Identifying protein interaction partners and cysteine oxidative modifications: case studies for peroxiredoxin 2 and STIM2

Onderzoeksoutput: PhD Thesis

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Samenvatting

Hydrogen peroxide (H2O2) acts as a signalling molecule by oxidizing specific cysteine thiols in proteins. Recent evidence has established a role for cytosolic peroxiredoxins in transmitting H2O2-based oxidation to a multitude of target proteins. Moreover, it is becoming clear that peroxiredoxins fulfil their function in organized microdomains, where not all interactors are covalently bound. However, previous studies aimed at identifying peroxiredoxin interactors were based on methods that only detect disulphide linked partners. Here, I applied two thiol-disulphide unbiased in-cell trapping methodological approaches for the identification of interaction partners of peroxiredoxin 2.First, I combined biotin-dependent proximity-labelling (BioID) in intact live cells with mass spectrometry. I identified 13 interactors under elevated H2O2conditions, among which CSN5, subunit five of the COP9 signalosome (CSN) complex, known to play a role in protein homeostasis and in the signalling of STAT3, an established Prdx2 interactor. Second, I used a chemical crosslinking protocol followed by immunoprecipitation, where the c-Jun protooncogene was revealed to interact with Prdx2. The CSN5-and c-Jun-Prdx2 interactions were then confirmed in a proximity ligation assay. Both methods identified known and novel Prdx2 interactors involved in markedly different biological processes. Taken together, my results demonstrated that BioID and crosslinker-IP are alternative methods in the identification of interactors of peroxiredoxins. In a second case study, I focused on the oxidative modifications of STIM2. STIM1 and STIM2 are responsible for sensing the Ca2+levels in the lumen of the endoplasmic reticulum (ER) and for the gating of the ORAI channels. It has been shown that oxidative modifications of cysteines of STIM1 and OARI1 have an impact on their function and lead to modulation of Store-operated calcium entry (SOCE). Recently, through FLIM and FRET microscopy as well as MD-simulations, Cys313 was identified as the main redox sensing residue of STIM2 and it was indicated that oxidative modifications of Cys313 alter the STIM2 activation dynamics, thereby hindering STIM2-mediated gating of ORAI1. As a collaborator on this project, I revealed the oxidative status of Cys313 using recombinant STIM2-GFP expressed in a mammalian cell line using nanobody-based GFP-trapping followed by target specific mass spectrometry. I showed that Cys313 forms a disulphidewithCys302 under normal Ca2+levels in the lumen of the ER. Further studies are needed to reveal the oxidative modifications of Cys313 under conditions of luminal Ca2+depletion of the ER and under oxidative stress
Originele taal-2English
Toekennende instantie
  • Vrije Universiteit Brussel
Begeleider(s)/adviseur
  • Messens, Joris, Promotor
  • Ezerina, Daria, Promotor
Datum van toekenning16 dec 2022
Plaats van publicatieBrussel
Uitgever
Gedrukte ISBN's9789464443448
StatusPublished - 2022

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