Characterization of essential domains in HSD17B13 for cellular localization and enzymatic activity

Yanling Ma,Suman Karki,Philip M Brown,Dennis D Lin,Maren C Podszun,Wenchang Zhou,Olga V Belyaeva,Natalia Y Kedishvili,Yaron Rotman

doi:10.1194/jlr.ra120000907

Abstract

Human genetic studies recently identified an association of SNPs in the 17-β hydroxysteroid dehydrogenase 13 (HSD17B13) gene with alcoholic and nonalcoholic fatty liver disease development. Mutant HSD17B13 variants devoid of enzymatic function have been demonstrated to be protective from cirrhosis and liver cancer, supporting the development of HSD17B13 as a promising therapeutic target. Previous studies have demonstrated that HSD17B13 is a lipid droplet (LD)-associated protein. However, the critical domains that drive LD targeting or determine the enzymatic activity have yet to be defined. Here we used mutagenesis to generate multiple truncated and point-mutated proteins and were able to demonstrate in vitro that the N-terminal hydrophobic domain, PAT-like domain, and a putative α-helix/β-sheet/α-helix domain in HSD17B13 are all critical for LD targeting. Similarly, we characterized the predicted catalytic, substrate-binding, and homodimer interaction sites and found them to be essential for the enzymatic activity of HSD17B13, in addition to our previous identification of amino acid P260 and cofactor binding site. In conclusion, we identified critical domains and amino acid sites that are essential for the LD localization and protein function of HSD17B13, which may facilitate understanding of its function and targeting of this protein to treat chronic liver diseases.

Highlights

Supplementary key words nonalcoholic fatty liver disease alcoholic liver disease retinoids lipid droplets enzyme regulation protein structure
The N terminus of HSD17B13 is predicted by hydropathy analysis to be a putative transmembrane domain and could serve to anchor the protein to lipid droplet (LD); amino acid (AA) 30–300 are likely to reside on the outside membrane surface
To test whether the hydrophobic domain is necessary for HSD17B13 to target LDs, we further generated an HSD17B13 devoid of the hydrophobic AAs 4–16 ( 4–16) and, as expected, found that without this hydrophobic sequence HSD17B13 does not target to LDs (Fig. 1A)

Summary

Introduction

All three protective variants generate protein products that are devoid, or predicted to be devoid of, enzymatic activity, confirming the importance of understanding the enzymatic function of HSD17B13. These data suggest that HSD17B13 activity can be modulated by large domain truncations and deletions, as well as by single or double critical AA mutations, implying the possibility of modulating the enzymatic activity therapeutically, either by interfering with gene expression (i.e., by using antisense oligonucleotides) or by inhibiting activity directly using small synthetic compounds.

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Conclusion