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Probing adenylation: using a fluorescently labelled ATP probe to directly label and immunoprecipitate VopS substrates
Daniel M Lewallen1, Caitlin J Steckler, Bryan Knuckley
1Department of Chemistry, The Scripps Research Institute, Scripps Florida, Jupiter, Florida 33458, USA.
Abstract:
The bacterial effector VopS from Vibrio parahaemolyticus modifies host Rho GTPases to prevent downstream signalling, which leads to cell rounding and eventually apoptosis. While previous studies have used [α-(32)P] ATP for studying this enzyme, we sought to develop a non-radioactive chemical probe of VopS function. To guide these studies, the kinetic parameters were determined for a variety of nucleotides and the results indicated that the C6 position of adenosine was amenable to modification. Since Fl-ATP is a commercially available ATP analogue that is fluorescently tagged at the C6 position, we tested it as a VopS substrate, and the results show that VopS uses Fl-ATP to label Cdc42 in vitro and in MCF7 whole cell extracts. The utility of this probe was further demonstrated by immunoprecipitating Fl-ATP labeled Cdc42 as well as several novel substrate proteins. The proteins, which were identified by LC-MS/MS, include the small GTPases Rac1 and Cdc42 as well as several proteins that are potential VopS substrates and may be important for V. parahaemolyticus pathology. In total, these studies identify Fl-ATP as a valuable chemical probe of protein AMPylation.
Insights
Researchers developed a non-radioactive fluorescent ATP (Fl-ATP) probe to study the bacterial effector VopS. This Fl-ATP probe successfully labeled host Rho GTPases, including Cdc42 and Rac1, revealing new insights into VopS function.
Area of Science:
- Biochemistry
- Molecular Biology
- Microbiology
Background:
- The bacterial effector VopS from Vibrio parahaemolyticus disrupts host cell signaling by modifying Rho GTPases.
- Previous studies on VopS function relied on radioactive ATP ([α-(32)P] ATP), posing limitations for research.
- Developing non-radioactive probes is crucial for safer and more accessible biochemical studies.
Purpose of the Study:
- To develop and validate a non-radioactive chemical probe for studying VopS activity.
- To identify novel VopS substrates and understand its role in host-pathogen interactions.
Main Methods:
- Kinetic parameter determination for VopS with various nucleotides.
- Utilizing fluorescent ATP (Fl-ATP) as a substrate for VopS-mediated protein labeling.
- In vitro and whole-cell extract assays using MCF7 cells.
- Immunoprecipitation of Fl-ATP labeled proteins.
- Protein identification using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).
Main Results:
- Kinetic studies indicated the C6 position of adenosine is suitable for modification.
- Vibrio parahaemolyticus VopS effectively uses Fl-ATP to label Cdc42 in vitro and in cell extracts.
- Fl-ATP labeling enabled the immunoprecipitation of Cdc42 and identification of novel substrate proteins.
- LC-MS/MS identified small GTPases Rac1 and Cdc42, along with other potential VopS substrates.
Conclusions:
- Fluorescent ATP (Fl-ATP) serves as a valuable non-radioactive chemical probe for studying protein AMPylation by VopS.
- This probe facilitates the identification of VopS substrates, including Rac1 and Cdc42.
- The findings contribute to understanding VopS-mediated host cell manipulation and Vibrio parahaemolyticus pathogenesis.
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