PPIL4 Human

What is PPIL4 and what structural domains does it contain?

PPIL4 (Peptidyl-prolyl cis-trans isomerase-like 4) is an enzyme encoded by the PPIL4 gene in humans, belonging to the cyclophilin family of peptidylprolyl isomerases . Structurally, PPIL4 contains an RNA recognition motif (RRM) domain and a cyclophilin-type peptidyl-prolyl cis-trans isomerase domain . The protein structure includes a PPIL4-like cyclophilin domain that distinguishes it from other family members. Domain analysis reveals that PPIL4 shares the nucleotide-binding alpha-beta plait domain superfamily and cyclophilin-like domain superfamily characteristics with other cyclophilins .

What are the primary biological functions of PPIL4?

PPIL4 functions primarily as a peptidyl-prolyl isomerase that catalyzes the cis-trans isomerization of proline imidic peptide bonds in oligopeptides, accelerating protein folding . It exhibits RNA binding activity and RNA polymerase II CTD heptapeptide repeat isomerase activity (both P3 and P6) . As a member of the highly conserved cyclophilin family, PPIL4 participates in protein folding mechanisms and may be involved in processes related to immunosuppression by cyclosporin A . Furthermore, PPIL4 has been identified as a critical factor in brain-specific angiogenesis through its role in potentiating Wnt signaling by binding to JMJD6, a known angiogenesis regulator .

How does PPIL4 contribute to the Wnt signaling pathway?

PPIL4 functions as a positive regulator of the Wnt signaling pathway through its interaction with JMJD6 (Jumonji domain-containing protein 6) . Experimental evidence indicates that wild-type PPIL4, but not intracranial aneurysm (IA)-mutant forms, potentiates Wnt signaling by binding to JMJD6, which is a known activator of the Wnt pathway . Methodologically, this interaction can be studied using co-immunoprecipitation assays and reporter gene assays measuring Wnt pathway activation. The PPIL4-JMJD6 interaction represents a novel mechanism in Wnt signaling that appears particularly important for brain-specific angiogenesis and maintenance of cerebrovascular integrity . Researchers investigating this pathway should consider both canonical and non-canonical Wnt signaling branches when designing experiments to fully characterize PPIL4's role.

What role does PPIL4 play in smooth muscle cell remodeling?

PPIL4 is a key modulator of smooth muscle cell (SMC) phenotypic switching and remodeling. Experimental downregulation of PPIL4 using lentiviral shRNA in human primary brain vascular smooth muscle cells (HPBVSMC) results in significant upregulation of genes encoding contractile proteins, including ACTA2, CNN1, TNNT1, and TNNT2 . This leads to increased cell size and favors a contractile SMC phenotype . Conversely, PPIL4 overexpression using ORF-lentiviral particles causes downregulation of these contractile proteins, increases expression of matrix metalloproteinases MMP1 and MMP3, and promotes a synthetic phenotype in SMCs characterized by cell elongation and spindle-like formation . These findings suggest that PPIL4 functions as a molecular switch controlling SMC phenotype, with potential implications for vascular diseases involving SMC dysfunction, including intracranial aneurysms.

How is PPIL4 involved in RNA processing mechanisms?

As a nuclear cyclophilin, PPIL4 likely participates in RNA processing mechanisms similar to other "spliceophilins" (spliceosome-associated cyclophilins) . While specific mechanisms for PPIL4 are not fully characterized in the search results, the protein contains an RNA recognition motif domain that enables nucleic acid binding . Other nuclear cyclophilins, such as PPIH, interact with spliceosomal components in ways that do not necessarily involve their canonical peptidyl-prolyl isomerase activity . These interactions can protect intrinsically disordered regions of spliceosomal proteins from unregulated post-translational modifications while also regulating the cyclophilin's activity . Research into PPIL4's RNA processing functions would benefit from techniques such as RNA immunoprecipitation, mass spectrometry to identify interaction partners, and in vitro splicing assays to determine functional outcomes.

What evidence links PPIL4 mutations to intracranial aneurysm development?

Genetic studies provide compelling evidence linking PPIL4 mutations to intracranial aneurysm (IA) pathogenesis. Analysis of IA cases identified rare loss-of-function (LoF) and deleterious missense (D-Mis) mutations in PPIL4 that were significantly enriched compared to control cohorts . Specifically, IA cases showed an odds ratio of 11.51 for harboring PPIL4 mutations compared to matched controls (Fisher's P < 0.001) . Three loss-of-function variants were identified in familial and index IA cases . Functional validation in vertebrate models demonstrated that Ppil4 depletion causes intracerebral hemorrhage and defects in cerebrovascular morphology , providing a mechanistic link between the genetic findings and the pathophysiology of IA. This genetic evidence, combined with PPIL4's role in brain angiogenesis and vascular integrity, establishes PPIL4 as an important contributor to IA development.

How do experimental models validate PPIL4's role in cerebrovascular development?

Multiple experimental approaches validate PPIL4's crucial role in cerebrovascular development. In zebrafish models, ppil4 expression increases significantly after 24 hours post-fertilization (hpf) and reaches peak levels at 3 months . Depletion of ppil4 in these models results in intracerebral hemorrhage and defects in cerebrovascular morphology . In vitro tube formation assays using human umbilical vein endothelial cells (HUVECs) demonstrate that shRNA-mediated downregulation of PPIL4 significantly reduces nodes, junctions, and branches during vascular network formation . This suggests that the cerebrovascular defects observed in animal models can be attributed to endothelial cell-autonomous effects. Additionally, restoration of wild-type PPIL4 expression, but not IA-mutant forms, in these models rescues the vascular phenotypes, further validating PPIL4's critical function in proper cerebrovascular development and maintenance .

What methodological approaches are most effective for studying PPIL4 in disease contexts?

For comprehensive investigation of PPIL4 in disease contexts, researchers should employ a multi-faceted approach:

• Genetic analysis: Targeted sequencing of PPIL4 in patient cohorts with relevant phenotypes (particularly cerebrovascular disorders), followed by in silico prediction of mutation effects and statistical analysis comparing mutation burden to matched controls .

• Animal models: Generation of PPIL4 knockout or knockdown models in zebrafish or mice, with careful evaluation of vascular phenotypes using techniques such as microangiography and confocal microscopy .

• Cellular assays: In vitro tube formation assays using endothelial cells with PPIL4 manipulation (via shRNA knockdown or overexpression) to assess angiogenic capacity . Cell size, morphology, and contractility measurements in smooth muscle cells with altered PPIL4 expression .

• Molecular interaction studies: Co-immunoprecipitation and pull-down assays to identify and validate protein-protein interactions, particularly with JMJD6 and components of the Wnt pathway .

• Signaling pathway analysis: Luciferase reporter assays for Wnt pathway activation, combined with Western blotting for downstream effectors, comparing wild-type versus mutant PPIL4 .

• Expression profiling: RNA-sequencing of tissues/cells with manipulated PPIL4 levels to identify global transcriptomic changes, with particular attention to contractile proteins in smooth muscle cells and angiogenic factors in endothelial cells .

How do the structural differences between PPIL4 and other nuclear cyclophilins influence their specialized functions?

PPIL4 contains both a cyclophilin-type peptidyl-prolyl isomerase domain and an RNA recognition motif domain, distinguishing it from some other nuclear cyclophilins . This dual-domain architecture likely enables PPIL4 to participate in both protein isomerization and RNA-related functions. To investigate the structural basis of PPIL4's specialized functions, researchers should employ:

• Structural biology approaches: X-ray crystallography or cryo-electron microscopy to determine the three-dimensional structure of PPIL4, particularly in complex with binding partners such as JMJD6 .

• Domain-specific mutagenesis: Creation of domain-specific mutants to dissect the contributions of the cyclophilin domain versus the RNA recognition motif in various cellular processes .

• Comparative analysis: Systematic comparison of PPIL4's structure and function with other nuclear cyclophilins ("spliceophilins") to identify unique features that explain its tissue-specific roles .

• Molecular dynamics simulations: Computational analysis of how disease-associated mutations affect PPIL4's structure and dynamics, particularly in relation to partner binding .

Understanding these structural determinants may reveal why PPIL4 has evolved specialized functions in brain angiogenesis and vascular maintenance compared to other cyclophilin family members.

What is the molecular basis for PPIL4's apparent tissue-specific effects in brain vasculature?

PPIL4 shows significantly higher expression in brain endothelial cells compared to those from other organs, and its depletion causes cerebrovascular-specific defects . The molecular basis for this tissue specificity may involve:

• Tissue-specific transcriptional regulation: Analysis of the PPIL4 promoter region and identification of brain endothelial-specific transcription factors regulating its expression.

• Interaction with brain-specific partners: Proteomic analysis comparing PPIL4 interactomes in brain versus non-brain endothelial cells to identify tissue-specific binding partners.

• Epigenetic regulation: Investigation of chromatin modifications and accessibility at the PPIL4 locus in different tissues using techniques such as ATAC-seq and ChIP-seq.

• Alternative splicing: Characterization of potential tissue-specific PPIL4 isoforms that may have altered functions or localization patterns.

• Post-translational modifications: Mass spectrometry analysis of PPIL4 from different tissues to identify brain-specific modifications that might alter its function.

Understanding these tissue-specific mechanisms would provide insight into why PPIL4 mutations particularly affect cerebral blood vessels and could identify new therapeutic targets for cerebrovascular diseases.

How might the contradictory effects of PPIL4 in smooth muscle cells versus endothelial cells be reconciled in the context of vascular pathophysiology?

PPIL4 appears to have seemingly contradictory roles in different vascular cell types. In smooth muscle cells, PPIL4
downregulation promotes a contractile phenotype, while in endothelial cells, PPIL4 is essential for normal angiogenic function . This apparent contradiction requires careful investigation:

• Cell-type specific signaling analysis: Comparative analysis of signaling pathways affected by PPIL4 in SMCs versus endothelial cells, with focus on Wnt signaling components.

• Co-culture experiments: Investigation of PPIL4 manipulation in co-culture systems containing both SMCs and endothelial cells to observe integrated effects on vascular modeling.

• Conditional knockout models: Generation of cell-type specific PPIL4 knockout mice (endothelial-specific versus SMC-specific) to determine the relative contributions of each cell type to vascular phenotypes.

• Temporal expression studies: Analysis of PPIL4 expression dynamics during different phases of vascular development and remodeling to identify potential stage-specific functions.

• Protein complex analysis: Identification of cell-type specific PPIL4 protein complexes that might direct its activity toward different downstream pathways in SMCs versus endothelial cells.

This paradox may reflect a sophisticated regulatory mechanism whereby PPIL4 coordinates the balance between vessel stability (SMC function) and remodeling capacity (endothelial function) in the cerebral vasculature.

What is the potential of PPIL4-targeted therapeutics for cerebrovascular diseases?

Given PPIL4's critical role in cerebrovascular development and its implication in intracranial aneurysm, it represents a potential therapeutic target. Research approaches should include:

• Small molecule screening: Development of high-throughput screens to identify compounds that can modulate PPIL4 activity or its interaction with key partners like JMJD6.

• Structure-based drug design: Utilizing structural information about PPIL4's active site and protein-protein interaction surfaces to design specific modulators.

• Peptide mimetics: Design of peptides that mimic PPIL4's interaction interfaces with key partners to competitively inhibit or enhance specific functions.

• Delivery systems: Development of brain-targeted delivery systems for PPIL4 modulators, potentially utilizing the blood-brain barrier's unique properties.

• Therapeutic window determination: Investigation of developmental versus adult requirements for PPIL4 function to identify safe intervention periods.

• Combination approaches: Testing PPIL4-targeted therapies in combination with modulators of related pathways, such as Wnt signaling inhibitors or activators.

The tissue-specific nature of PPIL4's critical functions in cerebral blood vessels may offer an advantage in developing therapies with limited systemic side effects, potentially opening new avenues for treating cerebrovascular disorders.