Functional perturbation of the Gab2 multiprotein complex by high affinity SH2-binding monobodies in chronic myelogenous leukemia
Başlık çevirisi mevcut değil.
- Tez No: 402988
- Danışmanlar: PROF. OLIVER HANTSCHEL
- Tez Türü: Doktora
- Konular: Biyoteknoloji, Biyomühendislik, Biotechnology, Bioengineering
- Anahtar Kelimeler: Chronic myeloid leukemia, Gab2, SHP2, Bcr-Abl, monobody, tandem affinity purification, interaction proteomics, tyrosine phosphatase, tyrosine kinase
- Yıl: 2015
- Dil: İngilizce
- Üniversite: Ecole Polytechnıque Federale de Lausanne (epfl)
- Enstitü: Yurtdışı Enstitü
- Ana Bilim Dalı: Belirtilmemiş.
- Bilim Dalı: Belirtilmemiş.
- Sayfa Sayısı: Belirtilmemiş.
Özet
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Özet (Çeviri)
Chronic myeloid leukemia is a myeloproliferative disorder that is characterized by a reciprocal chromosomal translocation between chromosome 9 and 22. This translocation results in the expression of the Bcr-Abl protein, which is a constitutively active tyrosine kinase. Despite the great improvement in patient survival using tyrosine kinase inhibitors (TKİ), resistance caused by point mutations in the Abl kinase domain is a majör drawback in TKİ therapy. Bcr-Abl forms a multi-protein complex and Gab2 (Grb2-associated binder 2] is one of the few functionally critical scaffold proteins and substrates of Bcr-Abl. The interaction of Gab2 with Bcr-Abl coordinates the activation of the MAP and PI3-kinase pathways and thus Bcr-Abl transformation. The goal of this thesis was to understand the contributions of the different pathways activated downstream of Gab2. In order to pursue this aim, we aimed at targeting the Gab2 protein complex with high-affinity and highly specific engineered fibronectin type III monobodies, which are recombinant single domain binding proteins that may act as protein interaction inhibitors, binding to the SH2 domains of Gab2 interactors. Firstly, the Gab2 complex was studied in an unbiased way by using interaction proteomics in order to confirm the pre-defined interactors of Gab2 and find out new interactors that could be possibly targetable by monobodies. To this end, Gab2 and the known interactors were affinity-tagged and expressed in CML cells, and interacting proteins were identified after tandem affinity purification by protein mass spectrometry. Many known interactors were confirmed and possible novel interactors were identified. Next, the SH2 domains of the Gab2 interactors SHP2 and PLCyl were targeted by monobodies and binding clones were functionally characterized. Initially, monobodies targeting the SH2 domains of SHP2, which is an oncogenic tyrosine phosphatase, were studied. Tandem affinity purification of the monobodies demonstrated that they are monospecific for their cognate target protein and no other SH2 domain-containing proteins were identified by mass spectrometric analysis. Upon monobody expression in celi lines, we were able to show a significant decrease of phosphorylation of tyrosine residues of SHP2 that are critical for its catalytic activity. Monobodies targeting the N-SH2 domain disrupted the interaction of SHP2 with its upstream activator, the Gab2 scaffold protein, suggesting the decoupling of SHP2 from the Bcr-Abl protein complex by disrupting the phosphotyrosine-SH2 mediated interaction. Moreover, SHP2 downstream signaling was altered upon monobody expression. HCC-1171 non-small celi lung cancer celi line was investigated for ERK phosphorylation due to its high ERK phosphorylation and the presence of a SHP2 activating mutation. Strikingly, ERK phosphorylation was abolished in the HCC-1171 cells upon monobody expression. The physiological effects of monobodies were investigated in an inducible expression system in HCC-1171 cells. However, we could not detect significant changes in celi proliferation or ERK phosphorylation when compared to the non-binding control monobody. Furthermore, tandem monobodies targeting both SH2 domains of SHP2 simultaneously resulted in decreased ERK phosphorylation in Bcr-Abl overexpressing cells. PLCyl targeting monobodies were also monospecific for their target protein in cells. In addition, PLCyl phosphorylation was decreased upon monobody expression in cells. Although monobodies against p85 subunit of PI3K remains to be evaluated and better monoobodies are required for PLCyl targeting, our results validate monobodies as potent and specific antagonists of protein-protein interactions in cells and they could be powerful tools to investigate the participation of different signaling pathways in malignant transformation.
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