Identification Of Angiogenin-interacting Proteins And Its Function In Cell Migration

Angiogenin (ANG) is an angiogenic factor which also promotes tumor cell proliferation directly. However, the underlying molecular mechanisms remain elusive. Protein-protein interactions play crucial roles in biological processes, therefore, we reason that identifying ANG-ineracting proteins is a feasible way to elucidate ANG's biological functions and mechanism of action.In the first part of the thesis, we identified ANG-interacting proteins in HeLa cells using co-immunoprecipitation (Co-IP) combined with mass spectrum (MS) analysis. The results showed 20 potential ANG-binding partners, two of which had been reported. Bioinformatics analysis of the proteins, including functional annotations and constructions of protein-protein interacting (PPI) networks, revealed IQ motif containing GTPase activating protein 1 (IQGAP1), non-muscle myosin heavy chain 9 (MYH9),α-actinin 4 (ACTN4), andβ-actin (ACTB) to be involved in actin cytoskeleton regulations and cell adhesions. The four proteins also organized an actin cytoskeleton regulatory pathway-related sub-PPI network together with Rac1, indicating ANG involved in the above mentioned pathways. The interactions between the four proteins and ANG were confirmed by immunoprecipitation assay.The interaction of ANG with actin cytoskeleton proteins MYH9, ACTN4, and ACTB suggests the possibility that ANG is involved in the processes of actin cytoskeleton dynamics and cell adhesion. Since the coordination of cytoskeleton dynamics and cell-matrix adhesion is particularly important for cell migration, potential ANG functions in the two processes may explain how the protein promotes cell migration. To verify this hypothesis, in the second part of the thesis, we first identified the co-localization of ANG with actin cytoskeleton proteins and found that ANG bond to actin cytoskeleton proteins at the focal adhesion (FA) sites. Next, we stained F-actin with TRITC-conjugated phalloidin, focal adhesion with paxillin antibody, and detected phosphorylated-FAK levels using Western-blot when ANG was down-regulated. Data showed that the formation of stress fibres was enhanced, focal adhesions were enlarged, and EGF-stimulated focal adhesion kinase activation was inhibited in ANG down-regulated cells. At last, we used wound healing assay to determine the migration speed, and found that EGF-stimulated migration was retarded in ANG down-regulated cells. Altogether, our data suggested that ANG might regulate actin cytoskeleton dynamics and cell adhesion through interacting with actin cytoskeleton proteins, thus promote cell migration.Another identified ANG-associated protein was damage-specific DNA binding protein 2 (DDB2), which bind to the damaged DNA sites immediately after UV irradiation, indicating ANG involved in the same process. Thus, we primarily detected that ANG might response to UV irradiation by degradation and decrease UV-induced cell apoptosis in ANG down-regulated cells. These results are presented in the supplement. Much more studies are undergoing.According to the results above, we conclude:1. ANG interacts with 20 potential proteins in HeLa cells.2. ANG interacts with IQGAP1, MYH9, ACTN4, ACTB and DDB2 in HeLa cells; 2. ANG binds to actin cytoskeleton proteins at focal adhesions to interrupt formations and activities of stress fibres and FAs, thus to enhance EGF stimulated cell migration.