HSD17B10 Antibody

What is HSD17B10 and why is it significant in biomedical research?

HSD17B10 (Hydroxysteroid 17-Beta Dehydrogenase 10) is a multifunctional mitochondrial enzyme involved in several critical biological processes. It functions as:

• A key component in fatty acid beta-oxidation, catalyzing the reversible conversion of (S)-3-hydroxyacyl-CoA to 3-ketoacyl-CoA

• An essential part of mitochondrial ribonuclease P (RNase P), which cleaves tRNA molecules at their 5'-ends

• A catalyst for the beta-oxidation at position 17 of androgens and estrogens

• A protein implicated in neurodegenerative disorders, particularly Alzheimer's disease through its interaction with amyloid-beta peptides

The significance of HSD17B10 extends beyond its enzymatic functions, as mutations in the HSD17B10 gene are associated with HSD10 Mitochondrial Disease and Syndromic X-Linked Intellectual Disability Type 10, making it a critical target for research in multiple fields .

What are the common synonyms and alternative names for HSD17B10?

HSD17B10 is known by numerous alternative names in the literature, which can sometimes create confusion when searching for relevant research. The most common synonyms include:

• 17-beta-hydroxysteroid dehydrogenase 10 (17-beta-HSD 10)

• 3-hydroxyacyl-CoA dehydrogenase type-2 or type II

• 3-hydroxy-2-methylbutyryl-CoA dehydrogenase

• Amyloid beta peptide binding alcohol dehydrogenase (ABAD)

• Endoplasmic reticulum-associated amyloid beta-peptide-binding protein (ERAB)

• Mitochondrial ribonuclease P protein 2 (MRPP2)

• Short chain dehydrogenase/reductase family 5C member 1 (SDR5C1)

• HADH2, HCD2, SCHAD, XH98G2

When designing experiments or searching literature, researchers should be aware of these alternative designations to ensure comprehensive coverage of available information .

How do I select the appropriate HSD17B10 antibody for my specific application?

Selection of the appropriate HSD17B10 antibody depends on several factors:

• Application compatibility: Verify that the antibody has been validated for your specific application (WB, IF, IP, FC, ELISA). For example, the antibody from Abcepta (AP22244a) is validated for IF (1:25), WB (1:8000), and FC (1:25) .

• Species reactivity: Ensure the antibody recognizes HSD17B10 in your species of interest. Most commercial antibodies target human HSD17B10, but cross-reactivity with mouse or rat should be confirmed if working with these models .

• Epitope location: Consider whether the target region matters for your research. For instance, the Abcepta antibody targets the N-terminal region (amino acids 14-48) of human HSD17B10 .

• Clonality: Polyclonal antibodies (like those from ARP, Abcepta, and Nordic Biosite) offer broader epitope recognition but may have batch-to-batch variability, while monoclonal antibodies provide more consistent results but may be less robust to protein modifications .

• Validation data: Request and review validation data for your specific application to ensure antibody specificity and sensitivity.

For complex studies examining protein interactions or post-translational modifications, it may be beneficial to validate results using antibodies targeting different epitopes of HSD17B10 .

What are the optimal protocols for using HSD17B10 antibodies in Western Blot analysis?

For optimal Western Blot results with HSD17B10 antibodies:

• Sample preparation:

  • Use RIPA buffer with protease inhibitors for protein extraction

  • Heat samples at 95°C for 5 minutes in reducing Laemmli buffer

  • Load 10-30 μg of total protein per lane

• Gel electrophoresis and transfer:

  • Use 12-15% SDS-PAGE gels (HSD17B10 has a calculated MW of ~27 kDa)

  • Transfer to PVDF membranes at 100V for 1 hour or 30V overnight

• Antibody incubation:

  • Block with 5% non-fat milk in TBST for 1 hour at room temperature

  • Dilute primary antibody according to manufacturer recommendations:

    • Abcepta HSD17B10 Antibody (N-Term): 1:8000 dilution

    • Other polyclonal antibodies typically: 1:1000-1:5000

  • Incubate overnight at 4°C with gentle rocking

  • Wash 3x with TBST, 5 minutes each

  • Incubate with appropriate HRP-conjugated secondary antibody (typically 1:5000-1:10000) for 1 hour at room temperature

  • Wash 3x with TBST, 5 minutes each

• Detection:

  • Use ECL substrate for visualization

  • Expected band at approximately 27 kDa

• Controls:

  • Include positive controls from tissues with known HSD17B10 expression (liver, brain)

  • Use CRISPR-Cas9 HSD17B10 knockout cells as negative controls (as described in reference )

Remember that protein lysates should be freshly prepared or properly stored at -80°C to preserve protein integrity .

How can I assess HSD17B10 enzymatic activity in experimental samples?

HSD17B10 enzymatic activity can be measured using a spectrophotometric assay that monitors the reduction of acetoacetyl-CoA to L-3-hydroxyacyl-CoA coupled with the oxidation of NADH to NAD+. The protocol involves:

• Assay mixture preparation:

  • 0.1 M potassium phosphate buffer (pH 7.0)

  • 0.2 mg/ml bovine serum albumin

  • 0.2 mM NADH

  • Various concentrations of acetoacetyl-CoA (0-300 μM)

• Reaction initiation:

  • Add 50 nM purified HSD17B10 or tissue/cell lysate to start the reaction

• Measurement:

  • Monitor the decrease in absorbance at 340 nm (indicative of NADH oxidation)

  • Establish an NADH standard curve to quantify substrate conversion

• Data analysis:

  • Calculate kinetic parameters (Km, Vmax) using non-linear regression analysis

  • Generate Michaelis-Menten plots using software like GraphPad Prism

This method allows for quantitative assessment of HSD17B10 enzymatic activity and can be used to compare wild-type versus mutant proteins or to evaluate the effects of potential inhibitors .

What approaches can be used to study HSD17B10 post-translational modifications?

HSD17B10 undergoes important post-translational modifications, particularly acetylation, which can be studied using the following approaches:

• Detection of acetylated HSD17B10:

  • Immunoprecipitate HSD17B10 using specific antibodies

  • Immunoblot with anti-acetyl-lysine antibodies to detect acetylation

  • Alternative approach: Mass spectrometry analysis of immunoprecipitated HSD17B10 to identify acetylation sites

• Study of acetyltransferases/deacetylases:

  • Co-immunoprecipitation assays to detect interactions between HSD17B10 and modifying enzymes (e.g., SIRT3, CBP, p300)

  • In vitro acetylation assays using purified enzymes

  • Manipulation of acetyltransferase/deacetylase activity:

    • Overexpression of CBP/p300 (acetyltransferases)

    • Treatment with Garcinol (CBP inhibitor)

    • siRNA knockdown of CBP

    • Overexpression or knockdown of SIRT3 (deacetylase)

• Functional impact assessment:

  • Compare enzymatic activities of acetylated versus deacetylated HSD17B10

  • Generate acetylation mimics (lysine to glutamine mutations) or non-acetylatable mutants (lysine to arginine mutations)

  • Assess impact on protein-protein interactions, subcellular localization, and stability

Research has shown that HSD17B10 acetylation is primarily mediated by CBP, while deacetylation is carried out by SIRT3. These modifications influence HSD17B10 function and may have implications for its role in cellular metabolism and disease pathogenesis .

How does HSD17B10 contribute to Alzheimer's disease pathology?

HSD17B10's contribution to Alzheimer's disease (AD) pathology involves several mechanisms:

• Direct interaction with amyloid-beta (Aβ):

  • HSD17B10 (also known as ABAD or ERAB) directly binds to intracellular Aβ

  • This interaction occurs within mitochondria and disrupts normal HSD17B10 function

  • The HSD17B10-Aβ complex contributes to mitochondrial dysfunction and oxidative stress

• Mitochondrial dysfunction:

  • Independent of its enzymatic activity, HSD17B10 is essential for maintaining mitochondrial structural and functional integrity

  • Loss of HSD17B10 function or its sequestration by Aβ leads to mitochondrial disintegration

  • This results in impaired energy metabolism and increased reactive oxygen species production

• Neural cell death:

  • Impairment of the non-enzymatic function of HSD17B10 in neural cells causes apoptotic cell death

  • This contributes to neurodegeneration observed in AD

• Therapeutic implications:

  • Disrupting the HSD17B10-Aβ interaction may represent a potential therapeutic approach

  • Enhancing HSD17B10's mitochondrial protective functions might delay neurodegeneration

Research indicates that the neuronal dysfunction associated with AD may be more related to the disruption of HSD17B10's structural role in mitochondria than to its enzymatic activity, suggesting that therapeutic approaches focused on preserving mitochondrial integrity may be more effective than those targeting metabolic pathways .

What is the connection between HSD17B10 mutations and intellectual disability syndromes?

HSD17B10 mutations cause a distinct neurodegenerative disorder known as HSD10 Mitochondrial Disease or Syndromic X-Linked Intellectual Disability Type 10. The connection involves:

The severity of clinical symptoms does not correlate with residual enzymatic activity of mutated HSD17B10, which supports the hypothesis that the primary pathogenic mechanism involves disruption of mitochondrial structure and function rather than metabolic dysregulation .

Beyond neurodegeneration, what other biological processes involve HSD17B10?

HSD17B10 participates in multiple biological processes beyond its role in neurodegeneration:

• RNA processing and modification:

  • Functions as a component of mitochondrial ribonuclease P (RNase P)

  • Part of a complex with MRPP1/TRMT10C and MRPP3/KIAA0391

  • Essential for 5' tRNA processing and maturation

  • Critical for mitochondrial translation and protein synthesis

• Steroid metabolism:

  • Catalyzes the beta-oxidation at position 17 of androgens and estrogens

  • Exhibits 3-alpha-hydroxysteroid dehydrogenase activity with androsterone

  • Performs oxidative conversions of bile acids (7-alpha-OH and 7-beta-OH)

  • Shows 20-beta-OH and 21-OH dehydrogenase activities with C21 steroids

  • May influence steroid hormone signaling in various tissues

• Fatty acid metabolism:

  • Catalyzes the third step in beta-oxidation of fatty acids

  • Preferentially acts on straight medium- and short-chain substrates

  • Contributes to energy production and lipid homeostasis

• Immune function and vascular biology:

  • Conditional knockout in immune cells (using Tie2-Cre) causes defects in spleen and vasculature

  • These mice survive to about 25 weeks but develop progressive defects

  • Suggests important roles in immune system development and function

• Embryonic development:

  • Complete knockout of Hsd17b10 in mice results in early embryonic lethality at gastrula stages

  • Indicates essential functions during early developmental processes

These diverse functions highlight HSD17B10 as a crucial metabolic enzyme with roles extending far beyond neurodegeneration, impacting fundamental cellular processes across multiple organ systems .

How can I generate and validate HSD17B10 knockout or knockdown models?

Several approaches for generating HSD17B10 knockout or knockdown models have been validated in the literature:

• CRISPR-Cas9 knockout in cell lines:

  • Design sgRNAs targeting HSD17B10 exons (validated sequences from literature):

    • HSD17B10 1#: 5′-CACCGCCACGGCGGAGCGACTTGT-3′

    • HSD17B10 5#: 5′-CACCGCATGCCCACTATTCCCCCCT-3′

  • Clone sgRNAs into LentiCRISPR-V2 vector

  • Co-transfect with packaging plasmids pSPAX2 and pMD.2G (4:3:1 ratio) into HEK293T cells

  • Collect viral supernatant after 48 hours

  • Infect target cells (e.g., U2OS, HCT116)

  • Select stable cell lines with 1 μg/ml puromycin for 2 weeks

• Conditional knockout mouse models:

  • Generate mice with floxed HSD17B10 allele (exon 1 flanked by loxP sites)

  • Cross with tissue-specific Cre expression lines:

    • DBH-Cre for noradrenergic neuron-specific knockout

    • Tie2-Cre for endothelial and hematopoietic cell knockout

  • Complete knockout is embryonic lethal, highlighting the need for conditional approaches

• siRNA/shRNA knockdown:

  • Design target sequences complementary to HSD17B10 mRNA

  • Transfect cells with siRNA or transduce with shRNA-expressing vectors

  • Typically achieves transient knockdown useful for short-term experiments

• Validation methods:

  • Western blot analysis to confirm protein depletion

  • qRT-PCR to verify mRNA reduction

  • Functional assays to assess HSD17B10 enzymatic activity

  • Microscopic analysis of mitochondrial morphology (critical given HSD17B10's role in mitochondrial integrity)

  • Phenotypic characterization (cell viability, growth, metabolism)

• Rescue experiments:

  • Reintroduce wild-type or mutant HSD17B10 to knockout/knockdown models

  • Compare ability to rescue phenotypes between enzymatically active versus inactive mutants

  • Critical for distinguishing enzymatic from non-enzymatic functions

These approaches have successfully demonstrated that HSD17B10 is essential for mitochondrial integrity independent of its enzymatic activity, providing crucial insights into its role in disease pathogenesis .

What strategies are effective for studying protein-protein interactions involving HSD17B10?

Several complementary strategies have proven effective for studying HSD17B10 protein-protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Overexpression system:

    • Transfect cells with tagged constructs (e.g., Flag-HSD17B10, HA-SIRT3)

    • Lyse cells in appropriate buffer (e.g., BC100 buffer)

    • Immunoprecipitate with anti-Flag/HA beads

    • Wash extensively (4× with BC100 buffer)

    • Elute with 0.1 M glycine followed by neutralization with 1 M Tris Base

    • Analyze by western blot with antibodies against the interaction partner

  • Endogenous system:

    • Lyse cells without overexpression

    • Incubate lysates with specific antibodies (anti-HSD17B10, anti-SIRT3) or control IgG at 4°C overnight

    • Add Protein A/G agarose beads and incubate at 4°C for 8 hours

    • Wash and elute as above

    • This approach confirms physiologically relevant interactions

• GST pull-down assays:

  • Express and purify GST-tagged HSD17B10 or interaction partners

  • Incubate with potential binding partners

  • Pull down with glutathione beads

  • Analyze by western blot

  • This technique confirms direct protein-protein interactions without cellular cofactors

• Proximity ligation assay (PLA):

  • Allows visualization of protein interactions in situ

  • Particularly useful for studying subcellular localization of interactions

  • Can detect endogenous protein interactions without overexpression

• Bimolecular fluorescence complementation (BiFC):

  • Tag HSD17B10 and potential partners with complementary fragments of a fluorescent protein

  • Interaction brings fragments together, restoring fluorescence

  • Enables live-cell imaging of interactions

• Mass spectrometry-based approaches:

  • Immunoprecipitate HSD17B10 followed by mass spectrometry analysis

  • Identifies novel interaction partners and post-translational modifications simultaneously

  • Can be combined with crosslinking for capturing transient interactions

Using these techniques, researchers have identified several important HSD17B10 interactions, including with SIRT3 (which regulates its acetylation) and amyloid-beta (which contributes to Alzheimer's disease pathogenesis) .

How can I troubleshoot non-specific binding when using HSD17B10 antibodies?

Non-specific binding is a common challenge when working with HSD17B10 antibodies. Here are systematic troubleshooting strategies:

• For Western blotting:

  • Optimize blocking conditions:

    • Test different blocking agents (5% non-fat milk, 5% BSA, commercial blockers)

    • Increase blocking time (2-3 hours at room temperature or overnight at 4°C)

  • Adjust antibody dilutions:

    • Use manufacturer-recommended dilutions as starting points (e.g., 1:8000 for Abcepta antibody)

    • Prepare multiple dilution series to determine optimal concentration

  • Modify washing protocol:

    • Increase number of washes (5-6 times instead of 3)

    • Extend washing time (10-15 minutes per wash)

    • Add 0.1-0.2% SDS to TBST for more stringent washing

  • Include competing peptides:

    • Pre-incubate antibody with the immunizing peptide to confirm specificity

  • Use knockout/knockdown controls:

    • Include CRISPR HSD17B10 knockout cell lysates as negative controls

• For immunofluorescence:

  • Optimize fixation method:

    • Compare 4% PFA, methanol, or acetone fixation

    • Test different fixation times

  • Enhance permeabilization:

    • Try different detergents (0.1-0.5% Triton X-100, 0.1% Saponin)

  • Reduce background:

    • Pre-adsorb antibody with acetone powder from tissues/cells

    • Include 0.1-0.3% BSA in antibody dilution buffer

    • Add 5-10% serum from the secondary antibody host species

  • Use specific counterstains:

    • Include mitochondrial markers (MitoTracker, TOM20) to confirm localization

• For immunoprecipitation:

  • Pre-clear lysates:

    • Incubate with Protein A/G beads and control IgG before adding specific antibody

  • Use crosslinking:

    • Crosslink antibody to beads to prevent heavy/light chain interference

  • Modify washing stringency:

    • Adjust salt concentration (150-500 mM NaCl)

    • Add low concentrations of detergents (0.1% NP-40, 0.1% Triton X-100)

• General validation approaches:

  • Peptide competition:

    • Pre-incubate antibody with immunizing peptide

  • Multiple antibodies:

    • Compare results using antibodies targeting different epitopes

  • Genetic validation:

    • Verify using knockout/knockdown systems

  • Antibody validation databases:

    • Check antibody validation resources (Antibodypedia, CiteAb)

These approaches systematically address non-specific binding issues while maintaining detection sensitivity for HSD17B10 .

What are emerging areas for HSD17B10 research beyond currently established functions?

Several emerging research areas represent the frontier of HSD17B10 investigation:

• Role in mitochondrial RNA processing beyond tRNA maturation:

  • Exploring HSD17B10's role in processing other mitochondrial RNAs

  • Investigating its potential involvement in mitochondrial ribosome assembly

  • Examining links between RNA processing defects and disease phenotypes

• Impact on cellular metabolic reprogramming:

  • Investigating how HSD17B10 influences metabolic shifts in cancer cells

  • Exploring its role in nutrient stress responses

  • Examining connections to NAD+ metabolism and mitochondrial redox balance

• Sex-specific functions and hormonal regulation:

  • Given its role in steroid metabolism, exploring sex-specific phenotypes

  • Investigating estrogen-mediated regulation of mitochondrial gene expression through HSD17B10

  • Examining potential roles in sex-specific neurodegeneration patterns

• Post-translational modification network:

  • Beyond acetylation, investigating other modifications (phosphorylation, SUMOylation)

  • Mapping the full post-translational modification landscape

  • Understanding how these modifications form an integrated regulatory network

• Therapeutic targeting approaches:

  • Developing small molecules to modulate specific HSD17B10 functions

  • Exploring peptide-based approaches to disrupt pathological interactions

  • Investigating mitochondrial-targeted interventions to rescue structural defects

• Role in immune regulation and inflammation:

  • Building on observations from Tie2-Cre conditional knockout mice

  • Investigating HSD17B10's role in immune cell metabolism and function

  • Exploring connections to inflammatory pathways in neurodegeneration

These emerging areas highlight the need for interdisciplinary approaches combining structural biology, systems biology, and translational research to fully understand HSD17B10's multifaceted functions and therapeutic potential .

How might single-cell approaches advance our understanding of HSD17B10 in tissue-specific contexts?

Single-cell approaches offer unprecedented opportunities to understand HSD17B10's tissue-specific functions:

• Single-cell transcriptomics:

  • Map HSD17B10 expression across cell types within tissues

  • Identify cell populations most vulnerable to HSD17B10 dysfunction

  • Correlate expression with other mitochondrial genes to identify regulatory networks

  • Track expression changes during development and in disease progression

• Single-cell proteomics:

  • Quantify HSD17B10 protein levels across cell types

  • Identify cell-specific interaction partners

  • Measure post-translational modifications in specific cellular contexts

  • Determine protein abundance correlations within mitochondrial complexes

• Spatial transcriptomics/proteomics:

  • Map HSD17B10 expression within tissue architecture

  • Identify regional heterogeneity in expression patterns

  • Correlate with mitochondrial distribution and morphology

  • Particularly valuable in brain regions affected in neurodegenerative disorders

• Single-cell metabolomics:

  • Measure metabolites processed by HSD17B10 at single-cell resolution

  • Identify cell-specific metabolic consequences of HSD17B10 dysfunction

  • Map metabolic heterogeneity within tissues

• Integrative multi-omics approaches:

  • Combine transcriptomic, proteomic, and metabolomic data from the same cells

  • Build comprehensive models of HSD17B10 function in specific cellular contexts

  • Identify cell-specific vulnerabilities and compensatory mechanisms

• Applications to disease models:

  • Apply to patient-derived tissues/organoids from HSD10 disease patients

  • Compare cellular heterogeneity in disease versus control samples

  • Identify early cellular changes preceding clinical manifestations

  • Discover potential biomarkers for early disease detection

These single-cell approaches would be particularly valuable for understanding why certain cell populations (e.g., specific neuronal subtypes) are more vulnerable to HSD17B10 dysfunction than others, potentially revealing new therapeutic targets for tissue-specific interventions .

What computational and structural biology approaches could advance HSD17B10 research?

Advanced computational and structural biology approaches offer significant potential for HSD17B10 research:

• Structural analysis techniques:

  • Cryo-electron microscopy (cryo-EM):

    • Determine high-resolution structures of HSD17B10 within the mitochondrial RNase P complex

    • Visualize conformational changes during catalysis

    • Analyze structural perturbations caused by disease-associated mutations

  • X-ray crystallography:

    • Obtain atomic-resolution structures of HSD17B10 with various substrates

    • Analyze binding modes of potential inhibitors

    • Study co-crystal structures with interaction partners (e.g., amyloid-beta)

  • Nuclear magnetic resonance (NMR):

    • Characterize dynamic properties and conformational changes

    • Study weak or transient interactions with partners

    • Analyze effects of post-translational modifications on protein dynamics

• Computational methods:

  • Molecular dynamics simulations:

    • Model protein flexibility and conformational changes

    • Simulate effects of mutations on protein stability and dynamics

    • Investigate binding mechanisms with various partners and substrates

  • Network analysis:

    • Model HSD17B10 in the context of protein-protein interaction networks

    • Identify hub proteins and potential regulatory nodes

    • Predict systemic effects of HSD17B10 perturbation

  • Machine learning approaches:

    • Develop predictive models for drug response

    • Identify patterns in multi-omics data related to HSD17B10 function

    • Predict functional consequences of novel mutations

• Drug discovery applications:

  • Virtual screening:

    • Screen compound libraries for potential HSD17B10 modulators

    • Identify molecules that disrupt pathological interactions (e.g., with amyloid-beta)

    • Design inhibitors with specificity for particular functions

  • Fragment-based drug design:

    • Identify chemical fragments that bind to HSD17B10

    • Optimize these fragments into lead compounds

    • Develop function-specific modulators

• Systems biology integration:

  • Genome-scale metabolic modeling:

    • Incorporate HSD17B10 into mitochondrial metabolic models

    • Predict metabolic consequences of altered HSD17B10 activity

    • Identify potential compensatory pathways

  • Multi-scale modeling:

    • Link molecular events to cellular and tissue-level phenotypes

    • Model therapeutic interventions across biological scales