Hsd17b13 Antibody

What is HSD17B13 and why is it a significant research target?

HSD17B13 is a liver-enriched, hepatocyte-specific, lipid droplet-associated protein that belongs to the hydroxysteroid 17β-dehydrogenase family with NAD(P)H/NAD(P)-binding function . Its significance stems from multiple genetic studies identifying HSD17B13 variants that are protective against liver diseases including NAFLD/MASLD, alcoholic liver disease, and hepatocellular carcinoma . The protein plays an important role in hepatic lipid metabolism, specifically in lipid droplet biogenesis and growth . HSD17B13's liver-specific expression pattern and its association with disease progression make it an attractive therapeutic target, with multiple pharmaceutical companies pursuing inhibition or knockdown strategies .

How is HSD17B13 expression regulated in normal versus diseased liver states?

• In choline-deficient diet (CD)-induced murine NASH/NAFLD models, HSD17B13 expression was significantly increased

• Type 2 diabetic db/db mice and high-fat diet (HFD)-induced obese mice show elevated liver HSD17B13 expression compared to controls

• RNA-seq analysis of steatotic liver from obese mice revealed HSD17B13 as a key differentially expressed gene

The protein's expression appears to be regulated by lipid status, as fenretinide treatment, which prevents obesity and steatotic liver, normalized HSD17B13 expression levels . Additionally, HSD17B13 may mediate LXRα activation-associated liver steatosis via a SREBP1-dependent mechanism .

What are the key structural domains of HSD17B13 essential for its function?

HSD17B13 contains several critical domains that determine its localization and enzymatic function:

• N-terminal hydrophobic domain - Essential for lipid droplet targeting

• PAT-like domain - Critical for protein stability, targeting to lipid droplets, and enzymatic function

• α-helix/β-sheet/α-helix domain - Required for correct lipid droplet localization

• NAD-binding domain - Necessary for enzymatic activity

Mutagenesis studies with truncated and point-mutated proteins have demonstrated that all these domains are required for proper function. The most prevalent IsoD variant of HSD17B13 (rs72613567:TA), which contains an insertion that causes premature truncation, maintains NAD-dependent catalytic activity towards some but not all substrates, contradicting earlier reports that the truncation results in complete loss-of-function .

What are the optimal conditions for immunofluorescence detection of HSD17B13?

For effective immunofluorescence detection of HSD17B13, researchers should consider:

• Primary antibody selection: Anti-FLAG antibodies (such as F3165) have been successfully used to detect FLAG-tagged HSD17B13 in transfection experiments . For endogenous protein detection, specific anti-HSD17B13 antibodies are required.

• Secondary antibody: Alexa Fluor 568 goat anti-mouse secondary antibodies have been effectively used to recognize mouse anti-FLAG antibodies for HSD17B13 detection .

• Co-staining markers: To confirm lipid droplet localization, lipid droplet-specific dyes should be used. For mitochondrial targeting studies of mutant HSD17B13, antibodies against apoptosis-inducing factor (AIF) have been used as mitochondrial markers (Cell Signaling Technology #5318) .

• Imaging technique: Confocal microscopy is recommended for high-resolution imaging of subcellular structures (e.g., Zeiss LSM 700) .

• Fixation protocol: Optimal fixation conditions should preserve both protein localization and lipid droplet structure, typically requiring paraformaldehyde fixation followed by careful permeabilization.

What animal models are commonly used to study HSD17B13 function?

Several animal models have been employed to investigate HSD17B13 function in liver disease:

• High-fat diet (HFD)-induced obese mice - Used to study the effects of HSD17B13 knockdown on liver steatosis. This model shows elevated expression of HSD17B13 and has demonstrated that shRNA-mediated knockdown of HSD17B13 significantly improves hepatic steatosis .

• Choline-deficient diet (CD)-induced murine NASH/NAFLD model - Shows significantly increased HSD17B13 expression and is useful for studying NASH progression .

• Type 2 diabetic db/db mice - Exhibits upregulated HSD17B13 expression and serves as a model for diabetes-associated liver disease .

• HSD17B13 knockout mice - Produced unexpected results, with some studies showing that HSD17B13 deficiency failed to protect against diet-induced steatotic damage, while others reported spontaneous late-onset fatty liver development at 9 months of age under normal chow .

• Adenovirus-mediated hepatic overexpression models - Used to demonstrate that increased HSD17B13 expression for just 4 days accelerates lipid droplet biogenesis and excessive neutral lipid accumulation in mouse liver .

These models have produced somewhat inconsistent results, particularly between knockout studies and human genetic findings, highlighting the complexity of HSD17B13 biology.

How can researchers distinguish between different HSD17B13 variants using antibody-based techniques?

Distinguishing between HSD17B13 variants requires careful antibody selection and experimental design:

• Epitope-specific antibodies: Select antibodies raised against epitopes that differ between variants. For the common protective variant (rs72613567:TA), antibodies targeting regions after the truncation site would not detect the shortened protein.

• Western blotting optimization: Use gradient gels (10-15%) to effectively separate closely related variants based on size differences. The rs72613567:TA variant produces a truncated protein that can be distinguished from wild-type by molecular weight .

• Immunoprecipitation followed by mass spectrometry: This approach can identify specific protein variants and their post-translational modifications.

• Variant-specific protocols: Different variants may require adjusted lysis conditions, as some variants (particularly truncated ones) may show altered solubility or stability in standard buffers.

• Validation with recombinant proteins: Include recombinant proteins representing each variant as positive controls to confirm antibody specificity and establish size references .

When interpreting results, researchers should be aware that the common rs72613567:TA variant maintains catalytic activity toward some substrates despite truncation, which complicates functional analyses .

How do researchers effectively measure HSD17B13 enzymatic activity in cellular systems?

Measuring HSD17B13 enzymatic activity presents several challenges that researchers have addressed through various approaches:

• Synthetic surrogate substrates: Structure-aided design has enabled the development of sulfonamide-based HSD17B13 inhibitors that can be transformed into synthetic surrogate substrates with distinct selectivity advantages over previously published substrates . These allow for quantitative measures of enzymatic inhibition in primary human hepatocytes.

• NAD-dependent activity assays: As HSD17B13 belongs to the short-chain dehydrogenase/reductase family and uses NAD as a cofactor, researchers can measure NAD+/NADH conversion spectrophotometrically.

• Cellular systems for activity measurement:

  • Primary human hepatocytes - Provide endogenous expression systems for measuring native enzyme activity

  • Cells expressing specific HSD17B13 variants - Allow comparison of wild-type versus mutant activities

  • Overexpression systems - Enable higher signal detection but may not reflect physiological conditions

• Activity toward multiple substrates: Testing activity against a panel of potential substrates is critical, as the IsoD variant maintains catalytic activity toward some but not all substrates .

• Bioluminescent assays: Specialized assays have been developed for specific activities, such as a bioluminescent retinol dehydrogenase assay for HSD17B13 .

When designing these experiments, researchers should consider that HSD17B13's enzymatic function may involve complex lipid metabolism pathways, including diacylglycerol processing and phospholipid metabolism .

What methodological approaches best address the contradictory findings between human and mouse HSD17B13 studies?

The contradictory findings between human genetic studies (where loss-of-function variants are protective) and mouse knockout models (showing either no protection or spontaneous fatty liver development) present a significant challenge. Researchers can address these contradictions through:

What techniques are most effective for studying HSD17B13-related lipid metabolism changes?

To effectively study HSD17B13's impact on lipid metabolism, researchers should consider:

• Global lipidomic analysis: This approach has revealed major changes in specific lipid species with HSD17B13 modulation. For example, HSD17B13 knockdown in HFD-obese mice demonstrated:

  • Decreased diacylglycerols (e.g., DAG 34:3)

  • Increased phosphatidylcholines containing polyunsaturated fatty acids (PUFAs), such as PC 34:3 and PC 42:10

• Transcriptomic analysis: RNA-Seq can identify changes in expression of genes involved in:

  • Phospholipid metabolism (e.g., Cept1)

  • Fatty acid transport (e.g., Cd36)

  • Fibrosis markers (e.g., Timp2)

• Metabolic flux analysis: Using labeled lipid precursors to track metabolic conversion rates in control versus HSD17B13-modulated systems.

• Microscopy techniques for lipid droplet analysis:

  • Confocal microscopy with lipid-specific dyes

  • Live-cell imaging to track lipid droplet dynamics

  • Super-resolution microscopy for detailed structural analysis

• Functional assays:

  • Measurement of serum markers like ALT and FGF21, which decrease with HSD17B13 knockdown

  • Analysis of fibrosis markers to assess disease progression

  • Examination of pyrimidine catabolism at the level of dihydropyrimidine dehydrogenase

What are the recommended protocols for studying HSD17B13 inhibition in experimental models?

For effective evaluation of HSD17B13 inhibition strategies, researchers should consider:

• Inhibition approaches:

  • Structure-based inhibitor design targeting the catalytic site or essential domains

  • RNA interference (shRNA, siRNA) for gene knockdown

  • Small molecule inhibitors of specific enzymatic functions

  • Indirect inhibition through dihydropyrimidine dehydrogenase inhibitors like gimeracil

• Experimental model selection:

  • HFD-induced obese mice have demonstrated significant improvements in liver steatosis with HSD17B13 knockdown

  • Primary human hepatocytes provide a physiologically relevant system for testing inhibitor efficacy on endogenous HSD17B13

  • Cell lines expressing specific HSD17B13 variants can help identify inhibitor selectivity

• Outcome measurements:

  • Liver histology to assess steatosis reduction

  • Serum markers (ALT, FGF21)

  • Fibrosis markers (e.g., Timp2 expression)

  • Lipid profiles focusing on diacylglycerols and phosphatidylcholines

  • Pyrimidine metabolism markers

• Control considerations:

  • Include both positive controls (known effective interventions) and negative controls

  • Test inhibitors in systems expressing protective variants to understand mechanism

  • Evaluate off-target effects through comprehensive profiling

• Timing considerations:

  • Evaluate both preventive (pre-disease) and therapeutic (established disease) inhibition paradigms

  • Consider long-term effects, as some mouse models show late-onset effects

Evidence suggests that direct inhibition of HSD17B13 or indirect targeting of downstream pathways like pyrimidine catabolism can protect against liver fibrosis . The finding that "pharmacological inhibition of pyrimidine catabolism by gimeracil phenocopies the HSD17B13-induced protection against liver fibrosis" provides a promising alternative therapeutic approach .

How can researchers validate HSD17B13 antibody specificity in experimental systems?

Validating antibody specificity is critical for generating reliable HSD17B13 research data. Recommended approaches include:

• Knockout/knockdown controls:

  • Use shRNA-mediated knockdown samples as negative controls

  • Compare staining in tissues from HSD17B13 knockout mice versus wild-type

  • Include isotype controls to assess non-specific binding

• Overexpression systems:

  • Test antibodies in cells transfected with tagged HSD17B13 constructs (e.g., FLAG-tagged) and confirm co-localization with tag-specific antibodies

  • Use Western blotting to confirm expected molecular weight

• Peptide competition assays:

  • Pre-incubate antibodies with immunizing peptides to block specific binding

  • Compare staining patterns with and without peptide competition

• Multiple antibody validation:

  • Use different antibodies targeting distinct epitopes of HSD17B13

  • Confirm consistent staining patterns across antibodies

• Variant-specific validation:

  • Test antibody recognition of known HSD17B13 variants (e.g., rs72613567:TA)

  • Include recombinant proteins of different variants as controls

• Subcellular localization confirmation:

  • Verify lipid droplet localization by co-staining with established lipid droplet markers

  • For mutant forms, confirm altered localization patterns (e.g., mitochondrial targeting)

• Mass spectrometry validation:

  • Confirm antibody-precipitated proteins by mass spectrometry to verify target identity

What are the most useful approaches for studying HSD17B13 interactions with other proteins and cellular structures?

Understanding HSD17B13's interactions with other proteins and cellular structures requires sophisticated approaches:

• Co-immunoprecipitation studies:

  • Use anti-HSD17B13 antibodies to pull down protein complexes

  • Identify interaction partners through mass spectrometry

  • Confirm interactions with reciprocal co-IP experiments

• Proximity labeling techniques:

  • BioID or APEX2 fusion proteins to identify proteins in close proximity to HSD17B13 on lipid droplets

  • TurboID for faster labeling kinetics and potential temporal studies

• Fluorescence microscopy approaches:

  • FRET or BRET to detect direct protein-protein interactions

  • FRAP to study dynamics of HSD17B13 on lipid droplets

  • Super-resolution microscopy for detailed localization studies

• Domain-specific interaction mapping:

  • Use truncated or point-mutated proteins to identify domains critical for specific interactions

  • Focus on the N-terminal hydrophobic domain, PAT-like domain, and α-helix/β-sheet/α-helix domain for lipid droplet interactions

• Functional interaction studies:

  • Investigate potential interactions with PNPLA3, as human genetic studies suggest possible interactions between HSD17B13-rs72613567:TA and PNPLA3-rs738409G alleles

  • Study interactions with LXRα and SREBP1 pathway components, which may mediate HSD17B13's effects on lipid metabolism

• Lipid droplet isolation techniques:

  • Isolate pure lipid droplet fractions to identify HSD17B13-associated proteins in this compartment

  • Compare protein complexes on lipid droplets between normal and steatotic conditions

When designing these studies, researchers should consider that HSD17B13's associations may be context-dependent, changing with disease state or metabolic conditions.

What are the advantages and limitations of different detection methods for HSD17B13?

Different detection methods offer varying benefits and limitations for HSD17B13 research:

For optimal results, researchers should consider combining multiple detection methods. For example, using immunofluorescence to confirm lipid droplet localization , Western blotting to verify protein size and expression levels, and activity assays with synthetic substrates to measure enzymatic function .

What are the key methodological considerations when designing HSD17B13 knockdown experiments?

Effective HSD17B13 knockdown studies require careful design considerations:

• Knockdown approach selection:

  • shRNA provides stable, long-term knockdown suitable for in vivo studies

  • siRNA offers transient knockdown with potentially fewer off-target effects

  • CRISPR/Cas9 enables complete knockout but may trigger compensatory mechanisms

• Delivery method optimization:

  • For liver-specific knockdown, consider adenoviral vectors which show high hepatocyte tropism

  • AAV8 vectors provide long-term expression for chronic studies

  • For cell culture, lipid-based transfection reagents are typically effective

• Target sequence selection:

  • Design targeting sequences to affect all relevant splice variants

  • Consider species differences when designing knockdown reagents

  • Avoid sequences with off-target potential

• Controls and validation:

  • Include scrambled sequence controls

  • Validate knockdown efficiency at both mRNA and protein levels

  • Consider rescue experiments with knockdown-resistant constructs

• Timing considerations:

  • For metabolic studies, determine optimal knockdown duration (short-term vs. long-term)

  • In HFD models, consider whether to implement knockdown before or after disease establishment

• Outcome measurements:

  • Assess multiple endpoints including histology, serum markers, gene expression, and lipidomic profiles

  • Consider pyrimidine metabolism as a key pathway affected by HSD17B13 modulation

Research has shown that shRNA-mediated knockdown of HSD17B13 in HFD-obese mice improves hepatic steatosis, decreases serum ALT and FGF21 levels, and reduces markers of liver fibrosis without affecting body weight or glycemia .

How do research findings on HSD17B13 variants inform therapeutic development strategies?

Human genetic studies of HSD17B13 variants provide crucial insights for therapeutic development:

• Target validation:

  • The protective effect of loss-of-function variants (particularly rs72613567:TA) against liver fibrosis, inflammation, and cancer provides strong human genetic validation for HSD17B13 as a therapeutic target

  • These genetic associations have been reproducibly demonstrated across different ethnic populations

• Mechanism insights:

  • Protection against liver fibrosis is associated with decreased pyrimidine catabolism at the level of dihydropyrimidine dehydrogenase

  • Effects on diacylglycerol and phosphatidylcholine metabolism suggest multiple potential mechanisms

  • Interaction with PNPLA3 pathways may contribute to hepatoprotection

• Therapeutic approaches:

  • Direct inhibition: Structure-aided design has enabled development of potent and selective sulfonamide-based HSD17B13 inhibitors

  • Indirect targeting: Inhibition of downstream pathways like pyrimidine catabolism by dihydropyrimidine dehydrogenase inhibitors (e.g., gimeracil) phenocopies HSD17B13-induced protection

  • RNA silencing: siRNA approaches targeting HSD17B13 are being pursued by pharmaceutical companies

• Biomarker development:

  • HSD17B13 protein levels may serve as biomarkers for disease progression

  • Specific lipid profiles (diacylglycerols, phosphatidylcholines) could function as pharmacodynamic markers

  • Pyrimidine metabolites might indicate therapeutic response

• Combination therapies:

  • Understanding interactions with PNPLA3 suggests potential for combination approaches targeting multiple genetic risk factors

Research challenges include the contradictory findings in mouse models and the fact that the IsoD variant maintains catalytic activity toward some substrates despite being protective , suggesting complex biology that must be carefully considered in drug development.

What methodological approaches best translate HSD17B13 research findings from experimental models to human applications?

Translating HSD17B13 research to human applications requires robust methodological approaches:

• Human-relevant models:

  • Primary human hepatocytes provide the most physiologically relevant cellular system for testing HSD17B13 inhibitors

  • Human liver organoids can model 3D tissue architecture and cell-cell interactions

  • Patient-derived xenografts maintain human liver disease characteristics

• Genetic validation:

  • Study carriers of protective variants (e.g., rs72613567:TA) to understand physiological effects

  • Compare metabolic profiles between variant carriers and non-carriers to identify relevant pathways

  • Use Mendelian randomization approaches to strengthen causal inference

• Biomarker development:

  • Identify lipid species altered by HSD17B13 modulation for potential biomarkers

  • Focus on pyrimidine metabolites as indicators of pathway modulation

  • Develop assays for relevant markers like ALT, FGF21, and fibrosis indicators

• Pharmacologic validation:

  • Test HSD17B13 inhibitors in multiple systems including:

    • Cell-free enzymatic assays with recombinant protein

    • Primary human hepatocytes

    • Human liver slice cultures

    • Humanized mouse models

• Combinatorial approaches:

  • Test HSD17B13 modulation in combination with other therapeutic strategies

  • Consider genetic background effects, particularly PNPLA3 status

• Systematic pathway analysis:

  • Use state-of-the-art metabolomics to identify pathway alterations

  • Perform comparative analysis between species to identify conserved mechanisms

  • Focus on pyrimidine catabolism, which appears to be a key mediator of protection

Research has demonstrated that "protection against liver fibrosis conferred by the HSD17B13 rs72613567-A variant in humans and by the Hsd17b13 knockdown in mice is associated with decreased pyrimidine catabolism at the level of dihydropyrimidine dehydrogenase" , providing a translationally relevant pathway for therapeutic development.

What emerging technologies might advance HSD17B13 antibody applications in research?

Several emerging technologies hold promise for enhancing HSD17B13 antibody applications:

• Single-cell proteomics:

  • Enables analysis of HSD17B13 expression at single-cell resolution

  • Can identify cell-specific responses to HSD17B13 modulation

  • Allows mapping of heterogeneous responses within liver tissue

• Spatially-resolved proteomics:

  • Technologies like imaging mass cytometry combine antibody specificity with spatial resolution

  • Can map HSD17B13 distribution within tissue microenvironments

  • Correlates HSD17B13 expression with disease features at microscopic scale

• Proximity labeling advances:

  • Next-generation TurboID or miniTurbo systems for faster labeling kinetics

  • Compartment-specific proximity labeling to identify lipid droplet-specific interactors

  • Multiplexed proximity labeling for simultaneous study of multiple proteins

• Engineered antibody formats:

  • Bispecific antibodies targeting HSD17B13 and lipid droplet markers

  • Intrabodies for tracking HSD17B13 in living cells

  • Nanobodies with superior tissue penetration for in vivo imaging

• Advanced imaging approaches:

  • Super-resolution techniques (STORM, PALM, STED) for nanoscale localization

  • Label-free imaging methods for unperturbed visualization

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

• Computational antibody design:

  • AI-driven epitope prediction for generating variant-specific antibodies

  • Structure-guided antibody engineering for improved specificity

  • In silico screening for antibodies with desired binding properties

These technologies could help address key research questions, including the structural basis of HSD17B13 variants' effects, the protein's dynamic interactions in disease states, and the spatial coordination of lipid metabolism in the liver.

What are the unresolved questions about HSD17B13 function that require new methodological approaches?

Despite significant progress, several key questions about HSD17B13 remain unresolved and require innovative methodological approaches:

• Endogenous substrate identification:

  • The natural substrates of HSD17B13 remain unclear despite its protective effects in liver disease

  • Approaches needed: Untargeted metabolomics with stable isotope tracing, activity-based protein profiling, and comprehensive substrate screening platforms

• Mechanistic basis of protective variants:

  • How the rs72613567:TA variant maintains activity toward some substrates while providing protection remains puzzling

  • Approaches needed: Structural biology techniques, hydrogen-deuterium exchange mass spectrometry, and variant-specific enzymatic profiling

• Species differences reconciliation:

  • Contradictory findings between human genetics and mouse models require explanation

  • Approaches needed: Comparative multi-omics across species, humanized mouse models, and advanced computational modeling

• Temporal dynamics of HSD17B13 function:

  • Whether HSD17B13's role changes throughout disease progression remains unknown

  • Approaches needed: Temporal knockdown/knockout systems, longitudinal imaging, and stage-specific intervention studies

• Interaction with genetic modifiers:

  • The reported interaction with PNPLA3 variants suggests complex genetic architecture

  • Approaches needed: Genetic interaction screens, systems biology approaches, and population-level studies with sufficient power

• Detailed mechanism of pyrimidine metabolism involvement:

  • How HSD17B13 affects dihydropyrimidine dehydrogenase activity remains unclear

  • Approaches needed: Enzyme complex analysis, structural studies of potential interactions, and metabolic flux analysis

• Tissue-specific roles beyond liver:

  • While primarily liver-expressed, potential functions in other tissues remain unexplored

  • Approaches needed: Single-cell atlasing across tissues, conditional tissue-specific expression models, and comparative proteomics

Addressing these questions will require interdisciplinary approaches combining advanced antibody technologies with state-of-the-art metabolomics, structural biology, and systems biology methods.