PLEKHF2 Human

What is the domain structure of PLEKHF2 and how does it relate to function?

PLEKHF2, also known as Phafin2 or EAPF, has a distinct multi-domain structure that defines its cellular functions. The protein contains:

• An N-terminal Pleckstrin Homology (PH) domain

• A central FYVE zinc finger domain

• A poly-aspartic acid (polyD) motif at the C-terminus

This architecture is functionally significant as the PH domain binds transient pools of PtdIns3P and PtdIns4P during early macropinosome formation, while the FYVE domain specifically associates with PtdIns3P and is required for both early and late steps of macropinosomal maturation . The protein adopts a moderately elongated monomeric structure composed of α-helical and β-strand elements, with significant random coil regions . This structure enables PLEKHF2 to function in membrane trafficking and protein transport processes.

What cellular processes is PLEKHF2 involved in?

PLEKHF2 participates in several key cellular processes:

• Endosome Organization: The protein is predicted to be involved in organizing early endosomes, contributing to the proper sorting and trafficking of endocytosed material .

• Endosome-to-Lysosome Transport: PLEKHF2 facilitates the movement of cargo from endosomes to lysosomes, a critical step in the degradative pathway .

• Macropinocytosis: During this process, PLEKHF2 binds to newly formed macropinosomes in a mechanism requiring two distinct pools of phosphoinositides .

• Cytoskeletal Interaction: The protein facilitates the transition of nascent macropinosomes through the subcortical actin network by directly interacting with F-actin .

These functions collectively suggest that PLEKHF2 serves as a phosphoinositide sensor that regulates membrane trafficking events in response to lipid signaling.

How does PLEKHF2 interact with phosphoinositides?

PLEKHF2 shows domain-specific interactions with different phosphoinositides:

The FYVE domain specifically binds to phosphatidylinositol 3-phosphate (PtdIns3P), which is enriched in early endosomal membranes . This interaction is critical for targeting PLEKHF2 to endosomal structures throughout the macropinosomal maturation process.

The PH domain exhibits more versatile binding capabilities, interacting with both PtdIns3P and phosphatidylinositol 4-phosphate (PtdIns4P) at different stages of macropinosome formation . This dual specificity allows PLEKHF2 to respond to changes in membrane composition during vesicle trafficking.

Research suggests that PLEKHF2's binding affinity for phosphoinositides is in the micromolar range, as indicated by studies using single-molecule pull-down (SiMPull) assays . Unlike many other PH domain-containing proteins, the PH domain of PLEKHF2 may not function independently of the full-length protein in terms of lipid binding, highlighting the importance of investigating full-length PLEKHF2 rather than isolated domains .

What experimental approaches are most effective for studying PLEKHF2's role in endosomal trafficking?

To effectively study PLEKHF2's function in endosomal trafficking, researchers should consider a multi-faceted approach:

• Fluorescence Microscopy Techniques:

  • Live-cell imaging with fluorescently tagged PLEKHF2 (e.g., GFP-PLEKHF2) to track its subcellular localization

  • Co-localization studies with endosomal markers like Rab5 (early endosomes) and Rab7 (late endosomes)

  • Super-resolution microscopy to visualize precise membrane interactions

• Biochemical Assays:

  • Liposome binding assays to characterize phosphoinositide interactions

  • Single-molecule pull-down (SiMPull) assays, which can detect protein-lipid interactions with affinities in the 10-20 μM range

  • Co-immunoprecipitation to identify protein binding partners

• Genetic Manipulation:

  • CRISPR-Cas9-mediated knockout or knockdown of PLEKHF2

  • Domain-specific mutations to disrupt PH or FYVE domain functions

  • Zebrafish models, which express plekhf2 in multiple tissues including brain, eye, and spleen

• Cargo Trafficking Assays:

  • Pulse-chase experiments with fluorescently labeled endocytic cargo

  • Quantification of endosome-to-lysosome transport rates in PLEKHF2-depleted cells

These methodologies can be combined to provide comprehensive insights into PLEKHF2's functional roles in endosomal dynamics.

How can researchers distinguish between the functions of the PH and FYVE domains in PLEKHF2?

Distinguishing the specific roles of PH and FYVE domains requires targeted experimental strategies:

• Domain Deletion and Mutation Studies:

  • Generate constructs expressing PLEKHF2 with either the PH or FYVE domain deleted

  • Create point mutations in critical residues of each domain to disrupt lipid binding

  • Express individual domains (PH-only or FYVE-only) to assess their independent functions

• Temporal Analysis During Macropinocytosis:

  • Time-lapse imaging of cells expressing wild-type and domain-mutant PLEKHF2 during macropinosome formation

  • This can reveal stage-specific requirements, as research has shown the FYVE domain is needed for both early and late steps of macropinosomal maturation, while the PH domain is essential only during early steps

• Lipid-Binding Specificity Assays:

  • Protein-lipid overlay assays using recombinant PLEKHF2 domains

  • Liposome sedimentation assays with vesicles containing different phosphoinositides

  • Surface plasmon resonance to measure binding kinetics of each domain with various lipids

• Structure-Function Analysis:

  • Use structural biology techniques (X-ray crystallography, cryo-EM) to resolve the three-dimensional structures of each domain

  • Molecular dynamics simulations to predict lipid-binding mechanisms

What techniques can be used to study the interaction between PLEKHF2 and the actin cytoskeleton?

To investigate PLEKHF2's interactions with the actin cytoskeleton, researchers can employ:

• Co-localization Studies:

  • Fluorescence microscopy with labeled PLEKHF2 and F-actin (using phalloidin)

  • Live-cell imaging to capture dynamic interactions during macropinosome formation

• Biochemical Interaction Assays:

  • F-actin co-sedimentation assays to test direct binding

  • Pull-down experiments using purified PLEKHF2 and actin

  • Proximity ligation assays to detect in situ protein-protein interactions

• Functional Perturbation Experiments:

  • Cytoskeletal disruption using agents like latrunculin or cytochalasin D to assess effects on PLEKHF2 localization

  • Domain mapping to identify actin-binding regions within PLEKHF2

  • Mutagenesis of putative actin-binding sites

• Advanced Microscopy Techniques:

  • Fluorescence resonance energy transfer (FRET) between labeled PLEKHF2 and actin

  • Single-particle tracking to follow PLEKHF2-containing vesicles through the actin network

  • Correlative light and electron microscopy to visualize ultrastructural details

Since PLEKHF2 facilitates the transition of macropinosomes through the subcortical actin network , these techniques can help elucidate the molecular mechanisms underlying this function.

What is known about PLEKHF2's role in cancer, particularly prostate cancer?

Research has revealed potential implications of PLEKHF2 in cancer progression:

Amplification of the PLEKHF2 gene has been associated with reduced survival in patients with prostate cancer . This suggests that PLEKHF2 might function as an oncogene in certain contexts, potentially by altering endosomal trafficking pathways that regulate signaling receptor turnover.

To investigate PLEKHF2's role in cancer, researchers can implement:

• Analysis of Cancer Genomic Databases:

  • Examination of PLEKHF2 expression, amplification, or mutation frequency across cancer types

  • Correlation of PLEKHF2 alterations with patient outcomes and clinical parameters

• Functional Studies in Cancer Cell Lines:

  • Overexpression and knockdown experiments to assess effects on proliferation, migration, and invasion

  • Rescue experiments with wild-type versus domain mutants to identify critical functional regions

• Signaling Pathway Analysis:

  • Investigation of how PLEKHF2 affects oncogenic signaling pathways

  • Assessment of receptor trafficking in cancer cells with altered PLEKHF2 expression

• In Vivo Cancer Models:

  • Generation of xenograft models with PLEKHF2-modified cancer cells

  • Analysis of tumor growth, metastasis, and response to therapy

These approaches can help elucidate the mechanisms by which PLEKHF2 contributes to cancer progression and potentially identify new therapeutic targets.

What approaches can be used to detect and analyze PLEKHF2 mutations in clinical samples?

For clinical genetic analysis of PLEKHF2, several methodologies are available:

• Next-Generation Sequencing (NGS):

  • Targeted gene panels including PLEKHF2

  • Whole exome sequencing to identify mutations across all exons

  • These approaches are used in clinical genetic testing of PLEKHF2

• Copy Number Variation Analysis:

  • Multiplex ligation-dependent probe amplification (MLPA)

  • Array comparative genomic hybridization (aCGH)

  • Quantitative PCR for detecting gene amplifications associated with cancer

• Expression Analysis:

  • Quantitative RT-PCR to measure PLEKHF2 mRNA levels

  • Immunohistochemistry to assess protein expression in tissue samples

  • Western blotting for semi-quantitative protein expression analysis

• Functional Characterization of Variants:

  • In silico prediction tools to assess mutation impact

  • Cell-based assays to test effects of patient-derived mutations on protein function

  • Protein stability and localization studies for missense variants

Clinical genetic testing for PLEKHF2 is available for diagnostic purposes and mutation confirmation , suggesting its potential relevance in hereditary diseases, though specific disease associations beyond prostate cancer were not detailed in the search results.

What are the evolutionary characteristics of the polyD motif in PLEKHF2?

The polyD (poly-aspartic acid) motif of PLEKHF2 exhibits interesting evolutionary patterns:

The polyD motif evolved specifically in Phafin2 (PLEKHF2) and other PH- or PH-FYVE-containing proteins in animals . Notably, this motif is absent in PH domain-free FYVE-containing proteins, which typically function in cellular trafficking or autophagy.

This evolutionary pattern suggests the co-evolution of the polyD motif with the PH domain, potentially enabling complex cellular functions that emerged specifically in animals . The combination of these domains may provide unique capabilities in membrane trafficking and protein interactions that were advantageous during animal evolution.

To study the evolutionary significance of the polyD motif, researchers could:

These approaches can provide insights into the functional significance of this evolutionarily conserved feature.

How can researchers effectively model PLEKHF2 function using animal models such as zebrafish?

Zebrafish (Danio rerio) represent an excellent model for studying PLEKHF2 function:

The zebrafish ortholog, plekhf2, shows expression in multiple tissues including brain, caudal fin, eye, spleen, and testis . This expression pattern partly overlaps with human PLEKHF2 expression, suggesting conserved functions.

To effectively utilize zebrafish for PLEKHF2 research:

• Gene Manipulation Techniques:

  • CRISPR-Cas9 genome editing to generate plekhf2 knockout or knock-in models

  • Morpholino-based knockdown for temporary gene suppression

  • Transgenic lines expressing fluorescently tagged Plekhf2 for in vivo imaging

• Functional Assays:

  • Analysis of endosomal trafficking in specific tissues like the brain or eye

  • Assessment of macropinocytosis in professional phagocytes

  • Evaluation of developmental phenotypes in plekhf2-deficient embryos

• High-Throughput Approaches:

  • Small molecule screening to identify modulators of Plekhf2 function

  • Suppressor/enhancer screens to identify genetic interactors

  • Transcriptomic analysis to identify downstream effects of Plekhf2 manipulation

• Comparative Studies:

  • Rescue experiments with human PLEKHF2 in zebrafish plekhf2 mutants

  • Structure-function analysis by expressing domain mutants

  • Investigation of tissue-specific functions based on expression patterns

These approaches leverage the advantages of zebrafish as a vertebrate model with optical transparency, rapid development, and genetic tractability.

What are the challenges in producing and purifying recombinant PLEKHF2 for structural studies?

Producing recombinant PLEKHF2 presents several technical challenges:

• Protein Solubility Issues:

  • The presence of both hydrophilic and hydrophobic domains can lead to aggregation

  • The FYVE domain contains zinc-binding sites that require proper folding

  • Expression strategies might include:

    • Using solubility-enhancing tags (MBP, SUMO, or GST)

    • Testing multiple expression systems (bacterial, insect, mammalian)

    • Optimizing growth and induction conditions

• Domain-Specific Considerations:

  • The FYVE domain requires zinc for proper folding, necessitating zinc supplementation

  • The PH domain may require phosphoinositide co-factors for stability

  • Consider domain-by-domain expression approach if full-length protein proves challenging

• Purification Strategies:

  • Multi-step purification combining affinity, ion exchange, and size exclusion chromatography

  • Buffer optimization to maintain protein stability

  • Addition of stabilizing agents like glycerol or specific lipids

• Quality Control:

  • Functional assays to verify lipid-binding activity of purified protein

  • Circular dichroism to assess proper folding

  • Dynamic light scattering to monitor aggregation state

Commercial recombinant PLEKHF2 proteins are available for research use , indicating successful production strategies have been developed, though specific protocols were not detailed in the search results.

What experimental designs best elucidate the specificity of PLEKHF2's phosphoinositide binding?

To accurately characterize PLEKHF2's phosphoinositide binding specificity:

• Comprehensive Lipid Panel Testing:

  • Test binding against all phosphoinositide species (PI, PI3P, PI4P, PI5P, PI(3,4)P₂, PI(3,5)P₂, PI(4,5)P₂, and PI(3,4,5)P₃)

  • Include other membrane lipids as controls

  • Use consistent assay conditions to enable direct comparisons

• Advanced Biophysical Techniques:

  • Surface plasmon resonance (SPR) to determine binding kinetics and affinity constants

  • Isothermal titration calorimetry (ITC) for thermodynamic characterization

  • Single-molecule pull-down assays which can detect interactions with Kd values in the 10-20 μM range

• Comparative Domain Analysis:

  • Test full-length PLEKHF2 versus isolated domains

  • Include domain mutants with disrupted lipid-binding sites

  • This approach is critical as research has shown that PH domains alone may not recapitulate the lipid binding of their full-length counterparts

• Membrane Context Considerations:

  • Use liposomes with compositions mimicking specific cellular membranes

  • Vary lipid ratios to determine minimum requirements for binding

  • Test effects of membrane curvature on binding efficiency

These experimental designs can help resolve discrepancies in reported phosphoinositide binding specificities, which have been noted in the literature .