Recombinant Seriola quinqueradiata SH3 domain-binding protein 4 (sh3bp4), partial

What is SH3BP4 and what are its key structural characteristics?

SH3BP4 (SH3 domain-binding protein 4) was initially identified as a novel cDNA through differential display analysis of cultured human corneal fibroblasts. The protein contains several important structural features that define its function:

• Three Asn-Pro-Phe (NPF) motifs

• An SH3 domain

• A PXXP motif

• A bipartite nuclear targeting signal

• A tyrosine phosphorylation site

These structural elements enable SH3BP4 to interact with specific endocytic proteins including clathrin, dynamin, and the transferrin receptor (TfR). The protein localizes to TfR-containing coated pits and vesicles, suggesting its involvement in cargo-specific control of clathrin-mediated endocytosis, specifically in the internalization of TfR. At least two isoforms of SH3BP4 are known to exist .

While most studies have focused on mammalian SH3BP4, the Seriola quinqueradiata (Japanese amberjack) homolog shares conserved domains that suggest similar functions in endocytic processes. Comparative studies between fish and mammalian SH3BP4 can provide evolutionary insights into protein function conservation.

How does phosphorylation affect SH3BP4 function and localization?

Phosphorylation plays a critical role in regulating SH3BP4 localization and function. Mass spectrometry analysis has identified multiple phosphorylation sites in SH3BP4, with serine-246 (S246) being the most frequently phosphorylated residue .

The phosphorylation status of S246 significantly impacts the targeting of SH3BP4 to clathrin-coated pits (CCPs). Experimental studies using phosphorylation-resistant (S246A) and phosphomimetic (S246D) mutations have demonstrated that:

• Non-phosphorylatable S246A mutants show enhanced colocalization with CCPs

• S246 is a target of Akt phosphorylation and fits an RxKRxxS Akt phosphorylation motif

• Phosphorylated SH3BP4 at S246 recruits 14-3-3 adaptor proteins

• 14-3-3 binding efficiently blocks SH3BP4 from CCP accumulation

These findings suggest a regulatory mechanism where phosphorylation acts as a molecular switch that controls SH3BP4's endocytic function. The phosphorylation-dependent interaction with 14-3-3 proteins represents a key regulatory mechanism for SH3BP4 activity in clathrin-mediated endocytosis .

What methods are recommended for expression and purification of recombinant SH3BP4?

Recombinant SH3BP4 can be expressed using several host systems, each with distinct advantages depending on research objectives:

For purification, a typical protocol would include:

• Cell lysis in buffer containing protease inhibitors

• Affinity chromatography using tagged recombinant protein

• Size exclusion chromatography for higher purity

• Storage in glycerol-containing buffer at -20°C or -80°C to maintain stability

Notably, repeated freeze-thaw cycles should be avoided, and working aliquots can be stored at 4°C for up to one week to maintain protein integrity.

How does SH3BP4 interact with the endocytic machinery?

SH3BP4 interacts with multiple components of the endocytic machinery, playing a crucial role in cargo-specific control of clathrin-mediated endocytosis. Immunoprecipitation studies have revealed a hierarchy of binding partners, with Eps15 showing the strongest interaction, followed by Dyn2 and clathrin heavy chain .

The interaction network is complex and regulated by several factors:

• SH3BP4 mutations in clathrin-mediated endocytosis (CME) domains reduce binding to Eps15 and Dyn2

• The temporal recruitment profile of SH3BP4 S246A more closely matches that of Eps15 than Dyn2

• 14-3-3 protein binding to phosphorylated SH3BP4 weakens the interaction with Eps15

These findings suggest a model where SH3BP4 serves as a regulatory node in endocytosis, with its activity modulated by phosphorylation and subsequent 14-3-3 binding. The specific timing of SH3BP4 recruitment to CCPs, particularly its resemblance to Eps15 recruitment patterns, indicates a role in the early stages of endocytic vesicle formation .

What is the role of SH3BP4 in melanogenesis and how can it be experimentally manipulated?

SH3BP4 has been identified as a melanogenesis-related regulatory target of miR-125b in melanocytic cells. Gene expression analysis across multiple microarray datasets has consistently shown an inverse relationship between SH3BP4 expression and miR-125b levels in melanoma .

Experimental manipulation of SH3BP4 through knockdown studies has demonstrated:

• Significant decrease in melanin content

• Downregulation of tyrosinase (TYR) expression

• Reduced TYR enzymatic activity

Compared to other potential miR-125b targets (IRF4 and SORT1), SH3BP4 knockdown produced the most pronounced effects on melanin production. Gene Set Enrichment Analysis (GSEA) of melanoma samples categorized by SH3BP4 expression levels further confirmed its role in pigmentation pathways .

For researchers interested in manipulating SH3BP4 to study melanogenesis, several approaches are recommended:

• siRNA or shRNA-mediated knockdown of SH3BP4

• miR-125b mimics or inhibitors to modulate SH3BP4 expression indirectly

• CRISPR/Cas9 gene editing to create cell lines with altered SH3BP4 expression or function

• Expression of phosphorylation-resistant (S246A) or phosphomimetic (S246D) mutants

These approaches provide versatile tools for investigating SH3BP4's role in pigmentation and related cellular processes.

How can recombinant SH3BP4 be used in structural biology studies?

Structural biology studies of SH3BP4 can provide critical insights into its mechanism of action and interactions with binding partners. Several experimental approaches are recommended:

For any structural study, careful consideration of SH3BP4's phosphorylation state is crucial, as this significantly affects its interactions and functional state .

What comparative differences exist between SH3BP4 across species?

Comparative analysis of SH3BP4 across species provides valuable insights into evolutionary conservation and functional adaptation. While mammalian SH3BP4 has been extensively studied, the Seriola quinqueradiata homolog represents an opportunity to understand functional conservation in teleost fish.

Key aspects to consider in comparative studies include:

• Domain conservation: The core functional domains (SH3 domain, NPF motifs, PXXP motif) are likely conserved across vertebrates, while regulatory regions may show greater divergence.

• Phosphorylation sites: The critical S246 phosphorylation site identified in human SH3BP4 may be conserved in fish homologs, suggesting similar regulatory mechanisms.

• Interaction partners: Comparing the binding affinities of fish versus mammalian SH3BP4 for endocytic proteins can reveal evolutionary adaptations in endocytic processes.

• Tissue-specific expression: While human SH3BP4 has been studied in contexts like melanogenesis , the expression pattern in fish may reflect species-specific adaptations.

Researchers should employ sequence alignment tools to identify conserved regulatory motifs and phosphorylation sites, followed by functional assays to compare activities between species.

What methods are recommended for studying SH3BP4 protein-protein interactions?

Several complementary approaches are recommended for studying SH3BP4 interactions:

• Co-immunoprecipitation (Co-IP): Effective for identifying stable protein interactions. Previous studies successfully used SH3BP4-GFP constructs to pull down endogenous 14-3-3ε and other interaction partners .

• Proximity-based labeling: BioID or APEX2 fusions with SH3BP4 can identify transient or context-dependent interactions in living cells.

• Yeast two-hybrid screening: Useful for systematic identification of novel binding partners.

• Fluorescence microscopy approaches:

  • Fluorescence Resonance Energy Transfer (FRET) to study direct protein interactions

  • Fluorescence recovery after photobleaching (FRAP) to examine dynamic association with CCPs

  • Total Internal Reflection Fluorescence (TIRF) microscopy for visualizing SH3BP4 recruitment to the plasma membrane during endocytosis

• In vitro binding assays: Using purified recombinant proteins (>90% purity) to determine direct binding and measure affinity constants.

When designing interaction studies, consider creating specific mutations in key domains. For example, the dCME mutation reduced SH3BP4 interaction with Eps15 and Dyn2 , providing valuable negative controls for binding specificity.

How can phosphorylation-dependent regulation of SH3BP4 be studied experimentally?

Phosphorylation-dependent regulation of SH3BP4 can be studied using several approaches:

• Phospho-specific antibodies: Develop antibodies that specifically recognize phosphorylated S246 to monitor SH3BP4 phosphorylation status under different conditions.

• Phospho-mimetic and phospho-resistant mutations: As demonstrated in previous work, S246A (non-phosphorylatable) and S246D (phosphomimetic) mutations provide valuable tools for understanding the functional consequences of phosphorylation .

• Mass spectrometry: Quantitative phosphoproteomics can identify additional phosphorylation sites and their relative abundance under different cellular conditions.

• Kinase inhibitors and activators: Akt inhibitors/activators can modulate S246 phosphorylation, allowing temporal control of SH3BP4 regulation.

• 14-3-3 binding assays: The R18 peptide approach, which introduces a 14-3-3 binding site into SH3BP4, provides an elegant tool for manipulating 14-3-3 binding independent of phosphorylation . This strategy allows researchers to distinguish between phosphorylation effects and 14-3-3 binding consequences.

What are the best methods for studying SH3BP4 in the context of melanogenesis?

To investigate SH3BP4's role in melanogenesis, researchers can employ the following methods:

• Gene expression analysis: Quantitative real-time PCR (RT-qPCR) using human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a normalization control has been successfully used to measure SH3BP4 expression in melanocytic cells .

• miRNA regulation studies: The inverse relationship between miR-125b and SH3BP4 can be studied using:

  • miR-125b mimics or inhibitors

  • Luciferase reporter assays to validate direct targeting

  • Bioinformatic analysis tools like TargetScan, PicTar, RNA22, PITA, and miRanda

• Functional melanogenesis assays:

  • Melanin content measurement following SH3BP4 knockdown or overexpression

  • Tyrosinase (TYR) expression and activity assays

  • Gene Set Enrichment Analysis (GSEA) to identify biological pathways associated with SH3BP4 expression levels in melanoma

• High-throughput approaches: Analyzing microarray datasets (such as GSE7553, GSE32474, and GSE36879) and ChIP-Seq data (like GSE50681) can provide valuable insights into SH3BP4's role in melanogenesis regulation and its relationship with transcription factors like MITF .

What recombinant protein expression systems are most suitable for studying fish SH3BP4?

The choice of expression system for recombinant Seriola quinqueradiata SH3BP4 depends on the specific research objectives:

• E. coli expression system:

  • Advantages: High yield, cost-effective, well-established protocols

  • Best for: Structural studies requiring large amounts of protein

  • Limitations: May lack post-translational modifications, potential protein folding issues with complex domains

• Yeast expression system:

  • Advantages: Eukaryotic folding machinery, some post-translational modifications

  • Best for: Functional studies requiring properly folded protein with basic modifications

  • Limitations: Lower yield than bacteria, more complex cultivation

• Baculovirus expression system:

  • Advantages: Better post-translational modifications, good for difficult-to-express proteins

  • Best for: Studies focusing on SH3BP4 regulatory mechanisms requiring authentic phosphorylation

  • Limitations: More technically demanding, longer production time

• Mammalian cell expression system:

  • Advantages: Most authentic post-translational modifications and folding

  • Best for: Studies of complex regulatory interactions and phosphorylation-dependent functions

  • Limitations: Lower yield, highest cost, most complex cultivation requirements

For optimal storage stability, recombinant SH3BP4 should be maintained in a liquid form containing glycerol at -20°C for regular use or -80°C for long-term storage. Working aliquots can be kept at 4°C for up to one week, but repeated freezing and thawing should be avoided as it may compromise protein integrity .

How can recombinant SH3BP4 contribute to understanding disease mechanisms?

Recombinant SH3BP4 serves as a valuable tool for investigating disease mechanisms across several biomedical fields:

• Cancer biology: The inverse regulation between miR-125b and SH3BP4 in melanoma suggests potential roles in cancer progression . Recombinant SH3BP4 can be used to:

  • Study altered endocytic trafficking in cancer cells

  • Investigate disrupted receptor internalization pathways

  • Examine melanoma cell pigmentation as a differentiation marker

• Endocytosis disorders: As SH3BP4 controls cargo-specific clathrin-mediated endocytosis, particularly for transferrin receptor internalization , it may contribute to disorders involving:

  • Iron metabolism

  • Receptor trafficking defects

  • Neurodegenerative diseases with endosomal-lysosomal dysfunction

• Signaling pathway dysregulation: The connection between SH3BP4 and Akt phosphorylation suggests potential roles in:

  • Insulin signaling disorders

  • Cancer-related signaling pathway alterations

  • Metabolic disorders

Recombinant proteins allow researchers to perform biochemical assays, develop screening platforms for therapeutic compounds, and establish in vitro models of disease mechanisms associated with SH3BP4 dysfunction.

What techniques are available for manipulating SH3BP4 in animal models?

Several approaches can be employed to study SH3BP4 function in animal models, with special considerations for Seriola quinqueradiata:

• CRISPR/Cas9 gene editing:

  • Generation of knockout or knockin fish models

  • Introduction of specific mutations (e.g., S246A) to study phosphorylation effects

  • Creation of reporter lines with fluorescently tagged SH3BP4

• Morpholino oligonucleotides:

  • Transient knockdown during early development

  • Allows temporal control of SH3BP4 expression

  • Useful for studying developmental roles before establishing stable genetic lines

• Recombinant protein delivery systems:

  • Similar to approaches used in recombinant vaccine development

  • Allows testing of wild-type or mutant SH3BP4 effects in vivo

  • Can be combined with tissue-specific targeting strategies

• Viral vector systems:

  • Adeno-associated virus (AAV) or lentiviral delivery of SH3BP4 variants

  • Enables tissue-specific or inducible expression

  • Useful for rescue experiments in knockout models

These approaches can be adapted from established protocols for model organisms, with appropriate modifications for Seriola quinqueradiata biology and experimental accessibility.

What are emerging technologies for studying SH3BP4 protein dynamics?

Emerging technologies offer new opportunities to study SH3BP4 dynamics with unprecedented resolution:

• Super-resolution microscopy:

  • Techniques like PALM, STORM, or STED microscopy can visualize SH3BP4 localization in clathrin-coated pits with nanometer precision

  • Multi-color imaging allows simultaneous tracking of SH3BP4 and interaction partners

  • Live-cell super-resolution approaches can capture dynamic recruitment during endocytosis

• Optogenetic control of SH3BP4 activity:

  • Light-inducible phosphorylation systems to temporally control SH3BP4 regulation

  • Optogenetic recruitment of SH3BP4 to specific cellular compartments

  • Photo-switchable protein-protein interaction modules to control 14-3-3 binding

• Single-molecule techniques:

  • Single-molecule FRET to study SH3BP4 conformational changes

  • Single-particle tracking to follow individual SH3BP4 molecules during endocytosis

  • Optical tweezers or AFM-based approaches to measure interaction forces

• Integrative structural biology:

  • Combining cryo-EM, X-ray crystallography, and computational modeling

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

  • AlphaFold2 and similar AI approaches to predict structural features of SH3BP4

These technologies will provide deeper insights into the dynamic regulation of SH3BP4 and its role in endocytic processes across different species.

How can multi-omics approaches enhance our understanding of SH3BP4 function?

Multi-omics approaches can provide a systems-level understanding of SH3BP4 function:

• Phosphoproteomics:

  • Comprehensive mapping of SH3BP4 phosphorylation sites beyond S246

  • Quantitative analysis of phosphorylation dynamics under different conditions

  • Identification of kinase and phosphatase networks regulating SH3BP4

• Interactomics:

  • Affinity purification-mass spectrometry (AP-MS) to identify the complete SH3BP4 interactome

  • BioID or APEX proximity labeling to capture transient interactions

  • Comparative interactomics between wild-type and phosphorylation mutants

• Transcriptomics and proteomics:

  • RNA-seq and proteomics analysis following SH3BP4 manipulation

  • Integration with existing datasets like GSE7553, GSE32474, and GSE36879

  • Identification of downstream effectors in different cellular contexts

• Evolutionary genomics:

  • Comparative analysis of SH3BP4 sequences and regulatory elements across species

  • Identification of conserved functional motifs versus species-specific adaptations

  • Correlation with species-specific endocytic or pigmentation mechanisms

By integrating these multi-omics approaches, researchers can build comprehensive models of SH3BP4 function in different cellular contexts and across evolutionary time.