ABSTRACT
Pathological deposition and crosslinking of collagen type I by activated myofibroblasts drives progressive tissue fibrosis. Therapies that inhibit collagen synthesis have potential as antifibrotic agents. We identify the collagen chaperone cyclophilin B as a major cellular target of the natural product sanglifehrin A (SfA) using photoaffinity labeling and chemical proteomics. Mechanistically, SfA inhibits and induces the secretion of cyclophilin B from the endoplasmic reticulum (ER) and prevents TGF-ß1-activated myofibroblasts from synthesizing and secreting collagen type I in vitro, without inducing ER stress or affecting collagen type I mRNA transcription, myofibroblast migration, contractility, or TGF-ß1 signaling. In vivo, SfA induced cyclophilin B secretion in preclinical models of fibrosis, thereby inhibiting collagen synthesis from fibrotic fibroblasts and mitigating the development of lung and skin fibrosis in mice. Ex vivo, SfA induces cyclophilin B secretion and inhibits collagen type I secretion from fibrotic human lung fibroblasts and samples from patients with idiopathic pulmonary fibrosis (IPF). Taken together, we provide chemical, molecular, functional, and translational evidence for demonstrating direct antifibrotic activities of SfA in preclinical and human ex vivo fibrotic models. Our results identify the cellular target of SfA, the collagen chaperone cyclophilin B, as a mechanistic target for the treatment of organ fibrosis.
Subject(s)
Cyclophilins , Animals , Humans , Mice , Cyclophilins/metabolism , Cyclophilins/antagonists & inhibitors , Collagen Type I/metabolism , Fibrosis , Myofibroblasts/metabolism , Myofibroblasts/drug effects , Myofibroblasts/pathology , Fibroblasts/metabolism , Fibroblasts/drug effects , Idiopathic Pulmonary Fibrosis/drug therapy , Idiopathic Pulmonary Fibrosis/pathology , Idiopathic Pulmonary Fibrosis/metabolism , Lung/pathology , Lung/drug effects , Lung/metabolism , Disease Models, Animal , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/drug effects , Male , Mice, Inbred C57BL , Transforming Growth Factor beta1/metabolism , Lactones , Spiro CompoundsABSTRACT
Cyclophilin B (CypB), a significant member of immunophilins family with peptidyl-prolyl cis-trans isomerase (PPIase) activity, is crucial for the growth and metabolism of prokaryotes and eukaryotes. Sporothrix globosa (S. globosa), a principal pathogen in the Sporothrix complex, causes sporotrichosis. Transcriptomic analysis identified the cypB gene as highly expressed in S. globosa. Our previous study demonstrated that the recombinant Escherichia coli strain containing SgcypB gene failed to produce sufficient product when it was induced to express the protein, implying the potential toxicity of recombinant protein to the bacterial host. Bioinformatics analysis revealed that SgCypB contains transmembrane peptides within the 52 amino acid residues at the N-terminus and 21 amino acids near the C-terminus, and 18 amino acid residues within the cytoplasm. AlphaFold2 predicted a SgCypB 3D structure in which there is an independent PPIase domain consisting of a spherical extracellular part. Hence, we chose to express the extracellular domain to yield high-level recombinant protein with PPIase activity. Finally, we successfully produced high-yield, truncated recombinant CypB protein from S. globosa (SgtrCypB) that retained characteristic PPIase activity without host bacterium toxicity. This study presents an alternative expression strategy for proteins toxic to prokaryotes, such as SgCypB. ONE-SENTENCE SUMMARY: The recombinant cyclophilin B protein of Sporothrix globosa was expressed successfully by retaining extracellular domain with peptidyl-prolyl cis-trans isomerase activity to avoid toxicity to the host bacterium.
Subject(s)
Cyclophilins , Escherichia coli , Recombinant Proteins , Sporothrix , Sporothrix/genetics , Sporothrix/enzymology , Sporothrix/drug effects , Sporothrix/metabolism , Cyclophilins/genetics , Cyclophilins/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Gene Expression , Computational Biology , Peptidylprolyl Isomerase/genetics , Peptidylprolyl Isomerase/metabolismABSTRACT
Disturbances in protein phase transitions promote protein aggregationâa neurodegeneration hallmark. The modular Ran-binding protein 2 (Ranbp2) is a cytosolic molecular hub for rate-limiting steps of phase transitions of Ran-GTP-bound protein ensembles exiting nuclear pores. Chaperones also regulate phase transitions and proteostasis by suppressing protein aggregation. Ranbp2 haploinsufficiency promotes the age-dependent neuroprotection of the chorioretina against phototoxicity by proteostatic regulations of neuroprotective substrates of Ranbp2 and by suppressing the buildup of polyubiquitylated substrates. Losses of peptidyl-prolyl cis-trans isomerase (PPIase) and chaperone activities of the cyclophilin domain (CY) of Ranbp2 recapitulate molecular effects of Ranbp2 haploinsufficiency. These CY impairments also stimulate deubiquitylation activities and phase transitions of 19S cap subunits of the 26S proteasome that associates with Ranbp2. However, links between CY moonlighting activity, substrate ubiquitylation, and proteostasis remain incomplete. Here, we reveal the Ranbp2 regulation of small heat shock chaperonesâcrystallins in the chorioretina by proteomics of mice with total or selective modular deficits of Ranbp2. Specifically, loss of CY PPIase of Ranbp2 upregulates αA-Crystallin, which is repressed in adult nonlenticular tissues. Conversely, impairment of CY's chaperone activity opposite to the PPIase pocket downregulates a subset of αA-Crystallin's substrates, γ-crystallins. These CY-dependent effects cause age-dependent and chorioretinal-selective declines of ubiquitylated substrates without affecting the chorioretinal morphology. A model emerges whereby inhibition of Ranbp2's CY PPIase remodels crystallins' expressions, subdues molecular aging, and preordains the chorioretina to neuroprotection by augmenting the chaperone capacity and the degradation of polyubiquitylated substrates against proteostatic impairments. Further, the druggable Ranbp2 CY holds pan-therapeutic potential against proteotoxicity and neurodegeneration.
Subject(s)
Cyclophilins , Molecular Chaperones , Nuclear Pore Complex Proteins , Peptidylprolyl Isomerase , Proteostasis , Animals , Molecular Chaperones/metabolism , Mice , Cyclophilins/metabolism , Proteostasis/physiology , Peptidylprolyl Isomerase/metabolism , Nuclear Pore Complex Proteins/metabolism , Crystallins/metabolismABSTRACT
Central for wound healing is the formation of granulation tissue, which largely consists of collagen and whose importance stretches past wound healing, including being implicated in both fibrosis and skin aging. Cyclophilin D (CyD) is a mitochondrial protein that regulates the permeability transition pore, known for its role in apoptosis and ischemia-reperfusion. To date, the role of CyD in human wound healing and collagen generation has been largely unexplored. Here, we show that CyD was upregulated in normal wounds and venous ulcers, likely adaptive as CyD inhibition impaired reepithelialization, granulation tissue formation, and wound closure in both human and pig models. Overexpression of CyD increased keratinocyte migration and fibroblast proliferation, while its inhibition reduced migration. Independent of wound healing, CyD inhibition in fibroblasts reduced collagen secretion and caused endoplasmic reticulum collagen accumulation, while its overexpression increased collagen secretion. This was confirmed in a Ppif-KO mouse model, which showed a reduction in skin collagen. Overall, this study revealed previously unreported roles of CyD in skin, with implications for wound healing and beyond.
Subject(s)
Collagen , Fibroblasts , Mice, Knockout , Peptidyl-Prolyl Isomerase F , Skin , Wound Healing , Animals , Female , Humans , Male , Mice , Cell Movement , Cell Proliferation , Collagen/metabolism , Cyclophilins/metabolism , Cyclophilins/genetics , Disease Models, Animal , Fibroblasts/metabolism , Granulation Tissue/metabolism , Granulation Tissue/pathology , Keratinocytes/metabolism , Peptidyl-Prolyl Isomerase F/metabolism , Peptidyl-Prolyl Isomerase F/genetics , Skin/metabolism , Skin/pathology , Swine , Wound Healing/physiologyABSTRACT
BACKGROUND AND HYPOTHESIS: Cognitive deficits in schizophrenia are linked to dysfunctions of the dorsolateral prefrontal cortex (DLPFC), including alterations in parvalbumin (PV)-expressing interneurons (PVIs). Redox dysregulation and oxidative stress may represent convergence points in the pathology of schizophrenia, causing dysfunction of GABAergic interneurons and loss of PV. Here, we show that the mitochondrial matrix protein cyclophilin D (CypD), a critical initiator of the mitochondrial permeability transition pore (mPTP) and modulator of the intracellular redox state, is altered in PVIs in schizophrenia. STUDY DESIGN: Western blotting was used to measure CypD protein levels in postmortem DLPFC specimens of schizophrenic patients (nâ =â 27) and matched comparison subjects with no known history of psychiatric or neurological disorders (nâ =â 26). In a subset of this cohort, multilabel immunofluorescent confocal microscopy with unbiased stereological sampling methods were used to quantify (1) numbers of PVI across the cortical mantle (20 unaffected comparison, 14 schizophrenia) and (2) PV and CypD protein levels from PVIs in the cortical layers 2-4 (23 unaffected comparison, 18 schizophrenia). STUDY RESULTS: In schizophrenic patients, the overall number of PVIs in the DLPFC was not significantly altered, but in individual PVIs of layers 2-4 PV protein levels decreased along a superficial-to-deep gradient when compared to unaffected comparison subjects. These laminar-specific PVI alterations were reciprocally linked to significant CypD elevations both in PVIs and total DLPFC gray matter. CONCLUSIONS: Our findings support previously reported PVI anomalies in schizophrenia and suggest that CypD-mediated mPTP formation could be a potential contributor to PVI dysfunction in schizophrenia.
Subject(s)
Interneurons , Parvalbumins , Peptidyl-Prolyl Isomerase F , Schizophrenia , Humans , Schizophrenia/metabolism , Schizophrenia/pathology , Parvalbumins/metabolism , Male , Peptidyl-Prolyl Isomerase F/metabolism , Female , Middle Aged , Adult , Aged , Interneurons/metabolism , Dorsolateral Prefrontal Cortex/metabolism , Prefrontal Cortex/metabolism , Cyclophilins/metabolism , Peptidyl-Prolyl Isomerase DABSTRACT
Mitochondrial dysfunction is implicated in neuropsychiatric disorders. Inhibition of mitochondrial permeability transition pore (mPTP) and thereby enhancement of mitochondrial Ca2+ retention capacity (CRC) is a promising treatment strategy. Here, we screened 1718 compounds to search for drug candidates inhibiting mPTP by measuring their effects on CRC in mitochondria isolated from mouse brains. We identified seco-cycline D (SCD) as an active compound. SCD and its derivative were more potent than a known mPTP inhibitor, cyclosporine A (CsA). The mechanism of action of SCD was suggested likely to be different from CsA that acts on cyclophilin D. Repeated administration of SCD decreased ischemic area in a middle cerebral artery occlusion model in mice. These results suggest that SCD is a useful probe to explore mPTP function.
Subject(s)
Mitochondrial Membrane Transport Proteins , Mitochondrial Permeability Transition Pore , Mice , Animals , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondria/metabolism , Cyclophilins/metabolism , Cyclosporine/pharmacology , Calcium/pharmacology , Brain/metabolismABSTRACT
Kashin-Beck disease is an endemic joint disease characterized by deep chondrocyte necrosis, and T-2 toxin exposure has been confirmed its etiology. This study investigated mechanism of T-2 toxin inducing mitochondrial dysfunction of chondrocytes through p53-cyclophilin D (CypD) pathway. The p53 signaling pathway was significantly enriched in T-2 toxin response genes from GeneCards. We demonstrated the upregulation of the p53 protein and p53-CypD complex in rat articular cartilage and ATDC5 cells induced by T-2 toxin. Transmission electron microscopy showed the damaged mitochondrial structure of ATDC5 cells induced by T-2 toxin. Furthermore, it can lead to overopening of the mitochondrial permeability transition pore (mPTP), decreased mitochondrial membrane potential, and increased reactive oxygen species generation in ATDC5 cells. Pifithrin-α, the p53 inhibitor, alleviated the increased p53-CypD complex and mitochondrial dysfunction of chondrocytes induced by T-2 toxin, suggesting that p53 played an important role in T-2 toxin-induced mitochondrial dysfunction. Mechanistically, T-2 toxin can activate the p53 protein, which can be transferred to the mitochondrial membrane and form a complex with CypD. The increased binding of p53 and CypD mediated the excessive opening of mPTP, changed mitochondrial membrane permeability, and ultimately induced mitochondrial dysfunction and apoptosis of chondrocytes.
Subject(s)
Mitochondrial Diseases , T-2 Toxin , Rats , Animals , Chondrocytes/metabolism , Mitochondrial Membrane Transport Proteins/genetics , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Permeability Transition Pore/metabolism , Tumor Suppressor Protein p53/metabolism , Peptidyl-Prolyl Isomerase F , Cyclophilins/genetics , Cyclophilins/metabolismABSTRACT
Mitochondrial dysfunction is an early pathological feature of Alzheimer disease and plays a crucial role in the development and progression of Alzheimer's disease. Strategies to rescue mitochondrial function and cognition remain to be explored. Cyclophilin D (CypD), the peptidylprolyl isomerase F (PPIase), is a key component in opening the mitochondrial membrane permeability transition pore, leading to mitochondrial dysfunction and cell death. Blocking membrane permeability transition pore opening by inhibiting CypD activity is a promising therapeutic approach for Alzheimer's disease. However, there is currently no effective CypD inhibitor for Alzheimer's disease, with previous candidates demonstrating high toxicity, poor ability to cross the blood-brain barrier, compromised biocompatibility and low selectivity. Here, we report a new class of non-toxic and biocompatible CypD inhibitor, ebselen, using a conventional PPIase assay to screen a library of â¼2000 FDA-approved drugs with crystallographic analysis of the CypD-ebselen crystal structure (PDB code: 8EJX). More importantly, we assessed the effects of genetic and pharmacological blockade of CypD on Alzheimer's disease mitochondrial and glycolytic bioenergetics in Alzheimer's disease-derived mitochondrial cybrid cells, an ex vivo human sporadic Alzheimer's disease mitochondrial model, and on synaptic function, inflammatory response and learning and memory in Alzheimer's disease mouse models. Inhibition of CypD by ebselen protects against sporadic Alzheimer's disease- and amyloid-ß-induced mitochondrial and glycolytic perturbation, synaptic and cognitive dysfunction, together with suppressing neuroinflammation in the brain of Alzheimer's disease mouse models, which is linked to CypD-related membrane permeability transition pore formation. Thus, CypD inhibitors have the potential to slow the progression of neurodegenerative diseases, including Alzheimer's disease, by boosting mitochondrial bioenergetics and improving synaptic and cognitive function.
Subject(s)
Alzheimer Disease , Isoindoles , Mitochondria , Organoselenium Compounds , Peptidyl-Prolyl Isomerase F , Alzheimer Disease/drug therapy , Alzheimer Disease/metabolism , Peptidyl-Prolyl Isomerase F/metabolism , Animals , Mitochondria/drug effects , Mitochondria/metabolism , Mice , Humans , Cognition/drug effects , Azoles/pharmacology , Azoles/therapeutic use , Cyclophilins/metabolism , Cyclophilins/antagonists & inhibitors , Mice, Transgenic , Mice, Inbred C57BL , Male , Disease Models, Animal , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic useABSTRACT
The mitochondrial permeability transition pore (mPTP) is a channel in the inner mitochondrial membrane whose sustained opening in response to elevated mitochondrial matrix Ca2+ concentrations triggers necrotic cell death. The molecular identity of mPTP is unknown. One proposed candidate is the mitochondrial ATP synthase, whose canonical function is to generate most ATP in multicellular organisms. Here, we present mitochondrial, cellular, and in vivo evidence that, rather than serving as mPTP, the mitochondrial ATP synthase inhibits this pore. Our studies confirm previous work showing persistence of mPTP in HAP1 cell lines lacking an assembled mitochondrial ATP synthase. Unexpectedly, however, we observe that Ca2+-induced pore opening is markedly sensitized by loss of the mitochondrial ATP synthase. Further, mPTP opening in cells lacking the mitochondrial ATP synthase is desensitized by pharmacological inhibition and genetic depletion of the mitochondrial cis-trans prolyl isomerase cyclophilin D as in wild-type cells, indicating that cyclophilin D can modulate mPTP through substrates other than subunits in the assembled mitochondrial ATP synthase. Mitoplast patch clamping studies showed that mPTP channel conductance was unaffected by loss of the mitochondrial ATP synthase but still blocked by cyclophilin D inhibition. Cardiac mitochondria from mice whose heart muscle cells we engineered deficient in the mitochondrial ATP synthase also demonstrate sensitization of Ca2+-induced mPTP opening and desensitization by cyclophilin D inhibition. Further, these mice exhibit strikingly larger myocardial infarctions when challenged with ischemia/reperfusion in vivo. We conclude that the mitochondrial ATP synthase does not function as mPTP and instead negatively regulates this pore.
Subject(s)
Mitochondrial Permeability Transition Pore , Mitochondrial Proton-Translocating ATPases , Mice , Animals , Mitochondrial Proton-Translocating ATPases/genetics , Mitochondrial Proton-Translocating ATPases/metabolism , Mitochondrial Permeability Transition Pore/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Cyclophilins/genetics , Cyclophilins/metabolism , Peptidyl-Prolyl Isomerase F , Mitochondria, Heart/genetics , Mitochondria, Heart/metabolism , Calcium/metabolismABSTRACT
Hepatitis E virus (HEV) is an emerging pathogen responsible for more than 20 million cases of acute hepatitis globally per annum. Healthy individuals typically have a self-limiting infection, but mortality rates in some populations such as pregnant women can reach 30â%. A detailed understanding of the virus lifecycle is lacking, mainly due to limitations in experimental systems. In this regard, the cyclophilins are an important family of proteins that have peptidyl-prolyl isomerase activity and play roles in the replication of a number of positive-sense RNA viruses, including hepatotropic viruses such as hepatitis C virus (HCV). Cyclophilins A and B (CypA/B) are the two most abundant Cyps in hepatocytes and are therefore potential targets for pan-viral therapeutics. Here, we investigated the importance of CypA and CypB for HEV genome replication using sub-genomic replicons. Using a combination of pharmacological inhibition by cyclosporine A (CsA), and silencing by small hairpin RNA we find that CypA and CypB are not essential for HEV replication. However, we find that silencing of CypB reduces replication of some HEV isolates in some cells. Furthermore, sensitivity to Cyp silencing appears to be partly conferred by the sequence within the hypervariable region of the viral polyprotein. These data suggest HEV is atypical in its requirements for cyclophilin for viral genome replication and that this phenomenon could be genotype- and sequence-specific.
Subject(s)
Hepatitis C , Hepatitis E virus , Pregnancy , Female , Humans , Cyclophilins/genetics , Cyclophilins/metabolism , Hepatitis E virus/genetics , Hepacivirus/genetics , Cyclosporine/pharmacology , Virus ReplicationABSTRACT
Penicillium species are an industrially important group of fungi. Cyclophilins are ubiquitous proteins and several members of this family exhibit peptidyl-prolyl cis-trans isomerase (PPIase) activity. We had earlier demonstrated that the salt-induced PPIase activity in a halotolerant strain of P. oxalicum was associated with enhanced expression of a cyclophilin gene, PoxCYP18. Cloning and characterization of PoxCYP18 revealed that its cDNA consists of 522 bp encoding a protein of 173 amino acid residues, with predicted molecular mass and pI values of 18.91 kDa and 8.87, respectively. The recombinant PoxCYP18 can catalyze cis-trans isomerization of peptidyl-prolyl bond with a catalytic efficiency of 1.46 × 107 M-1 s-1 and is inhibited specifically only by cyclosporin A, with an inhibition constant of 5.04 ± 1.13 nM. PoxCYP18 consists of two cysteine residues at positions - 45 and - 170, and loses its activity under oxidizing conditions. Substitution of these residues alone or together by site-directed mutagenesis revealed that the PPIase activity of PoxCYP18 is regulated through a redox mechanism involving the formation of disulfide linkages. Heterologous expression of PoxCYP18 conferred enhanced tolerance to salt stress in transgenic E. coli cells, implying that this protein imparts protection to cellular processes against salt-induced damage.
Subject(s)
Cyclophilins , Penicillium , Cyclophilins/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Peptidylprolyl Isomerase/genetics , Penicillium/genetics , Penicillium/metabolism , Cyclosporine/pharmacologyABSTRACT
This study tested whether human umbilical cord-derived mesenchymal stem cells (HUCDMSCs) treatment effectively protected the rat lung against acute respiratory distress syndrome (ARDS) injury, and benefits of early and dose-dependent treatment. Rat pulmonary epithelial cell line L2 (PECL2) were categorized into G1 (PECL2), G2 (PECL2 + healthy rat lung-derived extraction/50 mg/ml co-cultured for 24 h), G3 (PECL2 + ARDS rat lung-derived extraction/50 mg/ml co-cultured for 24 h), and G4 (condition as G3 + HUCDMSCs/1 × 105/co-cultured for 24 h). The result showed that the protein expressions of inflammatory (HMGB-1/TLR-2/TLR-4/MAL/TRAM/MyD88/TRIF/TRAF6/IkB/NF-κB/IL-1ß/TNF-α), oxidative-stress/mitochondrial-damaged (NOX-1/NOX-2/ASK1/p-MKK4/p-MKK7/JNKs/JUN/cytosolic-cytochrome-C/cyclophilin-D/DRP1), and cell-apoptotic/fibrotic (cleaved-caspase 3/cleaved-PARP/TGF-ß/p-Smad3) biomarkers were significantly increased in G3 than in G1/G2 and were significantly reversed in G4 (all P < 0.001), but they were similar between G1/G2. Adult male rats (n = 42) were equally categorized into group 1 (normal control), group 2 (ARDS only), group 3 [ARDS + HUCDMSCs/1.2 × 106 cells intravenous administration at 3 h after 48 h ARDS induction (i.e., early treatment)], group 4 [ARDS + HUCDMSCs/1.2 × 106 cells intravenous administration at 24 h after 48 h ARDS induction (late treatment)], and group 5 [ARDS + HUCDMSCs/1.2 × 106 cells intravenous administration at 3 h/24 h after-48 h ARDS induction (dose-dependent treatment)]. By day 5 after ARDS induction, the SaO2%/immune regulatory T cells were highest in group 1, lowest in group 2, significantly lower in group 4 than in groups 3/5, and significantly lower in group 3 than in group 5, whereas the circulatory/bronchioalveolar lavage fluid inflammatory cells (CD11b-c+/LyG6+/MPO+)/circulatory immune cells (CD3-C4+/CD3-CD8+)/lung-leakage-albumin level/lung injury score/lung protein expressions of inflammatory (HMGB-1/TLR-2/TLR-4/MAL/TRAM/MyD88/TRIF/TRAF6/IκB-ß/p-NF-κB/IL-1ß/TNF-α)/fibrotic (p-SMad3/TGF-ß), apoptosis (mitochondrial-Bax/cleaved-caspase-3)/oxidative-cell-stress (NOX-1/NOX-2/ASK1/p-MKK4/p-MKK7/p-JNKs/p-cJUN)/mitochondrial damaged (cyclophilin-D/DRP1/cytosolic-cytochrome-C) biomarkers displayed an opposite pattern of SaO2% among the groups (all P < 0.0001). Early administration was superior to and two-dose counterpart was even more superior to late HUCDMSCs treatment for protecting the lung against ARDS injury.
Subject(s)
Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells , Respiratory Distress Syndrome , Rats , Male , Humans , Animals , Rats, Sprague-Dawley , Rodentia/metabolism , Cyclophilins/metabolism , Tumor Necrosis Factor-alpha/metabolism , NF-kappa B/metabolism , Myeloid Differentiation Factor 88/metabolism , TNF Receptor-Associated Factor 6/metabolism , Toll-Like Receptor 2/metabolism , Toll-Like Receptor 4/metabolism , Inflammation/therapy , Inflammation/metabolism , Respiratory Distress Syndrome/therapy , Mesenchymal Stem Cells/metabolism , Oxidative Stress , Transforming Growth Factor beta/metabolism , Biomarkers/metabolism , Cytochromes/metabolism , HMGB Proteins/metabolism , Adaptor Proteins, Vesicular Transport/metabolismABSTRACT
BACKGROUND: A hallmark pathology of Alzheimer's disease (AD) is the construction of neurofibrillary tangles, which are made of hyperphosphorylated Tau. The cis-proline isomer of the pThr/Ser-Pro sequence has been suggested to act as an aggregation precursor according to the 'Cistauosis' hypothesis; however, this aggregation scheme is not yet completely approved. Various peptidyl-prolyl isomerases (PPIases) may specifically isomerize cis/trans-proline bonds and restitute Tau's ability to attach microtubules and may control Tau amyloid aggregation in AD. METHODS: In this study, we provided experimental evidence for indicating the effects of the plant Cyclophilin (P-Cyp) from Platanus orientalis pollens on the Tau aggregation by various spectroscopic techniques. RESULTS: Our findings disclosed that the rate/extent of amyloid formation in the Tau sample which is incubated with P-Cyp decreased and these observations do not seem to be due to the macromolecular crowding effect. Also, as proven that 80% of the prolines in the unfolded protein are in the trans conformation, urea-induced unfolding analyses confirmed this conclusion and showed that the aggregation rate/extent of urea-treated Tau samples decreased compared with those of the native protein. Also, XRD analysis indicated the reduction of scattering intensities and beta structures of amyloid fibrils in the presence of P-Cyp. Therefore, the ability of P-Cyp to suppress Tau aggregation probably depends on cis to trans isomerization of proline peptide bonds (X-Pro) and decreasing cis isomers in vitro. CONCLUSION: The findings of the current study may inspire possible protective/detrimental effects of various types of cyclophilins on AD onset/progression through direct regulation of intracellular Tau molecules and provides evidence that a protein from a plant source is able to enter the cell cytoplasm and may affect the behavior of cytoplasmic proteins.
Subject(s)
Alzheimer Disease , Cyclophilins , Cyclophilins/metabolism , Amyloid/metabolism , Allergens , tau Proteins/metabolism , Alzheimer Disease/drug therapy , Alzheimer Disease/metabolism , Pollen/metabolism , Proline/pharmacology , Proline/chemistry , Proline/metabolism , Urea , Amyloid beta-PeptidesABSTRACT
Inflammation is the leading subjacent cause of many chronic diseases. Despite several studies in the last decades, the molecular mechanism involving its pathophysiology is not fully known. Recently, the implication of cyclophilins in inflammatory-based diseases has been demonstrated. However, the main role of cyclophilins in these processes remains elusive. Hence, a mouse model of systemic inflammation was used to better understand the relationship between cyclophilins and their tissue distribution. To induce inflammation, mice were fed with high-fat diet for 10 weeks. In these conditions, serum levels of interleukins 2 and 6, tumour necrosis factor-α, interferon-Ï, and the monocyte chemoattractant protein 1 were elevated, evidencing a systemic inflammatory state. Then, in this inflammatory model, cyclophilins and CD147 profiles in the aorta, liver, and kidney were studied. The results demonstrate that, upon inflammatory conditions, cyclophilins A and C expression levels were increased in the aorta. Cyclophilins A and D were augmented in the liver, meanwhile, cyclophilins B and C were diminished. In the kidney, cyclophilins B and C levels were elevated. Furthermore, CD147 receptor was also increased in the aorta, liver, and kidney. In addition, when cyclophilin A was modulated, serum levels of inflammatory mediators were decreased, indicating a reduction in systemic inflammation. Besides, the expression levels of cyclophilin A and CD147 were also reduced in the aorta and liver, when cyclophilin A was modulated. Therefore, these results suggest that each cyclophilin has a different profile depending on the tissue, under inflammatory conditions.
Subject(s)
Cyclophilin A , Cyclophilins , Animals , Mice , Cyclophilins/metabolism , Cyclophilin A/pharmacology , Inflammation/metabolismABSTRACT
Pre-mRNA splicing follows a pathway driven by ATP-dependent RNA helicases. A crucial event of the splicing pathway is the catalytic activation, which takes place at the transition between the activated Bact and the branching-competent B* spliceosomes. Catalytic activation occurs through an ATP-dependent remodelling mediated by the helicase PRP2 (also known as DHX16)1-3. However, because PRP2 is observed only at the periphery of spliceosomes3-5, its function has remained elusive. Here we show that catalytic activation occurs in two ATP-dependent stages driven by two helicases: PRP2 and Aquarius. The role of Aquarius in splicing has been enigmatic6,7. Here the inactivation of Aquarius leads to the stalling of a spliceosome intermediate-the BAQR complex-found halfway through the catalytic activation process. The cryogenic electron microscopy structure of BAQR reveals how PRP2 and Aquarius remodel Bact and BAQR, respectively. Notably, PRP2 translocates along the intron while it strips away the RES complex, opens the SF3B1 clamp and unfastens the branch helix. Translocation terminates six nucleotides downstream of the branch site through an assembly of PPIL4, SKIP and the amino-terminal domain of PRP2. Finally, Aquarius enables the dissociation of PRP2, plus the SF3A and SF3B complexes, which promotes the relocation of the branch duplex for catalysis. This work elucidates catalytic activation in human splicing, reveals how a DEAH helicase operates and provides a paradigm for how helicases can coordinate their activities.
Subject(s)
Biocatalysis , RNA Splicing , Humans , Adenosine Triphosphate/metabolism , Cryoelectron Microscopy , Cyclophilins/metabolism , RNA Precursors/metabolism , RNA Splicing Factors/metabolism , RNA-Binding Proteins/metabolism , Spliceosomes/metabolismABSTRACT
Plants have evolved multiple mechanisms to cope with diverse types of light stress, particularly the regulation of the electron transport chain (ETC). Under high light (HL) conditions, the balance of electron flux in the ETC is disturbed, which leads to the overaccumulation of reactive oxygen species (ROS) and results in photodamage and photoinhibition. The cytochrome (Cyt) b6/f complex, which coordinates electron transfer between photosystems I and II (PSI and PSII), plays an essential role in regulating the ETC and initiating photoprotection. However, how the Cyt b6/f complex is maintained under HL conditions remains unclear. Here, we report that the activity of the Cyt b6/f complex is sustained by thylakoid-localized cyclophilin 37 (CYP37) in Arabidopsis (Arabidopsis thaliana). Compared with wild-type plants, cyp37 mutants displayed an imbalance in electron transport from Cyt b6/f to PSI under HL stress, which led to increased ROS accumulation, decreased anthocyanin biosynthesis, and increased chlorophyll degradation. Surprisingly, CYP37's role in regulating ETC balance was independent of photosynthesis control, which was indicated by a higher Y (ND), an indicator of P700 oxidation in PSI. Furthermore, the interaction between CYP37 and photosynthetic electron transfer A (PetA), a subunit of the Cyt b6/f complex, suggests that the central function of CYP37 is to maintain Cyt b6/f complex activity rather than to serve as an assembly factor. Our study provides insights into how plants balance electron flow between PSII and PSI via Cyt b6/f complex under HL.
Subject(s)
Arabidopsis , Electron Transport/physiology , Arabidopsis/genetics , Arabidopsis/metabolism , Cyclophilins/genetics , Cyclophilins/metabolism , Cytochromes b6/metabolism , Reactive Oxygen Species/metabolism , Chlorophyll/metabolism , Photosynthesis/physiology , Photosystem I Protein Complex/genetics , Photosystem I Protein Complex/metabolism , Photosystem II Protein Complex/metabolism , Cytochrome b6f Complex/genetics , Cytochrome b6f Complex/metabolism , Plants/metabolismABSTRACT
Procoagulant platelets are associated with an increased risk for thrombosis. Procoagulant platelet formation is mediated via Cyclophilin D (CypD) mediated opening of the mitochondrial permeability transition pore. Inhibiting CypD activity could therefore be an interesting approach to limiting thrombosis. In this study, we investigated the potential of two novel, non-immunosuppressive, non-peptidic small-molecule cyclophilin inhibitors (SMCypIs) to limit thrombosis in vitro, in comparison with the cyclophilin inhibitor and immunosuppressant Cyclosporin A (CsA). Both cyclophilin inhibitors significantly decreased procoagulant platelet formation upon dual-agonist stimulation, shown by a decreased phosphatidylserine (PS) exposure, as well as a reduction in the loss of mitochondrial membrane potential. Furthermore, the SMCypIs potently reduced procoagulant platelet-dependent clotting time, as well as fibrin formation under flow, comparable to CsA. No effect was observed on agonist-induced platelet activation measured by P-selectin expression, as well as CypA-mediated integrin αIIbß3 activation. Importantly, whereas CsA increased Adenosine 5'-diphosphate (ADP)-induced platelet aggregation, this was unaffected in the presence of the SMCypIs. We here demonstrate specific cyclophilin inhibition does not affect normal platelet function, while a clear reduction in procoagulant platelets is observed. Reducing platelet procoagulant activity by inhibiting cyclophilins with SMCypIs forms a promising strategy to limit thrombosis.
Subject(s)
Cyclophilins , Thrombosis , Mice , Animals , Humans , Cyclophilins/metabolism , Mice, Knockout , Blood Platelets/metabolism , Platelet Activation , Thrombosis/metabolism , Platelet Glycoprotein GPIIb-IIIa Complex/metabolismABSTRACT
The mitochondrial permeability transition pore (mtPTP) plays a vital role in altering the structure and function of mitochondria. Cyclophilin D (CypD) is a mitochondrial protein that regulates mtPTP function and a known drug target for therapeutic studies involving mitochondria. While the effect of aromatase inhibition on the mtPTP has been studied previously, the effect of anastrozole on the mtPTP has not been completely elucidated. The role of anastrozole in modulating the mtPTP was evaluated by docking, molecular dynamics and network-guided studies using human CypD data. The peripheral blood mononuclear cells (PBMCs) of patients with mitochondrial disorders and healthy controls were treated with anastrozole and evaluated for mitochondrial permeability transition pore (mtPTP) function and apoptosis using a flow cytometer. Spectrophotometry was employed for estimating total ATP levels. The anastrozole-CypD complex is more stable than cyclosporin A (CsA)-CypD. Anastrozole performed better than cyclosporine in inhibiting mtPTP. Additional effects included inducing mitochondrial membrane depolarization and a reduction in mitochondrial swelling and superoxide generation, intrinsic caspase-3 activity and cellular apoptosis, along with an increase in ATP levels. Anastrozole may serve as a potential therapeutic agent for mitochondrial disorders and ameliorate the clinical phenotype by regulating the activity of mtPTP. However, further studies are required to substantiate our preliminary findings.Communicated by Ramaswamy H. Sarma.
Subject(s)
Mitochondrial Diseases , Mitochondrial Permeability Transition Pore , Humans , Mitochondrial Permeability Transition Pore/metabolism , Mitochondrial Permeability Transition Pore/pharmacology , Anastrozole/pharmacology , Anastrozole/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Membrane Transport Proteins/pharmacology , Leukocytes, Mononuclear/metabolism , Mitochondria/metabolism , Peptidyl-Prolyl Isomerase F , Cyclophilins/genetics , Cyclophilins/metabolism , Adenosine Triphosphate/metabolism , Mitochondrial Diseases/metabolismABSTRACT
Cyclosporin (CsA) has antiparasite activity against the human pathogen Toxoplasma gondii. A possible mechanism of action involves CsA binding to T. gondii cyclophilins, although much remains to be understood. Herein, we characterize the functional and structural properties of a conserved (TgCyp23) and a more divergent (TgCyp18.4) cyclophilin isoform from T. gondii. While TgCyp23 is a highly active cis-trans-prolyl isomerase (PPIase) and binds CsA with nanomolar affinity, TgCyp18.4 shows low PPIase activity and is significantly less sensitive to CsA inhibition. The crystal structure of the TgCyp23:CsA complex was solved at the atomic resolution showing the molecular details of CsA recognition by the protein. Computational and structural studies revealed relevant differences at the CsA-binding site between TgCyp18.4 and TgCyp23, suggesting that the two cyclophilins might have distinct functions in the parasite. These studies highlight the extensive diversification of TgCyps and pave the way for antiparasite interventions based on selective targeting of cyclophilins.
Subject(s)
Cyclophilins , Toxoplasma , Binding Sites , Cyclophilins/chemistry , Cyclophilins/metabolism , Cyclosporine/pharmacology , Cyclosporine/metabolism , Protein IsoformsABSTRACT
Resistance of the human malaria parasites, Plasmodium falciparum, to artemisinins is now fully established in Southeast Asia and is gradually emerging in Sub-Saharan Africa. Although nonsynonymous SNPs in the pfk13 Kelch-repeat propeller (KREP) domain are clearly associated with artemisinin resistance, their functional relevance requires cooperation with other genetic factors/alterations of the P. falciparum genome, collectively referred to as genetic background. Here we provide experimental evidence that P. falciparum cyclophilin 19B (PfCYP19B) may represent one putative factor in this genetic background, contributing to artemisinin resistance via its increased expression. We show that overexpression of PfCYP19B in vitro drives limited but significant resistance to not only artemisinin but also piperaquine, an important partner drug in artemisinin-based combination therapies. We showed that PfCYP19B acts as a negative regulator of the integrated stress response (ISR) pathway by modulating levels of phosphorylated eIF2α (eIF2α-P). Curiously, artemisinin and piperaquine affect eIF2α-P in an inverse direction that in both cases can be modulated by PfCYP19B towards resistance. Here we also provide evidence that the upregulation of PfCYP19B in the drug-resistant parasites appears to be maintained by a short tandem repeat (SRT) sequence polymorphism in the gene's promoter region. These results support a model that artemisinin (and other drugs) resistance mechanisms are complex genetic traits being contributed to by altered expression of multiple genes driven by genetic polymorphism at their promoter regions.