Evaluating the potential of non-immunosuppressive cyclosporin analogs for targeting Toxoplasma gondii cyclophilin: Insights from structural studies.

Toxoplasmosis persists as a prevalent disease, facing challenges from parasite resistance and treatment side effects. Consequently, identifying new drugs by exploring novel protein targets is essential for effective intervention. Cyclosporin A (CsA) possesses antiparasitic activity against Toxoplasma gondii, with cyclophilins identified as possible targets. However, CsA immunosuppressive nature hinders its use as an antitoxoplasmosis agent. Here, we evaluate the potential of three CsA derivatives devoid of immunosuppressive activity, namely, NIM811, Alisporivir, and dihydrocyclosporin A to target a previously characterized cyclophilin from Toxoplasma gondii (TgCyp23). We determined the X-ray crystal structures of TgCyp23 in complex with the three analogs and elucidated their binding and inhibitory properties. The high resolution of the structures revealed the precise positioning of ligands within the TgCyp23 binding site and the details of protein-ligand interactions. A comparison with the established ternary structure involving calcineurin indicates that substitutions at position 4 in CsA derivatives prevent calcineurin binding. This finding provides a molecular explanation for why CsA analogs can target Toxoplasma cyclophilins without compromising the human immune response.

The structural basis for the collagen processing by human P3H1/CRTAP/PPIB ternary complex.

Collagen posttranslational processing is crucial for its proper assembly and function. Disruption of collagen processing leads to tissue development and structure disorders like osteogenesis imperfecta (OI). OI-related collagen processing machinery includes prolyl 3-hydroxylase 1 (P3H1), peptidyl-prolyl cis-trans isomerase B (PPIB), and cartilage-associated protein (CRTAP), with their structural organization and mechanism unclear. We determine cryo-EM structures of the P3H1/CRTAP/PPIB complex. The active sites of P3H1 and PPIB form a face-to-face bifunctional reaction center, indicating a coupled modification mechanism. The structure of the P3H1/CRTAP/PPIB/collagen peptide complex reveals multiple binding sites, suggesting a substrate interacting zone. Unexpectedly, a dual-ternary complex is observed, and the balance between ternary and dual-ternary states can be altered by mutations in the P3H1/PPIB active site and the addition of PPIB inhibitors. These findings provide insights into the structural basis of collagen processing by P3H1/CRTAP/PPIB and the molecular pathology of collagen-related disorders.

Nynrin preserves hematopoietic stem cell function by inhibiting the mitochondrial permeability transition pore opening.

Mitochondria are key regulators of hematopoietic stem cell (HSC) homeostasis. Our research identifies the transcription factor Nynrin as a crucial regulator of HSC maintenance by modulating mitochondrial function. Nynrin is highly expressed in HSCs under both steady-state and stress conditions. The knockout Nynrin diminishes HSC frequency, dormancy, and self-renewal, with increased mitochondrial dysfunction indicated by abnormal mPTP opening, mitochondrial swelling, and elevated ROS levels. These changes reduce HSC radiation tolerance and promote necrosis-like phenotypes. By contrast, Nynrin overexpression in HSCs diminishes irradiation (IR)-induced lethality. The deletion of Nynrin activates Ppif, leading to overexpression of cyclophilin D (CypD) and further mitochondrial dysfunction. Strategies such as Ppif haploinsufficiency or pharmacological inhibition of CypD significantly mitigate these effects, restoring HSC function in Nynrin-deficient mice. This study identifies Nynrin as a critical regulator of mitochondrial function in HSCs, highlighting potential therapeutic targets for preserving stem cell viability during cancer treatment.

Cyclophilin D induces necrotic core formation by mediating mitochondria-associated macrophage death in advanced atherosclerotic lesions.

BACKGROUND AND AIMS: In advanced atherosclerotic lesions, macrophage deaths result in necrotic core formation and plaque vulnerability. Cyclophilin D (CypD) is a mitochondria-specific cyclophilin involved in the process of cell death after organ ischemia-reperfusion. However, the role of CypD in atherosclerosis, especially in necrotic core formation, is unknown. Therefore, this experiment aims to clarify the role of CypD in necrotic core formation. METHODS: To clarify the specific role of CypD, encoded by Ppif in mice, apolipoprotein-E/CypD-double knockout (Apoe-/-Ppif-/-) mice were generated. These mice were fed a high-fat diet containing 0.15 % cholesterol for 24 weeks to accelerate atherosclerotic lesion development. RESULTS: Deletion of CypD decreased the necrotic core size, accompanied by a reduction of macrophage apoptosis compared to control Apoe-/- mice. In RAW264.7 cells, siRNA-mediated knockdown of CypD attenuated the release of cytochrome c from the mitochondria to the cytosol induced by endoplasmic reticulum stress inducer thapsigargin. In addition, necroptosis, induced by TNF-α and caspase inhibitor, was attenuated by knockdown of CypD. Ly-6Chigh inflammatory monocytes in peripheral blood leukocytes and mRNA expression of Il1b in the aorta were decreased by deletion of CypD. In contrast, siRNA-mediated knockdown of CypD did not significantly decrease Il1b nor Ccl2 mRNA expression in RAW264.7 cells treated with LPS and IFN-γ, suggesting that inhibition of inflammation in vivo is likely due to decreased cell death in the atherosclerotic lesions rather than a direct action of CypD deletion on the macrophage. CONCLUSIONS: These results indicate that CypD induces macrophage death and mediates necrotic core formation in advanced atherosclerotic lesions. CypD could be a novel therapeutic target for treating atherosclerotic vascular diseases.

Sanglifehrin A mitigates multiorgan fibrosis by targeting the collagen chaperone cyclophilin B.

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.

Optimized expression of Peptidyl-prolyl cis/transisomerase cyclophilinB with prokaryotic toxicity from Sporothrix globosa.

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.

Role of the mitochondrial protein cyclophilin D in skin wound healing and collagen secretion.

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.

Repair of CRISPR-guided RNA breaks enables site-specific RNA excision in human cells.

Genome editing with CRISPR RNA-guided endonucleases generates DNA breaks that are resolved by cellular DNA repair machinery. However, analogous methods to manipulate RNA remain unavailable. We show that site-specific RNA breaks generated with type-III CRISPR complexes are repaired in human cells and that this repair can be used for programmable deletions in human transcripts to restore gene function. Collectively, this work establishes a technology for precise RNA manipulation with potential therapeutic applications.

Proteostatic Remodeling of Small Heat Shock Chaperones─Crystallins by Ran-Binding Protein 2─and the Peptidyl-Prolyl cis-trans Isomerase and Chaperone Activities of Its Cyclophilin Domain.

Disturbances in protein phase transitions promote protein aggregation─a 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 chaperones─crystallins 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.

Cyclophilin D knockout significantly prevents HCC development in a streptozotocin-induced mouse model of diabetes-linked NASH.

A family of Peptidyl-prolyl isomerases (PPIases), called Cyclophilins, localize to numerous intracellular and extracellular locations where they contribute to a variety of essential functions. We previously reported that non-immunosuppressive pan-cyclophilin inhibitor drugs like reconfilstat (CRV431) or NV556 decreased multiple aspects of non-alcoholic fatty liver disease (NAFLD) in mice under two different non-alcoholic steatohepatitis (NASH) mouse models. Both CRV431 and NV556 inhibit several cyclophilin isoforms, among which cyclophilin D (CypD) has not been previously investigated in this context. It is unknown whether it is necessary to simultaneously inhibit multiple cyclophilin family members to achieve therapeutic benefits or if loss-of-function of one is sufficient. Furthermore, narrowing down the isoform most responsible for a particular aspect of NAFLD/NASH, such as hepatocellular carcinoma (HCC), would allow for more precise future therapies. Features of human diabetes-linked NAFLD/NASH can be reliably replicated in mice by administering a single high dose of streptozotocin to disrupt pancreatic beta cells, in conjunction with a high sugar, high fat, high cholesterol western diet over the course of 30 weeks. Here we show that while both wild-type (WT) and Ppif-/- CypD KO mice develop multipe severe NASH disease features under this model, the formation of HCC nodules was significantly blunted only in the CypD KO mice. Furthermore, of differentially expressed transcripts in a qPCR panel of select HCC-related genes, nearly all were downregulated in the CypD KO background. Cyclophilin inhibition is a promising and novel avenue of treatment for diet-induced NAFLD/NASH. This study highlights the impact of CypD loss-of-function on the development of HCC, one of the most severe disease outcomes.

IgE to cyclophilins in pollen-allergic children: Epidemiologic, clinical, and diagnostic relevance of a neglected panallergen.

BACKGROUND: Cyclophilins are ubiquitous panallergens whose epidemiologic, diagnostic, and clinical relevance is largely unknown and whose sensitization is rarely examined in routine allergy practice. OBJECTIVE: We investigated the epidemiologic, diagnostic, and clinical relevance of cyclophilins in seasonal allergic rhinitis and its comorbidities. METHODS: We examined a random sample of 253 (25%) of 1263 Italian children with seasonal allergic rhinitis from the Panallergens in Pediatrics (PAN-PED) cohort with characterized disease phenotypes. Nested studies of sensitization prevalence, correlation, and allergen extract inhibition were performed in patients sensitized to birch pollen extract but lacking IgE to Bet v 1/2/4 (74/1263) or with highest serum level of IgE to Bet v 1 (26/1263); and in patients with sensitization to various extracts (ragweed, mugwort, pellitory, Plantago, and plane tree), but not to their respective major allergenic molecule, profilins, and polcalcins. IgE to cyclophilin was detected with recombinant Bet v 7, and extract inhibition tests were performed with the same rBet v 7. RESULTS: IgE to rBet v 7 was detected in 43 (17%) of 253 patients. It was associated with asthma (P < .028) and oral allergy syndrome (P < .017) in univariate but not multivariate analysis adjusted for IgE to profilins (Phl p 12), PR-10s (Bet v 1), and lipid transfer proteins (Pru p 3). IgE to rBet v 7 was also highly prevalent (47/74, 63%) among patients with unexplained sensitization to birch pollen extract. In patients with unexplained sensitization to ragweed, mugwort, pellitory, Plantago and plane tree pollen, the levels of IgE to those extracts correlated with the levels of IgE to rBet v 7, and they were also significantly inhibited by rBet v 7 (inhibition range 45%-74%). CONCLUSIONS: IgE sensitization to cyclophilin is frequent in pollen-allergic patients living in temperate areas and can produce "false" positive outcomes in skin prick and IgE tests to pollen extracts. Molecular diagnostic guidelines should include this panallergen family.

Mice lacking cyclophilin B, but not cyclophilin A, are protected from the development of NASH in a diet and chemical-induced model.

Cyclophilins are a diverse family of peptidyl-prolyl isomerases (PPIases) of importance in a variety of essential cellular functions. We previously reported that the pan-cyclophilin inhibitor drug reconfilstat (CRV431) decreased disease in mice under the western-diet and carbon tetrachloride (CCl4) non-alcoholic steatohepatitis (NASH) model. CRV431 inhibits several cyclophilin isoforms, among which cyclophilin A (CypA) and B (CypB) are the most abundant. It is not known whether simultaneous inhibition of multiple cyclophilin family members is necessary for the observed therapeutic effects or if loss-of-function of one is sufficient. Identifying the responsible isoform(s) would enable future fine-tuning of drug treatments. Features of human liver fibrosis and complete NASH can be reliably replicated in mice by administration of intraperitoneal CCl4 alone or CCl4 in conjunction with high sugar, high cholesterol western diet, respectively. Here we show that while wild-type (WT) and Ppia-/- CypA KO mice develop severe NASH disease features under these models, Ppib-/- CypB KO mice do not, as measured by analysis of picrosirius red and hematoxylin & eosin-stained liver sections and TNFα immuno-stained liver sections. Cyclophilin inhibition is a promising and novel avenue of treatment for diet-induced NASH. In this study, mice without CypB, but not mice without CypA, were significantly protected from the development of the characteristic features of NASH. These data suggest that CypB is necessary for NASH disease progression. Further investigation is necessary to determine whether the specific role of CypB in the endoplasmic reticulum secretory pathway is of significance to its effect on NASH development.

Elevations in the Mitochondrial Matrix Protein Cyclophilin D Correlate With Reduced Parvalbumin Expression in the Prefrontal Cortex of Patients With Schizophrenia.

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 (n = 27) and matched comparison subjects with no known history of psychiatric or neurological disorders (n = 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.

Structural Insights into SARS-CoV-2 Nonstructural Protein 1 Interaction with Human Cyclophilin and FKBP1 to Regulate Interferon Production.

The ongoing coronavirus disease 2019 (COVID-19) pandemic caused by the SARS-CoV-2 coronavirus and the perpetual rise of new variants warrant investigation of the molecular and structural details of the infection process and modulation of the host defense by viral proteins. This Letter reports the combined experimental and computational approaches to provide key insights into the structural and functional basis of Nsp1's association with different cyclophilins and FKBPs in regulating COVID-19 infection. We demonstrated the real-time stability and functional dynamics of the Nsp1-CypA/FKBP1A complex and investigated the repurposing of potential inhibitors that could block these interactions. Overall, we provided insights into the inhibitory role Nsp1 in downstream interferon production, a key aspect for host defense that prevents the SARS-CoV-2 or related family of corona virus infection.

T-2 toxin induces mitochondrial dysfunction in chondrocytes via the p53-cyclophilin D pathway.

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.

An ex vivo screening using mouse brain mitochondria identified seco-cycline D as an inhibitor of mitochondrial permeability transition pore.

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.

New cyclophilin D inhibitor rescues mitochondrial and cognitive function in Alzheimer's disease.

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 ∼2000 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.

The mitochondrial ATP synthase is a negative regulator of the mitochondrial permeability transition pore.

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.

Retinitis pigmentosa with or without skeletal abnormalities due to homozygous mutations in the CWC27 gene: A case report.

RATIONALE: Retinitis pigmentosa with or without skeletal abnormalities (RPSKA) is an autosomal recessive disorder caused by mutations in the CWC27 gene. Skeletal dysplasia and non-syndromic retinitis pigmentosa are typical manifestations, and most patients present with retinopathy such as retinitis pigmentosa and limited visual field. Its clinical manifestations are complex and diverse, often involving multiple systems. Examples include short finger deformities, peculiar facial features, short stature, and neurodevelopmental abnormalities, and it is easy to misdiagnose clinically, and early diagnosis is crucial for prognosis. PATIENT CONCERNS: A 2-year and 2-month-old female child was admitted to the hospital due to "unsteady walking alone and slow reaction for more than half a year." After admission, the child was found to have delayed motor development, accompanied by special face, abnormal physical examination of the nervous system, cranial MRI Dandy-Walker malformation, considering developmental delay. DIAGNOSES: Whole exome sequencing of the family line revealed the presence of a c.617(exon7)C>A pure mutation in the CWC27 gene in the affected child (this locus has been reported in the clinical literature); the final diagnosis is RPSKA. INTERVENTIONS: Unfortunately, there is no specific drug for the disease; we give children rehabilitation training treatment. OUTCOMES: During follow-up process we found that children's condition is better than before. LESSONS SUBSECTIONS AS PER STYLE: We reported a case of RPSKA caused by mutations in the CWC27 gene. This study adds to our understanding of the clinical phenotype of TBL1XR1 mutations and provides a realistic and reliable basis for clinicians.