Protein S-palmitoylation is a post-translational modification (PTM) where a fatty acyl moiety (saturated 16C palmitate) is linked via a thioester bond to a cysteine residue on target proteins. Unlike myristoylation and prenylation, which are irreversible processes, palmitoylation is dynamic and reversible. Enzymes termed protein acyltransferases (PATs) attach the palmitate group to proteins, while acyl-protein thioesterases (APTs) remove the modification by hydrolysis of the thioester bond. Despite being a PTM that regulates a range of dynamic process including cell signaling, cell division and synapse formation, very few examples of reversibly palmitoylated proteins have been documented, reflecting the general lack of tools available for studying this dynamic process. We recently identified a class of compounds that enhance the process of host cell invasion by the parasite pathogen Toxoplasma gondii. We have determined that these compounds function by binding and inhibiting the parasite homolog of human acyl-protein thioesterase 1 (APT1), a hydrolase involved in depalmitoylation of a range of signaling proteins including Ras, eNOS and G proteins. Our compounds directly block the function of this enzyme, resulting in accumulation of palmitoylated substrates and alteration of parasite motility and organelle secretion. In addition, homology search and recent reports indicate that parasites may express three additional acyl-protein thioesterases. We hypothesize that reversible palmitoylation is a key regulatory process used by T. gondii and likely other human pathogens to regulate important processes and that understanding how regulated removal of palmitate groups on specific substrates will shed light on pathways that can be disrupted for therapeutic gain. Therefore, we propose to 1) determine the repertoire of depalmitoylating enzymes in T. gondii and develop small molecule inhibitors to study their function 2) Use a chemical proteomics strategy to identify candidate protein substrates regulated by dynamic palmitoylation and 3) develop chemical tools to validate the importance of specific depalmitoylation events. This proposal makes use of diverse chemical, biochemical, proteomic and cell biological methods to accomplish these aims.

Public Health Relevance

The proposal outlines plans to develop novel chemical tools and methods to study enzymes involved in the reversible modification of proteins by a palmitate lipid. This modification is used to control such important processes as cell motility, cell divisio and synapse function yet relatively little is known about how it is regulated for specific protein substrates. In particular, the human parasite pathogen Toxoplasma gondii uses palmitoylation to regulate processes that are essential for invasion of host cells. Thus, the methods developed and validated in this proposal will provide valuable information that can be used to guide future design of therapeutic agents for this and other important human pathogens.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM111703-03
Application #
9066755
Study Section
Synthetic and Biological Chemistry A Study Section (SBCA)
Program Officer
Gerratana, Barbara
Project Start
2014-09-01
Project End
2018-05-31
Budget Start
2016-06-01
Budget End
2017-05-31
Support Year
3
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Stanford University
Department
Pathology
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94304
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Amara, Neri; Foe, Ian T; Onguka, Ouma et al. (2018) Synthetic Fluorogenic Peptides Reveal Dynamic Substrate Specificity of Depalmitoylases. Cell Chem Biol :
Hewings, David S; Heideker, Johanna; Ma, Taylur P et al. (2018) Reactive-site-centric chemoproteomics identifies a distinct class of deubiquitinase enzymes. Nat Commun 9:1162
Di Cristina, Manlio; Dou, Zhicheng; Lunghi, Matteo et al. (2017) Toxoplasma depends on lysosomal consumption of autophagosomes for persistent infection. Nat Microbiol 2:17096
Dubey, Rashmi; Staker, Bart L; Foe, Ian T et al. (2017) Membrane skeletal association and post-translational allosteric regulation of Toxoplasma gondii GAPDH1. Mol Microbiol 103:618-634
Child, Matthew A; Garland, Megan; Foe, Ian et al. (2017) Toxoplasma DJ-1 Regulates Organelle Secretion by a Direct Interaction with Calcium-Dependent Protein Kinase 1. MBio 8:
Lentz, Christian S; Ordonez, Alvaro A; Kasperkiewicz, Paulina et al. (2016) Design of Selective Substrates and Activity-Based Probes for Hydrolase Important for Pathogenesis 1 (HIP1) from Mycobacterium tuberculosis. ACS Infect Dis 2:807-815
Foe, Ian T; Child, Matthew A; Majmudar, Jaimeen D et al. (2015) Global Analysis of Palmitoylated Proteins in Toxoplasma gondii. Cell Host Microbe 18:501-11