PPIL3 Human

What is PPIL3 and where is it located in the human genome?

PPIL3 (peptidylprolyl isomerase like 3) is a human gene that encodes a cyclophilin-like protein belonging to the peptidyl-prolyl cis-trans isomerase family . The PPIL3 gene has been mapped to chromosome 2q33, positioned between STS markers stSG2762 (proximal) and SHGC-3074 (distal) and oriented toward the telomere . Genomically, the PPIL3 gene consists of eight exons spanning more than 18 kb of DNA . This gene was initially isolated during large-scale sequencing analysis of a human fetal brain cDNA library .

The cyclophilin family, to which PPIL3 belongs, is notable for being the target of the immunosuppressive drug cyclosporin, which is commonly used in organ transplantation . PPIL3 is one of 17 human cyclophilin isoforms that have been identified, each containing a PPIase (peptidyl-prolyl isomerase) domain with varying degrees of sequence identity .

What is the expression pattern of PPIL3 in human tissues?

RT-PCR analysis has demonstrated that PPIL3 is ubiquitously expressed across adult human tissues . This widespread expression pattern suggests that PPIL3 may play a fundamental role in cellular processes rather than having tissue-specific functions. The ubiquitous expression of PPIL3 is consistent with other members of the cyclophilin family, many of which are broadly expressed throughout the body and participate in essential cellular functions such as protein folding and cellular signaling .

Unlike some other cyclophilins that show tissue-specific expression patterns, PPIL3's presence in all tested adult tissues indicates it may serve as a housekeeping protein with functions required by most cell types . This ubiquitous expression also suggests that studying PPIL3 function may require consideration of its role across multiple tissue contexts rather than focusing on a single organ system.

What is the structural architecture of PPIL3 protein?

Like all cyclophilins, PPIL3 shares a common fold architecture consisting of eight antiparallel β sheets and two α-helices that pack against the sheets . Additionally, there is a short α-helical turn containing the active site residue Trp121 found in the β6-β7 loop region . The structural similarity across the cyclophilin family is quite high, with root-mean-square deviation (RMSD) across all atoms for all PPIase domains being less than 2 Å .

Specifically, PPIL3 has been structurally characterized and shows high structural conservation with other family members. When aligned with the prototypical cyclophilin PPIA, PPIL3 shows an RMSD of between 0.4 Å to 1.0 Å, indicating very high structural similarity . This structural conservation suggests that PPIL3 likely functions through similar mechanisms as other cyclophilins, while potentially having unique substrate specificities determined by subtle structural differences.

What is the enzymatic function of PPIL3?

PPIL3 functions as a peptidyl-prolyl isomerase (PPIase), catalyzing the cis-trans isomerization of peptide bonds preceding proline residues in proteins . This isomerization is a rate-limiting step in protein folding for many proteins containing proline residues. The enzymatic activity of cyclophilins like PPIL3 is critical for proper protein folding and can influence protein structure, stability, and function .

The catalytic mechanism involves specific residues in the active site that are highly conserved across the cyclophilin family. For PPIL3, the active site includes the characteristic tryptophan residue (corresponding to Trp121 in PPIA) that is crucial for substrate binding and catalysis . The enzymatic function of PPIL3 can be studied using NMR-based assays that monitor the isomerization of proline-containing peptides, similar to methods used for other cyclophilins .

How does PPIL3 interact with Apoptin, and what are the implications?

One of the most significant protein-protein interactions identified for PPIL3 is with Apoptin, a small protein encoded by chicken anemia virus (CAV) that selectively induces cell death in cancer cells . Through a yeast two-hybrid screen, PPIL3 was identified as an Apoptin-associated protein that can bind directly to Apoptin . This interaction has important functional consequences for Apoptin's cellular localization and activity.

The interaction between PPIL3 and Apoptin leads to the cytoplasmic localization of Apoptin, even in tumor cells where Apoptin typically migrates to the nucleus . This is significant because Apoptin's nuclear localization in cancer cells is associated with its ability to induce cell death. Research has demonstrated that the nuclear-cytoplasmic distribution of Apoptin is related to the expression level of intrinsic PPIL3, and experimental modification of PPIL3 levels results in nuclear-cytoplasmic shuffling of Apoptin .

Mechanistically, the P109A mutant of Apoptin, located between the putative nuclear localization and export signals, can significantly impair the function of PPIL3 . This finding suggests that the specific interaction between PPIL3 and this region of Apoptin is critical for determining Apoptin's subcellular localization.

What is known about PPIL3 substrate specificity compared to other cyclophilins?

Cyclophilins, including PPIL3, have varying degrees of substrate specificity determined by their active site architecture and surrounding residues. Structural studies comparing the PPIase domains of different cyclophilins have revealed subtle differences that may contribute to distinct substrate preferences .

Research using in silico modeling and experimental validation has shown that cyclophilins can discriminate between different peptide sequences, particularly at the P2 and P3 positions (the second and third amino acids before the proline that undergoes isomerization) . For example, PPIA, the prototypical cyclophilin, shows preferences for aromatic residues at P2 and acidic residues at P3 .

While specific substrate preferences for PPIL3 have not been extensively characterized in the provided search results, the structural conservation between PPIL3 and other cyclophilins suggests it likely has its own unique substrate specificity profile. Experimental approaches using peptide test sets and NMR-based assays could be employed to determine PPIL3's substrate preferences, similar to methods used for other cyclophilins .

What techniques are available for studying PPIL3 isomerase activity?

Several experimental approaches can be used to study the peptidyl-prolyl isomerase activity of PPIL3:

• NMR-based assays: Nuclear magnetic resonance spectroscopy can directly monitor the cis-trans isomerization of proline-containing peptides catalyzed by PPIL3 . This technique provides detailed information about the kinetics of the isomerization reaction and can be used to assess substrate specificity.

• Peptide substrate libraries: Testing PPIL3 against libraries of peptides with varying sequences at the P2 and P3 positions can help determine substrate preferences . Synthetic peptides with sequences such as DEGPF, DFGPF, and DYGPF have been used to assess the catalytic activity of cyclophilins like PPIA and could be adapted for PPIL3 studies .

• Enzymatic coupled assays: These assays couple the isomerization reaction to another enzymatic reaction that produces a detectable signal, allowing for high-throughput screening of PPIL3 activity.

• Inhibitor studies: Using known cyclophilin inhibitors like cyclosporin A can help characterize PPIL3's enzymatic properties and compare them to other family members .

How can researchers investigate PPIL3 protein-protein interactions?

Several techniques are available for studying PPIL3's interactions with other proteins:

• Yeast two-hybrid screening: This method has successfully identified PPIL3 interaction with Apoptin and could be used to discover additional protein partners . The technique involves fusing PPIL3 to a DNA-binding domain and screening against a library of proteins fused to an activation domain.

• Co-immunoprecipitation: This technique can verify direct protein-protein interactions in cellular contexts by using antibodies to isolate PPIL3 along with its binding partners.

• Fluorescence-based interaction assays: Techniques such as FRET (Fluorescence Resonance Energy Transfer) or BiFC (Bimolecular Fluorescence Complementation) can be used to visualize PPIL3 interactions in living cells.

• Pull-down assays: Using purified recombinant PPIL3 as bait, researchers can identify interacting proteins from cell lysates, followed by mass spectrometry identification.

• Protein arrays: High-throughput screening of potential PPIL3 interactors can be performed using protein microarrays containing hundreds or thousands of candidate proteins.

What methods can be used to modulate PPIL3 expression for functional studies?

To investigate the cellular functions of PPIL3, researchers can employ various approaches to alter its expression:

• RNA interference (RNAi): Small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) can be designed to target PPIL3 mRNA, reducing its expression levels . This approach has been used to demonstrate that modifying PPIL3 levels affects Apoptin localization.

• CRISPR-Cas9 gene editing: This technology enables precise modification of the PPIL3 gene, allowing for knockout studies or the introduction of specific mutations to assess functional consequences.

• Overexpression systems: Transfection with expression vectors containing the PPIL3 coding sequence allows for increased protein levels and assessment of gain-of-function effects .

• Inducible expression systems: Systems such as Tet-On or Tet-Off allow for temporal control of PPIL3 expression, enabling studies of acute versus chronic changes in protein levels.

• Transgenic animal models: For in vivo studies, transgenic animals with altered PPIL3 expression can be developed to assess its function in complex physiological contexts.

What are the splice variants of PPIL3 and their potential functional differences?

The PPIL3 gene produces at least two different splicing variants, as identified during the sequencing analysis of a human fetal brain cDNA library . These two cDNA clones encode two novel proteins that show 52% and 72% identity to the cyclophilin isoform 10 of C. elegans, respectively .

How does the structural comparison of PPIL3 with other cyclophilins inform functional predictions?

Structural analysis of the cyclophilin family provides valuable insights into potential functional differences between members:

Despite the high structural conservation across the cyclophilin family, subtle differences in surface loops and the composition of the active site can lead to distinct substrate preferences and binding properties . Computational modeling approaches have been used to predict substrate specificities based on structural features, particularly focusing on the S2 pocket that accommodates the residue at the P2 position of the substrate .

What is the potential role of PPIL3 in disease processes?

While the direct involvement of PPIL3 in disease pathways is not extensively documented in the provided search results, its interaction with Apoptin suggests potential relevance to cancer biology . The finding that PPIL3 can regulate the subcellular localization of Apoptin, a protein that selectively induces death in cancer cells, indicates that PPIL3 may influence cancer cell survival pathways .

Cyclophilins as a family have been implicated in various diseases, including viral infections, inflammatory disorders, and cancer . Their roles in protein folding, cellular signaling, and immune regulation make them potential contributors to disease processes. For PPIL3 specifically, its ubiquitous expression pattern suggests it could have broad relevance to cellular homeostasis across multiple tissues .

Future research directions might include investigating whether PPIL3 expression levels are altered in specific disease states, whether genetic variants of PPIL3 are associated with disease risk, and whether modulating PPIL3 activity could have therapeutic potential, particularly in cancer contexts where its interaction with Apoptin may be relevant.

What are the promising avenues for further investigation of PPIL3 function?

Several research directions hold promise for advancing our understanding of PPIL3:

• Comprehensive substrate specificity profiling: Using peptide libraries and high-throughput assays to characterize the substrate preferences of PPIL3 compared to other cyclophilins .

• Identification of endogenous substrates: Proteomic approaches to identify proteins that undergo PPIL3-catalyzed isomerization in vivo.

• Characterization of splice variant functions: Investigating the expression patterns and potential functional differences between the two identified splice variants of PPIL3 .

• Expanded protein interaction network: Building upon the identified interaction with Apoptin to discover additional PPIL3 binding partners that might illuminate its cellular functions .

• Disease association studies: Examining potential links between PPIL3 expression levels or genetic variants and specific disease states.

How can structural biology approaches advance PPIL3 research?

Structural biology offers powerful tools for understanding PPIL3 function:

• Co-crystal structures with substrates: Determining the structure of PPIL3 bound to peptide substrates could reveal the molecular basis of its substrate specificity.

• Comparative structural analysis: More detailed comparisons between PPIL3 and other cyclophilins might identify subtle structural differences that account for functional specialization .

• Structure-based drug design: Using the structural information to design specific inhibitors or modulators of PPIL3 activity that could serve as research tools or potential therapeutics.

• Structural characterization of protein complexes: Investigating the structural basis of the PPIL3-Apoptin interaction and other protein-protein interactions .

• Molecular dynamics simulations: Computational approaches to understand the dynamics of PPIL3 during catalysis and protein interactions .