PIH1D2 Human

What is PIH1D2 and how does it differ structurally from other PIH family proteins?

PIH1D2 is a member of the PIH (Protein Interacting with Hsp90) protein family that shares limited sequence homology with other family members. Human PIH1D1 and PIH1D2 share very low sequence identity (only 21%), suggesting distinct functional roles . Unlike PIH1D1, PIH1D2 is not a component of the R2TP complex, which serves as an HSP90 cochaperone facilitating the assembly of protein and ribonucleoprotein complexes . The structural differences likely underpin their divergent functions, with PIH1D2 potentially having specialized roles in specific tissues or developmental contexts.

What is the expression pattern of PIH1D2 in human tissues and cell types?

PIH1D2 shows a tissue-specific expression pattern that differs from the more ubiquitously expressed PIH1D1. Based on data from model organisms like zebrafish, PIH proteins including those homologous to PIH1D2 are predominantly expressed in ciliated organs such as Kupffer's vesicle, floor plate, otic vesicle, and pronephric duct . The Human Protein Atlas provides comprehensive expression data across various cancer and non-cancerous cell lines . This tissue-specific expression suggests PIH1D2 may have specialized functions in particular cell types, especially those with ciliary structures.

How evolutionarily conserved is PIH1D2 across species?

PIH1D2 shows evolutionary conservation across vertebrate species, though with varying degrees of sequence similarity. Functional studies in zebrafish have demonstrated that PIH proteins have distinct but related roles across species . The functional divergence observed between human and zebrafish homologs suggests that while core functions may be conserved, species-specific adaptations have occurred. Comparative genomics approaches can help researchers understand which domains and motifs are most conserved, potentially highlighting functionally critical regions.

What are the recommended methods for studying PIH1D2 protein-protein interactions?

For investigating PIH1D2 protein interactions, researchers should consider:

• Co-immunoprecipitation (Co-IP): Use PIH1D2-specific antibodies to pull down protein complexes from cell lysates, followed by mass spectrometry to identify interacting partners. This approach was successfully used for PIH1D1 to identify its binding partners .

• Yeast two-hybrid screening: This can be employed to identify direct protein interactions with PIH1D2.

• Proximity-dependent biotin identification (BioID): This method can capture both stable and transient interactions in living cells.

• Phosphorylation-dependent interaction studies: Given that PIH1D1 interactions are often phosphorylation-dependent through its PIH-N domain, researchers should investigate whether PIH1D2 also exhibits phosphorylation-dependent binding properties, despite the sequence differences .

How can researchers effectively detect and quantify PIH1D2 expression in different experimental systems?

For optimal detection and quantification of PIH1D2:

• RT-qPCR: Design primers specific to PIH1D2 mRNA, avoiding cross-reactivity with other PIH family members due to potential sequence similarities.

• Western blotting: Use validated antibodies specific to PIH1D2. Caution is needed as antibodies may cross-react with other PIH proteins due to structural similarities.

• Immunohistochemistry/Immunofluorescence: For tissue localization studies, use optimized fixation protocols as protein detection may be sensitive to fixation methods.

• RNA-seq analysis: For transcriptome-wide studies, ensure sufficient sequencing depth to detect PIH1D2, which may have low expression in certain tissues.

• Single-cell RNA-seq: Particularly valuable for identifying cell-type specific expression patterns within heterogeneous tissues.

What CRISPR-Cas9 strategies are most effective for PIH1D2 knockout studies?

When designing CRISPR-Cas9 knockout strategies for PIH1D2:

• Guide RNA design: Target early exons to ensure complete loss of function. Multiple guide RNAs should be designed to increase efficiency.

• Validation methods: Confirm knockouts by sequencing, western blot, and RT-qPCR to ensure complete protein loss.

• Control considerations: Include appropriate controls such as non-targeting guides and rescue experiments with wild-type PIH1D2 to confirm phenotype specificity.

• Inducible systems: Consider doxycycline-inducible CRISPR systems if constitutive knockout affects cell viability.

• Domain-specific editing: For structure-function studies, design in-frame deletions of specific domains rather than complete gene knockout.

Based on zebrafish studies, where CRISPR/Cas9 was successfully used to generate mutations in PIH family genes, similar approaches could be applied to human cell lines .

How does PIH1D2 differ functionally from PIH1D1 in humans?

The functional divergence between PIH1D2 and PIH1D1 is significant:

• Complex formation: While PIH1D1 is a component of the R2TP complex (RUVBL1, RUVBL2, RPAP3/Tah1, and PIH1D1) that interacts with HSP90, PIH1D2 does not participate in this complex .

• Phosphopeptide binding: PIH1D1 contains a PIH-N domain that functions as a phosphopeptide binding domain essential for substrate recognition. Whether PIH1D2 shares this capability remains to be thoroughly investigated .

• Cellular distribution: PIH1D1 has ubiquitous cellular functions beyond ciliary roles, while PIH1D2 may have more specialized functions in ciliated cells based on expression patterns observed in model organisms .

• Substrate specificity: The low sequence identity (21%) suggests PIH1D2 likely has distinct substrate preferences compared to PIH1D1 .

What is the potential role of PIH1D2 in ciliary function and ciliopathies?

Based on studies in model organisms, PIH1D2 may have significant roles in ciliary function:

• Expression pattern: In zebrafish, PIH protein homologs including those related to PIH1D2 are expressed in ciliated organs .

• Dynein assembly: Other PIH family proteins are involved in the assembly of axonemal dynein motors, which are crucial for ciliary movement. For instance, Drosophila Dnaaf4 and Dnaaf6 form an R2TP-like complex with a conserved role in dynein assembly .

• Ciliopathy connection: Mutations in genes encoding dynein assembly factors cause Primary Ciliary Dyskinesia (PCD), suggesting PIH1D2 could potentially be involved in ciliopathies if it has similar functions .

• Experimental approach: To investigate PIH1D2's role in ciliary function, researchers should examine ciliary structure and motility in PIH1D2-depleted cells, particularly in cell types with motile cilia such as respiratory epithelial cells or sperm.

What evidence exists for PIH1D2 involvement in cancer progression?

While direct evidence is limited in the provided search results, several aspects suggest potential involvement in cancer:

• Expression data: The Human Protein Atlas contains information on PIH1D2 expression across various cancer cell lines, indicating possible differential expression in malignant contexts .

• Functional implications: If PIH1D2 functions in protein complex assembly similar to other PIH proteins, dysregulation could potentially affect multiple cellular processes relevant to cancer, including ribosome biogenesis or signaling pathway integrity.

• Research direction: Investigators should consider analyzing PIH1D2 expression levels across cancer databases such as TCGA and correlating expression with clinical outcomes and molecular subtypes.

How can researchers differentiate between direct and indirect effects when studying PIH1D2 function?

To distinguish direct from indirect effects:

• Acute depletion systems: Use degron-tagged PIH1D2 or inducible knockdown systems to observe immediate consequences of protein loss before compensatory mechanisms activate.

• Domain mutants: Generate specific mutations in functional domains rather than complete knockouts to identify domain-specific functions.

• Temporal analysis: Perform time-course experiments after PIH1D2 depletion to distinguish primary from secondary effects.

• Direct binding assays: Use in vitro binding assays with purified components to confirm direct interactions.

• Proximity labeling: Methods like TurboID or APEX2 can identify proteins in close proximity to PIH1D2 in living cells.

What are the challenges in developing specific antibodies for PIH1D2 and how can they be overcome?

Developing specific antibodies for PIH1D2 presents several challenges:

• Sequence similarity: The 21% sequence identity with PIH1D1 means some epitopes may be shared between family members .

• Recommended approaches:

  • Target unique regions identified through careful sequence alignment

  • Validate antibodies against overexpressed PIH1D2 and in knockout cell lines

  • Use multiple antibodies targeting different epitopes

  • Consider monoclonal antibodies for increased specificity

  • Perform competition assays with recombinant protein to confirm specificity

• Alternative methods: For challenging projects, epitope tagging of endogenous PIH1D2 using CRISPR/Cas9 may provide a reliable detection alternative when antibodies prove problematic.

How might post-translational modifications regulate PIH1D2 function?

Post-translational modifications could be critical for PIH1D2 function:

• Phosphorylation: Given that PIH1D1 functions through phosphopeptide binding and its interactions with TEL2 are phosphorylation-dependent , researchers should investigate whether PIH1D2 is subject to regulatory phosphorylation or if it recognizes phosphorylated substrates.

• Experimental approach: Use phosphoproteomic analysis of immunoprecipitated PIH1D2 to identify modification sites, followed by site-directed mutagenesis to assess functional significance.

• Kinase prediction: In silico analysis to predict potential kinases that might modify PIH1D2, followed by kinase inhibitor studies or kinase knockdown experiments.

• Other modifications: Beyond phosphorylation, consider investigating ubiquitination, SUMOylation, or acetylation, which might regulate protein stability, localization, or interactions.

How does the tissue expression pattern of PIH1D2 compare across different model organisms?

Based on the limited information available, a comparative analysis suggests:

*Note: This refers to related PIH proteins in Drosophila, as direct PIH1D2 ortholog information is limited.

The conserved expression in ciliated tissues across species suggests evolutionary conservation of function in these specialized cell types.

What is known about PIH1D2 interaction with the molecular chaperone system compared to other PIH proteins?

The interaction with molecular chaperones differs between PIH family members:

• PIH1D1: Functions as part of the R2TP complex, which is an HSP90 cochaperone involved in the assembly of various protein complexes .

• PIH1D2: Not a component of the R2TP complex, suggesting a distinct relationship with the chaperone system if any exists .

• Research direction: Investigators should perform co-immunoprecipitation experiments with HSP90 and other chaperones to determine whether PIH1D2 interacts with different components of the chaperone system.

• Functional implications: If PIH1D2 does interact with molecular chaperones, it may serve as an adapter for specific substrates distinct from those recognized by PIH1D1.

What high-throughput approaches might reveal novel insights into PIH1D2 function?

Several high-throughput approaches could advance our understanding of PIH1D2:

• Interactome profiling:

  • BioID or APEX proximity labeling followed by mass spectrometry

  • IP-mass spectrometry under various cellular conditions

  • Yeast two-hybrid screening against cDNA libraries

• Functional genomics:

  • CRISPR screens to identify synthetic lethal interactions with PIH1D2

  • Transcriptome analysis (RNA-seq) after PIH1D2 modulation

  • Ribosome profiling to assess effects on translation if involved in ribosome biogenesis

• Structural biology:

  • Cryo-EM of PIH1D2-containing complexes

  • X-ray crystallography of PIH1D2 domains

  • Hydrogen-deuterium exchange mass spectrometry to map binding interfaces

• Single-cell approaches:

  • Single-cell RNA-seq to identify cell populations where PIH1D2 is actively expressed

  • Single-cell proteomics to detect cell-type specific protein interactions

How might PIH1D2 function in the context of ciliary assembly and maintenance?

Building on the evidence from model organisms, researchers should investigate:

• Dynein arm assembly: Determine if PIH1D2 contributes to the assembly of outer dynein arms (ODAs) or inner dynein arms (IDAs) similar to the Drosophila Dnaaf4/Dnaaf6 complex .

• Ciliary localization: Examine whether PIH1D2 localizes to the cytoplasm where dynein assembly occurs or if it has any ciliary localization.

• Ciliary phenotyping: Assess ciliary structure and motility in PIH1D2-depleted cells using high-speed videomicroscopy, transmission electron microscopy, and immunofluorescence for ciliary markers.

• Rescue experiments: Test whether PIH1D2 can rescue defects in model organisms with mutations in orthologous genes to establish functional conservation.

• Patient cohorts: Screen PCD patients with unidentified genetic causes for mutations in PIH1D2 to establish clinical relevance.