| Vascular endothelial cell growth inhibitor ( VEGI ) is a recently discovered novel member of the TNF superfamily, which is expressed predominantly in endothelial cells. The VEGI gene encodes a protein of 174 amino acids that exhibits a 20-30% overall sequence homology to the TNF superfamily. VEGI is a type II membrane protein with a brief intracellular N-terminal segment and most of the protein (residues26-174) being an extracellular domain. VEGI can markedly inhibit the growth of endothelial cells but not that of other types of cells examined. The protein also inhibited formation of capillary-like structures by endothelial cells in vitro. In vivo, a secreted form of VEGI significantly inhibited the growth of colon carcinoma in mice and the growth of the breast cancer xenograft rumors. As an endothelial cell-specific negative regulator of angio genesis, VEGI will be a promising candidate for therapeutic interventions of tumor.It is known that the structure of protein determines its biological function. However, the relationship between structure and function of VEGI is not understood at present, which hampers the further structure optimization of VEGI. Additionally, very little is known about the molecular mechanism of this protein, which limits the development of VEGI as a promising therapeutic antiangiogenesis drug.In this study, some basic research about the relationship between structure and function and the mechanism of VEGI were explored.Part I Studies on the relationship between structure and function of VEGI1. Sequence alignment between VEGI and the some other members of TNF superfamily, TNF-A,TNF-B,CD40L, TRAIL were performed. From the sequence alignment, the structure and function key amino acids of TNF-A,TNF-B,CD40L, TRAIL were compared and analyzed, then the structure key amino acids of VEGI were deduced.2. Based on the X-ray crytal strctures of TNF-A,TNF-B,CD40L, TRAIL, the 3-D structure of VEGI were modeled . The structure of VEGI is entirely compatible with the jelly-roll β-strand structure characteristic of the TNFs. The model predicts the distribution of the struture key amino aicd of VEGI and which regions of VEGI could interact with its receptors.3. In order to study the effect of the N-terminus of VEGI on biological activity, two truncated forms of VEGI were constructed, in which 43 and 51 amino acids fromN-terminus were deleted and named VEGI131 and VEGI123. Recombinant proteins were generated from E.coli. The proliferation of HUVEC and chick choriallantic membrane assay were performed to study the activity of two mutants. The results showed that VEGI131 has the similar activity with wild type VEGI, but VEGI123 have marked lower activity compared with the wild type VEGI. It suggested that the first 43 amino acids from N-terminus of VEGI have no significant effect on biological activity, but the amino acids 44-51 at N-terminus are required for full biological activity.4. In order to explore the functional key amino acids of VEGI, four mutants of VEGI(E45→R, G47→A, Y111 →F, Yl 11 →T) were constraced by site-directed mutagenesis, and recombinant proteins were generated from E.coli. Four mutant proteins behaved similar to the wild type VEGI in various physico-chemical assays. The proliferation of HUVEC and chick choriallantic membrane assay were performed to study the activity of four mutants. The results showed that the mutant E45→R significantly decreased the biological activity, and the mutant G47→A caused a slight drop on activity, but the mutants Y1 11→F, Y111→T almost completely abolished biological activity. It suggests that Y111 is an important residue in biological activity, which may play a direct role in receptor recognition. Moreover, the tyrosine ring and hydroxy group of the amino acid are important determinant of biological activity. Additionally, E45 also plays an important role in biological activity of VEGI.Part II Study on the biological activity and molecular mechanism of VEGI1. In order to explore the biological activi...
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