Trypanosoma brucei Acyl-Protein Thioesterase-like (TbAPT-L) Is a Lipase with Esterase Activity for Short and Medium-Chain Fatty Acids but Has No Depalmitoylation Activity

• Published in: Pathogens (Basel) Vol. 11; no. 11; p. 1245
• Main Authors: Brown, Robert W B, Sharma, Aabha I, Villanueva, Miguel Rey, Li, Xiaomo, Onguka, Ouma, Zilbermintz, Leeor, Nguyen, Helen, Falk, Ben A, Olson, Cheryl L, Taylor, Joann M, Epting, Conrad L, Kathayat, Rahul S, Amara, Neri, Dickinson, Bryan C, Bogyo, Matthew, Engman, David M
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 27.10.2022; MDPI
• Subjects: Acylation; Acyltransferase; alpha/beta hydrolase; Antibiotics; Cloning; depalmitoylation; Enzymes; Epitopes; Esterase; Fatty acids; Gene expression; Homeostasis; Hydrolase; Lipase; Lipids; Localization; Lysates; mRNA; Overexpression; Palmitic acid; palmitoylation; Parasites; Post-translation; post-translational modification; Proteins; RNA polymerase; Substrates; Tagging; Thioesterase; Trypanosoma brucei; trypanosome; United States > US; trypanosome; lipase; thioesterase; depalmitoylation; esterase; post-translational modification; Trypanosoma brucei; alpha/beta hydrolase; palmitoylation
• ISSN: 2076-0817; 2076-0817
• DOI: 10.3390/pathogens11111245

Dynamic post-translational modifications allow the rapid, specific, and tunable regulation of protein functions in eukaryotic cells. -acylation is the only reversible lipid modification of proteins, in which a fatty acid, usually palmitate, is covalently attached to a cysteine residue of a protein by a zDHHC palmitoyl acyltransferase enzyme. Depalmitoylation is required for acylation homeostasis and is catalyzed by an enzyme from the alpha/beta hydrolase family of proteins usually acyl-protein thioesterase (APT1). The enzyme responsible for depalmitoylation in parasites is currently unknown. We demonstrate depalmitoylation activity in live bloodstream and procyclic form trypanosomes sensitive to dose-dependent inhibition with the depalmitoylation inhibitor, palmostatin B. We identified a homologue of human APT1 in which we named TbAPT-like (TbAPT-L). Epitope-tagging of TbAPT-L at N- and C- termini indicated a cytoplasmic localization. Knockdown or over-expression of TbAPT-L in bloodstream forms led to robust changes in TbAPT-L mRNA and protein expression but had no effect on parasite growth , or cellular depalmitoylation activity. Esterase activity in cell lysates was also unchanged when TbAPT-L was modulated. Unexpectedly, recombinant TbAPT-L possesses esterase activity with specificity for short- and medium-chain fatty acid substrates, leading to the conclusion, TbAPT-L is a lipase, not a depalmitoylase.