PPIL1 Human

What is the primary function of PPIL1 in human cells?

PPIL1 serves two primary functions in human cells. First, it functions as a peptidyl-prolyl isomerase (PPIase) that catalyzes the cis-trans isomerization of proline peptide bonds in proteins . Second, it acts as an integral component of the spliceosome, the ribonucleoprotein complex responsible for removing introns from pre-mRNA . The protein is expressed ubiquitously across human tissues, suggesting its fundamental importance in cellular processes . Recent research has focused on characterizing its active site and enzymatic mechanisms, with particular attention to how mutations affect both stability and catalytic function.

Research methodological approach: To study PPIL1's PPIase activity, NMR spectroscopy with model substrates has proven effective for measuring the catalytic efficiency of both wild-type and mutant proteins . Comparative analysis of enzymatic parameters (kcat, KM) between wild-type and mutant forms provides insights into functional consequences of disease-associated variants.

How does PPIL1 interact with other components of the spliceosome?

PPIL1 primarily interacts with SNW1/SKIP within the spliceosome, which is critical for its recruitment to the splicing machinery . Surface plasmon resonance (SPR) experiments have demonstrated that this interaction is abolished by certain patient mutations, particularly p.R131Q . Additionally, cryo-EM structural studies have revealed potential interactions with PRP17, which may be a biological target of PPIL1's PPIase activity within the spliceosome .

Research approach: Protein-protein interactions can be investigated using a combination of:

• SPR to measure binding kinetics and affinity

• Co-immunoprecipitation to confirm interactions in cellular contexts

• Proximity ligation assays to validate interactions in situ

• Yeast two-hybrid screening to identify novel binding partners

What is the spectrum of neurodevelopmental phenotypes associated with PPIL1 variants?

Biallelic pathogenic variants in PPIL1 cause a distinctive neurodevelopmental disorder characterized by profound microcephaly (7-9 SD below mean), severe cortical dysplasia, cerebellar hypoplasia affecting both vermis and hemispheres, and brainstem hypoplasia . Clinical manifestations include:

Research approach: For genotype-phenotype correlation studies, researchers should collect comprehensive clinical data, including detailed neuroimaging, and correlate specific variants with phenotypic severity using standardized assessment tools. Functional studies of each variant's impact on protein stability, enzymatic activity, and protein interactions provide mechanistic insights.

How do different PPIL1 variants affect protein function and stability?

Different PPIL1 variants exhibit distinct effects on protein function and stability. Research has identified four main mechanisms by which pathogenic variants disrupt PPIL1 function :

• Reduced protein stability: The p.A99T and p.G109C;A101_D106dup mutations significantly decrease protein stability.

• Disrupted protein interactions: The p.R131Q variant abolishes interaction with SKIP, preventing proper recruitment to the spliceosome.

• Decreased enzymatic activity: Certain mutations reduce PPIase activity on model substrates, as demonstrated using NMR techniques.

• Altered splicing patterns: Mutations likely affect alternative splicing patterns, contributing to tissue-specific manifestations.

Research approach: To characterize novel variants, researchers should employ thermal shift assays to assess protein stability, enzymatic assays to measure PPIase activity, and interaction studies to evaluate binding to known partners. RNA-seq analysis of patient-derived cells can reveal splicing defects.

What methodologies are most effective for studying PPIL1's role in the spliceosome?

Investigating PPIL1's function within the spliceosome requires multidisciplinary approaches:

• Structural studies: Cryo-EM has been instrumental in visualizing PPIL1 within the spliceosome complex, revealing its spatial relationships with other components . NMR studies have complemented these findings by providing dynamic information about protein-protein interactions.

• Splicing assays: In vitro splicing assays using minigene constructs and reporter systems can detect alterations in splicing efficiency and fidelity when PPIL1 is mutated or depleted.

• Proteomic approaches: Proximity-dependent biotin identification (BioID) or APEX labeling can identify proteins that interact with PPIL1 during various stages of spliceosome assembly.

• CRISPR-based methods: Gene editing to introduce specific patient variants into cellular models, followed by RNA-seq analysis, can reveal global impacts on the transcriptome.

Research approach: For comprehensive characterization, researchers should integrate multiple methodologies. For example, combining cryo-EM structural data with biochemical interaction studies and functional splicing assays provides the most complete picture of how PPIL1 variants affect spliceosome function.

How can researchers effectively model PPIL1-associated disorders?

Modeling PPIL1-associated disorders requires multiple complementary approaches:

• Patient-derived iPSCs: These can be differentiated into neural progenitors and neurons to study neurodevelopmental phenotypes. Time-course analyses during differentiation can reveal when and how developmental trajectories diverge.

• CRISPR-engineered cellular models: Introduction of specific patient variants into appropriate cell lines using CRISPR/Cas9 enables precise examination of variant effects.

• Organoid models: Brain organoids derived from patient iPSCs or CRISPR-edited stem cells can recapitulate aspects of cortical development and cerebellar formation.

• Animal models: While complete knockouts may be lethal, conditional or hypomorphic models may recapitulate aspects of the human phenotype.

Research approach: For neural phenotypes, researchers should focus on developmental timelines, analyzing effects on neural progenitor proliferation, migration, and differentiation. Real-time imaging of developing neurons in 3D cultures can capture dynamic phenotypes that might be missed in endpoint analyses.

What is the evidence for PPIL1's involvement in cancer progression?

There is emerging evidence for PPIL1's role in cancer:

• Expression levels: PPIL1 shows elevated expression in several cancer types, including colorectal, gastric, lung, pancreatic, and breast cancers .

• Functional effects: In vitro studies have demonstrated that wild-type PPIL1 has growth-promoting effects in NIH3T3 and HEK293 cells, while siRNA-mediated knockdown of PPIL1 retarded growth of colon cancer cells .

• Interacting partners: PPIL1 interacts with SNW1/SKIP (involved in transcription regulation and mRNA splicing) and stathmin (involved in microtubule stabilization), suggesting potential mechanisms by which it could influence cancer cell proliferation .

Research approach: To investigate PPIL1's role in cancer, researchers should:

• Analyze PPIL1 expression levels across cancer datasets

• Perform knockout/knockdown experiments in cancer cell lines

• Investigate effects on critical cancer pathways including cell cycle regulation, apoptosis, and migration

• Evaluate splicing patterns of cancer-related genes in the presence/absence of functional PPIL1

How does PPIL1 function differ between neural and non-neural tissues?

While PPIL1 is ubiquitously expressed, its functional consequences appear to be tissue-specific, with pronounced effects in the developing brain . This tissue specificity may result from:

• Differential splicing requirements: Neural tissues may have unique splicing demands during development that make them particularly vulnerable to PPIL1 dysfunction.

• Tissue-specific interaction partners: PPIL1 may interact with proteins that are predominantly expressed in neural tissues.

• Developmental timing: The requirement for precise splicing may be especially critical during specific windows of brain development.

Research approach: Comparative proteomics and transcriptomics across different tissues at various developmental stages can identify tissue-specific PPIL1 interactors and splicing targets. Single-cell RNA-seq of developing brain tissues from animal models with PPIL1 mutations can reveal cell-type specific vulnerabilities.

What are the most promising therapeutic approaches for PPIL1-associated disorders?

While current treatments for PPIL1-associated disorders are supportive rather than curative, several therapeutic approaches warrant investigation:

• Antisense oligonucleotides (ASOs): These could potentially modulate splicing of specific genes affected by PPIL1 dysfunction.

• Small molecule stabilizers: For variants that reduce protein stability, small molecules that bind and stabilize the mutant protein might restore function.

• Gene therapy: Delivery of functional PPIL1 to affected tissues during critical developmental windows could theoretically prevent or mitigate disease manifestations.

• Stem cell approaches: For postnatal treatment, neural stem cell transplantation might provide trophic support and partial functional restoration.

Research approach: Initial therapeutic development should focus on identifying the most critical splicing events disrupted by PPIL1 mutations, followed by high-throughput screening for compounds that can correct these specific defects. Early intervention will likely be essential, necessitating prenatal diagnosis and potential in utero treatments for optimal outcomes.

What technological advances are needed to better understand PPIL1 function?

Several technological advancements would accelerate PPIL1 research:

• Improved spatial transcriptomics: Higher resolution techniques to map splicing changes in developing brain tissues at single-cell resolution.

• Real-time splicing visualization: Methods to observe spliceosome dynamics and PPIL1 function in living cells.

• Tissue-specific conditional models: More sophisticated animal models with tissue-specific and temporally controlled PPIL1 manipulation.

• High-throughput functional assays: Systems to rapidly test the functional consequences of PPIL1 variants of uncertain significance.

Research approach: Interdisciplinary collaboration between structural biologists, developmental neurobiologists, and clinicians is essential to leverage these technological advances for meaningful insights into PPIL1 biology and pathology.