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Detailed understanding of how conformational dynamics orchestrates function in allosteric regulation of recognition and catalysis remains ambiguous. Here, we simulate CypA using multiple-microsecond-long atomistic molecular dynamics in explicit solvent and carry out NMR experiments. We analyze a large amount of time-dependent multidimensional data with a coarse-grained approach and map key dynamical features within individual macrostates by defining dynamics in terms of residue–residue contacts. The effects of substrate binding are observed to be largely sensed at a location over 15 Å from the active site, implying its importance in allostery. Using NMR experiments, we confirm that a dynamic cluster of residues in this distal region is directly coupled to the active site. Furthermore, the dynamical network of interresidue contacts is found to be coupled and temporally dispersed, ranging over 4 to 5 orders of magnitude. Finally, using network centrality measures we demonstrate the changes in the communication network, connectivity, and influence of CypA residues upon substrate binding, mutation, and during catalysis. We identify key residues that potentially act as a bottleneck in the communication flow through the distinct regions in CypA and, therefore, as targets for future mutational studies. Mapping these dynamical features and the coupling of dynamics to function has crucial ramifications in understanding allosteric regulation in enzymes and proteins, in general.

The APOE4-mediated proinflammatory pathway is shown to initiate blood–brain barrier breakdown and resulting neurodegeneration in transgenic mice. Restoring the blood–brain barrier There are known connections between the Alzheimer's-disease-linked APOE4 gene and cerebrovascular integrity. However, the mechanisms that drive known blood–brain-barrier dysfunction both in rodent models and in APOE4-associated neurological disorders are unknown. Here, Berislav Zlokovic and colleagues report that APOE4 activates a matrix metalloproteinase pathway in cells forming the blood–brain barrier in mice, leading to its breakdown and the neuronal uptake of blood-derived neurotoxic proteins. In turn, microvascular and cerebral blood flow are reduced; together, these deficits can initiate neurodegenerative changes in rodents. The authors suggest that cyclophilin A (CypA), a component of the APOE4-activated pathway, is a potential target for treating APOE4-mediated neuronal dysfunction. Treatment with the CypA inhibitor cyclosporine A restores the blood–brain barrier in APOE4 mice. Human apolipoprotein E has three isoforms: APOE2, APOE3 and APOE4 1 . APOE4 is a major genetic risk factor for Alzheimer’s disease 2 , 3 and is associated with Down’s syndrome dementia and poor neurological outcome after traumatic brain injury and haemorrhage 3 . Neurovascular dysfunction is present in normal APOE4 carriers 4 , 5 , 6 and individuals with APOE4 -associated disorders 3 , 7 , 8 , 9 , 10 . In mice, lack of Apoe leads to blood–brain barrier (BBB) breakdown 11 , 12 , whereas APOE4 increases BBB susceptibility to injury 13 . How APOE genotype affects brain microcirculation remains elusive. Using different APOE transgenic mice, including mice with ablation and/or inhibition of cyclophilin A (CypA), here we show that expression of APOE4 and lack of murine Apoe, but not APOE2 and APOE3, leads to BBB breakdown by activating a proinflammatory CypA–nuclear factor-κB–matrix-metalloproteinase-9 pathway in pericytes. This, in turn, leads to neuronal uptake of multiple blood-derived neurotoxic proteins, and microvascular and cerebral blood flow reductions. We show that the vascular defects in Apoe- deficient and APOE4 -expressing mice precede neuronal dysfunction and can initiate neurodegenerative changes. Astrocyte-secreted APOE3, but not APOE4, suppressed the CypA–nuclear factor-κB–matrix-metalloproteinase-9 pathway in pericytes through a lipoprotein receptor. Our data suggest that CypA is a key target for treating APOE4-mediated neurovascular injury and the resulting neuronal dysfunction and degeneration.

The entire hepatitis C virus life cycle can be recapitulated in an inbred mouse model, allowing preclinical assessment of antiviral therapeutics and vaccines. Humanized mouse model for hepatitis C infection In a 2009 Nature paper, Alexander Ploss and colleagues showed that transient expression of the human genes CD81 and occludin ( OCLN ) constituted a minimal set of cellular factors required for uptake of hepatitis C virus (HCV) into immune-competent mouse cells. Now they report that transgenic immune-deficient mice stably expressing CD81 and OCLN can sustain the complete HCV replication cycle with measurable viraemia. The availability of this genetically humanized mouse model opens the way to closer study of HCV infection in vivo and should provide a valuable platform for testing potential therapeutics. More than 130 million people worldwide chronically infected with hepatitis C virus (HCV) are at risk of developing severe liver disease. Antiviral treatments are only partially effective against HCV infection, and a vaccine is not available. Development of more efficient therapies has been hampered by the lack of a small animal model. Building on the observation that CD81 and occludin (OCLN) comprise the minimal set of human factors required to render mouse cells permissive to HCV entry 1 , we previously showed that transient expression of these two human genes is sufficient to allow viral uptake into fully immunocompetent inbred mice 2 . Here we demonstrate that transgenic mice stably expressing human CD81 and OCLN also support HCV entry, but innate and adaptive immune responses restrict HCV infection in vivo . Blunting antiviral immunity in genetically humanized mice infected with HCV results in measurable viraemia over several weeks. In mice lacking the essential cellular co-factor cyclophilin A (CypA), HCV RNA replication is markedly diminished, providing genetic evidence that this process is faithfully recapitulated. Using a cell-based fluorescent reporter activated by the NS3-4A protease we visualize HCV infection in single hepatocytes in vivo . Persistently infected mice produce de novo infectious particles, which can be inhibited with directly acting antiviral drug treatment, thereby providing evidence for the completion of the entire HCV life cycle in inbred mice. This genetically humanized mouse model opens new opportunities to dissect genetically HCV infection in vivo and provides an important preclinical platform for testing and prioritizing drug candidates and may also have utility for evaluating vaccine efficacy.

Cyclophilin (Cyp) inhibitors are of clinical interest in respect to their antiviral activities in the context of many viral infections including chronic hepatitis B and C. Cyps are a group of enzymes with peptidyl-prolyl isomerase activity (PPIase), known to be required for replication of diverse viruses including hepatitis B and C viruses (HBV and HCV). Amongst the Cyp family, the molecular mechanisms underlying the antiviral effects of CypA have been investigated in detail, but potential roles of other Cyps are less well studied in the context of viral hepatitis. Furthermore, most studies investigating the role of Cyps in viral hepatitis did not investigate the potential therapeutic effects of their inhibition in already-established infections but have rather been performed in the context of neo-infections. Here, we investigated the effects of genetically silencing Cyps on persistent HCV and HBV infections. We confirm antiviral effects of CypA and CypD knock down and demonstrate novel roles for CypG and CypH in HCV replication. We show, furthermore, that CypA silencing has a modest but reproducible impact on persistent HBV infections in cultured human hepatocytes.

The pathogenesis of aortic aneurysms involves inflammatory cell recruitment and increased levels of reactive oxygen species and matrix metalloproteases. Kimio Satoh et al . now mechanistically link the protein cyclophilin A—expressed in vascular smooth muscle cells—to these known mediators of aortic aneurysm formation and provide evidence in both mice and humans for the importance of cyclophilin A in aortic aneurysm formation. Inflammation and oxidative stress are pathogenic mediators of many diseases, but molecules that could be therapeutic targets remain elusive. Inflammation and matrix degradation in the vasculature are crucial for abdominal aortic aneurysm (AAA) formation. Cyclophilin A (CypA, encoded by Ppia ) is highly expressed in vascular smooth muscle cells (VSMCs), is secreted in response to reactive oxygen species (ROS) and promotes inflammation. Using the angiotensin II (AngII)-induced AAA model in Apoe −/− mice, we show that Apoe −/− Ppia −/− mice are completely protected from AngII–induced AAA formation, in contrast to Apoe −/− Ppia +/+ mice. Apoe −/− Ppia −/− mice show decreased inflammatory cytokine expression, elastic lamina degradation and aortic expansion. These features were not altered by reconstitution of bone marrow cells from Ppia +/+ mice. Mechanistic studies showed that VSMC-derived intracellular and extracellular CypA are required for ROS generation and matrix metalloproteinase-2 activation. These data define a previously undescribed role for CypA in AAA formation and suggest CypA as a new target for treating cardiovascular disease.

Cyclophilin A (CyPA) is a ubiquitously distributed protein belonging to the immunophilin family. CyPA has peptidyl prolyl cis - trans isomerase (PPIase) activity, which regulates protein folding and trafficking. Although CyPA was initially believed to function primarily as an intracellular protein, recent studies have revealed that it can be secreted by cells in response to inflammatory stimuli. Current research in animal models and humans has provided compelling evidences supporting the critical function of CyPA in several human diseases. This review discusses recently available data about CyPA in cardiovascular diseases, viral infections, neurodegeneration, cancer, rheumatoid arthritis, sepsis, asthma, periodontitis and aging. It is believed that further elucidations of the role of CyPA will provide a better understanding of the molecular mechanisms underlying these diseases and will help develop novel pharmacological therapies.

Background Heart failure (HF) represents the end stage of cardiovascular diseases with high mortality and limited treatment options. Cyclophilin B (CypB), known mainly as an endoplasmic reticulum chaperone, has been implicated in cardiovascular diseases. But the role of CypB in HF remains unclear. Methods Transverse aortic constriction (TAC) surgery on mice in vivo was conducted to model cardiac hypertrophy (CH) and HF, and angiotensin II (Ang II) was applied to neonatal rat cardiomyocytes in vitro to mimic cardiomyocyte hypertrophy. The effects of CypB deficiency on CH/HF were evaluated by echocardiography, tissue staining, and molecular expression assays. The mechanism of CypB action was elucidated by RNA sequencing, bioinformatics analysis, mitochondrial function assay, immunofluorescence staining, glucose uptake assay, PET/CT scan, transcription factor analysis, dual luciferase reporter assay, Cut&Run-qPCR assay, STAT3 inhibitor, and overexpression virus. Results Increased expression of CypB has been observed in hypertrophied and failing hearts. CypB deficiency improves cardiac function, reduces hypertrophy after TAC surgery, and attenuates Ang II-induced cardiomyocyte hypertrophy. Mechanistically, CypB deletion increases AMPK phosphorylation, enhances the expression of glucose transporter type 1 (GLUT1), glucose transporter type 4 (GLUT4), peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), and downstream signaling molecules, thereby promoting cardiac glucose catabolism and mitochondrial function. STAT3 transcriptionally activates CypB expression, STAT3 inhibition ameliorates TAC-induced heart failure, and CypB deficiency reverses STAT3 overexpression-induced HF. Conclusions CypB deficiency ameliorates CH and HF by enhancing cardiac energy production, providing a potential therapeutic target for CH and HF. Graphical Abstract

The mitochondrial permeability transition pore (mPTP) significantly impacts mitochondrial responses to cell death signals through its structural opening. Cyclophilin D (CypD) serves as a key regulator of the mPTP and plays a pivotal role in governing mitochondrial responses to cell death. In this study, we have demonstrated that Toxoplasma expresses a homolog of cyclophilin D, named TgCypD, which is localized in the mitochondria. Depletion of TgCypD resulted in a modest inhibition of tachyzoite invasion and proliferation, with no notable effect on mitochondrial morphology. However, TgCypD deficiency led to the inhibition of cytochrome c release from mitochondria into the cytosol, thereby imparting resistance to oxidative stress-induced cell death. Our findings suggest that T. gondii contains the mPTP component protein TgCypD, which is intricately involved in regulating mitochondrial responses to cell death.

Coronaviruses (CoVs) are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. The outbreak of the severe acute respiratory syndrome (SARS) in 2002/2003 has demonstrated human vulnerability to (Coronavirus) CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biology approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B) as interaction partners of the CoV non-structural protein 1 (Nsp1). These molecules modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as observed in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA) blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivatives of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock.

Vascular contributions to dementia and Alzheimer’s disease are increasingly recognized 1 – 6 . Recent studies have suggested that breakdown of the blood–brain barrier (BBB) is an early biomarker of human cognitive dysfunction 7 , including the early clinical stages of Alzheimer’s disease 5 , 8 – 10 . The E4 variant of apolipoprotein E ( APOE4 ), the main susceptibility gene for Alzheimer’s disease 11 – 14 , leads to accelerated breakdown of the BBB and degeneration of brain capillary pericytes 15 – 19 , which maintain BBB integrity 20 – 22 . It is unclear, however, whether the cerebrovascular effects of APOE4 contribute to cognitive impairment. Here we show that individuals bearing APOE4 (with the ε3/ε4 or ε4/ε4 alleles) are distinguished from those without APOE4 (ε3/ε3) by breakdown of the BBB in the hippocampus and medial temporal lobe. This finding is apparent in cognitively unimpaired APOE4 carriers and more severe in those with cognitive impairment, but is not related to amyloid-β or tau pathology measured in cerebrospinal fluid or by positron emission tomography 23 . High baseline levels of the BBB pericyte injury biomarker soluble PDGFRβ 7 , 8 in the cerebrospinal fluid predicted future cognitive decline in APOE4 carriers but not in non-carriers, even after controlling for amyloid-β and tau status, and were correlated with increased activity of the BBB-degrading cyclophilin A-matrix metalloproteinase-9 pathway 19 in cerebrospinal fluid. Our findings suggest that breakdown of the BBB contributes to APOE4 -associated cognitive decline independently of Alzheimer’s disease pathology, and might be a therapeutic target in APOE4 carriers. Breakdown of the blood–brain barrier in individuals carrying the ε4 allele of the APOE gene, but not the ε3 allele, increases with and predicts cognitive impairment and is independent of amyloid β or tau pathology.