PhD Scientific Days 2023

Molecular Sciences I.

Revealing Hidden Connections: Screening the Neutrophilic Granulocyte Interactome for Lesser-Known Interactions

Előadó neve

Sasvári, Péter

Neptun code

BEH4Y8

Előadó munkahelye

Department of Physiology, Semmelweis University

Előadó telefonszáma

06309650396

Előadó e-mail címe

sasvari.peter@phd.semmelweis.hu

Az előadás címe

Revealing Hidden Connections: Screening the Neutrophilic Granulocyte Interactome for Lesser-Known Interactions

Szerző(k) neve és munkahelye

Péter Sasvári1, Aladár Pettkó-Szandtner2, Éva Wisniewski1, Domonkos Czárán1, Roland Csépányi-Kömi1
1Semmelweis University, Department of Physiology, Budapest
2Biological Research Centre, Proteomics lab, Szeged Introduction

Bemutatás módja

Szóbeli

Szekció

Molecular Sciences I.

Language of the presentation

English

Preferred session

Molecular Sciences

Összefoglaló szövege

Introduction
Protein-protein interactions (PPIs) are vital for all intracellular processes. While in silico prediction of PPIs is still a developing field, experimental data remains a cornerstone in our understanding. Over the last decade, many methods have emerged to detect transient or weak interactions that occur in physiological conditions. However, we may have overlooked an essential factor in interactome research: tissue or cell type specificity of certain proteins. ARHGAP25 is a leukocyte-specific protein that is typically undetected in PPI searches, due to the use of cell lines that lack this protein. Despite its crucial role in immunological processes, we know very little about its interactions and the pathways it leads to.
Aims
Using co-immunoprecipitation and pulldown assays, we aim to uncover the intracellular interactions and potential protein complexes formed within human neutrophils in order to understand the mechanisms underlying the effects of ARHGAP25.
Method
In our study, we used human neutrophils obtained from healthy adults and either endogenous or GST-fused recombinant ARHGAP25 to capture interaction partners. To increase sensitivity and detect a wide range of proteins, we employed mass spectrometry (MS) to identify eluted proteins, followed by the quantification of our data with the MaxQuant program.
Results
We isolated over 100 different proteins associated with GST-ARHGAP25 to varying degrees. Quantification of MS data provided a large number of proteins that were significantly enriched in the GST-ARHGAP25 concentrated samples. We detected Rac2, the only known interaction partner of ARHGAP25, along with proteins that fit well with what we know about ARHGAP25’s function: proteins involved in phagocytosis, superoxide production, and cell migration. Surprisingly, many other candidates arose that participate in other cellular processes (e.g., metabolism), so we compared our results with those obtained via co-immunoprecipitation with endogenous ARHGAP25. The results confirmed our findings, and out of the many new partners, we validated the ARHGAP25-IMPDH2 interaction via Western blot.
Conclusion
Our two different approaches provided a wide range of proteins that appear to form stable protein complexes with ARHGAP25. Based on both sets of results, IMPDH2, ARCP4, and constituents of the NADPH oxidase complex are promising candidates for further research. Interactions with Rac isoforms are well-studied, and ARCP4 and NCF proteins participate in processes that are affected by ARHGAP25. However, the ARHGAP25-IMPDH2 interaction in neutrophils has not yet been described in the literature.
Funding
EFOP-3.6.3-VEKOP-16-2017-00009, NKFIH FK_18/128376, TKP2021-EGA-24, Semmelweis 250+ Kiválósági ösztöndíj.

University and Doctoral School

Semmelweis University, Doctoral School of Molecular Medicine

Supervisor

Dr. Roland Csépányi-Kömi

Publication of my abstract

I do not give consent to the publication of my abstract on the website of the congress.

Kind

Szabad

Status

elfogadva

Accepted presentation method

szóbeli

Előadás fájl jóváhagyás

nem rendelkezett róla

Előadó

6060

Start

09:45

End

10:00

Authors (legacy)

Péter Sasvári1, Aladár Pettkó-Szandtner2, Éva Wisniewski1, Domonkos Czárán1, Roland Csépányi-Kömi1
1Semmelweis University, Department of Physiology, Budapest
2Biological Research Centre, Proteomics lab, Szeged Introduction