Methods and kits using a molecular interaction between a Smurf-1 WW domain and LIM mineralization protein isoforms

Abstract

The instant application provides kits and methods for identifying agents which induce or inhibit the osteogenic effect of LMP or BMP proteins. The kits are directed to methods which measure either an amount of a complex between a Smurf protein or a fragment thereof and an LMP protein or a fragment thereof. Alternatively, the kits are directed to methods of measuring an amount of the ubiquitinated Smad protein or a fragment thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a scheme of ubiquitination of the Smad proteins by Smurf1.

FIG. 2 illustrates predicted three-dimentional structures for the WW-2 domain of Smurf1 (FIG. 2A, FIG. 2B), a model of the peptide motif structures of WW domain interacting site B of LMP-1 (FIG. 2C), and a model of binding between WW-2 domain of Smurf1 and the WW domain interacting site B of LMP-1 (FIG. 2D).

FIG. 3 is a photography of an SDS-PAGE gel demonstrating the purity and integrity of recombinant proteins (Smurf1, LMP wild type, LMP-1 mutant form A, and LMP-1 mutant form B) (FIG. 3A) and the results of the binding assays in ligand blots using biotin-labeled Smurf1 (FIG. 3B).

FIG. 4 is a photography of an SDS-PAGE gel demonstrating that LMP-1 inhibits ubiquitination of Smad1 by Smurf1 by competing with Smad1 for Smurf1 binding when used at 10 uM concentration.

Claims

1. A method of identifying an agent affecting a binding between an LMP protein and a Smurf1 protein comprising: providing a first composition comprising: a first amino acid sequence comprising an amino acid sequence of SEQ. ID NO. 1;a second amino acid sequence comprising an amino acid sequence at least 70% identical to SEQ. ID. NO. 2 and capable of binding the amino acid sequence of SEQ ID NO 1; andthe agent; andmeasuring an amount of a complex formed between the first amino acid sequence and the second amino acid sequence in the first composition.

2. The method of claim 1, further comprising comparing the amount of the complex in the first composition with an amount of a complex formed between a third amino acid sequence and a fourth amino acid sequence in a second composition, wherein said second composition does not include the agent;the third amino acid sequence comprises an amino acid sequence of SEQ. ID. NO. 1; andthe fourth amino acid sequence comprises an amino acid sequence at least 70% identical to SEQ. ID. NO. 2 and capable of binding the amino acid sequence of SEQ. ID. NO. 1.

3. The method of claim 2, wherein the increased amount of the complex in the first composition indicates that the agent induces binding between the amino acid sequence of SEQ. ID. NO. 1 and the amino acid sequence of SEQ. ID. NO. 2; andthe decreased amount of the complex in the first composition indicates that the agent inhibits binding between the amino acid sequence of SEQ. ID. NO. 1 and the amino acid sequence of SEQ. ID. NO. 2.

4. The method of claim 2, wherein the increased amount of the complex in the first composition indicates that the agent inhibits ubiquitination of a Smad protein by Smurf1; andthe decreased amount of the complex in the first composition indicates that the agent induces ubiquitination of a Smad protein by Smurf1.

5. The method of claim 4, wherein the Smad protein is selected from the group consisting of a Smad1 protein, a Smad5 protein, and a Smad6 protein.

6. The method of claim 2, wherein the increased amount of the complex in the first composition indicates that the agent induces an osteogenic effect of an LMP protein or a fragment thereof; andthe decreased amount of the complex in the first composition indicates that the agent inhibits an osteogenic effect of an LMP protein or a fragment thereof.

7. The method of claim 6, wherein the LMP protein or the fragment thereof is selected from the group consisting of LMP-1, corresponding to SEQ. ID. NO. 53, LMP-3 corresponding to SEQ. ID. NO. 54, and LMP-1s corresponding to SEQ. ID. NO. 55.

8. The method of claim 2, wherein the first amino acid sequence is identical to the third amino acid sequence, orthe second amino acid sequence is identical to the fourth amino acid sequence, orthe first amino acid sequence is identical to the third amino acid sequence and the second amino acid sequence is identical to the fourth amino acid sequence.

9. The method of claim 1, wherein the SEQ. ID NO. 1 is identical to a SEQ. ID. NO. 5.

10. The method of claim 1, wherein the SEQ. ID NO. 1 identical to a SEQ. ID. NO. 6.

11. The method of claim 1, wherein the SEQ. ID NO. 1 is incorporated within SEQ. ID. NO. 4.

12. The method of claim 1, wherein the amino acid sequence at least 70% identical to SEQ. ID. NO. 2 and capable of binding the amino acid sequence of SEQ. ID. NO. 1 is 100% identical to SEQ. ID. NO. 2.

13. The method of claim 1, wherein the amino acid sequence at least 70% identical to SEQ. ID. NO. 2 comprises: leucine at a position corresponding to position 3 of SEQ. ID. NO. 2;proline at a position corresponding to position 4 of SEQ. ID. NO. 2;tryptophan at a position corresponding to position 7 of SEQ. ID. NO. 2;glutamic acid at a position corresponding to position 8 of SEQ. ID. NO. 2;phenylalanine or isoleucine at a position corresponding to position 18 of SEQ. ID. NO. 2;phenylalanine or isoleucine at a position corresponding to position 19 of SEQ. ID. NO. 2;valine or isoleucine at a position corresponding to position 20 of SEQ. ID. NO. 2;asparagine or aspartic acid at a position corresponding to position 21 of SEQ. ID. NO. 2;histidine at a position corresponding to position 22 of SEQ. ID. NO. 2;asparagine, valine, proline or serine at a position corresponding to position 23 of SEQ. ID. NO. 2;arginine or lysine at a position corresponding to position 25 of SEQ. ID. NO. 2;serine or threonine at a position corresponding to position 27 of SEQ. ID. NO. 2;aspartic acid at a position corresponding to position 31 of SEQ. ID. NO. 2;proline at a position corresponding to position 32 of SEQ. ID. NO. 2; andarginine at a position corresponding to position 33 of SEQ. ID. NO. 2.

14. The method of claim 1, wherein the amino acid sequence at least 70% identical to SEQ. ID. NO. 2 comprises: arginine at a position corresponding to position 10 of SEQ. ID. NO. 2;tyrosine at a position corresponding to position 18 of SEQ. ID. NO. 2;arginine at a position corresponding to position 25 of SEQ. ID. NO. 2;threonine at a position corresponding to position 27 of SEQ. ID. NO. 2;glutamine at a position corresponding to position 28 of SEQ. ID. NO. 2.

15. The method of claim 2, wherein the first composition and the second composition are cell-free systems.

16. A method of identifying an agent affecting ubiquitination of a Smad protein by Smurf1 comprising: providing a first composition comprising: a first amino acid sequence comprising an amino acid sequence of SEQ. ID. NO. 1;a second amino acid sequence comprising an amino acid sequence at least 70% identical to SEQ. ID. NO. 2 (WW domain of Smurf1), said second amino acid sequence capable of binding the amino acid sequence of SEQ. ID. NO. 1 and of ubiquitinating the Smad protein;the Smad protein;a source of ubiquitin;a source of ATP;the agent; andmeasuring an amount of a ubiquitinated Smad protein in the first composition.

17. The method of claim 16, wherein the SEQ. ID NO. 1 is identical to a SEQ. ID. NO. 5.

18. The method of claim 16, wherein the SEQ. ID NO. 1 is identical to a SEQ. ID. NO. 6.

19. The method of claim 16, wherein the SEQ. ID NO. 1 is incorporated within SEQ. ID. NO. 4.

20. The method of claim 16, wherein the amino acid sequence at least 70% identical to SEQ. ID. NO. 2 and capable of binding the amino acid sequence of SEQ ID. NO. 1 is 100% identical to SEQ. ID. NO. 2.

21. The method of claim 16, wherein the amino acid sequence at least 70% identical to SEQ. ID. NO. 2 comprises: leucine at a position corresponding to position 3 of SEQ. ID. NO. 2;proline at a position corresponding to position 4 of SEQ. ID. NO. 2;tryptophan at a position corresponding to position 7 of SEQ. ID. NO. 2;glutamic acid at a position corresponding to position 8 of SEQ. ID. NO. 2;phenylalanine or isoleucine at a position corresponding to position 18 of SEQ. ID. NO. 2;phenylalanine or isoleucine at a position corresponding to position 19 of SEQ. ID. NO. 2;valine or isoleucine at a position corresponding to position 20 of SEQ. ID. NO. 2;asparagine or aspartic acid at a position corresponding to position 21 of SEQ. ID. NO. 2;histidine at a position corresponding to position 22 of SEQ. ID. NO. 2;asparagine, valine, proline or serine at a position corresponding to position 23 of SEQ. ID. NO. 2;arginine or lysine at a position corresponding to position 25 of SEQ. ID. NO. 2;serine or threonine at a position corresponding to position 27 of SEQ. ID. NO. 2;aspartic acid at a position corresponding to position 31 of SEQ. ID. NO. 2;proline at a position corresponding to position 32 of SEQ. ID. NO. 2; andarginine at a position corresponding to position 33 of SEQ. ID. NO. 2.

22. The method of claim 16, wherein the amino acid sequence at least 70% identical to SEQ. ID. NO. 2 comprises: arginine at a position corresponding to position 10 of SEQ. ID. NO. 2;tyrosine at a position corresponding to position 18 of SEQ. ID. NO. 2;arginine at a position corresponding to position 25 of SEQ. ID. NO. 2;threonine at a position corresponding to position 27 of SEQ. ID. NO. 2;glutamine at a position corresponding to position 28 of SEQ. ID. NO. 2.

23. The method of claim 16, wherein the second amino acid sequence is identical to an amino acid sequence derived from a human Smurf1 protein, a rat Smurf1 protein, a mouse Smurf1 protein, or a chimpanzee Smurf1 protein.

24. The method of claim 16, wherein the Smad protein is selected from the group consisting of a Smad1 protein, a Smad5 protein, and a Smad6 protein.

25. The method of claim 16, further comprising comparing the amount of the complex in the first composition with an amount of a complex formed between a third amino acid sequence and a fourth amino acid sequence in a second composition, wherein said second composition comprises a source of ubiquitin, a source of ATP, and the Smad protein or the fragment thereof capable of being ubiquitinated by the Smurf1 protein;said second composition does not include the agent;the third amino acid sequence comprises an amino acid sequence of SEQ. ID NO. 1; andthe fourth amino acid sequence comprises an amino acid sequence at least 70% identical to SEQ. ID. NO. 2, said second amino acid sequence capable of binding the amino acid sequence of SEQ. ID. NO. 1 and of ubiquitinating the Smad protein.

26. The method of claim 25, wherein the first amino acid sequence is identical to the third amino acid sequence, orthe second amino acid sequence is identical to the fourth amino acid sequence, orthe first amino acid sequence is identical to the third amino acid sequence and the second amino acid sequence is identical to the fourth amino acid sequence.

27. The method of claim 25, wherein an increased amount of the ubiquitinated Smad protein in the first composition indicates that the agent induces ubiquitination of the Smad protein by Smurf1; anda decreased amount of the ubiquitinated Smad protein in the first composition indicates that the agent inhibits ubiquitination of the Smad protein by Smurf1.

28. The method of claim 25, wherein an increased amount of the ubiquitinated Smad protein in the first composition indicates that the agent inhibits an osteogenic effect of a BMP protein; andan decreased amount of the ubiquitinated Smad protein in the first composition indicates that the agent induces an osteogenic effect of the BMP protein.

29. The method of claim 25, wherein the first composition and the second composition are cell-free compositions.

30. A method of identifying an agent affecting ubiquitination of a Smad protein by a Smurf1 protein comprising: providing a first composition comprising: the Smurf1 protein or a fragment thereof capable of ubiquitinating the Smad protein;a source of ubiquitin;a source of ATP;the Smad protein or a fragment thereof capable of being ubiquitinated by the Smurf1 protein;the agent; andmeasuring an amount of ubiquitinated Smad protein or the fragment thereof in the first composition.

31. The method of claim 30, wherein the Smad protein is selected from the group consisting of a Smad1 protein, a Smad5 protein, and a Smad6 protein.

32. The method of claim 30, wherein the Smurf1 protein is selected from the group consisting of a human Smurf1 protein, a rat Smurf1 protein, a mouse Smurf-1 protein, and a chimpanzee Smurf1 protein.

33. The method of claim 30, further comprising comparing the amount of the ubiquitinated Smad protein or the fragment thereof in the first composition with an amount of amount of ubiquitinated Smad protein or a fragment thereof in a second composition, wherein said second composition does not include the agent; andsaid second composition comprises the Smurf1 protein or a fragment thereof capable of ubiquitinating the Smad protein;a source of ubiquitin;a source of ATP;the Smad protein or a fragment thereof capable of being ubiquitinated by the Smurf1 protein.

34. The method of claim 33, wherein: (a) the fragment of the Smurf1 protein in the first composition is identical to the fragment of the Smurf1 protein in the second composition;(b) the source of ubiquitin in the first composition is identical to the source of ubiquitin in the second composition;(c) the fragment of the Smad protein in the first composition is identical to the fragment of the Smad protein in the second composition; or(d) any combination of (a), (b), and (c).

35. The method of claim 33, wherein the first composition and the second composition are cell-free systems.

36. The method of claim 33, wherein an increased amount of the ubiquitinated Smad protein indicates that the agent increases ubiquitination of the Smad protein by Smurf1; anda decreased amount of the ubiquitinated Smad protein indicates that the agent decreases ubiquitination of the Smad protein by Smurf1.

37. The method of claim 33, wherein an increased amount of the ubiquitinated Smad protein indicates that the agent inhibits an osteogenic effect of a BMP protein; anda decreased amount of the ubiquitinated Smad protein indicates that the agent induces an osteogenic effect of the BMP protein.

38. A kit comprising: a first amino acid sequence comprising an amino acid sequence of SEQ. ID. NO. 1; anda second amino acid sequence comprising an amino acid sequence at least 70% identical to SEQ. ID. NO. 2 (WW domain of SMURF1) and capable of binding the amino acid sequence of SEQ. ID. NO. 1.

39. The kit of claim 38 further comprising a detection means capable of detecting an amount of a complex between the first amino acid sequence and the second amino acid sequence.

40. The kit of claim 38, wherein the second amino acid sequence is capable of ubiquitinating a Smad protein or a fragment thereof capable of being ubiquitinated by the second amino acid sequence.

41. The kit of claim 40, further comprising the Smad protein or the fragment thereof capable of being ubiquitinated by the second amino acid sequence;a source of ubiquitin; anda source of ATP.

42. The kit of claim 40, wherein the Smad protein is selected from the group consisting of a Smad1 protein, a Smad5 protein, and a Smad6 protein.

43. The kit of claim 40, further comprising a detection means capable of detecting an amount of the Smad protein or the fragment thereof which is ubiquitinated.

44. The kit of claim 38 further comprising a set of instructions.

45. A kit comprising a Smurf1 protein or a fragment thereof capable of ubiquitinating a Smad protein;a source of ubiquitin;a source of ATP;the Smad protein or a fragment thereof capable of being ubiquitinated by the Smurf1 protein.

46. The kit of claim 45 further comprising a detection means.

47. The kit of claim 46, wherein the detection means is capable of detecting an amount of the Smad protein or the fragment thereof which is ubiquitinated.

48. The kit of claim 45, wherein the Smad protein is selected from the group consisting of a Smad1 protein, a Smad5 protein, and a Smad6 protein.

49. The kit of claim 45, further comprising a set of instructions.

50. A method of identifying an agent affecting a binding between an LMP protein and a Smurf1 protein comprising: a) obtaining coordinates for a three-dimensional structure of a Smurf1 protein or a WW-2 motif containing fragment thereof;b) selecting the agent by performing a rational drug design with the three-dimensional coordinates, wherein said selecting is performed in conjunction with computer modeling of a complex between an LMP protein or a fragment thereof and the Smurf1 protein or the fragment thereof.

51. The method of claim 50, further comprising testing said agent by methods described in at least one of the claims 1, 16, or 30.