Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and  GTPase-linked Immunosorbent Assay

Javad Alizadeh; Shahla Shojaei; Simone da Silva Rosa; Adel Rezaei Moghadam; Amir A. Zeki; Mohammad Hashemi; Marek J. Los; Joseph W. Gordon; Saeid Ghavami

doi:10.3791/57646

Rilevazione di piccolo GTPase prenilazione e GTP Binding mediante frazionamento di membrana e GTP...

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Biochemistry
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JoVE Journal Biochemistry

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and GTPase-linked Immunosorbent Assay

Rilevazione di piccolo GTPase prenilazione e GTP Binding mediante frazionamento di membrana e GTPase-collegata dell'immunosorbente

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13:51 min

November 11, 2018

DOI: 10.3791/57646-v

Javad Alizadeh^1,2, Shahla Shojaei^1,2,3, Simone da Silva Rosa¹, Adel Rezaei Moghadam^1,2, Amir A. Zeki⁴, Mohammad Hashemi⁵, Marek J. Los^6,7,8, Joseph W. Gordon^1,9, Saeid Ghavami^1,2,10

¹Department of Human Anatomy and Cell Science, Rady Faculty of Health Sciences, Max Rady College of Medicine,University of Manitoba, ²Biology of Breathing Theme, Children Hospital Research Institute of Manitoba,University of Manitoba, ³Department of Biochemistry, School of Pharmacy and Pharmaceutical Sciences,Isfahan University of Medical Sciences, ⁴Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Internal Medicine,Center for Comparative Respiratory Biology and Medicine, ⁵Department of Clinical Biochemistry, School of Medicine,Zahedan University of Medical Sciences, ⁶Department of Molecular Biology, School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec,Medical University of Silesia, ⁷Centre de biophysique moléculaire - UPR 4301,Centre national de la recherche scientifique (CNRS) CS80054, ⁸LinkoCare Life Sciences AB, ⁹College of Nursing and Children's Hospital Research Institute of Manitoba, Rady Faculty of Health Sciences,University of Manitoba, ¹⁰Health Policy Research Center, Institute of Health,Shiraz University of Medical Sciences

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Overview

This article presents a protocol for investigating the prenylation and GTP-loading of Rho GTPase proteins. The methods described include membrane fractionation and a GTPase-linked immunosorbent assay, which can be applied to various small GTPases.

Key Study Components

Area of Science

Neuroscience
Cell Biology
Biochemistry

Background

Small Rho GTPases are part of a superfamily with diverse cellular functions.
They regulate cell fate, stress responses, motility, and the cell cycle.
Post-translational modifications, such as prenylation and GTP binding, tightly regulate their activity.
Understanding these modifications is crucial for elucidating their roles in cellular processes.

Purpose of Study

To develop a simple method for investigating post-translational modifications of small Rho GTPases.
To measure prenylation and GTP loading in these proteins.
To enhance understanding of the regulatory mechanisms governing Rho GTPase activity.

Methods Used

Membrane fractionation to isolate GTPases.
GTPase-linked immunosorbent assay for quantifying GTP loading.
Application of the protocol to various small GTPases.
Analysis of post-translational modifications.

Main Results

Successful isolation and analysis of Rho GTPases.
Demonstrated the effectiveness of the developed methods.
Provided insights into the prenylation and GTP loading processes.
Facilitated further research on small GTPases in cellular functions.

Conclusions

The protocol offers a reliable approach to study Rho GTPase modifications.
It can be adapted for other small GTPases, broadening its applicability.
Understanding these modifications can lead to insights into their regulatory roles in cells.

Frequently Asked Questions

What are Rho GTPases?

Rho GTPases are small proteins that play critical roles in various cellular processes, including cell motility and cycle regulation.

Why is prenylation important?

Prenylation is a post-translational modification that helps anchor Rho GTPases to cell membranes, which is essential for their function.

What methods are used in this study?

The study utilizes membrane fractionation and GTPase-linked immunosorbent assays to analyze Rho GTPases.

How can this protocol be applied?

It can be used to investigate the prenylation and GTP loading of various small GTPases beyond Rho GTPases.

What are the implications of this research?

This research enhances our understanding of the regulatory mechanisms of Rho GTPases, which could have implications in cell biology and disease.

Qui descriviamo un protocollo per studiare la prenilazione e guanosina-5'-trifosfato (GTP)-caricamento di Rho GTPasi. Questo protocollo è costituito da due metodi dettagliati, vale a dire del kit membrana frazionamento e un GTPase-collegata dell'immunosorbente. Il protocollo può essere utilizzato per misurare la prenilazione e GTP caricamento di diverse altre piccole GTPasi.

La piccola Rho GTPasi è una proteina di una superfamiglia con diverse funzioni cellulari. Ad esempio, queste proteine sono coinvolte nella regolazione del destino cellulare, della risposta cellulare a uno stress, della motilità cellulare e del ciclo cellulare. L'attività di una piccola proteina Rho GTPasi è regolata dalla modifica post-traslazionele.

Queste modifiche sono coinvolte in una stretta regolamentazione della loro attività. Ad esempio, la prenilazione proteica e il legame GTP sono la più importante modifica post-traslazione di queste proteine. Il mio laboratorio ha recentemente modificato e sviluppato un metodo semplice per indagare la modifica post-traslazione di queste proteine della superfamiglia.

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