PhD Scientific Days 2019

Oral presentations: Molecular sciences

Development of a BRET-based GTPase assay for the investigation of ARHGAP25 regulation

Előadó neve

Dr. Wisniewski, Éva

Előadó munkahelye

Semmelweis University, Department of Physiology

Előadó telefonszáma

+3614591500/60458

Előadó e-mail címe

wisniewski.eva@med.semmelweis-univ.hu

Az előadás címe

Development of a BRET-based GTPase assay for the investigation of ARHGAP25 regulation

Szerző(k) neve és munkahelye

Éva Wisniewski1, Fanni Kovács1, Enikő Bahurek1, Roland Csépányi-Kömi1, Erzsébet Ligeti1
1Semmelweis University, Department of Physiology, Budapest, Hungary

Bemutatás módja

Szóbeli

Szekció

Oral presentations: Molecular sciences

Language of the presentation

English

Témacsoport

molecular sciences

Összefoglaló szövege

Introduction: Small G-proteins regulate numerous immune cell functions. ARHGAP25, a Rac-specific GTPase activating protein is a key factor in the regulation of phagocytosis, leukocyte migration and extravasation according to our previous results.

Aims: To further investigate its precise enzymatic function and effect on Rac, finding a reliable GTPase assay with high temporal resolution was crucial. Since the currently available methods were not suited for our purposes, we considered that using bioluminescence resonance energy transfer (BRET), the active, GTP-bound Rac could be monitored by measuring the distance between Rac and CRIB, a domain which binds active Rac only.

Method: Luciferase-tagged CRIB (CRIB-Rluc) and Venus-tagged Rac (Venus-Rac) as well as ARHGAP25 were produced in E.coli. BRET ratio was measured every 30 s under in vitro conditions in 96-well plates for 15 minutes. (Optimal donor (CRIB-Rluc) to acceptor (Venus-Rac) ratio was determined in advance.) GTPγS or GDPβS-loaded Rac were used to determine BRETmax and BRETmin, resp. Phosphorylation of ARHGAP25 was carried out with activated and heat-inactivated neutrophil cytosol. All results were validated by both radioactive and commercially available endpoint GTPase activity assays.

Results: BRET signal between Venus-Rac and CRIB-Rluc displayed a continuous decrease demonstrating the endogenous GTPase activity of Rac. BRET signal decrease was accelerated by ARHGAP25 in a dose-dependent manner, verifying its effect as a RacGAP. Importantly, phosphorylation of ARHGAP25 with neutrophil cytosol attenuated its GAP activity.

Conclusion: We developed a simple and cost-effective in vitro BRET-based assay, which measures Rac GTPase activity in a real-time manner and without the use of radioactive labelling. Using this novel approach we demonstrated that phosphorylation of ARHGAP25 with neutrophil cytosol reduces its GAP activity.

Funding sources: NKFIH, Hungary (K108382, FK128376), ÚNKP-18-3-III-SE-37

Kind

Szabad

Status

elfogadva

Accepted presentation method

szóbeli

Előadás fájl jóváhagyás

nem rendelkezett róla

Előadó

4109

Authors (legacy)

Éva Wisniewski1, Fanni Kovács1, Enikő Bahurek1, Roland Csépányi-Kömi1, Erzsébet Ligeti1
1Semmelweis University, Department of Physiology, Budapest, Hungary

Data of the presenter

Doctoral School: Molecular Medicine
Program: Cellular and Molecular Physiology
Supervisor: Erzsébet Ligeti, Roland Csépányi-Kömi
E-mail address: wisniewski.eva@med.semmelweis-univ.hu