HSD17B8 Antibody

What is HSD17B8 and what is its primary function in cellular metabolism?

HSD17B8 is an NAD-dependent 17-beta-hydroxysteroid dehydrogenase that demonstrates highest catalytic activity toward estradiol. Its primary function involves converting estradiol (E2) to estrone (E1) through oxidative activity . Additionally, when complexed with CBR4, it exhibits NADH-dependent 3-ketoacyl-acyl carrier protein reductase activity, suggesting a potential role in mitochondrial fatty acid biosynthesis .

Methodologically, researchers investigating HSD17B8 function should consider:

• Measuring enzyme activity using NAD as a cofactor

• Evaluating both steroid conversion (E2→E1) and potential fatty acid metabolic functions

• Examining protein-protein interactions, particularly with CBR4 and PTEN

How should researchers select the appropriate HSD17B8 antibody for their experimental design?

Selection criteria should be based on:

For optimal results, researchers should:

• Validate antibody specificity using positive controls (HeLa cells, human testis tissue)

• Consider cross-reactivity requirements based on species (human, mouse, rat)

• Select antibodies targeting specific domains based on research questions (e.g., AA 28-127 vs. full-length protein)

How does HSD17B8 influence breast cancer proliferation, and what experimental approaches are optimal for studying this relationship?

Recent findings demonstrate that HSD17B8 plays a crucial role in breast cancer cell proliferation through estrogen metabolism regulation. HSD17B8 converts estradiol (E2) to estrone (E1), with these hormones having opposing effects on cancer cells .

Key experimental findings:

• E1 stimulates breast cancer cell proliferation while E2 has inhibitory effects

• HSD17B8 knockdown significantly suppresses growth of MCF-7 cells

• E2 treatment and HSD17B8 knockdown arrest tumor cells in G2/M phase

Recommended experimental approaches:

• siRNA-mediated knockdown of HSD17B8 in ER+ breast cancer cell lines

• Flow cytometry for cell cycle analysis following E1/E2 treatment

• Western blot analysis of phosphorylated ERK levels as a downstream indicator

• Colony formation assays to assess long-term proliferative effects

What is the relationship between PTEN and HSD17B8, and how can researchers effectively study this interaction?

The PTEN-HSD17B8 interaction represents a novel regulatory mechanism in cell proliferation. Research indicates that:

• PTEN physically interacts with HSD17B8 to inhibit its enzymatic conversion of E2 to E1

• Loss of PTEN results in released HSD17B8 activity, leading to decreased E2 levels

• This regulatory relationship affects ERK/MAPK activation pathways

Methodological approaches for studying this interaction:

• Co-immunoprecipitation assays to confirm physical interaction

• In vitro enzyme activity assays with purified proteins

• Proximity ligation assays in intact cells

• Comparative analysis of E1:E2 ratios in PTEN-deficient versus normal cells

• Combined knockdown experiments to establish epistatic relationships

How can researchers effectively measure and interpret changes in HSD17B8 enzymatic activity in experimental models?

Quantifying HSD17B8 activity requires specialized approaches:

For robust results, researchers should:

• Include appropriate controls (NAD+ vs. NADH dependency)

• Correlate expression levels with activity measurements

• Consider the heterotetramer formation with CBR4 when studying fatty acid metabolism functions

• Account for post-translational modifications that may affect enzyme activity

What are the critical parameters for successful Western blotting of HSD17B8?

Optimized Western blot protocols for HSD17B8 detection should address:

• Sample preparation: Effective lysis in tissues with high expression (liver, small intestine, testis)

• Expected molecular weight: 27 kDa (calculated) vs. 34 kDa (observed)

• Antibody selection: Polyclonal antibodies (e.g., 16752-1-AP) at 1:500-1:2000 dilution

• Blocking conditions: Optimize based on antibody manufacturer recommendations

• Positive controls: HeLa cells, mouse liver tissue, human testis tissue

Technical validation approaches:

• Knockdown/knockout controls to confirm band specificity

• Use of recombinant HSD17B8 as positive control

• Comparison of multiple antibodies targeting different epitopes

What approaches should researchers use to resolve contradictory findings in HSD17B8 functional studies?

Contradictory results have been reported regarding HSD17B8 function, particularly in metabolic contexts . To address these contradictions, researchers should:

• Consider tissue-specific effects:

  • Analyze expression patterns across different tissues

  • Perform conditional knockout studies rather than global deletion

• Evaluate experimental model differences:

  • Compare in vitro vs. in vivo systems

  • Assess acute vs. chronic manipulation of HSD17B8 levels

  • Consider compensatory mechanisms in knockout models

• Examine dual enzymatic functions:

  • Separately assess steroid metabolism vs. fatty acid metabolism roles

  • Investigate context-dependent protein complex formation (e.g., with CBR4)

• Control for background genetic differences:

  • Use isogenic cell lines for comparative studies

  • Consider strain background in mouse models

How can researchers effectively validate knockdown or inhibition of HSD17B8 in their experimental systems?

Validation of HSD17B8 manipulation requires multi-level confirmation:

For siRNA approaches:

• Use multiple siRNA sequences to control for off-target effects

• Implement dose-response studies to determine optimal knockdown conditions

• Establish time course for protein depletion after siRNA treatment

• Include appropriate negative controls (scrambled siRNA)

Beyond estrogen metabolism, what other metabolic roles of HSD17B8 warrant investigation?

Current evidence suggests broader metabolic functions that deserve further research:

• Mitochondrial fatty acid biosynthesis: The heteroteramer with CBR4 exhibits NADH-dependent 3-ketoacyl-acyl carrier protein reductase activity

• Potential involvement in liver metabolism and non-alcoholic fatty liver disease pathways

• Possible roles in metabolic syndrome and insulin resistance related to steroid hormone balance

Methodological considerations for these studies:

• Mitochondrial isolation techniques for functional assays

• Lipidomic analysis to profile fatty acid changes

• Metabolic flux analysis using isotope-labeled precursors

• Tissue-specific conditional knockout models focusing on liver, adipose tissue, and muscle

How can researchers address the technical challenges in studying protein-protein interactions involving HSD17B8?

HSD17B8 forms functional complexes with multiple proteins, including CBR4 and PTEN . Effective study of these interactions requires:

• In vitro interaction analysis:

  • Recombinant protein co-immunoprecipitation

  • Surface plasmon resonance to determine binding kinetics

  • Isothermal titration calorimetry for thermodynamic parameters

• Structural studies:

  • X-ray crystallography of protein complexes

  • Cryo-EM for larger assemblies

  • Hydrogen-deuterium exchange mass spectrometry for interaction interfaces

• Cellular confirmation techniques:

  • Förster resonance energy transfer (FRET)

  • Bimolecular fluorescence complementation

  • Proximity ligation assays in fixed cells

• Functional validation:

  • Mutagenesis of predicted interaction domains

  • Activity assays with reconstituted complexes

  • Cellular phenotypes with interaction-deficient mutants

These approaches provide complementary data to fully characterize the interactions that mediate HSD17B8's diverse cellular functions.

What are the most promising therapeutic applications emerging from HSD17B8 research?

Recent findings highlight several potential therapeutic directions:

• Breast cancer treatment: Inhibiting HSD17B8 may suppress ER+ breast cancer cell proliferation by altering the E1:E2 ratio

• Metabolic disorders: Modulating HSD17B8 activity could potentially address aspects of fatty liver disease

• Cell cycle regulation: HSD17B8 manipulation affects G2/M phase arrest, suggesting applications in cancer therapy approaches

Future research should focus on:

• Development of specific inhibitors of HSD17B8 enzymatic activity

• Tissue-specific delivery systems to target HSD17B8 in cancer cells

• Combination approaches with existing therapies like anti-estrogens

• Clinical correlations between HSD17B8 expression/activity and patient outcomes