Cyclophilin D Human, His

What is Cyclophilin D and what are its primary functions in mitochondria?

Cyclophilin D (CypD) is a mitochondrial peptidyl-prolyl cis-trans isomerase that catalyzes the isomerization of proline imidic peptide bonds in oligopeptides, which speeds up protein folding . Its primary function involves regulating the mitochondrial permeability transition pore (MPTP), an inner membrane channel that plays a significant role in cell death execution . Additionally, CypD interacts with the F-ATP synthase through binding to its OSCP subunit, which inhibits catalysis and favors the transition of the enzyme complex to the permeability transition pore . CypD also demonstrates the ability to prevent protein aggregation, particularly of α-synuclein, suggesting a potential protective role in certain neurodegenerative contexts .

How is the structure of human Cyclophilin D characterized?

Human Cyclophilin D contains a conserved catalytic domain known as the cyclophilin-like domain (CLD), consisting of a β-barrel arranged by 8 antiparallel β-sheets forming a compact hydrophobic core . This core is connected by several loops and three helical elements: two α-helices (H1 and H3) that pack against the barrel at opposite sides, and a small H2 between strands S6 and S7 (sometimes reported as a 3₁₀ helix) . The active site presents two distinct pockets: the S1' pocket containing highly conserved proline-binding residues, and the S2 pocket responsible for interacting with residues P2 and P3 relative to the substrate proline . Recent NMR studies have revealed that while the globular part of CypD is rigid, the N-terminus is highly flexible, which affects its binding properties .

What is the enzymatic activity of Cyclophilin D and how is it measured?

Cyclophilin D possesses peptidyl-prolyl cis-trans isomerase (PPIase) activity, which can be measured using synthetic tetrapeptide substrates such as succinyl-Ala-Ala-Phe-Pro-p-nitroanilide (suc-AAFP-pNA) . In this assay, the peptide is first set in the cis conformation, and upon addition of α-chymotrypsin, only the trans form is cleaved, releasing p-nitroaniline that can be detected spectrophotometrically . Standard experimental conditions include a temperature of 25°C in Tris-HCl buffer at pH 8.0 . The specific activity of high-quality recombinant human CypD preparations typically exceeds 700 nmol/min/mg, defined as the amount of enzyme that cleaves 1 nmole of substrate per minute . This activity can be inhibited by nanomolar concentrations of Cyclosporin A (CsA), which serves as a confirmation of specificity .

What is known about recombinant human Cyclophilin D production for research purposes?

Recombinant human Cyclophilin D for research applications is typically produced in E. coli expression systems . The protein is engineered as a single, non-glycosylated polypeptide chain containing amino acids 1-370, with a total of 390 amino acids including a 20-amino acid His-Tag at the N-terminus for purification purposes . The resulting protein has a molecular mass of approximately 42.9 kDa . After expression, the protein is purified using chromatographic techniques and formulated in solutions typically containing phosphate-buffered saline (PBS) and 10% glycerol for stability . For optimal stability, the purified protein should be stored at 4°C if used within 2-4 weeks, or at -20°C for longer periods, with the addition of carrier proteins like HSA or BSA (0.1%) recommended for long-term storage .

What is the significance of the N-terminal cleavage of Cyclophilin D?

Recent research has identified a form of Cyclophilin D lacking the first 10 (in mouse) or 13 (in human) N-terminal residues, termed ΔN-CyPD . This discovery has significant implications for CypD function. NMR studies comparing full-length CypD (FL-CyPD) and ΔN-CyPD reveal that while the globular part is rigid, the N-terminus is highly flexible . Importantly, this N-terminal cleavage substantially affects CypD's binding properties with the OSCP subunit of F-ATP synthase . While FL-CyPD does not readily bind OSCP in saline media, ΔN-CyPD shows significantly higher binding affinity, indicating that the N-terminus acts as a negative regulator of this interaction . Evidence indicates that calpain 1 is responsible for generating ΔN-CyPD in cells . This novel mechanism of CypD modulation through N-terminal cleavage may have critical pathophysiological implications, particularly in conditions involving mitochondrial dysfunction and permeability transition .

How does Cyclophilin D interact with α-Synuclein and what are the implications for neurodegenerative diseases?

Cyclophilin D interacts directly with α-Synuclein via the latter's acidic proline-rich C-terminus region, binding at the putative ligand binding pocket of CypD . This interaction has profound implications for protein aggregation dynamics relevant to neurodegenerative diseases like Parkinson's disease . Studies demonstrate that CypD binding with soluble α-Synuclein prevents its aggregation . Moreover, adding CypD to preformed α-Synuclein fibrils leads to their disassembly . Experiments with enzymatically-compromised CypD mutants show reduced abilities to dissociate α-Synuclein aggregates, suggesting that fibril disassembly is mechanistically linked to peptidyl-prolyl isomerization catalyzed by CypD . This provides strong evidence for a potential protective role of CypD against certain protein aggregation pathologies in mitochondria . The significance of this interaction is further supported by studies showing delayed onset of Parkinson's disease and extended lifespan in PD mouse models with genetic ablation of CypD .

What is the role of Cyclophilin D in the mitochondrial permeability transition pore (MPTP) and cell death?

Cyclophilin D serves as a key regulator of the mitochondrial permeability transition pore (MPTP), an inner membrane channel central to cell death mechanisms . The MPTP, when persistently open, allows the free passage of solutes up to 1.5 kDa in size, leading to mitochondrial swelling, outer membrane rupture, and release of pro-apoptotic factors . CypD promotes MPTP opening in response to elevated matrix Ca²⁺, oxidative stress, and other cellular stressors . This regulatory role has been conclusively demonstrated through multiple approaches: pharmacological inhibition with CsA desensitizes the pore to Ca²⁺, and genetic ablation of CypD in Ppif⁻/⁻ mice renders mitochondria resistant to Ca²⁺-induced permeability transition . Despite this clear role, the molecular composition of the MPTP remains debated, with evidence suggesting that CypD interacts with the F-ATP synthase to facilitate its conversion to a pore-forming structure . Importantly, while CypD regulates MPTP sensitivity, the pore can still open through CypD-independent mechanisms under severe stress conditions, indicating multiple regulatory pathways .

How does Cyclophilin D contribute to disease pathophysiology?

Cyclophilin D has been implicated in the pathophysiology of multiple diseases, with both detrimental and protective roles depending on the context . In ischemia-reperfusion injury of the heart and brain, genetic ablation of CypD (Ppif⁻/⁻ mice) results in reduced infarct size compared to wild-type controls, demonstrating CypD's contribution to cell death in these conditions . Studies using CsA in ischemic isolated hearts and in infarcted patients have corroborated these findings . CypD also plays roles in neurodegenerative conditions: Ppif⁻/⁻ mice display resistance to development of axonopathy in autoimmune encephalomyelitis models and slower disease progression when crossed with SOD1 mutant mice (an ALS model) . Interestingly, the role of CypD appears to change during development, as neonatal Ppif⁻/⁻ mice are paradoxically more sensitive to ischemia-reperfusion injury of the brain compared to adult knockouts, suggesting age-dependent functions . Additionally, recent findings about CypD's protective role in protein disaggregation highlight the complexity of its contributions to disease states .

What are the most effective approaches for studying the interaction between Cyclophilin D and F-ATP synthase?

Studying the CypD-F-ATP synthase interaction requires a multi-methodological approach for comprehensive characterization. For biochemical characterization, co-immunoprecipitation experiments using antibodies against CypD or the OSCP subunit of F-ATP synthase in mitochondrial extracts can confirm their association in native contexts . Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) with purified recombinant proteins provides quantitative binding parameters (KD, kon, koff) . To understand structural aspects, NMR spectroscopy has proven particularly valuable for mapping binding interfaces, especially given the flexible nature of CypD's N-terminus . Comparing the binding properties of full-length CypD versus ΔN-CyPD reveals mechanistic insights into how the N-terminal region modulates this interaction . Functionally, researchers should assess how CypD binding affects F-ATP synthase activity by measuring ATP synthesis rates in isolated mitochondria with and without CsA, or comparing wild-type and Ppif⁻/⁻ mitochondria . Crosslinking experiments followed by mass spectrometry can identify precise contact points between the proteins. To validate physiological relevance, mitochondrial membrane potential measurements during Ca²⁺ challenges can link the interaction to permeability transition susceptibility .

How should researchers design experiments to distinguish between the roles of Cyclophilin D in protein folding versus MPTP regulation?

Separating CypD's roles in protein folding from MPTP regulation requires experiments that can selectively target each function. Start by generating structure-guided CypD mutants that maintain PPIase activity but cannot bind to F-ATP synthase, or vice versa . These can be characterized biochemically to confirm the selective loss of function before cellular or in vivo studies. To assess protein folding/disaggregation functions independent of MPTP effects, conduct in vitro aggregation assays with aggregation-prone substrates like α-synuclein in the presence of wild-type CypD, PPIase-deficient mutants, and OSCP-binding deficient mutants . These experiments should be performed under conditions that prevent MPTP formation. For cellular studies, use mitochondrially-targeted aggregation-prone reporter proteins in cells expressing different CypD variants, and monitor their folding state without inducing cell death . To isolate MPTP-specific effects, perform calcium retention capacity assays in isolated mitochondria expressing the same CypD variants and measure parameters unrelated to protein folding, such as membrane potential changes and matrix swelling . Time-course experiments can help distinguish immediate effects (likely MPTP-related) from delayed effects (potentially related to protein folding). Additionally, combining CsA treatment with conditions that specifically induce either protein misfolding or MPTP opening can help parse these distinct functions .

What techniques can effectively distinguish between full-length and N-terminally cleaved Cyclophilin D?

Distinguishing between full-length CypD and ΔN-CyPD requires techniques that can detect their subtle structural differences. High-resolution SDS-PAGE with 15-20% gradient gels can resolve the small molecular weight difference (approximately 1-1.5 kDa) between these forms, followed by western blotting with anti-CypD antibodies . For enhanced specificity, develop and use antibodies that recognize either the N-terminal region (detecting only full-length CypD) or the neo-N-terminus created by cleavage (specific for ΔN-CyPD) . Mass spectrometry provides superior resolution: targeted LC-MS/MS can identify and quantify specific peptides unique to each form, while intact protein MS can distinguish the precise molecular weights of the two species . NMR spectroscopy has been particularly valuable for characterizing these isoforms, as it can detect the structural differences in the N-terminal region and provide information about the dynamic properties of both variants . Two-dimensional gel electrophoresis can separate the forms based on both molecular weight and potential isoelectric point differences. To specifically study calpain 1-mediated generation of ΔN-CyPD, in vitro cleavage assays using purified components can be combined with calpain inhibitors in cellular systems to validate the processing mechanism .

How can researchers study Cyclophilin D's role in protein disaggregation?

To investigate CypD's role in protein disaggregation, particularly with α-synuclein, researchers should employ a combination of in vitro and cellular approaches. For in vitro studies, prepare α-synuclein fibrils under standardized conditions that yield consistent morphologies, confirmed by thioflavin T fluorescence and electron microscopy . Incubate these preformed fibrils with purified recombinant CypD (both full-length and ΔN-CyPD forms) and monitor disaggregation through thioflavin T fluorescence decrease, light scattering, sedimentation assays, or direct visualization by electron microscopy . Include controls with enzymatically inactive CypD mutants to establish the connection between PPIase activity and disaggregation ability . To determine if the effect is specific to CypD versus other PPIases, compare with other cyclophilin family members . For cellular studies, express fluorescently-tagged aggregation-prone proteins in cells with modified CypD levels (overexpression, knockdown, or knockout) and monitor aggregate formation and clearance by live-cell imaging . Additionally, isolate mitochondria from these cells to assess whether aggregation-prone proteins that localize to mitochondria show different aggregation propensities depending on CypD status . Use fluorescence recovery after photobleaching (FRAP) to examine the dynamics of protein aggregates in living cells with different CypD levels . Combine these approaches with CsA treatment to pharmacologically inhibit CypD's PPIase activity and confirm its role in the disaggregation process .

What animal models are most appropriate for studying Cyclophilin D-dependent pathologies?

The foundation for studying CypD-dependent pathologies is the global Ppif⁻/⁻ mouse (CypD knockout), which allows researchers to determine if a disease process is CypD-dependent by comparing pathology progression in knockout versus wild-type animals . For more refined investigations, conditional knockout models using Cre-loxP systems enable tissue-specific or temporally controlled CypD deletion, particularly valuable for distinguishing developmental versus acute roles of CypD, as evidenced by the different responses of neonatal versus adult Ppif⁻/⁻ mice to ischemia-reperfusion injury . To model gain-of-function scenarios, transgenic animals overexpressing wild-type or mutant CypD (especially the ΔN-CyPD form) under tissue-specific promoters can be developed . These genetic models should be combined with disease-specific challenges: for cardiovascular studies, ischemia-reperfusion injury protocols; for neurodegenerative disease models, MPTP administration, α-synuclein overexpression (Parkinson's), or crossing with SOD1 mutant backgrounds (ALS) . For pharmacological studies in these models, administer CsA or non-immunosuppressive analogs (NIM811, Debio 025) that target CypD specifically, while controlling for their effects on other cyclophilins . Key readouts should include mitochondrial function parameters (membrane potential, calcium retention capacity, ATP production), markers of cell death, and disease-specific pathological outcomes .

How should researchers interpret contradictory findings regarding Cyclophilin D's roles in health and disease?

Interpreting contradictory findings about CypD requires careful consideration of several factors. First, developmental timing significantly affects CypD's role—neonatal Ppif⁻/⁻ mice show increased susceptibility to ischemia-reperfusion injury, while adult knockouts are protected, suggesting age-dependent functions that must be accounted for when comparing studies . Second, the dual nature of CypD activities must be recognized: while its promotion of MPTP opening can drive cell death, its PPIase activity contributes to protein disaggregation and potentially cellular protection . These seemingly contradictory roles may predominate under different stress conditions or in different tissues. Third, methodological differences between studies significantly impact outcomes—the specific cell type or tissue examined, the disease model employed, the severity and duration of stress, and whether acute pharmacological inhibition or chronic genetic deletion was used . When CsA is employed, its effects on other cyclophilins and calcineurin must be distinguished from CypD-specific actions . Additionally, compensatory mechanisms in chronic knockout models may mask CypD's effects compared to acute inhibition . To reconcile contradictions, integrate findings across multiple experimental systems, from in vitro biochemical studies to cellular and in vivo models, focusing on identifying the specific conditions under which particular CypD functions predominate .

What are promising therapeutic strategies targeting Cyclophilin D for mitochondrial dysfunction-related diseases?

Promising therapeutic strategies targeting CypD are advancing beyond traditional CsA approaches. Next-generation CypD inhibitors with improved specificity are being developed, including non-immunosuppressive CsA derivatives optimized for CypD binding and small molecules identified through structure-based drug design . These compounds aim to inhibit CypD without affecting other cyclophilins or calcineurin . Novel delivery systems such as mitochondria-targeted nanoparticles conjugated to CypD inhibitors enhance drug delivery to the mitochondrial matrix . Beyond direct inhibition, strategies targeting the regulation of CypD are emerging, including approaches to modulate its post-translational modifications or to prevent specific protein-protein interactions, such as selective disruption of the CypD-OSCP interaction without affecting PPIase activity . The discovery of ΔN-CyPD opens therapeutic possibilities focused on controlling calpain 1-mediated N-terminal cleavage, potentially modulating CypD's ability to bind F-ATP synthase and trigger permeability transition . For protein aggregation disorders, harnessing CypD's disaggregase activity through PPIase enhancement or targeted delivery of recombinant CypD to affected tissues represents a novel approach distinct from MPTP inhibition . Development of biomarkers reflecting CypD activity status or ΔN-CyPD levels could enable personalized therapeutic approaches and treatment monitoring . Future therapeutic strategies will likely be disease-specific, balancing inhibition of detrimental MPTP-related functions while preserving beneficial protein quality control activities of CypD .

What methodological advances could improve the study of Cyclophilin D's multiple functions in mitochondria?

Advancing CypD research requires methodological innovations across multiple domains. For structural studies, applying cryo-electron microscopy to visualize CypD-F-ATP synthase complexes at near-atomic resolution would provide unprecedented insights into how CypD binding induces conformational changes in its partners . Developing improved mitochondrially-targeted optogenetic tools to acutely modulate CypD activity or interactions in specific cellular compartments would allow temporal precision currently lacking in pharmacological approaches . For monitoring CypD dynamics, genetically-encoded biosensors that report on CypD conformational changes, post-translational modifications, or protein-protein interactions in real-time within living cells could transform our understanding of its regulation . To study the newly discovered ΔN-CyPD, developing specific antibodies or fluorescent probes that distinguish between full-length and cleaved forms would enable tracking of their differential localization and function . Advanced proteomics approaches combining proximity labeling with mass spectrometry could comprehensively map the CypD interactome under various physiological and pathological conditions . For functional studies, microfluidic systems that allow simultaneous measurement of multiple mitochondrial parameters (membrane potential, calcium flux, ATP production, ROS generation) in response to CypD modulation would provide integrated datasets currently difficult to obtain . Finally, developing organoid or microphysiological systems incorporating patient-derived cells with genetic CypD variants could bridge the gap between reductionist models and human disease relevance .