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. 2022 Dec;11(6):e1334.
doi: 10.1002/mbo3.1334.

A yeast-based tool for screening mammalian diacylglycerol acyltransferase inhibitors

Peter Gajdoš  1 , Rodrigo Ledesma-Amaro  2 , Jean-Marc Nicaud  3 , Tristan Rossignol  3
Affiliations

A yeast-based tool for screening mammalian diacylglycerol acyltransferase inhibitors

Peter Gajdoš et al. Microbiologyopen. 2022 Dec.

Abstract

Dysregulation of lipid metabolism is associated with obesity and metabolic diseases but there is also increasing evidence of a relationship between lipid body excess and cancer. Lipid body synthesis requires diacylglycerol acyltransferases (DGATs) which catalyze the last step of triacylglycerol synthesis from diacylglycerol and acyl-coenzyme A. The DGATs and in particular DGAT2, are therefore considered potential therapeutic targets for the control of these pathologies. Here, the murine and the human DGAT2 were overexpressed in the oleaginous yeast Yarrowia lipolytica deleted for all DGAT activities, to evaluate the functionality of the enzymes in this heterologous host and DGAT activity inhibitors. This work provides evidence that mammalian DGATs expressed in Y. lipolytica are a useful tool for screening chemical libraries to identify potential inhibitors or activators of these enzymes of therapeutic interest.

Keywords: DGAT; Yarrowia lipolytica; acyl-CoA:diacylglycerol acyltransferase; heterologous expression; inhibitor screening.

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Conflict of interest statement

None declared.

Figures

Figure 1
Figure 1
Phenotype complementation of strains overexpressing diacylglycerol acyltransferases (DGAT). (a) Strain Y1880 Q4. (b) Strain Y1884 Q4 + YlDGAT1. (c) Strain Y1892 Q4 + YlDGAT2. (d) Strain Y3137 Q4 + MmDGAT2. (e) Strain Y4592 Q4 + UrDGAT2. (f) Strain Y7378 Q4 + HsDGAT2.
Figure 2
Figure 2
Evaluation of lipid body) formation inhibition in Y7378 Q4 + HsDGAT2 using different concentrations of PF‐06424439. (a) 0 µg/mL, (b) 6.25 µg/mL, (c) 12.5 µg/mL, (d) 25 µg/mL, (e) 50 µg/mL.
Figure 3
Figure 3
Y7378 (Q4 + HsDGAT2) representative growth curve on microtiter plate with increasing concentration of PF‐06424439.
Figure 4
Figure 4
Total lipid content of Y7378 (Q4 + HsDGAT2) exposed to different concentrations of PF‐06424439 inhibitor in flask culture, evaluated by gas chromatography. Each concentration of inhibitor was evaluated against the control (0 µg/mL). Asterisks correspond to p‐value < 0.05.
Figure 5
Figure 5
Evaluation of lipid body formation inhibition in the strains overexpressing different diacylglycerol acyltransferases (DGATs) using PF‐06424439 and PF‐046020110 as inhibitors at 25 µg/mL.
Figure 6
Figure 6
Ratio of relative fluorescence/OD600 of strains Y1892 (Q4 + YlDGAT2), Y3137 (Q4 + MmDGAT2), and Y7378 (Q4 + HsDGAT2) exposed to 25 µg/mL of inhibitors. Asterisks correspond to p‐value < 0.05.
Figure A1
Figure A1
(a) Protein sequence alignment. (b) Phylogenetic tree.
Figure A2
Figure A2
Representative growth curve on microtiter plate for the different strains exposed to inhibitors PF‐06424439 and PF‐04620110.
Figure A3
Figure A3
The ratio of relative fluorescence/OD600 of strains Y1880, Y1892, Y3137, and Y7378 exposed to 25 µg/mL of inhibitors. Asterisks correspond to p‐value < 0.05.
See this image and copyright information in PMC

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