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%0 Thesis
%A Ahmed, Nabil
%T Comprehensive analysis of the IRE1 interactome using turboID in the human mast cell leukemia cell line HMC-1.2
%I RWTH Aachen University
%V Dissertation
%C Aachen
%M RWTH-2024-09585
%P 1 Online-Ressource : Illustrationen
%D 2024
%Z Veröffentlicht auf dem Publikationsserver der RWTH Aachen University
%Z Dissertation, RWTH Aachen University, 2024
%X The unfolded protein response (UPR) is essential for reducing the impact of misfolded proteins in the endoplasmic reticulum (ER) of cells. Misfolded proteins are recognized by the chaperone BIP, which subsequently releases the three ER stress sensors IRE1, PERK, and ATF6, leading to their activation. These ER stress sensors initiate the production of transcription factors XBP1s, ATF4, and ATF6. The target genes of these transcription factors contribute to restoring proteostasis or induce apoptosis, depending on the cellular conditions. This is facilitated by enhancing the expression of chaperones, reducing translation, and initiating ER-associated degradation pathways. In cancer, the UPR plays a pivotal role in tumor development, progression, and resistance to therapy through its complex signaling networks. UPR is involved in multiple types of cancer, including Mast Cell Leukemia (MCL), which is characterized by a median survival time of only six months after diagnosis. In the presence of misfolded proteins, the ER stress sensor protein IRE1, forms dimers or oligomers and undergoes trans-autophosphorylation by its kinase domain, resulting in the activation of its RNase domain. The RNase domain of IRE1 initiates the removal of a 26-nucleotide intron from the mRNA encoding XBP1, a crucial transcription factor for UPR target genes. We could demonstrate the expression of the IRE1β isoform in the human mast cell lines HMC-1 and ROSA by RT-qPCR. In addition, XBP1 splicing was slightly reduced in IRE1β knockout cells, suggesting minor participation in XBP1 splicing. This strongly confirms the distinguishing function between IRE1α and IRE1β based on their RNase domain.IRE1α is essential for the proliferation and survival of the MCL cell line HMC-1.2, prompting further investigation into the identification of IRE1α interactors/regulators through proteomic analysis. For this purpose, proteomic analysis of IRE1 in the HMC-1.2 cells with and without ER stress condition was performed, and IRE1α-regulated factors were identified using the TurboID proximity labelling technique combined with MS-analysis. We could classify the function of the identified proteins using Gene Ontology (GO) and pathway enrichment analyses, which are implicated in vesicle-mediated transport, protein stabilization, and ubiquitin-dependent ER-associated protein degradation. IRE1α interactions with VAPA and Emerin potentially could be due to membrane contact sites. Interactions with the oncoproteins MTDH and SND1, members of the RISC complex, and the protein degradation-associated AAA ATPase VCP were confirmed via co-immunoprecipitation experiments. In HEK293 cells, the overexpression of VCP and IRE1α resulted in increased stability of IRE1α. Additionally, this study characterized the amino acid residue T973 at the C-terminus of IRE1α. We demonstrated that the mutation of T973 is associated with the phosphorylation of S724 in the kinase domain of IRE1α under ER stress conditions.
%F PUB:(DE-HGF)11
%9 Dissertation / PhD Thesis
%R 10.18154/RWTH-2024-09585
%U https://publications.rwth-aachen.de/record/994915