Identification And Functional Study Of The Interaction Between Progranulin And Angiogenin

Angiogenin is a potent angiogenic growth factor, originally isolated from theconditioned medium of HT-29 human colon adenocarcinoma cells. Elevated serumangiogenin levels in many types of cancers suggest that it is closely related to tumorgrowth, progression and metastasis. Recently, angiogenin was found to directly act ontumor cells and promote proliferation. However, the molecular mechanismsunderlying the functions of angiogenin remain elusive.Since protein interactions are critical in every biological process, we reason thatinteractions between angiogenin and other proteins should mediate a series of cellularevents in angiogenesis and tumor cell growth. But until now only a few proteins havebeen identified as angiogenin interacting partners, which is not enough to clarify themolecular mechanism of ANG-induced angiogenesis and tumorigenesis.To identify additional mediators or modulators of angiogenin, we have carried out ayeast two-hybrid screening and identified progranulin, which is an autocrine growthfactor and plays an important role in tube formation, wound repair, and tumor cellproliferation, as a potential angiogenin-binding protein. In this thesis, we carried outfurther studies to confirm the interactions between progranulin and angiogenin, andthen explore the biological implication of the interaction.After reassuring progranulin did interact with angiogenin in yeast cells, weconfirmed it by an in vitro co-immunoprecipitation experiment. First the progranulinwas expressed in an in vitro TNT^? T7 expression system and labeled with ³⁵[S]-Met.Then angiogenin was incubated with the in vitro expressed products, andco-immunoprecipitated by anti-angiogenin antibody. The pellets were subjected to SDS-PAGE and then autoradiography. The results showed that progranulin wasco-immunoprecipitated with angiogenin, indicating that there is direct interactionbetween progranulin and angiogenin.To document if there is a specific interaction between progranulin and angiogeninin living cells, we firstly examined their intracellular localization. The images of theCFP-tagged progranulin and/or YFP-tagged angiogenin were monitored by a confocalmicroscope. Data revealed that single transfection of progranulin into HeLa cellsshowed a predominant distribution in the cytoplasm; Angiogenin was present mainlyin the nucleus. When the two proteins were co-expressed in HeLa cells, however, thedistribution pattern of angiogenin was altered: it moved into the cytoplasm andco-localized with the progranulin. When exogenous angiogenin was applied to theculture media of HeLa cells pre-transfected with progranulin, its nuclear translocationwas blocked. These data provide evidence to reveal that progranulin interacts withangiogenin in a living cell. Then, we further confirmed their interactions in thecytoplasm using fluorescence resonance energy transfer (FRET) methodology.To narrow down the binding domain with ANG in progranulin, we constructed aseries of truncated fragments of progranulin into vector pEXP-AD502 and evaluatedtheir interactions with angiogenin in yeast cells. Data showed that either theN-terminal PGF domain or C-terminal DE domain interacted with angiogenin,indicating that binding of angiogenin requires at least two tandem domains. Thefollowing FRET analysis and GST pull-down assay further confirmed the interactionsbetween grnPGF or grnDE with angiogenin. Meanwhile, the binding sites and keyamino acids in angiogenin were explored. The results showed that the smallestfragment binding to progranulin in angiogenin was from amino acid 63 to 110, but nokey amino acids were detected.We were then curious about the biological consequences of the interaction betweenangiogenin and progranulin. Based on the fact that progranulin inhibits angiogeinnuclear translocation, we speculated that progranulin or its binding domains mayregulate angiogenin's nuclear function. To test that hypothesis, an ABE-luciferasereporter system was applied. The results showed that the expression level of luciferase in HeLa cells transfected with pGL3E-ABE was nearly 30 times higher than thosetransfected with pGL3E vector, indicating ABE acted as a promoter. Whenco-transfected with angiogenin-expressing plasmid, the activity of luciferaseincreased greatly, indicating that ABE's promoter activity was ANG-dependent.Progranulin had no effect on ABE's promotor activity, as the luciferase expressionlevel did not change much in progranulin co-transfection group. When the cells wereco-transfected with both angiogenin and progranulin, the ABE activity reduced to thebasic level, indicating that progranulin inhibited angiogenin's ABE-driven activity.Correspondingly, grnPGF and grnDE both inhibited angiogenin's ABE-drivenactivity. All these evidence suggested that progranulin or its domains may play as anangiogenin regulator.Therefore, innovative conclusions could be drawn based on the data:1. Progranulin is one of the angiogenin-interacting proteins.2. Either grnPGF or grnDE is responsible for angiogenin binding.3. The smallest fragment interacting with progranulin in angiogenin is from aminoacid 63 to 110.4. Progranulin, grnPGF or grnDE is a functional regulator of angiogenin.