HSD17B7 Antibody

What is HSD17B7 and what are its primary biological functions?

HSD17B7 is a bifunctional enzyme with two distinct catalytic activities:

• Steroid metabolism: Converts estrone to the more potent estradiol by reducing the keto group at the 17β position using NAD(P)H as a cofactor .

• Cholesterol biosynthesis: Functions as a 3-ketosteroid reductase in the cholesterol biosynthesis pathway, specifically converting zymosterone to zymosterol .

This dual functionality makes HSD17B7 important in both reproductive biology and cellular metabolism. The protein belongs to the Short-chain dehydrogenases/reductases (SDR) family, specifically the ERG27 subfamily . Notably, knockout studies in mice have demonstrated that HSD17B7 is essential for embryonic development, with homozygous null mice exhibiting embryonic lethality .

What is the tissue distribution and subcellular localization of HSD17B7?

HSD17B7 shows tissue-specific expression patterns with highly variable levels:

At the subcellular level, HSD17B7 exhibits a distinctive particulate perinuclear distribution pattern that does not overlap with mitochondria . It is predominantly found as a cell membrane-associated single-pass membrane protein . In certain cell types, it may also associate with the endoplasmic reticulum, consistent with its role in sterol metabolism.

What applications are HSD17B7 antibodies commonly used for?

HSD17B7 antibodies are employed in multiple research applications:

Positive control samples include MCF-7 cells, HEK-293 cells, and Jurkat cells for Western blot applications, while adrenal gland tissue serves as an excellent positive control for IHC .

How can researchers validate the specificity of HSD17B7 antibodies?

Rigorous validation of HSD17B7 antibodies is essential for reliable experimental outcomes. A comprehensive validation approach should include:

• Positive and negative tissue controls:

  • Use tissues with known high expression (adrenal gland, liver) as positive controls

  • Include non-steroidogenic tissues (certain brain regions, skeletal muscle) as negative controls

• Cell line validation:

  • MCF-7 breast cancer cells show detectable HSD17B7 expression

  • Compare against cell lines with lower expression

• Genetic validation approaches:

  • siRNA knockdown of HSD17B7 should reduce antibody signal

  • Overexpression systems should show increased signal intensity

  • If possible, validate with samples from conditional knockout models

• Multiple detection methods:

  • Compare results between Western blot, IHC, and IF

  • Correlate protein detection with mRNA expression data

  • Consider mass spectrometry confirmation for novel findings

• Epitope competition:

  • Pre-incubate antibody with immunizing peptide to block specific binding

  • Signal should be significantly reduced or eliminated

Researchers should note that HSD17B7 is detected at 32-38 kDa in Western blots, slightly lower than its calculated molecular weight of 38.2 kDa .

Western Blot Analysis

For optimal Western blot detection of HSD17B7:

• Sample preparation: Use lysis buffers containing 1-2% detergent (Triton X-100 or NP-40) to efficiently extract this membrane-associated protein

• Protein loading: Load 20-50 μg total protein per lane

• Gel percentage: 10-12% acrylamide gels provide good resolution around 38 kDa

• Transfer conditions: Standard semi-dry or wet transfer protocols are suitable

• Blocking: 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Primary antibody: Dilute according to manufacturer recommendations (typically 1:500-1:2000)

• Secondary antibody: Anti-rabbit HRP conjugates work well for most commercial antibodies

• Expected band size: 32-38 kDa

Immunohistochemistry

For IHC detection of HSD17B7:

• Fixation: Standard 10% neutral-buffered formalin is appropriate

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0)

• Blocking: 1-5% serum from secondary antibody species

• Antibody incubation: Overnight at 4°C in a humidified chamber

• Detection systems: DAB chromogen systems provide good signal-to-noise ratio

• Expected pattern: Particulate perinuclear distribution with some membrane association

Immunofluorescence

For immunofluorescence detection:

• Fixation: 4% paraformaldehyde (10-15 minutes)

• Permeabilization: 0.1-0.5% Triton X-100 (5-10 minutes)

• Counterstaining: DAPI for nuclear visualization

• Controls: Include ER markers to confirm subcellular localization

• Imaging: Confocal microscopy recommended for precise localization

How is HSD17B7 expression related to cancer biology?

HSD17B7 has emerged as an important player in cancer biology, particularly in hormone-dependent cancers and squamous cell carcinomas:

• Breast cancer implications:

  • HSD17B7 is highly expressed in ductal carcinoma

  • Estradiol enhances HSD17B7 expression in breast cancer cells, creating a positive feedback loop

  • This estradiol-induced stimulation occurs through ERα activation and NF1 recruitment to the HSD17B7 promoter

• Squamous cell carcinoma (SCC):

  • HSD17B7 is the top-ranked differentially expressed gene in both keratinocytes and Head/Neck SCCs when comparing samples from individuals of Black African versus Caucasian ancestries

  • Higher HSD17B7 expression correlates with poor HNSCC patient survival across ancestries

  • This association remains significant after adjusting for patient sex, age, and ancestry

• Functional impact on cancer cells:

  • HSD17B7 overexpression enhances clonogenicity and proliferation of keratinocytes and SCC cell lines

  • It plays a positive role in controlling keratinocyte stem cell and oncogenic potential

  • Interestingly, it suppresses OXPHOS (oxidative phosphorylation) activity

• Genetic associations:

  • Several ancestry-specific expression quantitative trait loci (eQTLs) have been linked to HSD17B7 expression

  • These genetic variants show different allele frequencies between Black and White populations (FST > 0.3)

These findings suggest HSD17B7 could be a targetable determinant of cancer susceptibility and progression, potentially amenable to prevention and treatment strategies across different human populations.

What are the optimal conditions for immunoprecipitation using HSD17B7 antibodies?

For successful immunoprecipitation (IP) of HSD17B7:

Essential controls include:

• Input sample (5-10% of starting material)

• IgG control from same species as primary antibody

• Beads-only control to identify non-specific binding

• Peptide competition control where appropriate

When investigating protein interactions, consider cross-linking the antibody to beads to prevent co-elution of heavy and light chains that may interfere with detection of proteins of similar molecular weight.

What approaches can be used to measure HSD17B7 enzymatic activity?

HSD17B7 has dual enzymatic functions that can be assessed using several approaches:

Estrone to Estradiol Conversion

• Radiometric assays:

  • Incubate samples with [³H]-labeled estrone

  • Extract steroids and separate by TLC or HPLC

  • Quantify radioactive estradiol formation

• LC-MS/MS methods:

  • Higher specificity and sensitivity than radiometric assays

  • Allows simultaneous measurement of multiple steroids

  • No radioactive materials required

• Enzyme immunoassays:

  • Commercial kits available for estradiol quantification

  • Less specific but more accessible than LC-MS/MS

Zymosterone to Zymosterol Conversion

• Gas chromatography-mass spectrometry (GC-MS):

  • Direct measurement of zymosterol formation

  • High specificity for cholesterol intermediates

• Complementation assays:

  • Utilize Erg27p-deficient yeast strains

  • Measure growth restoration in cholesterol-deficient medium

Indirect Activity Measures

NADH/NADPH consumption can be monitored using:

• Spectrophotometric assays (340 nm)

• Fluorescence-based detection

• Luminescence-based NAD(P)H detection with commercial kits

For all enzymatic assays, appropriate controls include enzyme inhibitors, heat-inactivated samples, and siRNA knockdown of HSD17B7 .

How should ancestry-specific expression differences in HSD17B7 be considered in experimental design?

Research has identified HSD17B7 as the top-ranked differentially expressed gene in samples from individuals of Black African versus Caucasian ancestry, with significant implications for experimental design:

• Genetic considerations:

  • Multiple ancestry-specific eQTLs influence HSD17B7 expression

  • Six eQTLs have significantly different allele frequencies between Black and White populations (FST > 0.3)

  • Strong linkage disequilibrium exists between these variants (D′ > 0.5)

• Experimental design recommendations:

  • Include demographically diverse donor samples

  • Document and report ancestry information for human samples

  • Consider ancestry as a variable in data analysis

  • Include ancestry-informative markers in genetic studies

  • Evaluate expression in the context of relevant eQTLs

• Cell line selection:

  • Use cell lines derived from diverse populations

  • Document the ancestry of cell line donors

  • Consider how cell line ancestry may influence results

  • Include multiple cell lines representing different ancestries

• Clinical correlations:

  • Stratify patient outcome analyses by ancestry

  • Perform multivariate analyses adjusting for ancestry

  • Consider ancestry-specific responses in therapeutic studies

These considerations are particularly important given the associations between HSD17B7 expression and clinical outcomes in cancer studies, where higher expression correlates with poor survival in HNSCC patients .

How should researchers troubleshoot unexpected HSD17B7 antibody staining patterns?

When encountering unexpected staining patterns with HSD17B7 antibodies, a systematic troubleshooting approach is recommended:

• Unexpected subcellular localization:

  • Confirm antibody specificity with additional validation methods

  • Verify fixation conditions (over-fixation can alter localization patterns)

  • Test different permeabilization methods for better epitope access

  • Compare with published reports showing perinuclear distribution

  • Co-stain with compartment markers to identify precise localization

• Absence of signal:

  • Verify sample expression using RT-qPCR

  • Optimize antigen retrieval methods (try both citrate and EDTA buffers)

  • Increase antibody concentration or incubation time

  • Test multiple antibodies targeting different epitopes

  • Check species reactivity of antibody

• Excessive background:

  • Increase blocking time/concentration

  • Optimize antibody dilution (typically 1:500-1:2000)

  • Include additional washing steps

  • Use more specific detection systems

  • Consider tissue-specific autofluorescence quenching methods

• Multiple bands in Western blot:

  • Determine if bands represent isoforms (compare to expected sizes)

  • Check for post-translational modifications (phosphorylation known to occur)

  • Optimize SDS-PAGE conditions for better separation

  • Perform peptide competition to identify specific bands

  • Consider siRNA validation to confirm specific bands

• Inconsistent results between applications:

  • Some epitopes may be accessible only in certain applications

  • Native vs. denatured protein structure affects antibody binding

  • Different fixation methods may preserve different epitopes

  • Application-specific optimization may be required

How should researchers interpret HSD17B7 expression data in relation to estrogen signaling?

Interpreting HSD17B7 expression in the context of estrogen signaling requires consideration of several complex regulatory relationships:

• Estradiol-induced regulation:

  • Estradiol strongly stimulates HSD17B7 mRNA and protein expression in MCF-7 cells

  • This creates a positive feedback loop where estradiol enhances its own synthesis

  • The stimulation is mediated by ERα and requires NF1 recruitment to the HSD17B7 promoter

  • ER antagonists (ICI 182,780 and 4-hydroxytamoxifen) block this stimulation

• Promoter regulation:

  • A conserved -185 bp region of the HSD17B7 promoter confers estradiol regulation

  • This region lacks a classical estradiol response element but contains an essential NF1 site

  • Knockdown of NF1 family members (NF1B, NF1A, NF1X) prevents estradiol induction of HSD17B7

• Therapeutic implications:

  • ER antagonists not only block estradiol action but also its production by inhibiting HSD17B7 expression

  • This dual mechanism may contribute to their effectiveness in hormone-dependent cancers

  • HSD17B7 expression may serve as a biomarker for estrogen dependence

• Experimental considerations:

  • Estrogen status of experimental systems should be documented

  • Phenol red (estrogenic) vs. phenol red-free media can influence results

  • Charcoal-stripped serum eliminates endogenous estrogens

  • Timing of estrogen exposure is critical for interpreting expression changes

This regulatory relationship highlights the importance of considering hormonal context when designing experiments and interpreting HSD17B7 expression data, particularly in breast cancer and other estrogen-responsive tissues.