain mitochondria (BM) prepared from total rat brain (BM(total)) or from cerebral subregions under basal and Ca(2+) overload conditions in order to evaluate the effects of cyclosporine A (CsA) in a regiospecific manner. CsA-induced effects were compared with those of two derivatives-the none-immunosuppressive [O-(NH(2)(CH2)(5)NHC(O)CH(2))-D-Ser](8)-CsA (Cs9) and its congener, the immunosuppressive [D-Ser](8)-CsA. The glutamate/malate-dependent state 3 respiration of mitochondria (state 3(glu/mal)) differed in region-specific manner (cortex > striatum = cerebellum > substantia nigra > hippocampus), but was significantly increased by 1μM CsA (+21±5%) in all regions. Ca(2+) overload induced by addition of 20μM Ca(2+) caused a significant decrease of state 3(glu/mal) (-45 to -55%) which was almost completely prevented in the presence of 1μM CsA, 1μM Cs9 or 1μM [D-Ser](8)-CsA. Mitochondrial Ca(2+) accumulation thresholds linked to permeability transition (PT) as well as the rate and completeness of mitochondrial Ca(2+) accumulation differed between different brain regions. For the first time, we provide a detailed, regiospecific analysis of Ca(2+)-dependent properties of brain mitochondria. Regardless of their immunosuppressive impact, CsA and its analogues improved mitochondrial functional properties under control conditions. They also preserved brain mitochondria against Ca(2+) overload-mediated PT and functional impairments. Since Cs9 does not mediate immunosuppression, it might be used as a more specific PT inhibitor than CsA.
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http://dx.doi.org/10.1016/j.mito.2010.12.012DOI Listing
May 2011

A cell-impermeable cyclosporine A derivative reduces pathology in a mouse model of allergic lung inflammation.

J Immunol 2010 Dec 5;185(12):7663-70. Epub 2010 Nov 5.

Department of Microbiology, Immunology, and Tropical Medicine, The George Washington University Medical Center, Washington, DC 20037, USA.

Although the main regulators of leukocyte trafficking are chemokines, another family of chemotactic agents is cyclophilins. Intracellular cyclophilins function as peptidyl-prolyl cis-trans isomerases and are targets of the immunosuppressive drug cyclosporine A (CsA). Cyclophilins can also be secreted in response to stress factors, with elevated levels of extracellular cyclophilins detected in several inflammatory diseases. Extracellular cyclophilins are known to have potent chemotactic properties, suggesting that they might contribute to inflammatory responses by recruiting leukocytes into tissues. The objective of the present study was to determine the impact of blocking cyclophilin activity using a cell-impermeable derivative of CsA to specifically target extracellular pools of cyclophilins. In this study, we show that treatment with this compound in a mouse model of allergic lung inflammation demonstrates up to 80% reduction in inflammation, directly inhibits the recruitment of Ag-specific CD4(+) T cells, and works equally well when delivered at 100-fold lower doses directly to the airways. Our findings suggest that cell-impermeable analogs of CsA can effectively reduce inflammatory responses by targeting leukocyte recruitment mediated by extracellular cyclophilins. Specifically blocking the extracellular functions of cyclophilins may provide an approach for inhibiting the recruitment of one of the principal immune regulators of allergic lung inflammation, Ag-specific CD4(+) T cells, into inflamed airways and lungs.
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http://dx.doi.org/10.4049/jimmunol.1001707DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3603141PMC
December 2010

The protein-free IANUS peptide array uncovers interaction sites between Escherichia coli parvulin 10 and alkyl hydroperoxide reductase.

Biochemistry 2010 Oct 9;49(39):8626-35. Epub 2010 Sep 9.

Max Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, 06120 Halle/Saale, Germany.

The reliable identification of interacting structural elements without prior isolation of interacting proteins can be achieved by using the novel fluorescence resonance energy transfer-coupled IANUS (Induced orgANization of strUcture by matrix-assisted togethernesS) peptide array. Here we report that parvulin 10 (Par10), an abundant Escherichia coli peptidyl prolyl cis/trans isomerase (PPIase), physically interacts with the alkyl hydroperoxide reductase subunit C (AhpC) in bacterial cell extracts, as determined by affinity chromatography and chemical cross-linking experiments. A Par10-negative E. coli strain showed increased sensitivity toward hydrogen peroxide compared to the wild-type strain. The IANUS experiment revealed three segments of the peroxiredoxin AhpC chain as potential Par10 binding partners. Inhibition of the Par10 PPIase activity by the corresponding AhpC-derived peptides as well as NMR data of (15)N-labeled Par10 in the presence of the AhpC(115-132) peptide or full-length AhpC confirmed that the putative Par10 active site is involved in the Par10-AhpC interaction. Moreover, NMR-based docking calculations as well as NOESY exchange peaks between the proline cis and trans isomers revealed the Asp125-Pro126 moiety of the AhpC segment G115-A132 as a substrate for Par10 enzymatic action. On the basis of these data, we conclude that Par10 catalytic activity is involved in the cellular protection against oxidative stress.
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http://dx.doi.org/10.1021/bi101015pDOI Listing
October 2010