PROTEIN DEMETHYLASES COMPRISING A JMJC DOMAIN

• US 20090203057A1
• Filed: 10/27/2006
• Published: 08/13/2009
• Est. Priority Date: 10/28/2005
• Status: Abandoned Application

• Current Assignee: University of North Carolina At Chapel Hill (University of North Carolina System)
• Original Assignee: University of North Carolina At Chapel Hill (University of North Carolina System)
• Inventors: Tsukada, Yuichi, Klose, Robert John, Yamane, Kenichi, Zhang, Yi
• Application Number:
  US12/091,205
• Publication Number:
  US 20090203057A1
• Time in Patent Office: Days
• US Class Current: 435/18
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• CPC Class Codes: G01N 2333/90245 acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: G01N 2500/00 Screening for compounds of potential therapeutic value
• CPC Class Codes: G01N 2500/04 Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/6875 Nucleoproteins

Abstract

Post-translational modification, including protein methylation, plays an important role in regulating protein function. The present invention provides a novel assay for evaluating demethylase activity and the discovery of a family of protein demethylases comprising a novel demethylase motif.

Claims

1-23. -23. (canceled)

24. A method of identifying a compound that modulates the demethylase activity of a demethylase comprising a JmjC domain, the method comprising:

(a) contacting the demethylase with a methylated protein substrate in the presence of a test compound; and

(b) detecting the level of demethylation of the protein substrate under conditions sufficient for demethylation, wherein a change in demethylation of the protein substrate as compared with the level of demethylation in the absence of the test compound indicates that the test compound is a modulator of the demethylase activity of the demethylase.

25. The method of claim 24, wherein the methylated protein substrate is a methylated histone substrate.

26. The method of claim 25, wherein the methylated histone substrate is a methylated histone H3 substrate.

27. The method of claim 26, wherein the demethylation at lysine 36 of histone H3 (H3-K36) is detected.

28. The method of claim 26, wherein the demethylation at lysine 9 of histone H3 (H3-K9) is detected.

29. The method of claim 26, wherein the demethylation at lysine 4 of histone H3 (H3-K4) is detected.

30. The method of claim 25, wherein the methylated histone substrate is a methylated core histone substrate, mononucleosome substrate, dinucleosome substrate, oligonucleosome substrate or peptide substrate.

31. The method of claim 24, wherein the demethylase is a JHDM1 protein.

32. The method of claim 31, wherein the demethylase is JHDM1A or JHDM1B.

35. The method of claim 24, wherein the demethylase is a JHDM2 protein.

36. The method of claim 35, wherein the demethylase is a JHDM2A protein, a JHDM2B protein, or a JHDM2C protein.

39. The method of claim 24, wherein the demethylase is a JHDM3 protein.

40. The method of claim 39, wherein the demethylase is a JHDM3A protein.

43. The method of claim 24, wherein the demethylase is a JARID protein.

44. The method of claim 43, wherein the demethylase is a JARID1 protein.

47. The method of claim 24, wherein a reduction in demethylation activity as compared with the level of demethylation in the absence of the test compound indicates that the test compound is an inhibitor of the demethylase activity of the demethylase.

48. The method of claim 24, wherein an enhancement of demethylation activity as compared with the level of demethylation in the absence of the test compound indicates that the test compound is an activator of the demethylase activity of the demethylase.

49. The method of claim 24, wherein the method is a cell-based method.

50. The method of claim 24, wherein the method is a cell-free method.

64. The method of claim 24, wherein the demethylase comprising a JmiC domain is a Hairless protein and

wherein a change in demethylation of the protein substrate as compared with the level of demethylation in the absence of the test compound indicates that the test compound is a candidate compound for the treatment of hair loss.

33-34. -34. (canceled)

37-38. -38. (canceled)

41-42. -42. (canceled)

45-46. -46. (canceled)

51. A method of identifying a candidate compound for treating cancer, the method comprising:

(a) contacting a histone demethylase comprising a JmjC domain with a methylated histone substrate in the presence of a test compound; and

(b) detecting the level of demethylation of the histone substrate under conditions sufficient for demethylation, wherein a change in demethylation of the histone substrate as compared with the level of demethylation in the absence of the test compound indicates that the test compound is a candidate compound for the treatment of cancer.

52. The method of claim 51, wherein the methylated histone substrate is methylated at lysine 36 of histone H3 (H3-K36), lysine 9 of histone H3 (H3-K9) or lysine 4 of histone H3 (H3-K4).

53. The method of claim 51, wherein the demethylase is a JHDM1 protein.

54. The method of claim 53, wherein the demethylase is JHDM1A or JHDM1B.

56. The method of claim 51, wherein the demethylase is a JHDM2 protein or a JHDM3 protein.

57. The method of claim 56, wherein the demethylase is JHDM2A, JHDM2B or JHDM2C.

58. The method of claim 56, wherein the demethylase is JHDM3A.

60. The method of claim 51, wherein the demethylase is a JARID protein.

61. The method of claim 60, wherein the demethylase is a JARID1 protein.

63. The method of claim 51, wherein the methylated histone substrate is a methylated core histone substrate, mononucleosome substrate, dinucleosome substrate, oligonucleosome substrate or peptide substrate.

96. The method of claim 51, the method further comprising testing said candidate compound identified in (b) for the ability to inhibit the growth of a demethylase dependent cancer cell.

55. (canceled)

59. (canceled)

62. (canceled)

65-79. -79. (canceled)

80. A method of demethylating a methylated protein, the method comprising contacting the methylated protein with a demethylase comprising a JmjC domain under conditions sufficient for demethylation.

81-95. -95. (canceled)