Recombinant Human 17-beta-hydroxysteroid dehydrogenase 13 (HSD17B13)

What is HSD17B13 and what are its primary tissue expression patterns?

HSD17B13 is a liver-enriched, hepatocyte-specific protein belonging to the hydroxysteroid dehydrogenase family. It functions as an NAD(P)H/NAD(P)+-dependent oxidoreductase . The protein is primarily expressed in the liver, with significantly higher expression levels observed in patients with non-alcoholic fatty liver disease (NAFLD) .

Methodology for expression analysis:

• RNA-seq and RT-PCR have demonstrated that HSD17B13 is liver-enriched, though recent research has detected expression in the kidney, ureter, and urinary bladder .

• Quantitative studies show HSD17B13 expression is elevated in multiple models of MASLD (metabolic dysfunction-associated steatotic liver disease) and normalizes with the prevention of obesity and steatotic liver .

What is the subcellular localization of HSD17B13 and how can it be verified?

HSD17B13 is a lipid droplet (LD)-associated protein with specific domains required for this localization .

Methods to verify subcellular localization:

• Fluorescence microscopy using GFP-tagged HSD17B13 constructs has confirmed its lipid droplet localization .

• Subcellular fractionation followed by Western blotting of lipid droplet fractions.

• Immunofluorescence studies in primary human hepatocytes show HSD17B13 targeting to lipid droplets under various conditions, including oleate or palmitate treatment .

What enzymatic activities has HSD17B13 been demonstrated to possess?

HSD17B13 exhibits multiple enzymatic activities involving:

• Steroid metabolism (particularly 17β-hydroxysteroid dehydrogenase activity)

• Retinol metabolism (retinol dehydrogenase activity)

• Lipid metabolism

• Pyrimidine catabolism at the level of dihydropyrimidine dehydrogenase

Methods for measuring enzymatic activity:

• MALDI-TOF-MS-based assays using estrone as substrate

• RapidFire MS enzyme activity assays with estradiol, LTBR4, or retinol as substrates

• Synthetic substrates with improved LogD properties have been developed to measure selective HSD17B13 activity

How does HSD17B13 contribute to liver pathophysiology?

HSD17B13 plays dual roles in liver disease:

The protein induces G1/S cell cycle delay in HCC cells by upregulating P21, P27, and MMP2 expression , while potentially promoting lipid accumulation in NAFLD through its enzymatic activity.

Which genetic variants of HSD17B13 confer protection against liver disease?

Several loss-of-function variants of HSD17B13 have been identified as protective:

Detection methods include:

• Exome-wide association studies

• Targeted genotyping of rs72613567 using PCR-based methods

• RNA splicing analysis to detect variant transcripts

What structural domains are critical for HSD17B13 lipid droplet targeting?

Mutagenesis studies have identified three critical domains required for proper LD targeting:

• N-terminal hydrophobic domain (amino acids 4-16): Deletion of this region (Δ4-16) abolishes LD targeting and results in mitochondrial proximity localization .

• PAT-like domain (amino acids 22-28): Essential for stability and LD targeting .

• α-helix/β-sheet/α-helix structure (amino acids 69-106):

  • First α-helix (aa 69-84)

  • β-sheet (aa 85-93)

  • Second α-helix (aa 94-106)

The entire intact structure is required, as deletion of any of these three components impairs LD localization. Without these domains, HSD17B13 is retained in the endoplasmic reticulum .

Experimental approach: Generate truncated and point-mutated proteins, express them in cell lines using fluorescent protein tags, and analyze subcellular localization using confocal microscopy.

What mechanisms underlie HSD17B13's protective effects when inhibited or mutated?

Multiple mechanisms have been identified:

• Cell cycle regulation: HSD17B13 induces accumulation of cells in G1 phase and reduction in S and G2 phases via upregulation of P21, P27, and MMP2 .

• Pyrimidine metabolism: Protection against liver fibrosis by the rs72613567-A variant in humans and Hsd17b13 knockdown in mice is associated with decreased pyrimidine catabolism at the level of dihydropyrimidine dehydrogenase .

• Lipid metabolism modulation:

  • Hsd17b13 knockdown in high-fat diet mice decreases diacylglycerols (e.g., DAG 34:3)

  • Increases phosphatidylcholines containing polyunsaturated fatty acids (e.g., PC 34:3 and PC 42:10)

  • Reciprocally regulates expression of lipid metabolism genes like Cd36 and phospholipid metabolism genes like Cept1

Methodology: Combine genetic (knockdown/overexpression), metabolomic (LC-MS/MS), and transcriptomic (RNA-seq) approaches to comprehensively map the pathways affected by HSD17B13 modulation.

How do structural features of HSD17B13 inform inhibitor development?

Crystal structures of HSD17B13 have revealed:

• Cofactor binding: HSD17B13 is NAD+-dependent, with the cofactor crucial for proper protein folding and inhibitor binding .

• Inhibitor binding sites: Two distinct series of inhibitors interact with the active site residues and bound cofactor similarly, but occupy different paths leading to the active site .

• Membrane association mechanism: Structures provide insights into how lipid droplet-associated proteins anchor to membranes .

• NAD+ dependency: The phenol lead series of inhibitors, including BI-3231, showed strong NAD+ dependency for binding and inhibition of HSD17B13 .

Approach: Structure-based drug design using crystallography data can inform the development of selective inhibitors. The first potent and selective HSD17B13 inhibitor, BI-3231 (compound 45), has been identified through high-throughput screening followed by optimization .

How can recombinant HSD17B13 be effectively produced and utilized in enzymatic assays?

Production of high-quality recombinant HSD17B13:

• Expression systems:

  • E. coli (for tag-free protein)

  • Yeast (for N-terminal 6xHis-tagged and C-terminal Myc-tagged protein)

• Protein regions: Expression region 20-300aa has been successfully used .

• Purity assessment: >85-90% purity can be achieved as determined by SDS-PAGE .

For enzymatic assays:

• Use NAD+ as a cofactor (0.5 mM for human HSD17B13, 10 mM for mouse HSD17B13)

• Substrates include estradiol/estrone (30 μM), retinol (30 μM), or synthetic substrates with improved properties

• Detection methods include RapidFire MS and MALDI-TOF-MS

What is the relationship between HSD17B13 knockdown and hepatic lipid profiles?

Liver-specific shRNA-mediated knockdown of Hsd17b13 in high-fat diet obese mice produces specific changes in hepatic lipidome:

These changes are associated with:

• Improved hepatic steatosis without effects on body weight, adiposity, or glycemia

• Decreased serum ALT and FGF21 levels

• Reduced markers of liver fibrosis (e.g., Timp2 expression)

• Altered expression of key genes in phospholipid and PUFA metabolism (e.g., Cept1)

Methodology: Combine liver-specific knockdown with global lipidomic analysis (LC-MS/MS) and transcriptomic profiling to identify affected pathways and potential mechanisms.

How do HSD17B13 inhibitors differ in their mechanisms and selectivity?

The development of HSD17B13 inhibitors has revealed important features:

• BI-3231 (compound 45):

  • First potent and selective chemical probe reported for HSD17B13

  • Shows NAD+ dependency for binding and inhibition

  • Exhibits extensive liver tissue accumulation despite high clearance

  • Available through the opnMe platform for research use

• Synthetic substrates:

  • Compounds with modified polarity (LogD 0.5 and 0.4) show excellent substrate activity

  • Similar Km and Vmax to reference compounds

  • Minimal turnover in hepatocytes, indicating selectivity for HSD17B13

Analysis approach: Determine compound potencies by fitting dose-response data to a four-parameter logistical equation, with calculations of fm (fraction metabolized by HSD17B13) and fm1 (fraction of specific product generated by HSD17B13) .

What experimental models are most appropriate for studying HSD17B13 function?

Methodology considerations:

• For subcellular localization studies, primary human hepatocytes provide more relevant context than immortalized cell lines

• For metabolic studies, high-fat diet mouse models capture the complex interplay of metabolic pathways affected by HSD17B13

• For mechanistic insights, combine in vitro enzymatic assays with targeted gene expression studies

How can crystal structures of HSD17B13 guide understanding of disease-associated variants?

Crystal structures of HSD17B13 have provided significant insights:

• Structure-function relationship: The structures reveal how disease-associated variants might disrupt function:

  • The rs72613567 variant (IsoD) produces a truncated P274del protein that lacks stability

  • The A192Lfs variant creates a truncated protein distinct from IsoD

  • The P260S variant likely disrupts protein folding, leading to instability

• Active site architecture: Crystal structures with NAD+ cofactor and small molecule inhibitors from two distinct series in the ligand binding pocket reveal:

  • Different paths leading to the active site

  • Interaction patterns with active site residues

  • Basis for developing structure-guided inhibitors

Approach: Use structural information to design mutagenesis experiments that can validate the functional importance of specific residues and domains in HSD17B13.

Human genetic and crystal structure studies together provide complementary evidence for therapeutic targeting of HSD17B13 for liver disease treatment.