HDGFL1 Human

What is HDGFL1 and what is its molecular structure?

HDGFL1 is a member of the hepatoma-derived growth factor family with a full-length protein sequence spanning 251 amino acids. The sequence begins with MSAYGMPMYK and contains several functional domains including a PWWP motif . This nuclear-targeted protein shares structural features with other HDGF family members which are known for their roles in cell proliferation and development .

For structural studies, researchers should note that the protein contains:

• A PWWP domain important for protein-protein interactions

• Nuclear localization signals for nuclear targeting

• Multiple potential post-translational modification sites

The first residue of the PWWP motif has been shown to be particularly critical, as it modulates HATH domain binding, stability, and protein-protein interactions .

What expression patterns does HDGFL1 exhibit in human tissues?

HDGFL1 shows tissue-specific expression patterns that change throughout development. During early development, HDGFL1 expression is high in the nucleus and cytoplasm of smooth muscle and endothelial cells . Interestingly, the expression typically declines after birth but has been observed to increase again during vascular injury .

In adult tissues, HDGFL1 can be detected in testis as demonstrated by immunohistochemical analysis using specific antibodies . When studying expression patterns, researchers should consider:

• Using validated antibodies targeting specific epitopes (e.g., amino acids 150-250 of human HDGFL1)

• Employing multiple detection methods to confirm expression

• Examining both protein and mRNA levels to identify potential post-transcriptional regulation

What are the optimal methods for detecting HDGFL1 in human samples?

For reliable detection of HDGFL1 in experimental settings, multiple validated methods are available:

• Immunohistochemistry (IHC-P): For tissue sections, using antibodies such as those targeting amino acids 150-250 of human HDGFL1 at optimized dilutions (1/2500 has shown good results) . Proper fixation and antigen retrieval protocols are essential.

• Western Blotting (WB): For protein lysates, recombinant HDGFL1 with tags (GST or His) can serve as positive controls .

• ELISA: For quantitative measurement in solution phase samples .

• qPCR: For mRNA expression analysis, with careful primer design to avoid cross-reactivity with other HDGF family members.

When validating antibodies, confirm specificity using positive controls such as testis tissue, which has shown reliable HDGFL1 expression .

How should recombinant HDGFL1 proteins be handled for optimal results?

When working with recombinant HDGFL1:

• Storage: Store at -80°C and aliquot to avoid repeated freeze-thaw cycles .

• Buffer conditions: Optimal stability is achieved in 50 mM Tris-HCl buffer with 10 mM reduced Glutathione at pH 8.0 .

• Use timeline: For best experimental results, use within three months of receipt .

• Quality assessment: Verify protein integrity using 12.5% SDS-PAGE stained with Coomassie Blue .

Available recombinant proteins include:

• GST-tagged HDGFL1 (AA 1-251) expressed in wheat germ in vitro system

• His-tagged HDGFL1 expressed in E. coli or HEK-293 cells with >90-95% purity

Select the appropriate recombinant protein based on your experimental requirements, considering the expression system and tag compatibility with your downstream applications.

How does HDGFL1 differ functionally from other HDGF family members?

While HDGFL1 shares structural similarities with other HDGF family members, its functional profile appears distinct:

• HDGF (the founding family member) functions as a secreted mitogen from hepatoma cells and stimulates proliferation in vascular smooth muscle cells .

• HDGF is involved in organ development and lung remodeling following injury .

• HDGFL1's specific functions are still being elucidated, but structural differences, particularly in the PWWP domain, suggest unique molecular interactions .

When designing comparative studies:

• Use multiple family members as controls

• Focus on tissue-specific expression differences

• Investigate unique binding partners for HDGFL1 through interaction proteomics

• Examine differential responses to vascular injury between HDGF and HDGFL1

What are effective strategies for studying HDGFL1's role in signaling pathways?

To elucidate HDGFL1's role in cellular signaling:

Gene Modulation Approaches:

• siRNA or shRNA knockdown using validated reagents available from commercial sources

• CRISPR-Cas9 gene editing for complete knockout

• Overexpression studies using tagged constructs to track localization

Pathway Analysis Methods:

• Phosphoproteomic analysis following HDGFL1 modulation

• Transcriptome profiling (RNA-seq) to identify downstream effectors

• Chromatin immunoprecipitation to identify potential DNA binding sites

• Protein-protein interaction studies using co-immunoprecipitation

Functional Assays:

• Proliferation assays in vascular smooth muscle cells

• Migration and invasion assays, particularly in the context of vascular injury models

• Angiogenesis assays for potential roles in vascular development

Consider examining HDGFL1 in the context of vascular injury models where its expression has been shown to increase despite normally declining after birth .

What is known about HDGFL1's role in epigenetic regulation networks?

HDGFL1 (PWWP1) contains a PWWP domain, which in other proteins is associated with chromatin binding and epigenetic regulation. For investigating HDGFL1's epigenetic functions:

• Chromatin Association Studies:

  • Chromatin immunoprecipitation followed by sequencing (ChIP-seq)

  • DNA adenine methyltransferase identification (DamID)

  • Assay for transposase-accessible chromatin (ATAC-seq)

• Interaction Analysis:

  • Co-immunoprecipitation with known epigenetic regulators

  • Proximity ligation assays to confirm interactions in situ

  • Mass spectrometry to identify chromatin-associated binding partners

• Data Integration:

  • Posterior Association Networks (PANs) have been used to study diverse epigenetic strategies in cellular differentiation and may be applicable to HDGFL1 studies

  • Beta-mixture modeling for identifying significant interactions

  • Integration with protein-protein interaction data for network analysis

What methods should be employed to investigate HDGFL1's potential role in disease contexts?

Given the relationship between HDGF family members and various pathologies, investigating HDGFL1 in disease contexts requires:

Patient Sample Analysis:

• Immunohistochemistry on tissue microarrays from various pathologies

• Analysis of expression levels in publicly available disease datasets

• Correlation of expression with clinical outcomes

Disease Models:

• Vascular injury models where HDGFL1 expression changes have been documented

• Potential cancer models, given the role of related family members in tumorigenesis

• Developmental models to explore roles in organogenesis

Therapeutic Potential Assessment:

• Target validation through knockdown/knockout in disease models

• Development of inhibitory antibodies or peptides

• Screen for small molecules that modulate HDGFL1 function

What experimental approaches can reveal HDGFL1's subcellular localization and trafficking?

Understanding HDGFL1's intracellular dynamics requires:

Microscopy Techniques:

• Immunofluorescence with co-localization markers for different organelles

• Live-cell imaging with fluorescently tagged HDGFL1

• Super-resolution microscopy for precise localization

Biochemical Fractionation:

• Nuclear/cytoplasmic fractionation followed by Western blotting

• Chromatin association assays

• Membrane versus soluble protein separation

Trafficking Studies:

• Photoactivatable or photoconvertible tagged HDGFL1 to track movement

• Nuclear import/export inhibitors to determine transport mechanisms

• Mutagenesis of potential localization signals

HDGFL1 contains nuclear targeting signals similar to HDGF, which requires nuclear targeting for its mitogenic activity in vascular smooth muscle cells . This suggests nuclear functions that should be carefully investigated.

How can protein-protein interactions of HDGFL1 be comprehensively mapped?

For mapping HDGFL1's interactome:

Affinity-Based Methods:

• Immunoprecipitation followed by mass spectrometry

• GST-pulldown using recombinant HDGFL1 with GST tag

• Proximity-dependent biotin identification (BioID)

Library Screening Approaches:

• Yeast two-hybrid screening

• Protein complementation assays

• Phage display with HDGFL1 as bait

Validation Methods:

• Co-immunoprecipitation of endogenous proteins

• FRET/BRET assays for direct interactions

• Surface plasmon resonance for binding kinetics

Pay particular attention to the PWWP domain, as the first residue has been shown to modulate binding interactions . Different tags (GST, His) may affect binding properties, so using multiple tagged versions is recommended .

What techniques are most appropriate for studying HDGFL1 post-translational modifications?

To characterize HDGFL1 post-translational modifications:

Identification Methods:

• Mass spectrometry-based proteomics for comprehensive PTM mapping

• Western blotting with modification-specific antibodies

• Phos-tag gels for detecting phosphorylated species

Functional Analysis:

• Site-directed mutagenesis of modified residues

• Phosphatase/kinase inhibitors to modulate modification states

• Correlation of modifications with subcellular localization and activity

Temporal Dynamics:

• Pulse-chase experiments to determine modification turnover

• Stimulation time courses to identify rapid changes in modification

• Cell cycle synchronization to detect cell cycle-dependent modifications

Given HDGFL1's nuclear localization and potential roles in vascular responses, examining phosphorylation events following growth factor stimulation or vascular injury would be particularly informative.

What considerations are important when designing HDGFL1 knockout or transgenic animal models?

When creating HDGFL1 animal models:

Design Considerations:

• Complete knockout versus conditional systems (Cre-loxP)

• Tissue-specific expression using appropriate promoters

• Knockin reporter constructs for expression tracking

• CRISPR-Cas9 versus traditional homologous recombination

Validation Requirements:

• Confirmation of gene/protein absence or modification

• Assessment of compensatory changes in other HDGF family members

• Phenotypic characterization focused on vascular development

• Molecular profiling of affected tissues

Experimental Applications:

• Vascular injury models to examine HDGFL1's role in repair

• Developmental studies focusing on periods of high expression

• Challenge models to reveal conditional phenotypes

• Cross-breeding with disease models to assess modifier effects

Since HDGFL1 expression changes during development and in response to vascular injury , these models would be particularly valuable for understanding its functional significance in these contexts.

Available Research Tools for HDGFL1 Studies