Česká společnost pro hmotnostní spektrometrii

(5. konference České společnosti pro hmotnostní spektrometrii - ThO-015)
Interactome of human DNA damage-inducible protein 2 (Ddi2). What is the effect of different taging strategies and how to increase the confidence of the emerging interaction map?

Martin Hubálek ^1 *, Michal Svoboda ¹, Jan Belza ¹, Klára Grantz Šašková ^2,1, Jan Konvalinka ¹

1. Ústav organické chemie a biochemie AV ČR, v.v.i.
2. Katedra Biochemie, Přf UK Praha

Abstrakt

DNA damage-inducible protein 1 (Ddi1) is an eukaryotic protein whose role has been implicated in DNA damage response (DDR) pathway. In spite of several structural and functional studies, the biochemical function of Ddi1 has so far remained elusive. The Ddi1 protein family is characterized by a highly conserved central domain homologous to retroviral aspartyl proteases called retroviral protease-like domain (RVP) of Ddi1. It also possess an N-terminal ubiquitin-associated (UBA) domain that binds selectively to Lys48-linked polyubiquitin chains and helical domain of Ddi1 (HDD) that is probably an interaction module that mediates substrate recognition of protease-like domain. In yeast the protein is indeed induced by DNA damage, plays role in cell cycle control and exocytosis. In human, the domain structure varies slightly and the function of the protein is not known at all. We performed a quantitative mass spectrometric analysis of potential binding partners of human Ddi2 by FLAG tagging first N-terminus then C-terminus of the polypeptide chain. Proteins binding to FLAG-Ddi2 were compared to the control cell lines during affinity purification procedure followed by label free data independent analysis (SWATH). The measurement was performed on 5600 TripleTOF, analysis in PeakView 2.2 and statistical analysis was carried out in MarkerView. To confirm the newly found interactions we selected one of the identified proteins (UV excision repair protein RAD23 homolog B) and reverse the experiment. The results confirm the specific interaction of these proteins that links Ddi2 into the process of ubiquitin dependent regulatory proteolysis.

* Korespondující autor: hubalek@uochb.cas.cz