PPIF Human

What is PPIF and what cellular functions does it regulate in humans?

PPIF (Peptidyl-prolyl cis-trans isomerase F) is a mitochondrial enzyme encoded by the PPIF gene in humans. It functions as a major component of the mitochondrial permeability transition pore (MPTP) in the inner mitochondrial membrane. As a member of the peptidyl-prolyl cis-trans isomerase (PPIase) family, PPIF catalyzes the cis-trans isomerization of proline imidic peptide bonds in oligopeptides, thereby accelerating protein folding and repair . The protein exhibits a β-barrel structure with a hydrophobic core, composed of eight anti-parallel β-strands capped by two α-helices at the top and bottom, weighing approximately 17.5 kDa .

PPIF regulates several critical cellular processes including:

• Mitochondrial metabolism and bioenergetics

• Mitochondrial permeability transition

• Apoptotic and necrotic cell death pathways

• Inflammatory responses

• Cellular stress responses

Methodologically, researchers can study PPIF function using mitochondrial isolation protocols followed by permeability transition assays, where calcium retention capacity serves as a key readout for MPTP activity.

How do I differentiate between PPIF and other cyclophilins in experimental setups?

When designing experiments to study PPIF specifically, researchers should be aware that PPIF is often incorrectly referred to as cyclophilin D (CypD), which is actually encoded by the PPID gene . To differentiate PPIF from other cyclophilins:

To validate specificity, always perform parallel experiments with other cyclophilin family members (PPIA, PPIB, PPID) to confirm that observed effects are PPIF-specific rather than general cyclophilin-mediated phenomena.

What are the most reliable methods for quantifying PPIF expression in human samples?

For robust quantification of PPIF in human samples, researchers should employ multiple complementary techniques:

• RNA-level quantification: RT-qPCR using PPIF-specific primers, including appropriate housekeeping genes for normalization (GAPDH, ACTB, 18S rRNA). RNA-seq can provide more comprehensive analysis and allow for isoform detection.

• Protein-level quantification: Western blotting with validated PPIF-specific antibodies, normalizing to mitochondrial markers like VDAC or COX IV rather than whole-cell markers . ELISA-based methods can provide more quantitative results.

• Tissue-specific analysis: Immunohistochemistry (IHC) for spatial distribution, using appropriate controls to ensure specificity . Single-cell RNA sequencing can reveal cell-type specific expression patterns.

• Activity assays: Functional PPIase activity assays using specific PPIF substrates can complement expression data.

For clinical samples, TPM (Transcripts Per Million) normalization of RNA-seq data represents the current gold standard for cross-study comparisons, as demonstrated in TCGA and GEO database analyses of PPIF expression in cancer samples .

How does PPIF expression vary across different cancer types, and what are the implications for cancer research?

PPIF expression varies significantly across cancer types, with notable overexpression documented in several malignancies:

Methodologically, researchers investigating PPIF in cancer should:

• Utilize multiple patient-derived datasets (TCGA, GEO) to validate expression patterns

• Perform survival analyses stratified by PPIF expression levels

• Conduct gain- and loss-of-function experiments to determine causality in cancer progression

• Analyze PPIF expression in relation to immune cell infiltration profiles

The functional implications of PPIF overexpression in cancer include enhanced proliferation, advanced cell-cycle progression, and impaired mitophagy through modulation of the FOXO3a/PINK1–Parkin signaling pathway . These findings suggest PPIF as a potential therapeutic target in multiple cancer types.

What role does PPIF play in modulating the immune microenvironment, particularly in cancer contexts?

PPIF significantly influences immune cell infiltration and function within the tumor microenvironment (TME). Recent research reveals:

• T-cell balance regulation: PPIF modulates the T helper 1–T helper 2 (Th1/Th2) cell balance, potentially influencing anti-tumor immune responses .

• Macrophage phenotype modulation: PPIF upregulation in Macrophage-C1-C1QC cells enhances their anti-inflammatory phenotype, potentially contributing to immune suppression in the TME .

• Immune evasion mechanisms: PPIF promotes F11R-F11R signaling, helping cancer cells evade macrophage-mediated cytotoxicity .

To study these relationships experimentally:

• Conduct single-cell RNA sequencing (scRNA-seq) to analyze immune cell heterogeneity and PPIF expression patterns within specific immune cell populations

• Perform co-culture experiments between PPIF-modulated cancer cells and various immune cell types

• Utilize flow cytometry with immune cell subtype markers to quantify infiltration patterns

• Apply trajectory analysis to map functional evolution of immune cells based on PPIF expression levels

Pseudo-time analysis has revealed that PPIF_low macrophages exhibit pro-inflammatory, anti-tumor phenotypes associated with phagocytosis and T-cell activation, while PPIF_high macrophages transition to a suppressive phenotype that supports tumor progression through processes like angiogenesis .

How can researchers effectively study PPIF's role in mitochondrial permeability transition in disease models?

Studying PPIF's role in mitochondrial permeability transition (MPT) requires specialized techniques focusing on mitochondrial function:

• Calcium retention capacity (CRC) assays: This gold-standard approach measures mitochondrial calcium uptake until MPTP opening. Implementation requires:

  • Freshly isolated mitochondria from cell cultures or tissue samples

  • Calcium-sensitive fluorescent dyes (e.g., Calcium Green-5N)

  • Sequential calcium additions with fluorescence monitoring

  • Comparison between wild-type and PPIF-modulated systems

• Mitochondrial swelling assays: Measures the decrease in light scattering as mitochondria swell following MPTP opening.

• Membrane potential assessments: Using potential-sensitive dyes like TMRM or JC-1 to monitor mitochondrial depolarization associated with MPTP opening.

• In vivo disease models:

  • PPIF knockout or knockdown models show protection against ischemia-reperfusion injury

  • Tissue-specific conditional PPIF models can isolate effects to particular organs

  • Humanized models expressing human PPIF variants provide translational relevance

• Drug-based approaches: Cyclosporin A (CsA) and its non-immunosuppressive derivatives inhibit PPIF, offering pharmacological tools to study MPT in disease models .

When working with disease models, researchers should implement appropriate controls for each pathological condition, as PPIF's impact on MPT may vary contextually depending on the specific disease environment, redox status, and energy state of the affected tissues.

What approaches can researchers use to investigate PPIF-mediated regulation of mitophagy in cancer progression?

The investigation of PPIF's role in mitophagy regulation, particularly its inhibitory effect through the FOXO3a/PINK1–Parkin pathway , requires sophisticated experimental approaches:

• Mitophagy flux assessment:

  • Utilize mt-Keima or mito-QC reporter systems that change fluorescence properties upon mitophagosome-lysosome fusion

  • Quantify colocalization of mitochondrial markers (TOM20, COXIV) with autophagosomal (LC3) and lysosomal (LAMP1) markers by confocal microscopy

  • Monitor mitochondrial mass using MitoTracker dyes coupled with flow cytometry in PPIF-modulated cells

• Molecular pathway analysis:

  • Assess FOXO3a phosphorylation status and nuclear localization by Western blotting and immunofluorescence

  • Quantify PINK1 stabilization on depolarized mitochondria and subsequent Parkin recruitment

  • Measure ubiquitination of outer mitochondrial membrane proteins (MFN1/2, VDAC)

• Functional consequences:

  • Evaluate mitochondrial network morphology using super-resolution microscopy

  • Assess mitochondrial function (OCR, ECAR) using Seahorse analyzer in PPIF-overexpressing vs. knockdown cancer cells

  • Perform rescue experiments by modulating key components of the FOXO3a/PINK1–Parkin pathway in PPIF-altered cells

• In vivo verification:

  • Use xenograft models with PPIF-modulated cancer cells to assess tumor growth and mitophagy markers

  • Analyze patient samples for correlations between PPIF expression and mitophagy markers

This methodological framework allows researchers to establish not only correlative but causal relationships between PPIF expression, mitophagy impairment, and cancer progression.

How can researchers resolve contradictory findings regarding PPIF's pro-survival versus pro-death roles in different cellular contexts?

The dual role of PPIF in promoting either cell survival or death represents one of the field's most challenging paradoxes. To resolve contradictory findings:

• Contextual experimental design:

  • Simultaneously examine multiple cell types (cancer vs. normal, proliferating vs. differentiated)

  • Test varying stress conditions (hypoxia, nutrient deprivation, genotoxic stress) within the same experimental framework

  • Quantify PPIF-dependent effects across a time course to capture temporal dynamics

• Molecular interaction mapping:

  • Employ proximity labeling techniques (BioID, APEX) to identify PPIF interactors under different conditions

  • Use co-immunoprecipitation coupled with mass spectrometry to detect context-specific binding partners

  • Perform cross-linking mass spectrometry to capture transient interactions

• Post-translational modification analysis:

  • Investigate how phosphorylation, acetylation, or other modifications of PPIF alter its function

  • Utilize site-directed mutagenesis to create PPIF variants mimicking or preventing specific modifications

  • Apply phospho-proteomics to map signaling networks connecting PPIF to survival or death pathways

• Reconciliation framework:

  • Measure the activation threshold for MPTP opening under different conditions

  • Quantify the balance between transient versus sustained MPTP opening

  • Determine how PPIF concentration and localization within mitochondrial compartments affects outcomes

An integrated approach combining these strategies can help distinguish when PPIF promotes survival (as in cancer cells resisting apoptosis) versus when it facilitates cell death (as in neurodegenerative conditions or ischemic injury).

What cutting-edge techniques are advancing our understanding of PPIF's structure-function relationships?

Recent methodological advances have significantly enhanced our ability to probe PPIF's structure-function relationships:

• Cryo-electron microscopy (cryo-EM) applications:

  • High-resolution structures of PPIF in complex with the MPTP components

  • Visualization of conformational changes upon binding to inhibitors or activators

  • Structural analysis of PPIF interactions with the FOXO3a/PINK1–Parkin pathway components

• Molecular dynamics simulations:

  • Modeling PPIF's β-barrel flexibility under various physiological conditions

  • Predicting binding interfaces with novel interacting partners

  • Simulating the effects of post-translational modifications on protein dynamics

• In situ structural biology:

  • Proximity-based protein interaction mapping within intact mitochondria

  • Single-molecule tracking of PPIF mobility and clustering in live cells

  • Correlative light and electron microscopy to localize PPIF relative to MPTP components

• Integrative proteomics approaches:

  • Thermal proteome profiling to identify direct and indirect PPIF interactors

  • Crosslinking mass spectrometry to map interaction interfaces

  • Hydrogen-deuterium exchange mass spectrometry to detect conformational changes upon ligand binding

• High-throughput functional screening:

  • CRISPR-based screens for genetic modifiers of PPIF activity

  • Chemical library screening for novel PPIF modulators with therapeutic potential

  • Synthetic lethality screens in PPIF-high cancer cells to identify targetable vulnerabilities

These techniques are particularly valuable for rational drug design targeting PPIF in disease contexts and for elucidating how structural features translate to PPIF's diverse functional roles.

What methodologies should researchers employ to assess PPIF as a prognostic biomarker in cancer?

To rigorously evaluate PPIF's potential as a prognostic biomarker in cancer, researchers should implement a comprehensive methodological framework:

Evidence already suggests that high PPIF expression correlates with poor outcomes in lung adenocarcinoma and head and neck/laryngeal squamous cell carcinoma , making this a promising area for further translational research.

How can researchers design effective experiments to evaluate PPIF-targeting compounds for therapeutic potential?

Designing robust experiments to evaluate PPIF-targeting therapeutic compounds requires a systematic approach:

• Compound screening and validation pipeline:

  • Primary screening: PPIase enzymatic activity assays using recombinant PPIF

  • Secondary screening: MPTP opening assays in isolated mitochondria

  • Tertiary screening: Cell-based viability and function assays in disease-relevant models

  • Specificity testing: Counter-screening against other cyclophilins (PPIA, PPIB, PPID)

• Mechanism of action characterization:

  • Binding site identification through mutagenesis or structural biology approaches

  • Conformational effects using hydrogen-deuterium exchange mass spectrometry

  • Impact on PPIF interactions with MPTP components and signaling partners

  • Effects on PPIF-regulated pathways (mitophagy, apoptosis, inflammation)

• Disease-specific efficacy assessment:

  • Cancer models: Anti-proliferative effects, sensitivity in PPIF-high vs. PPIF-low tumors

  • Neurodegenerative disease models: Neuroprotection, mitochondrial function preservation

  • Inflammatory disease models: Modulation of macrophage phenotypes and T-cell responses

  • Ischemia-reperfusion models: Protection against tissue damage

• Delivery and pharmacokinetic considerations:

  • Mitochondrial targeting strategies to enhance compound accumulation

  • Assessment of tissue distribution, particularly to affected organs

  • Stability and metabolism studies in physiologically relevant systems

• Combination therapy approaches:

  • Synergy testing with standard-of-care treatments

  • Sequential treatment protocols to maximize therapeutic windows

  • Biomarker-guided patient selection strategies based on PPIF expression

Cyclosporin A derivatives that selectively target PPIF without immunosuppressive effects represent one of the most promising therapeutic approaches currently under investigation .

What are the key considerations for studying PPIF in exosomes and their potential as liquid biopsy biomarkers?

The emerging field of PPIF in exosomes presents unique methodological challenges and opportunities for liquid biopsy applications:

• Exosome isolation optimization:

  • Compare ultracentrifugation, size exclusion chromatography, and precipitation-based methods

  • Validate isolation purity using nanoparticle tracking analysis and exosome markers (CD9, CD63, CD81)

  • Standardize pre-analytical variables (collection, storage, processing) to ensure reproducibility

  • Consider tissue/cell-specific exosome isolation using immunoaffinity approaches

• PPIF detection and quantification in exosomes:

  • Develop sensitive detection methods for low-abundance PPIF in exosomal fractions

  • Compare RNA-seq, proteomic, and functional assays for exosomal PPIF

  • Establish normalization strategies using exosome quantity and quality metrics

  • Validate findings using multiple technical approaches

• Clinical correlation studies:

  • Analyze exosomal PPIF levels across disease stages and treatment responses

  • Perform longitudinal sampling to capture dynamic changes

  • Compare exosomal PPIF with tissue-based expression and circulating tumor cells

  • Correlate exosomal PPIF with patient outcomes and treatment responses

• Functional characterization of exosomal PPIF:

  • Investigate recipient cell uptake of PPIF-containing exosomes

  • Assess functional transfer of PPIF activity to recipient cells

  • Examine how exosomal PPIF influences immune cell functions and phenotypes

  • Determine the impact on tumor microenvironment remodeling

Recent findings showing PPIF upregulation in cancer-derived exosomes suggest their potential utility as minimally invasive biomarkers, particularly in cancers where tissue biopsies are challenging or for longitudinal monitoring during treatment.

How might single-cell analysis techniques be optimized to study PPIF heterogeneity in complex tissues?

Single-cell analysis represents a frontier for understanding PPIF's heterogeneous expression and function across different cell types within complex tissues. Researchers should consider:

• Single-cell RNA sequencing (scRNA-seq) optimization:

  • Preservation protocols that maintain mitochondrial integrity during sample preparation

  • Enrichment strategies for low-abundance mitochondrial transcripts

  • Integration of spatial transcriptomics to maintain tissue context information

  • Computational approaches for identifying PPIF co-expression networks at single-cell resolution

• Single-cell proteomics approaches:

  • Development of ultra-sensitive mass spectrometry protocols for PPIF detection

  • Optimization of CyTOF (mass cytometry) panels incorporating PPIF antibodies

  • Single-cell Western blotting for PPIF protein level heterogeneity assessment

  • Proximity ligation assays to detect PPIF interactions in intact cells

• Functional single-cell analysis:

  • Adaptation of mitochondrial function assays to single-cell formats

  • Live-cell imaging of PPIF activity using fluorescent reporters

  • Correlation of PPIF levels with single-cell metabolic profiles

  • Single-cell CRISPR perturbations to assess PPIF function across different cell types

• Integrative analysis frameworks:

  • Multi-omics integration at single-cell level (transcriptome, proteome, metabolome)

  • Trajectory analysis to map cellular states based on PPIF expression and activity

  • Cell-cell communication analysis to identify PPIF-dependent intercellular signaling

Single-cell analysis has already revealed important insights into PPIF's role in specific immune cell populations like Macrophage-C1-C1QC cells in cancer contexts , and further methodological refinements will expand our understanding of PPIF heterogeneity in complex tissues.

What methodological approaches can resolve the dual role of PPIF in inflammation and immune regulation?

PPIF demonstrates intriguing dual roles in inflammation and immune regulation that require sophisticated experimental approaches to resolve:

• Temporal dynamics investigation:

  • Time-course analyses of PPIF expression during inflammatory progression

  • Live-cell imaging of PPIF activity during immune cell activation and resolution

  • Pulse-chase experiments to track PPIF-dependent signaling cascades

• Cell type-specific analysis:

  • Conditional PPIF knockout/knockin models targeting specific immune cell populations

  • Co-culture systems with defined PPIF expression in different immune cell types

  • Analysis of PPIF's effects on polarization states (M1/M2 macrophages, Th1/Th2 T cells)

• Pathway dissection approaches:

  • Selective inhibition of downstream signaling components to isolate PPIF-dependent effects

  • Phospho-proteomic mapping of inflammatory signaling networks with/without PPIF

  • Metabolic profiling to link PPIF activity to pro- versus anti-inflammatory metabolic states

• In vivo models with readout optimization:

  • Non-invasive imaging of inflammation with PPIF reporters

  • Multiplex cytokine profiling in PPIF-modulated systems

  • Flow cytometric immune phenotyping with high-dimensional analysis (20+ parameters)

• Translational validation strategies:

  • Ex vivo analysis of patient-derived immune cells with varying PPIF levels

  • Correlation of PPIF polymorphisms with inflammatory disease phenotypes

  • Testing PPIF modulators in humanized mouse models of inflammatory diseases

The recent discovery that PPIF regulates macrophage phenotypes and T helper cell balance provides a foundation for these investigations, potentially leading to novel therapeutic strategies for inflammatory and immune-mediated diseases.