HRASLS3 Human

What is HRASLS3 and what is its primary function in human cells?

HRASLS3 (HRAS-like suppressor 3) is a gene that encodes a protein involved in cellular growth regulation and differentiation. Based on experimental evidence, HRASLS3 functions as a downstream mediator of PPARγ signaling, particularly in adipocyte differentiation . The protein demonstrates antiproliferative properties and may antagonize Ras signaling pathways, suggesting roles in terminal differentiation of cells and potentially tumor suppression .

To characterize HRASLS3 function in human cells, researchers should employ:

• Expression analysis across different tissues and cell types

• Loss-of-function studies using siRNA or CRISPR-Cas9 gene editing

• Gain-of-function studies with overexpression systems

• Comparative analysis between mouse models (which show high adipose tissue expression) and human systems

How is HRASLS3 expression regulated at the transcriptional level?

HRASLS3 expression is primarily regulated by PPARγ through direct binding to a PPAR response element (PPRE) in the HRASLS3 promoter region . In mouse studies, electrophoretic mobility shift assays confirmed that PPARγ and RXRα bind as a heterodimer to a site approximately 6 kb upstream of the HRASLS3 transcriptional start site . Furthermore, transfection assays verified that this binding drives transcription in a ligand-dependent manner .

Methodological approaches to study human HRASLS3 transcriptional regulation include:

• Chromatin immunoprecipitation (ChIP) to confirm PPARγ binding to the human HRASLS3 promoter

• Reporter gene assays with the human HRASLS3 promoter to identify regulatory elements

• Treatment of human adipocyte models with PPARγ agonists and antagonists to observe effects on HRASLS3 expression

• Analysis of promoter methylation status and histone modifications

What experimental models are most appropriate for studying human HRASLS3 function?

For studying human HRASLS3 function, researchers should consider these experimental models:

• Human cell models:

  • Primary human preadipocytes isolated from subcutaneous or visceral adipose tissue

  • SGBS (Simpson-Golabi-Behmel Syndrome) human preadipocyte cell line

  • Human adipose-derived stem cells (hASCs)

  • HEK293T cells for initial molecular characterization studies

• Genetic manipulation approaches:

  • Lentiviral expression systems for overexpression or knockdown studies

  • siRNA for transient knockdown (validated in the referenced study)

  • CRISPR-Cas9 for gene editing and knockout studies

• Translational approaches:

  • Adipose tissue samples from patients with different metabolic profiles

  • Correlation studies between HRASLS3 expression and clinical parameters

  • Ex vivo culture of human adipose tissue explants

When comparing with animal models, researchers should note that while mouse studies show adipose-specific upregulation of HRASLS3 , expression patterns and regulatory mechanisms may differ in humans and require independent verification.

How does HRASLS3 mechanistically contribute to adipocyte differentiation?

The molecular mechanisms by which HRASLS3 contributes to adipocyte differentiation are still being elucidated, but research suggests several potential pathways:

• Ras signaling modulation: HRASLS3 has been linked to Ras signaling, and several reports indicate an antagonistic role for Ras in adipocyte differentiation . Specifically, Ras-induced activation of MAPK signaling impairs adipogenesis in cell models . HRASLS3 may promote adipogenesis by inhibiting this Ras-MAPK pathway.

• Cell cycle regulation: HRASLS3 demonstrates antiproliferative properties that might contribute to the cell cycle arrest necessary for terminal differentiation of preadipocytes .

Experimental approaches to investigate these mechanisms include:

• Phosphoproteomic analysis before and after HRASLS3 manipulation

• Co-immunoprecipitation to identify protein interaction partners

• RNA-seq to identify transcriptional networks regulated by HRASLS3

• Pathway inhibition studies to test specific signaling mechanisms

• Time-course analysis during differentiation to determine when HRASLS3 exerts its effects

How do post-translational modifications affect HRASLS3 function in human adipogenesis?

While specific post-translational modifications (PTMs) of HRASLS3 have not been extensively characterized in the literature, understanding these modifications is critical for elucidating its functional regulation.

Methodological approaches to investigate HRASLS3 PTMs:

• Mass spectrometry analysis:

  • Immunoprecipitate HRASLS3 from differentiating adipocytes at various timepoints

  • Perform LC-MS/MS to identify phosphorylation, ubiquitination, acetylation, or other modifications

  • Compare PTM profiles between basal and PPARγ ligand-stimulated conditions

• Site-directed mutagenesis studies:

  • Generate mutants of predicted modification sites

  • Test their functional capacity in adipogenic assays

  • Assess protein stability, localization, and interaction patterns

• PTM-specific antibodies:

  • Develop antibodies against predicted modified forms of HRASLS3

  • Monitor changes in specific modifications during differentiation

  • Correlate modifications with functional outcomes

• Enzyme identification:

  • Screen kinase, phosphatase, or other enzyme inhibitors for effects on HRASLS3 function

  • Use proximity labeling approaches to identify modifying enzymes

  • Perform co-immunoprecipitation studies to confirm enzyme interactions

How does HRASLS3 expression correlate with PPARγ activity in different human metabolic states?

The relationship between HRASLS3 and PPARγ signaling in various human metabolic states represents an important area for investigation. Based on animal studies showing HRASLS3 as a PPARγ target gene , several research approaches can be employed:

• Clinical sample analysis:

  • Compare HRASLS3 expression in adipose tissue from insulin-sensitive versus insulin-resistant subjects

  • Measure expression before and after thiazolidinedione (TZD) treatment in diabetic patients

  • Correlate HRASLS3 levels with markers of adipose tissue function

• Genetic association studies:

  • Identify HRASLS3 polymorphisms in human populations

  • Assess their association with metabolic traits and PPARγ-related phenotypes

  • Perform functional characterization of variants with significant associations

• Mechanistic studies in human adipocytes:

  • Create dose-response curves for PPARγ ligands and HRASLS3 expression

  • Measure the temporal relationship between PPARγ activation and HRASLS3 induction

  • Compare HRASLS3 regulation across different adipose tissue depots

What are the most effective approaches for manipulating HRASLS3 expression in human cells?

Based on experimental approaches described in the literature, several methods can be adapted for manipulating HRASLS3 expression in human cells:

For knockdown/silencing:

• siRNA approach (as validated in the referenced study) :

  • Design multiple siRNA sequences targeting different regions of human HRASLS3 mRNA

  • Include appropriate controls (non-targeting siRNA, mutated siRNA)

  • Transfect using optimized lipid-based reagents for the specific cell type

  • Validate knockdown efficiency by qPCR and Western blot

  • Optimal for short-term experiments (3-5 days)

• shRNA stable expression:

  • Clone effective siRNA sequences into lentiviral vectors

  • Generate stable cell lines through antibiotic selection

  • Verify sustained knockdown over multiple passages

  • Suitable for long-term differentiation studies (14+ days)

For overexpression:

What assays are most appropriate for evaluating HRASLS3's role in human adipocyte differentiation?

Based on experimental approaches described in the literature, several complementary assays should be employed:

• Morphological and lipid accumulation assessment:

  • Oil Red O staining to visualize and quantify lipid droplet accumulation

  • BODIPY or Nile Red staining for fluorescence-based quantification

  • Transmission electron microscopy for detailed ultrastructural analysis

• Molecular markers of differentiation:

  • qPCR analysis of adipogenic markers (e.g., FABP4/aP2, PLIN1/perilipin, ADIPOQ)

  • Western blot analysis of key adipogenic proteins

  • Immunofluorescence to visualize protein expression patterns and subcellular localization

• Functional assays:

  • Glucose uptake measurements to assess insulin sensitivity

  • Lipolysis assays in response to adrenergic stimulation

  • Adipokine secretion profiling (ELISA, multiplex assays)

• Cell cycle analysis:

  • Flow cytometry for DNA content analysis

  • EdU incorporation to measure proliferation rates

  • Expression analysis of cell cycle regulators

Experimental design should include:

• Multiple timepoints (early, middle, and late differentiation)

• Both gain-of-function and loss-of-function approaches

• Appropriate positive controls (e.g., PPARγ manipulation)

• Comparison between different adipose depots when using primary cells

How can researchers identify and validate HRASLS3 protein interaction partners?

To comprehensively identify and validate HRASLS3 protein interaction partners, researchers should employ multiple complementary approaches:

• Affinity purification-mass spectrometry (AP-MS):

  • Express epitope-tagged HRASLS3 in relevant cell types

  • Perform immunoprecipitation under various conditions (basal, during differentiation, after PPARγ activation)

  • Identify co-purifying proteins by mass spectrometry

  • Filter against appropriate controls to remove non-specific interactions

• Proximity labeling approaches:

  • Generate BioID or TurboID fusion with HRASLS3

  • Express in adipocyte models and activate labeling at different differentiation stages

  • Purify biotinylated proteins and identify by mass spectrometry

  • Provides information about transient or weak interactions in the native cellular context

• Co-immunoprecipitation validation:

  • Perform reciprocal co-IPs for top candidate interactors

  • Test interactions under different conditions (ligand treatment, differentiation stages)

  • Include appropriate controls (IgG control, HRASLS3 knockdown cells)

• Domain mapping:

  • Generate truncation or point mutants of HRASLS3

  • Identify domains required for specific protein interactions

  • Correlate interaction defects with functional outcomes in adipogenesis assays

• Functional validation:

  • Knockdown or overexpress identified interaction partners

  • Assess effects on HRASLS3-mediated adipogenesis

  • Test for colocalization using immunofluorescence or proximity ligation assays

What is the potential significance of HRASLS3 in human metabolic diseases?

Based on HRASLS3's role in adipocyte differentiation and its regulation by PPARγ , several potential implications for human metabolic diseases can be proposed:

• Obesity and adipose tissue dysfunction:

  • As HRASLS3 promotes adipocyte differentiation, alterations might affect adipose tissue expandability

  • Dysregulation could contribute to ectopic lipid deposition and metabolic complications

  • Expression changes might correlate with adipose tissue inflammation status

• Type 2 diabetes and insulin resistance:

  • HRASLS3 may mediate some insulin-sensitizing effects of PPARγ activation

  • Altered HRASLS3 function could contribute to adipose insulin resistance

  • TZD responsiveness might correlate with HRASLS3 expression or regulation

• Adipose tissue distribution disorders:

  • Depot-specific HRASLS3 expression might influence regional adipose tissue development

  • Potential role in sex-specific fat distribution patterns

  • Possible involvement in lipodystrophic syndromes

Research approaches to investigate these roles:

• Case-control studies comparing HRASLS3 expression in metabolically healthy vs. unhealthy obesity

• Genetic association studies examining HRASLS3 variants in large cohorts

• Correlation of adipose HRASLS3 expression with metabolic parameters

• Analysis of HRASLS3 regulation in response to weight loss interventions

How might HRASLS3's antiproliferative properties relate to cancer biology?

The documented antiproliferative properties of HRASLS3 and its downregulation in various tumors and tumor cell lines suggest potential roles in cancer biology:

• Tumor suppressor functions:

  • HRASLS3 may inhibit cell proliferation pathways relevant to cancer development

  • Its role in promoting terminal differentiation could counteract dedifferentiation in malignancy

  • Antagonism of oncogenic Ras signaling might suppress transformation

• Research approaches to investigate cancer relevance:

  • Analysis of HRASLS3 expression, mutation, and copy number variation across cancer databases

  • Correlation of expression levels with patient outcomes and treatment responses

  • Functional studies in human cancer cell lines with manipulated HRASLS3 expression

  • Investigation of HRASLS3 regulation by oncogenic signaling pathways

• Experimental methods for cancer studies:

  • Colony formation and soft agar assays to assess transformation

  • Xenograft models to evaluate in vivo tumor growth

  • Cell cycle and apoptosis analysis in response to HRASLS3 manipulation

  • Assessment of sensitivity to chemotherapeutic agents

• Potential mechanistic interactions:

  • HRASLS3 effects on MAPK signaling in cancer contexts

  • Relationship with other known tumor suppressors in the Ras pathway

  • Impact on cancer cell metabolism and differentiation state

What challenges exist in translating HRASLS3 research from animal models to human applications?

Several significant challenges must be addressed when translating HRASLS3 research from animal models to human applications:

• Species-specific differences:

  • While mouse studies show high HRASLS3 expression in adipose tissue , human tissue expression patterns require verification

  • Regulatory elements controlling HRASLS3 expression may differ between species

  • Protein interaction networks might not be fully conserved

• Methodological challenges:

  • Limited availability of well-characterized human adipose tissue samples

  • Variability in primary human preadipocyte isolation and culture

  • Differences in differentiation protocols between mouse and human systems

  • Need for time-course studies throughout the longer human adipogenesis process

• Context-dependent functions:

  • Human metabolic diseases involve complex genetic and environmental interactions

  • The relative importance of HRASLS3 in human adipogenesis might differ from mouse models

  • Human adipose depots have distinct properties not fully mirrored in animal models

• Strategic approaches to address translation challenges:

  • Systematic comparison of HRASLS3 function in mouse and human cell systems

  • Development of humanized mouse models

  • Utilization of human induced pluripotent stem cells (iPSCs) differentiated to adipocytes

  • Integration of human genetic and gene expression data with functional studies

  • Single-cell analysis to identify cell type-specific roles in human adipose tissue

These approaches will help determine whether findings from mouse HRASLS3 studies translate effectively to human biology and disease mechanisms, ultimately informing the potential therapeutic relevance of targeting this pathway.

What emerging technologies could advance understanding of HRASLS3 function?

Several cutting-edge technologies hold promise for deepening our understanding of HRASLS3 biology:

• CRISPR-based functional genomics:

  • Genome-wide CRISPR screens to identify genetic interactions with HRASLS3

  • CRISPRa/CRISPRi for precise temporal control of HRASLS3 expression

  • Base editing for studying effects of specific HRASLS3 variants

  • CRISPR knock-in approaches for endogenous tagging of HRASLS3

• Advanced imaging techniques:

  • Live-cell imaging of fluorescently tagged HRASLS3 during adipogenesis

  • Super-resolution microscopy to examine subcellular localization

  • Label-free imaging technologies to track lipid metabolism in real-time

  • Correlative light and electron microscopy to link HRASLS3 localization with ultrastructure

• Single-cell technologies:

  • scRNA-seq to identify cell populations dependent on HRASLS3 function

  • Spatial transcriptomics to map HRASLS3 expression in adipose tissue architecture

  • CyTOF or spectral flow cytometry for multiparameter protein analysis

  • Single-cell proteomics to detect low-abundance HRASLS3-related signaling events

• Organoid and advanced tissue culture systems:

  • 3D adipose tissue organoids for physiologically relevant testing

  • Microphysiological systems that incorporate multiple cell types

  • Bioprinting approaches for creating structured adipose tissue models

  • Co-culture systems to study adipocyte-immune cell interactions

How can multi-omics approaches be integrated to study HRASLS3 in human adipose biology?

Multi-omics integration offers powerful approaches to comprehensively understand HRASLS3 function:

• Experimental design for multi-omics studies:

  • Parallel sampling from the same experimental system for different omics analyses

  • Time-course designs capturing HRASLS3 manipulation effects

  • Inclusion of both bulk and single-cell approaches

  • Comparison across different adipose depots and metabolic states

• Data types to integrate:

  • Transcriptomics: RNA-seq to identify gene expression changes

  • Proteomics: Global and phospho-proteomics to detect signaling changes

  • Metabolomics: Analysis of lipid species and metabolic intermediates

  • Epigenomics: ATAC-seq, ChIP-seq for regulatory landscape

  • Interactomics: Protein-protein interaction networks

• Computational integration strategies:

  • Pathway enrichment and network analysis across multiple data types

  • Machine learning approaches to identify patterns across datasets

  • Causal network inference to establish directional relationships

  • Integration with public databases on adipose tissue biology

• Validation approaches:

  • Targeted experimental validation of key predictions

  • Perturbation experiments to test inferred regulatory relationships

  • Development of computational models that predict HRASLS3 function

What are the most promising strategies for therapeutic targeting of the HRASLS3 pathway?

Based on HRASLS3's role in adipocyte differentiation and potential metabolic implications, several therapeutic targeting strategies could be considered:

• Direct HRASLS3 modulation approaches:

  • Small molecule screening to identify HRASLS3 activity modulators

  • Stabilization of HRASLS3 protein in contexts where it is downregulated

  • RNA therapeutics (antisense oligonucleotides, siRNAs) for specific targeting

  • Targeted protein degradation approaches (PROTACs) if inhibition is desired

• Pathway-based interventions:

  • Targeting upstream regulators of HRASLS3 beyond PPARγ

  • Modulation of HRASLS3 interaction partners identified through proteomics

  • Combination approaches with existing PPARγ modulators

  • Tissue-specific delivery systems to target adipose HRASLS3 signaling

• Translation considerations:

  • Development of biomarkers to identify patients likely to benefit

  • Consideration of depot-specific effects on different adipose tissues

  • Assessment of potential off-target effects in non-adipose tissues

  • Establishment of appropriate therapeutic windows and dosing regimens

• Drug development pipeline recommendations:

  • Initial screening in human adipocyte cell models

  • Validation in primary human adipocytes from diverse donors

  • Testing in advanced tissue models (organoids, microphysiological systems)

  • Careful assessment in preclinical models with humanized adipose tissue

These approaches should be pursued with careful consideration of the complex role of adipose tissue in metabolic health and the potential for both beneficial and adverse effects of manipulating adipocyte differentiation pathways.