Modulation of angiogenesis through targeting of arginyl transferase (ATE1)

Abstract

The invention provides methods and compositions for modulating angiogenesis in a subject. The methods of modulating angiogenesis in a subject include administering to the subject a modulator of N-terminal arginylation activity. The invention also provides a method of identifying such a modulator and a method of in vitro screening for modulators of N-terminal arginylation activity. Additionally, the invention provides a method of treating an angiogenesis-related disorder.

What is claimed is:  
     
         1 . A method of modulating angiogenesis in a subject, comprising administering to a subject a modulator of N-terminal protein arginylation, thereby modulating angiogenesis in the subject.  
     
     
         2 . The method of  claim 1 , wherein the modulator inhibits ATE1 gene product activity.  
     
     
         3 . The method of  claim 1 , wherein the subject is a mammal.  
     
     
         4 . A method of identifying a modulator of N-terminal arginylation activity, comprising: 
 a) providing a cell that expresses a reporter protein with an N-terminal aspartic acid, glutamic acid or cysteine residue, wherein the half-life of the reporter protein is affected by arginylation of the N-terminal protein;    b) contacting at least one potential modulator with the cell; and    c) measuring the level of the reporter protein expressed in the presence and absence of the modulator, wherein a change in the level of reporter protein expressed is indicative of modulation of N-terminal arginylation activity.    
     
     
         5 . The method of  claim 4 , wherein the cell is a cultured mammalian cell.  
     
     
         6 . The method of  claim 4 , wherein the cell is a yeast cell.  
     
     
         7 . The method of  claim 4 , wherein the cell is a bacterial cell.  
     
     
         8 . The method of  claim 4 , wherein the reporter protein is expressed as a cleavable fusion protein comprising a reporter protein and a ubiquitin domain functionally linked to the reporter protein, wherein the reporter protein comprises an N-terminal aspartic acid, glutamic acid or cysteine.  
     
     
         9 . The method of  claim 4 , wherein the reporter protein comprises a selectable marker protein, and the measuring comprises a selective genetic screen.  
     
     
         10 . The method of  claim 9 , wherein the selectable marker protein is an antibiotic resistance protein.  
     
     
         11 . The method of  claim 4 , wherein the measuring comprises fluorescence detection of the reporter protein.  
     
     
         12 . The method of  claim 4 , wherein the reporter portion comprises a fluorescent protein.  
     
     
         13 . The method of  claim 12 , wherein the fluorescent protein is Aequorea green fluorescent protein.  
     
     
         14 . The method of  claim 4 , wherein the reporter portion comprises a light-generating protein.  
     
     
         15 . The method of  claim 13 , wherein the light-generating protein is luciferase.  
     
     
         16 . The method of  claim 4 , wherein the reporter portion comprises an enzyme.  
     
     
         17 . The method of  claim 16 , wherein the enzyme is beta-galactosidase.  
     
     
         18 . The method of  claim 4 , wherein the expression of the reporter protein is inducible.  
     
     
         19 . The method of  claim 4 , wherein the reporter protein has a half-life in the first cell of less than about an hour in the absence of a modulator.  
     
     
         20 . The method of  claim 4 , wherein the reporter protein has a half-life in the first cell of less than about  10  minutes in the absence of a modulator.  
     
     
         21 . The method of  claim 4 , wherein the change in the level of reporter protein expressed is increased expression.  
     
     
         22 . The method of  claim 4 , further comprising: 
 d) providing a second cell that expresses a second reporter protein, where the half-life of the second reporter protein is affected by N-terminal arginylation of the protein;    e) contacting at least one potential modulator of ATE1 gene product activity with the second cell; and    f) measuring the level of second reporter protein expressed within the second cell relative to the level of reporter protein expressed within the first cell.    
     
     
         23 . The method of  claim 22 , wherein the cell in step a) and the second cell are the same cell.  
     
     
         24 . A modulator of N-terminal arginylation activity identified by the method of  claim 4 .  
     
     
         25 . The modulator of  claim 24 , wherein the modulator functions by modulating ATE1 gene activity.  
     
     
         26 . The modulator of  claim 24 , wherein the modulator functions by modulating ATE1 gene product activity.  
     
     
         27 . The method of  claim 4 , wherein the modulator of N-terminal arginylation activity also modulates angiogenesis.  
     
     
         28 . A method of modulating angiogenesis in a subject, comprising administration of a modulator identified by the method of  claim 4 .  
     
     
         29 . An in vitro method of screening for modulators of N-terminal arginylation activity, comprising: 
 a) providing an incubation mixture comprising protein substrate, ATE1 arginy1 transferase source, and at least one potential modulator of ATE1 arginy1 transferase activity; and    b) determining the level of arginylation of the peptide substrate in the presence and absence of the modulator, wherein a change in the level of arginylation of the peptide substrate is indicative of modulation of N-terminal arginylation activity.    
     
     
         30 . The method of  claim 29 , wherein the protein substrate further comprising an N-terminal amino acid selected from the group consisting of aspartic acid and glutamic acid.  
     
     
         31 . The method of  claim 29 , wherein the ATE1 arginyl transferase source is a cellular extract.  
     
     
         32 . The method of  claim 29 , wherein the ATE1 arginyl transferase source is a purified enzyme.  
     
     
         33 . The method of  claim 29 , wherein the incubation mixture further comprises ATP and arginyl-tRNA.  
     
     
         34 . The method of  claim 29 , wherein said peptide substrate is immobilized to a solid surface.  
     
     
         35 . The method of  claim 29 , wherein said solid surface comprises a microtiter plate.  
     
     
         36 . The method of  claim 29 , wherein the determining of the level of arginylation comprises electrophoresis, chromatography, mass spectrometry or imunoassay.  
     
     
         37 . The method of  claim 29 , wherein the determining of the level of arginylation comprises reaction with an antibody that distinguishes between the substrate peptide and an arginylated substrate peptide.  
     
     
         38 . The method of  claim 29 , wherein the modulator of N-terminal arginylation activity also modulates angiogenesis.  
     
     
         39 . The method of  claim 29 , wherein the change in the level of arginylation of the peptide substrate is increased arginylation.  
     
     
         40 . A modulator of N-terminal arginylation activity identified by the method of  claim 29 .  
     
     
         41 . A method of modulating angiogenesis in a subject, comprising administration of a modulator identified by the method of  claim 28 .  
     
     
         42 . A method of treating an angiogenesis-related disorder in a subject comprising administering to a subject a modulator of angiogenesis, thereby treating the angiogenesis-related disorder in the subject.  
     
     
         43 . The method of  claim 42 , wherein the modulator inhibits N-terminal protein arginylation.  
     
     
         44 . The method of  claim 43 , wherein the modulator inhibits ATE1 gene product activity.  
     
     
         45 . The method of  claim 42 , wherein the subject is a mammal.  
     
     
         46 . The method of  claim 42 , wherein the modulator of angiogenesis inhibits angiogenesis in the subject.  
     
     
         45 . The method of  claim 42 , wherein the angiogenesis-related disorder is cancer, tumors, rheumatoid arthritis, psoriasis or metastasis of cancerous cells in the subject.  
     
     
         48 . The method of  claim 42 , wherein the modulator of angiogenesis stimulates angiogenesis in the subject.  
     
     
         49 . The method of  claim 42 , wherein the angiogenesis-related disorder is coronary artery disease, stroke or delayed wound healing.