Acyl Protein Thioesterase Research Articles

Large scale structural rearrangement provides dual control over the catalytic and membrane binding activity of a bacterial serine hydrolase

The serine hydrolase FTT258 from the highly pathogenic bacterium Francisella tularensis exists in distinct open and closed conformations. Based on comparison to the homologous human acyl protein thioesterase, this large structural rearrangement was proposed to provide connected control over the catalytic and membrane binding activity of FTT258. Using comprehensive mutagenesis and differential kinetic and membrane‐binding measurements, we determined the contribution of a key flexible loop to controlling the structural rearrangement and biological activity of FTT258. For the catalytic activity, a centrally located tryptophan residue (Trp66) was deemed essential, with the resulting alanine variant showing complete ablation of enzyme activity. Other amino acids localized near this essential tryptophan residue, including Met63, Arg64, Tyr67, and Asp68, also significantly decreased the hydrolase activity, indicating a critical target area for controlling the enzyme activity and structural rearrangement of FTT258. Combinatorial variants containing the Trp66 substitution with other important residues showed enzymatic activity similar to the Trp66 variant alone, suggesting a controlling role for this residue. Liposome‐binding experiments with the tryptophan variants did not however show significant changes in membrane binding activity. Instead, removal of a proximal positively‐charged arginine residue (Arg64) significantly decreased the membrane binding activity of FTT258. Together, the conformational change in FTT258 dually controlled the catalytic and membrane binding activity of FTT258, but through distinct subsections of a key flexible loop.Support or Funding InformationFunding provided by a Butler University Holcomb research award and a Senior Research Grant from the Indiana Academy of Sciences.

New Insight into the Catalytic and Inhibition Mechanism of the Human Acyl Protein Thioesterase

Reference EPFL-CONF-219863View record in Web of Science Record created on 2016-07-19, modified on 2017-05-12

Acyl protein thioesterase 1 and 2 (APT-1, APT-2) inhibitors palmostatin B, ML348 and ML349 have different effects on NRAS mutant melanoma cells.

Oncogenic NRAS mutations are frequent in melanoma and lead to increased downstream signaling and uncontrolled cell proliferation. Since the direct inhibition of NRAS is not possible yet, modulators of NRAS posttranslational modifications have become an area of interest. Specifically, interfering with NRAS posttranslational palmitoylation/depalmitoylation cycle could disturb proper NRAS localization, and therefore decrease cell proliferation and downstream signaling. Here, we investigate the expression and function of NRAS depalmitoylating acyl protein thioesterases 1 and 2 (APT-1, APT-2) in a panel of NRAS mutant melanoma cells. First, we show that all melanoma cell lines examined express APT-1 and APT-2. Next, we show that siRNA mediated APT-1 and APT-2 knock down and that the specific APT-1 and -2 inhibitors ML348 and ML349 have no biologically significant effects in NRAS mutant melanoma cells. Finally, we test the dual APT-1 and APT-2 inhibitor palmostatin B and conclude that palmostatin B has effects on NRAS downstream signaling and cell viability in NRAS mutant melanoma cells, offering an interesting starting point for future studies.

ABHD17 proteins are novel protein depalmitoylases that regulate N-Ras palmitate turnover and subcellular localization.

Dynamic changes in protein S-palmitoylation are critical for regulating protein localization and signaling. Only two enzymes - the acyl-protein thioesterases APT1 and APT2 - are known to catalyze palmitate removal from cytosolic cysteine residues. It is unclear if these enzymes act constitutively on all palmitoylated proteins, or if additional depalmitoylases exist. Using a dual pulse-chase strategy comparing palmitate and protein half-lives, we found knockdown or inhibition of APT1 and APT2 blocked depalmitoylation of Huntingtin, but did not affect palmitate turnover on postsynaptic density protein 95 (PSD95) or N-Ras. We used activity profiling to identify novel serine hydrolase targets of the APT1/2 inhibitor Palmostatin B, and discovered that a family of uncharacterized ABHD17 proteins can accelerate palmitate turnover on PSD95 and N-Ras. ABHD17 catalytic activity is required for N-Ras depalmitoylation and re-localization to internal cellular membranes. Our findings indicate that the family of depalmitoylation enzymes may be substantially broader than previously believed.

Global Analysis of Palmitoylated Proteins in Toxoplasma gondii

Post-translational modifications (PTMs) such as palmitoylation are critical for the lytic cycle of the protozoan parasite Toxoplasma gondii. While palmitoylation is involved in invasion, motility, and cell morphology, the proteins that utilize this PTM remain largely unknown. Using a chemical proteomic approach, we report a comprehensive analysis of palmitoylated proteins in T. gondii, identifying a total of 282 proteins, including cytosolic, membrane-associated, and transmembrane proteins. From this large set of palmitoylated targets, we validate palmitoylation of proteins involved in motility (myosin light chain 1, myosin A), cell morphology (PhIL1), and host cell invasion (apical membrane antigen 1, AMA1). Further studies reveal that blocking AMA1 palmitoylation enhances the release of AMA1 and other invasion-related proteins from apical secretory organelles, suggesting a previously unrecognized role for AMA1. These findings suggest that palmitoylation is ubiquitous throughout the T. gondii proteome and reveal insights into the biology of this important human pathogen.

MiRs-138 and -424 control palmitoylation-dependent CD95-mediated cell death by targeting acyl protein thioesterases 1 and 2 in CLL

AbstractResistance toward CD95-mediated apoptosis is a hallmark of many different malignancies, as it is known from primary chronic lymphocytic leukemia (CLL) cells. Previously, we could show that miR-138 and -424 are downregulated in CLL cells. Here, we identified 2 new target genes, namely acyl protein thioesterase (APT) 1 and 2, which are under control of both miRs and thereby significantly overexpressed in CLL cells. APTs are the only enzymes known to promote depalmitoylation. Indeed, membrane proteins are significantly less palmitoylated in CLL cells compared with normal B cells. We identified APTs to directly interact with CD95 to promote depalmitoylation, thus impairing apoptosis mediated through CD95. Specific inhibition of APTs by siRNAs, treatment with miRs-138/-424, and pharmacologic approaches restore CD95-mediated apoptosis in CLL cells and other cancer cells, pointing to an important regulatory role of APTs in CD95 apoptosis. The identification of the depalmitoylation reaction of CD95 by APTs as a microRNA (miRNA) target provides a novel molecular mechanism for how malignant cells escape from CD95-mediated apoptosis. Here, we introduce palmitoylation as a novel posttranslational modification in CLL, which might impact on localization, mobility, and function of molecules, survival signaling, and migration.

Enzymatic protein depalmitoylation by acyl protein thioesterases.

Protein palmitoylation is a dynamic post-translational modification, where the 16-carbon fatty acid, palmitate, is added to cysteines of proteins to modulate protein sorting, targeting and signalling. Palmitate removal from proteins is mediated by acyl protein thioesterases (APTs). Although initially identified as lysophospholipases, increasing evidence suggests APT1 and APT2 are the major APTs that mediate the depalmitoylation of diverse cellular substrates. Here, we describe the conserved functions of APT1 and APT2 across organisms and discuss the possibility that these enzymes are members of a larger family of depalmitoylation enzymes.

Differential proteomic analysis of the anti-depressive effects of oleamide in a rat chronic mild stress model of depression

Depression is a complex psychiatric disorder, and its etiology and pathophysiology are not completely understood. Depression involves changes in many biogenic amine, neuropeptide, and oxidative systems, as well as alterations in neuroendocrine function and immune-inflammatory pathways. Oleamide is a fatty amide which exhibits pharmacological effects leading to hypnosis, sedation, and anti-anxiety effects. In the present study, the chronic mild stress (CMS) model was used to investigate the antidepressant-like activity of oleamide. Rats were exposed to 10weeks of CMS or control conditions and were then subsequently treated with 2weeks of daily oleamide (5mg/kg, i.p.), fluoxetine (10mg/kg, i.p.), or vehicle. Protein extracts from the hippocampus were then collected, and hippocampal maps were generated by way of two-dimensional gel electrophoresis (2-DE). Altered proteins induced by CMS and oleamide were identified through mass spectrometry and database searches. Compared to the control group, the CMS rats exhibited significantly less body weight gain and decreased sucrose consumption. Treatment with oleamide caused a reversal of the CMS-induced deficit in sucrose consumption. In the proteomic analysis, 12 protein spots were selected and identified. CMS increased the levels of adenylate kinase isoenzyme 1 (AK1), nucleoside diphosphate kinase B (NDKB), histidine triad nucleotide-binding protein 1 (HINT1), acyl-protein thioesterase 2 (APT-2), and glutathione S-transferase A4 (GSTA4). Compared to the CMS samples, seven spots changed significantly following treatment with oleamide, including GSTA4, glutathione S-transferase A6 (GSTA6), GTP-binding nuclear protein Ran (Ran-GTP), ATP synthase subunit d, transgelin-3, small ubiquitin-related modifier 2 (SUMO2), and eukaryotic translation initiation factor 5A-1 (eIF5A1). Of these seven proteins, the level of eIF5A1 was up-regulated, whereas the remaining proteins were down-regulated. In conclusion, oleamide has antidepressant-like properties in the CMS rat model. The identification of proteins altered by CMS and oleamide treatment provides support for targeting these proteins in the development of novel therapies for depression.

Mirs-138 and -424 Control Palmitoylation-Dependent CD95-Mediated Cell Death By Targeting Acyl Protein Thioesterases 1 and 2 in Chronic Lymphocytic Leukemia

Introduction: Resistance towards CD95-mediated apoptosis is a hallmark of many different malignancies, like it is known from primary chronic lymphocytic leukemia (CLL) cells. Moreover, apoptosis mediated through CD95 is an essential mechanism to eliminate e.g. auto-reactive or virally infected cells. However, its mode of action is still not fully understood. Recently, it could be shown that palmitoylation of CD95 can influence its signaling properties. Nevertheless, the role and regulation of palmitoylated CD95 still needs to be determined.Methods and results: Previously, we could show that miR-138 and -424 are down-regulated in CLL cells. By applying luciferase reporter assays, mutations of the binding sites qRT-PCR and immunoblots after transfection of both miRs, we identified two new target genes, namely acyl protein thioesterase (APT) 1 and 2, which are under control of both miRs and thereby are significantly over-expressed in CLL cells. Interestingly, our data reveal that expression of APTs is already controlled by miRs on mRNA level. This way APT1 is regulated by miR-138 and expression of APT2 is controlled by miR-424. So far, APTs are the only enzymes known to promote de-palmitoylation. Indeed, membrane proteins are significantly less palmitoylated in CLL cells compared to normal B cells as we determined by click-chemistry, which is a non-radioactive method to determine palmitoylated proteins. Importantly, via acyl-biotin exchange assays with subsequent immunoprecipitation of CD95 and fluorescence lifetime imaging microscopy (FLIM) to Foerster resonance energy transfer (FRET) in living cells we identified APTs to directly interact with CD95 to promote de-palmitoylation, thus impairing apoptosis mediated through CD95. As proof of concept APTs were inhibited specifically by siRNAs, miRs-138/-424 or our pharmacological inhibitor Palmostatin B. Thereby we could restore CD95-mediated apoptosis in CLL cells and other cancers, pointing to a central regulatory role of APTs in CD95 apoptosis.Conclusion: The identification of the de-palmitoylation reaction of CD95 by APTs as a miRNA target provides a novel molecular mechanism how malignant cells escape from CD95-mediated apoptosis. Here, we introduce palmitoylation as a novel post-translational modification in CLL. In light of global palmitoylome studies, which show that potentially palmitoylated proteins are involved in all central cellular processes, such as protein transport, survival, migration, apoptosis and B-cell receptor signaling, this emphasizes the importance of palmitoylation and might put it on par with modifications like phosphorylation. DisclosuresNo relevant conflicts of interest to declare.

Involvement of RARRES3 in the regulation of Wnt proteins acylation and signaling activities in human breast cancer cells.

The Wnt/β-catenin signaling pathway has emerged as a key regulator of complex biological processes, such as embryonic development, cell proliferation, cell fate decision and tumorigenesis. Recent studies have shown that the deregulation of Wnt/β-catenin signaling is frequently observed and leads to abnormal cell growth in human breast cancer cells. In this study, we identified a novel regulatory mechanism of Wnt/β-catenin signaling through RARRES3 that targets and modulates the acylation status of Wnt proteins and co-receptor low-density lipoprotein receptor-related protein 6, resulting in the suppression of epithelial-mesenchymal transition and cancer stem cell properties. Mutation of the conserved active site residues of RARRES3 indicates that RARRES3 serves as an acyl protein thioesterase that tethers its target proteins and modulates their acylation status. Furthermore, the functions of p53 in cell proliferation and Wnt/β-catenin signaling are significantly associated with the induction of RARRES3. Thus our findings provide a new insight into the molecular link between p53, protein acylation and Wnt/β-catenin signaling whereby RARRES3 plays a pivotal role in modulating the acylation status of signaling proteins.

Abstract 5276: Phospholipase A/acyltransferase enzyme activity of H-rev107 inhibits the H-RAS signal pathway

Abstract H-rev107, also called HRASLS3 or PLA2G16, is a member of the HREV107 type II tumor suppressor gene family that includes HREV107, Retinoid-inducible gene 1, and HRASLS. The HREV107 protein family contains an NC domain, with unknown function at the N-terminus, and a hydrophobic membrane-anchoring domain at the C-terminus. Previous study showed that the H-rev107 gene specifically expressed in H-ras resistant fibroblasts. However, the mode of action and the site of inhibition of H-rev107 are still unknown. Our results revealed that H-rev107 reduced the levels of activated RAS (RAS-GTP) or Elk1-mediated transactivation in epidermal growth factor stimulated HtTA cells that expressed H-RAS. Further, we found that H-rev107 reduced H-RAS palmitoylation determined by acyl-biotin exchange assay. Palmitoylation or S-acylation is the post-translational attachment of fatty acids to cysteine residues and continuous cycles of de- and reacylation reactions accounts for the specific subcellular distribution of RAS. H-rev107 was shown as a phospholipase A/Acyltransferase (PLA/AT) that involved in the phospholipid metabolism. We next determined whether H-rev107 affected H-RAS palmitoylation through deacylation/acylation by acyl-protein thioesterases (APT1) or PLA/AT using APT1 inhibitor (palmostatin B) or PLA/AT inhibitor (arachidonyl trifluoromethyl ketone, AACOCF3). Treating cells with AACOCF3 can increase H-rev107-induced acylated H-RAS suppression of HtTA cells. Also, AACOCF3 significantly increased the levels of activated RAS and Elk-mediated transactivation in H-rev107 expressed cells. However, treating cells with palmostatin B enhanced H-rev107-induced acylated H-RAS suppression in H-rev107 expressing cells. In conclusion, H-rev107 can inhibit H-RAS palmitoylation to reduce its GTP-form levels and further suppress the downstream signal pathway. Our study suggested that PLA/AT activity of H-rev107 might play an important role in H-rev107 mediated RAS suppression. Note: This abstract was not presented at the meeting. Citation Format: Fu-Ming Tsai, Chang-Chieh Wu, Chun-Hua Wang, Tzung-Chieh Tsai, Rong-Yaun Shyu, Shun-Yuan Jiang. Phospholipase A/acyltransferase enzyme activity of H-rev107 inhibits the H-RAS signal pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5276. doi:10.1158/1538-7445.AM2014-5276

Palmitoylation and depalmitoylation defects.

Palmitoylation describes the enzymatic attachment of a 16-carbon atom fatty acid to a target protein. Such lipidation events occur in all eukaryotes and can be of reversible (S-palmitoylation) or irreversible (N-palmitoylation) nature. In particular S-palmitoylation is dynamically regulated by two opposing types of enzymes which add (palmitoyl acyltransferases - PAT) or remove (acyl protein thioesterases) palmitate from proteins. Protein palmitoylation is an important process that dynamically regulates the assembly and compartmentalization of many neuronal proteins at specific subcellular sites. Enzymes that regulate protein palmitoylation are critical for several biological processes. To date, eight palmitoylation related genes have been reported to be associated with human disease. This review intends to give an overview on the pathological changes which are associated with defects in the palmitoylation/depalmitoylation process.

Phospholipase A/Acyltransferase enzyme activity of H-rev107 inhibits the H-RAS signaling pathway

BackgroundH-rev107, also called HRASLS3 or PLA2G16, is a member of the HREV107 type II tumor suppressor gene family. Previous studies showed that H-rev107 exhibits phospholipase A/acyltransferase (PLA/AT) activity and downregulates H-RAS expression. However, the mode of action and the site of inhibition of H-RAS by H-rev107 are still unknown.ResultsOur results indicate that H-rev107 was co-precipitated with H-RAS and downregulated the levels of activated RAS (RAS-GTP) and ELK1-mediated transactivation in epidermal growth factor-stimulated and H-RAS-cotransfected HtTA cervical cancer cells. Furthermore, an acyl-biotin exchange assay demonstrated that H-rev107 reduced H-RAS palmitoylation. H-rev107 has been shown to be a PLA/AT that is involved in phospholipid metabolism. Treating cells with the PLA/AT inhibitor arachidonyl trifluoromethyl ketone (AACOCF3) or methyl arachidonyl fluorophosphate (MAFP) alleviated H-rev107-induced downregulation of the levels of acylated H-RAS. AACOCF3 and MAFP also increased activated RAS and ELK1-mediated transactivation in H-rev107-expressing HtTA cells following their treatment with epidermal growth factor. In contrast, treating cells with the acyl-protein thioesterase inhibitor palmostatin B enhanced H-rev107-mediated downregulation of acylated H-RAS in H-rev107-expressing cells. Palmostatin B had no effect on H-rev107-induced suppression of RAS-GTP levels or ELK1-mediated transactivation. These results suggest that H-rev107 decreases H-RAS activity through its PLA/AT activity to modulate H-RAS acylation.ConclusionsWe made the novel observation that H-rev107 decrease in the steady state levels of H-RAS palmitoylation through the phospholipase A/acyltransferase activity. H-rev107 is likely to suppress activation of the RAS signaling pathway by reducing the levels of palmitoylated H-RAS, which decreases the levels of GTP-bound H-RAS and also the activation of downstream molecules. Our study further suggests that the PLA/AT activity of H-rev107 may play an important role in H-rev107-mediated RAS suppression.

Identification of Bioactivating Enzymes Involved in the Hydrolysis of Laninamivir Octanoate, a Long-Acting Neuraminidase Inhibitor, in Human Pulmonary Tissue

Laninamivir octanoate (LO) is an octanoyl ester prodrug of the neuraminidase inhibitor laninamivir. After inhaled administration, LO exhibits clinical efficacy for both treatment and prophylaxis of influenza virus infection, resulting from hydrolytic bioactivation into its pharmacologically active metabolite laninamivir in the pulmonary tissue. In this study, we focused on the identification of LO-hydrolyzing enzymes from human pulmonary tissue extract using proteomic correlation profiling-a technology integration of traditional biochemistry and proteomics. In a single elution step by gel-filtration chromatography, LO-hydrolyzing activity was separated into two distinct peaks, designated as peak I and peak II. By mass spectrometry, 1160 and 1003 proteins were identified and quantitated for peak I and peak II, respectively, and enzyme candidates were ranked based on the correlation coefficient between the enzyme activity and the proteomic profiles. Among proteins with a high correlation value, S-formylglutathione hydrolase (esterase D; ESD) and acyl-protein thioesterase 1 (APT1) were selected as the most likely candidates for peak I and peak II, respectively, which was confirmed by LO-hydrolyzing activity of recombinant proteins. In the case of peak II, LO-hydrolyzing activity was completely inhibited by treatment with a specific APT1 inhibitor, palmostatin B. Moreover, immunohistochemical analysis revealed that both enzymes were mainly localized in the pulmonary epithelia, a primary site of influenza virus infection. These findings demonstrate that ESD and APT1 are key enzymes responsible for the bioactivation of LO in human pulmonary tissue.

The Autodepalmitoylating Activity of APT Maintains the Spatial Organization of Palmitoylated Membrane Proteins

The localization and signaling of S-palmitoylated peripheral membrane proteins is sustained by an acylation cycle in which acyl protein thioesterases (APTs) depalmitoylate mislocalized palmitoylated proteins on endomembranes. However, the APTs are themselves reversibly S-palmitoylated, which localizes thioesterase activity to the site of the antagonistc palmitoylation activity on the Golgi. Here, we resolve this conundrum by showing that palmitoylation of APTs is labile due to autodepalmitoylation, creating two interconverting thioesterase pools: palmitoylated APT on the Golgi and depalmitoylated APT in the cytoplasm, with distinct functionality. By imaging APT-substrate catalytic intermediates, we show that it is the depalmitoylated soluble APT pool that depalmitoylates substrates on all membranes in the cell, thereby establishing its function as release factor of mislocalized palmitoylated proteins in the acylation cycle. The autodepalmitoylating activity on the Golgi constitutes a homeostatic regulation mechanism of APT levels at the Golgi that ensures robust partitioning of APT substrates between the plasma membrane and the Golgi.

Acyl protein thioesterase inhibitors as probes of dynamic S-palmitoylation.

Protein palmitoylation describes the hydrophobic post-translational modification of cysteine residues in certain proteins, and is required for the spatial organization and composition of cellular membrane environments. Certain palmitoylated proteins are processed by acyl protein thioesterase (APT) enzymes, which catalyze thioester hydrolysis of palmitoylated cysteine residues. Inhibiting APT enzymes disrupts Ras trafficking and attenuates oncogenic growth signaling, highlighting these enzymes as potential therapeutic targets. As members of the serine hydrolase enzyme family, APT enzymes can be assayed by fluorophosphonate activity-based protein profiling (ABPP) methods, allowing rapid profiling of inhibitor selectivity and potency. In this review, we discuss recent progress in the development of potent and selective inhibitors to APT enzymes, including both competitive and non-competitive chemotypes. These examples highlight how ABPP methods integrate with medicinal chemistry for the discovery and optimization of inhibitors in complex proteomes.

2-Bromopalmitate Reduces Protein Deacylation by Inhibition of Acyl-Protein Thioesterase Enzymatic Activities

S-acylation, the covalent attachment of palmitate and other fatty acids on cysteine residues, is a reversible post-translational modification that exerts diverse effects on protein functions. S-acylation is catalyzed by protein acyltransferases (PAT), while deacylation requires acyl-protein thioesterases (APT), with numerous inhibitors for these enzymes having already been developed and characterized. Among these inhibitors, the palmitate analog 2-brompalmitate (2-BP) is the most commonly used to inhibit palmitoylation in cells. Nevertheless, previous results from our laboratory have suggested that 2-BP could affect protein deacylation. Here, we further investigated in vivo and in vitro the effect of 2-BP on the acylation/deacylation protein machinery, with it being observed that 2-BP, in addition to inhibiting PAT activity in vivo, also perturbed the acylation cycle of GAP-43 at the level of depalmitoylation and consequently affected its kinetics of membrane association. Furthermore, 2-BP was able to inhibit in vitro the enzymatic activities of human APT1 and APT2, the only two thioesterases shown to mediate protein deacylation, through an uncompetitive mechanism of action. In fact, APT1 and APT2 hydrolyzed both the monomeric form as well as the micellar state of the substrate palmitoyl-CoA. On the basis of the obtained results, as APTs can mediate deacylation on membrane bound and unbound substrates, this suggests that the access of APTs to the membrane interface is not a necessary requisite for deacylation. Moreover, as the enzymatic activity of APTs was inhibited by 2-BP treatment, then the kinetics analysis of protein acylation using 2-BP should be carefully interpreted, as this drug also inhibits protein deacylation.

Characterization of a Serine Hydrolase Targeted by Acyl-protein Thioesterase Inhibitors in Toxoplasma gondii

In eukaryotic organisms, cysteine palmitoylation is an important reversible modification that impacts protein targeting, folding, stability, and interactions with partners. Evidence suggests that protein palmitoylation contributes to key biological processes in Apicomplexa with the recent palmitome of the malaria parasite Plasmodium falciparum reporting over 400 substrates that are modified with palmitate by a broad range of protein S-acyl transferases. Dynamic palmitoylation cycles require the action of an acyl-protein thioesterase (APT) that cleaves palmitate from substrates and conveys reversibility to this posttranslational modification. In this work, we identified candidates for APT activity in Toxoplasma gondii. Treatment of parasites with low micromolar concentrations of β-lactone- or triazole urea-based inhibitors that target human APT1 showed varied detrimental effects at multiple steps of the parasite lytic cycle. The use of an activity-based probe in combination with these inhibitors revealed the existence of several serine hydrolases that are targeted by APT1 inhibitors. The active serine hydrolase, TgASH1, identified as the homologue closest to human APT1 and APT2, was characterized further. Biochemical analysis of TgASH1 indicated that this enzyme cleaves substrates with a specificity similar to APTs, and homology modeling points toward an APT-like enzyme. TgASH1 is dispensable for parasite survival, which indicates that the severe effects observed with the β-lactone inhibitors are caused by the inhibition of non-TgASH1 targets. Other ASH candidates for APT activity were functionally characterized, and one of them was found to be resistant to gene disruption due to the potential essential nature of the protein.

The role of palmitoyl-protein thioesterases in T cell activation (P1398)

Abstract Reversible protein palmitoylation is a posttranslational modification important for transport, localization and function of proteins. The acyl-protein thioesterase 1 (APT1) also known as lysophospholipase 1 (LYPLA1) is a cytosolic depalmitoylating enzyme. Another thioesterase APT2 (LYPLA2) is highly homologous to APT1. Jurkat T cells were chosen to analyze the role of APT1 and APT2 in T cell activation. APT1/2 mRNA expression was measured by qPCR and knockdown of APT1/2 gene expression was accomplished by RNA interference, which consisted of transient siRNA expression by electroporation or by stable expression of shRNA by lentiviral infection. Palmostatin B was used to inhibit the APT thioesterase activity pharmacologically. The functional effects of APT1/2 knockdown or inhibition were measured by IL-2 secretion (ELISA) and CD69 upregulation (flow cytometry) after CD3/CD28 stimulation. Jurkat cells expressed APT2 mRNA 3.5-fold higher than APT1 mRNA. Even greater differences were found in primary human and murine T cells. Both, transient and stable knockdown of each of the APTs alone reduced IL-2 secretion and CD69 upregulation following CD3/CD28 stimulation. The effect of APT2 knockdown was always stronger than that of APT1. Inhibition with palmostatin B blocked cell activation in Jurkat cells and in human peripheral T cells. Thus, both palmitoyl-protein thioesterases APT1 and APT2 are involved in T cell activation, but APT2 seems to be the predominant enzyme in T cells.

Measuring bacterial S‐acylation in Pseudomonas putida

In eukaryotes, reversible palmitoylation of cysteine residues via a thioester linkage allows dynamic shuttling of proteins between intracellular compartments. Regulation of protein localization by palmitoylation is involved in key cellular processes including cell signaling, ion transport, and neuronal communication. These dynamic cycles of palmitoylation and depalmitoylation are controlled by two classes of cellular enzymes, protein acyl transferases and acyl‐protein thioesterases. While bacterial proteins are S‐acylated and homologues of human acyl protein thioesterase are present in many gram‐negative bacterium, dynamic cycles of S‐acylation have not been observed in prokaryotes. In this study, we utilized azide mimics of palmitic acid to measure the level of protein S‐acylation in Pseudomonas putida. S‐acylated proteins were then detected by click chemistry using either biotin or fluorophore labeled alkynes. Specific S‐acylated proteins were identified in P. putida and their identities are currently being confirmed. Using deletion strains for potential bacterial palmitoylation machinery, we plan to search for proteins that regulate levels of S‐acylation in P. putida. Funding provided by Butler University Holcomb research fund.