Recombinant Mouse Uncharacterized protein FLJ45252 homolog

What is known about the structure and basic properties of mouse FLJ45252 homolog?

The mouse uncharacterized protein FLJ45252 homolog (UniProt ID: Q6PIU9) is classified under the UniProt ID YJ005_MOUSE. According to the Protein Ontology (PRO) database, it is defined as "an AAK1 gene translation product (mouse) that is a translation product of some mRNA whose exon structure and start site selection renders it capable of giving rise to a protein with the amino acid sequence represented by UniProtKB:Q6PIU9-1" .

The protein has been identified in proteomic analyses of mouse tissues, particularly in neuronal cell types. While the full tertiary structure remains unresolved, sequence analysis reveals multiple domains with functional significance, particularly sites susceptible to post-translational modifications which may regulate its activity and interactions with other cellular components.

What post-translational modifications (PTMs) have been identified in mouse FLJ45252 homolog?

Numerous PTMs have been identified in the mouse FLJ45252 homolog through proteomic analyses. Below is a comprehensive table of currently documented PTMs:

This diverse pattern of modifications suggests complex regulation mechanisms that may influence protein function, localization, and interactions with other cellular components .

How is recombinant mouse FLJ45252 homolog typically expressed and purified for research studies?

Recombinant mouse FLJ45252 homolog is typically expressed using eukaryotic expression systems that maintain proper post-translational modifications. Common methodologies include:

• Expression System Selection: Mammalian cell lines (particularly HEK293 or CHO cells) are preferred over bacterial systems to ensure proper folding and post-translational modifications, especially given the multiple phosphorylation sites identified in the native protein.

• Vector Design: Construction of expression vectors containing the full-length mouse FLJ45252 cDNA sequence, typically with a fusion tag (His-tag, GST, or FLAG) to facilitate purification and detection.

• Purification Strategy: Sequential chromatography approaches using affinity chromatography based on the fusion tag, followed by ion exchange chromatography and size exclusion chromatography to achieve high purity.

• Quality Control: Verification of protein identity by mass spectrometry and Western blotting, with particular attention to PTM retention that may be critical for biological activity.

Researchers should carefully consider the expression system and purification protocol based on downstream applications, particularly if studying PTM-dependent functions or protein-protein interactions.

What signaling pathways might mouse FLJ45252 homolog participate in based on current proteomic data?

While direct functional characterization remains limited, proteomic analyses suggest potential involvement of mouse FLJ45252 homolog in several signaling cascades:

• Possible Connection to Wnt Signaling: Though not directly established, the identification of E3 ubiquitin ligases like Pdzrn3 as regulatory targets in Wnt5a-Ror signaling pathways suggests potential parallel regulatory mechanisms for other uncharacterized proteins in similar contexts . The phosphorylation of mouse FLJ45252 at multiple sites might indicate regulation through kinase cascades potentially downstream of Wnt signaling.

• Phosphorylation-Dependent Pathways: The extensive phosphorylation profile (at sites S115, S174, Y291, S292, and others) suggests regulation by various kinases, potentially implicating it in multiple signaling networks . The tyrosine phosphorylation at Y291 particularly suggests possible involvement in receptor tyrosine kinase signaling pathways.

• Connection to AAK1 (AP2 Associated Kinase 1): Given its classification as an AAK1 gene product, it may participate in pathways related to clathrin-mediated endocytosis and membrane protein trafficking, which are critical processes in neuronal development and function.

Further investigation through interaction studies and functional assays would be necessary to confirm these potential pathway associations.

How do the phosphorylation patterns of mouse FLJ45252 homolog compare across different developmental stages or cellular conditions?

Research into developmental and condition-specific phosphorylation patterns of mouse FLJ45252 homolog reveals dynamic regulation that may correspond to functional states:

• Developmental Regulation: Phosphorylation sites S115 and S174 have been identified in multiple studies (PMID: 19144319, 17242355, 21183079), suggesting conservation and potential developmental importance . Comparative proteomic analyses across embryonic, postnatal, and adult mouse tissues would further elucidate stage-specific regulation.

• Cellular Conditions: The pattern of phosphorylation may vary under different cellular conditions such as:

  • Neuronal differentiation states

  • Response to growth factors

  • Cellular stress conditions

  • Activity-dependent changes

• Spatial Distribution: Phosphorylation patterns may differ between neuronal compartments (soma, dendrites, axons), suggesting compartment-specific functions.

Researchers investigating these patterns should employ quantitative phosphoproteomics with temporal and spatial resolution to capture these dynamics. Cell-type specific analyses using sorted populations or single-cell approaches would provide greater resolution of condition-specific modifications.

What are the challenges in studying protein-protein interactions involving mouse FLJ45252 homolog?

Investigating protein-protein interactions (PPIs) for uncharacterized proteins like mouse FLJ45252 homolog presents several significant challenges:

• PTM-Dependent Interactions: The diverse PTM profile of mouse FLJ45252 (12 identified sites) suggests that interactions may be highly dependent on specific modification states . This necessitates preserving native PTM patterns during interaction studies or engineering recombinant proteins with site-specific modifications.

• Transient Interactions: If involved in signaling pathways, many interactions may be transient and context-dependent, requiring specialized techniques for detection:

  • Proximity labeling approaches (BioID, APEX)

  • Crosslinking mass spectrometry

  • Time-resolved interaction studies

• Detection Sensitivity: Low abundance of the native protein may limit detection in conventional co-immunoprecipitation approaches, requiring more sensitive methodologies.

• Specificity Validation: Given its uncharacterized nature, distinguishing biologically relevant interactions from artifactual associations requires rigorous validation through:

  • Reciprocal pulldowns

  • Domain mapping

  • Functional validation of interaction consequences

  • In vivo confirmation of interactions identified in vitro

• Structural Considerations: Without resolved structural information, rational design of interaction studies is challenging. Computational prediction of functional domains may guide targeted approaches.

What mass spectrometry approaches are most effective for characterizing PTMs in recombinant mouse FLJ45252 homolog?

For comprehensive PTM characterization of recombinant mouse FLJ45252 homolog, a multi-faceted mass spectrometry approach is recommended:

• Sample Preparation:

  • Enrichment strategies for specific PTMs (e.g., TiO₂ for phosphopeptides, lectin affinity for glycopeptides)

  • Multiple proteolytic enzymes beyond trypsin (e.g., chymotrypsin, AspN) to increase sequence coverage

  • Preservation of labile modifications through gentle handling

• MS Instrumentation and Techniques:

  • High-resolution instruments (Orbitrap, Q-TOF) for accurate mass determination

  • Electron transfer dissociation (ETD) or electron capture dissociation (ECD) for preserving labile modifications during fragmentation

  • Parallel reaction monitoring (PRM) for targeted analysis of known modification sites

  • Data-independent acquisition (DIA) for comprehensive detection

• Data Analysis Pipeline:

  • Open search parameters allowing for multiple variable modifications

  • Site localization probability scoring (e.g., Ascore for phosphorylation)

  • Cross-validation using complementary fragmentation techniques

  • Integration with PTM databases (PhosphoSitePlus, GlyGen) for annotation

Given the diversity of modifications identified on FLJ45252 (phosphorylation, acetylation, O-glycosylation), a sequential or parallel enrichment strategy would be most effective for comprehensive characterization .

How can researchers design effective antibodies for studying mouse FLJ45252 homolog in various applications?

Designing effective antibodies for an uncharacterized protein requires strategic epitope selection and validation:

• Epitope Selection Strategies:

  • Avoid regions with high PTM density (e.g., S285-S292 region) for general detection antibodies

  • Consider generating modification-specific antibodies targeting key PTM sites (e.g., phospho-S115, phospho-Y291)

  • Prioritize regions with high predicted antigenicity and surface accessibility

  • Analyze sequence conservation if cross-reactivity with human FLJ45252 is desired

• Antibody Development Approaches:

  • Recombinant antibody technologies (phage display) for difficult epitopes

  • Multiple immunization strategies (peptide vs. protein) to maximize epitope diversity

  • Development of both polyclonal (for sensitivity) and monoclonal (for specificity) antibodies

• Rigorous Validation Protocol:

  • Western blot against recombinant protein and tissue lysates

  • Immunoprecipitation followed by mass spectrometry

  • Immunofluorescence with knockout controls

  • Peptide competition assays

  • Cross-validation with orthogonal detection methods

• Application-Specific Considerations:

  • For immunohistochemistry: fixation-resistant epitopes

  • For live imaging: extracellular epitopes or development of intrabodies

  • For ChIP applications: accessibility in native chromatin context

What CRISPR/Cas9 strategies would be most effective for functional studies of mouse FLJ45252 homolog?

For functional genomic studies of mouse FLJ45252 homolog, the following CRISPR/Cas9 approaches would be most effective:

• Complete Knockout Strategy:

  • Design of guide RNAs targeting early exons to ensure complete loss of function

  • Multiple guide RNA approach to increase efficiency

  • Verification of knockout by genomic sequencing, RT-PCR, and Western blotting

  • Phenotypic assessment across multiple cell types to account for potential compensatory mechanisms

• Domain-Specific Editing:

  • Targeted modification of specific functional domains

  • Generation of PTM-null variants by mutating key modification sites (e.g., S115A, Y291F)

  • Introduction of phosphomimetic mutations (S→D, T→E) to study constitutively active states

• Endogenous Tagging:

  • Knock-in of fluorescent proteins or affinity tags for live imaging and purification

  • Strategic tag placement to minimize functional interference

  • Generation of PTM-specific biosensors through proximity-based systems

• Inducible Systems:

  • Implementation of auxin-inducible degron (AID) system for temporal control

  • Conditional knockout strategies using tissue-specific Cre expression

  • Degron-based approaches for acute protein depletion

• High-Throughput Screening:

  • CRISPR interference (CRISPRi) or activation (CRISPRa) approaches for phenotypic screens

  • Pooled CRISPR screening with relevant phenotypic readouts

  • Single-cell analysis of knockout effects on signaling pathways

Validation of functional outcomes should include comprehensive assessment of compensatory mechanisms and potential off-target effects.

What experimental approaches would best elucidate the role of mouse FLJ45252 homolog in neuronal contexts?

Given the identification of mouse FLJ45252 homolog in neuronal contexts , the following experimental approaches would be most informative:

• Cell Type-Specific Expression Analysis:

  • Single-cell RNA sequencing of neuronal subtypes

  • Immunohistochemistry across brain regions using validated antibodies

  • Subcellular localization studies to determine distribution in neuronal compartments

  • Developmental expression profiling during neurogenesis and maturation

• Functional Perturbation in Neuronal Models:

  • CRISPR knockout or knockdown in neuronal cell lines (e.g., Neuro2a )

  • Primary neuron cultures from conditional knockout mice

  • Assessment of effects on:

    • Neurite outgrowth and morphology

    • Synapse formation and function

    • Electrophysiological properties

    • Response to neurotrophin signaling

• Proteomic Approaches in Neuronal Contexts:

  • Proximity labeling (BioID/APEX) in specific neuronal compartments

  • Quantitative phosphoproteomics following neuronal activity

  • PTM dynamics during neuronal differentiation

  • Comparison between normal and pathological states

• In Vivo Studies:

  • Generation of conditional knockout mouse models

  • Behavioral assessment focusing on functions associated with implicated brain regions

  • Electrophysiological recordings in intact circuits

  • In vivo imaging of protein dynamics with endogenously tagged variants

• Disease Relevance Assessment:

  • Analysis in models of neurodevelopmental and neurodegenerative disorders

  • Correlation of expression or PTM patterns with disease progression

  • Therapeutic potential of modulating activity or expression

What are the most promising future research directions for mouse FLJ45252 homolog?

Based on current knowledge and technological capabilities, several promising research directions emerge:

• Comprehensive Functional Characterization:

  • Systematic phenotypic analysis of knockout models across tissues and developmental stages

  • Identification of genetic and physical interaction networks

  • Determination of enzymatic activities or structural roles

• PTM-Function Relationships:

  • Site-directed mutagenesis of key PTM sites followed by functional assessment

  • Identification of the kinases, phosphatases, and other enzymes responsible for the diverse PTM profile

  • Temporal dynamics of modifications under various stimuli

• Structural Biology Approaches:

  • Cryo-EM or X-ray crystallography studies of the full-length protein

  • NMR studies of dynamic regions

  • Structure-based functional prediction and drug design

• Translational Potential:

  • Investigation of human ortholog (LOC112268437) in relevant disease contexts

  • Development of small molecule modulators if functional significance is established

  • Biomarker potential based on PTM signatures

• Systems Biology Integration:

  • Network analysis placing FLJ45252 in broader cellular pathways

  • Multi-omics integration to correlate expression, PTMs, and functional outcomes

  • Mathematical modeling of dynamic regulation