BCL-XL-interacting protein and use thereof

• US 20020177692A1
• Filed: 04/15/2002
• Published: 11/28/2002
• Est. Priority Date: 04/16/2001
• Status: Abandoned Application

• Current Assignee: Myriad Genetics
• Original Assignee: Myriad Genetics
• Inventors: Bartel, Paul
• Application Number:
  US10/122,573
• Publication Number:
  US 20020177692A1
• Time in Patent Office: Days
• US Class Current: 530/350
• CPC Class Codes: C07K 14/4747 Apoptosis related proteins
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: G01N 2500/02 Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
• CPC Class Codes: G01N 2500/10 involving cells
• CPC Class Codes: G01N 2500/20 cell-free systems
• 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 33/5011 for testing antineoplastic activity
• CPC Class Codes: G01N 33/502 for testing non-proliferative effects
• CPC Class Codes: G01N 33/5091 for testing the pathological state of an organism

Abstract

Protein complexes are provided comprising BCL-XL and TCTP. The protein complexes are useful in screening assays for identifying compounds effective in modulating the protein complexes and in treating and/or preventing diseases and disorders associated with BCL-XL and TCTP. In addition, methods for detecting the protein complexes and modulating the functions and activities of the protein complexes or interacting members thereof are also provided.

Claims

1. An isolated protein complex having a first protein which is BCL-XL or a homologue or derivative or fragment thereof interacting with a second protein which is TCTP or a homologue or derivative or fragment thereof.

2. The isolated protein complex of claim 1, wherein said first protein is BCL-XL and said second protein is TCTP.

3. The isolated protein complex of claim 1, wherein said first protein is a first fusion protein containing BCL-XL or a BCL-XL homologue or fragment.

4. The isolated protein complex of claim 1, wherein said second protein is a second fusion protein containing TCTP or a TCTP homologue or fragment.

5. An isolated protein complex comprising a first protein interacting with a second protein, wherein:

(a) said first protein is selected from the group consisting of

(i) BCL-XL,

(ii) a BCL-XL fragment capable of interacting with TCTP, and

(iii) a fusion protein containing BCL-XL or said BCL-XL fragment; and

(b) said second protein is selected from the group consisting of

(1) TCTP,

(2) a TCTP fragment capable of interacting with BCL-XL, and

(3) a fusion protein containing TCTP or said TCTP fragment.

6. A protein microarray comprising the protein complex according to claim 5.

9. A method for selecting modulators of the protein complex of claim 5, comprising:

providing the protein complex;

contacting said protein complex with a test compound; and

detecting the binding of said test compound to said protein complex.

10. The method of claim 9, further comprising a step of generating a data set defining one or more selected test compounds, said data set being embodied in a transmittable form.

18. A method for selecting modulators of the protein complex of claim 5, comprising:

contacting said protein complex with a test compound; and

detecting the interaction between said first protein and said second protein.

35. A method for providing modulators of a protein-protein interaction comprising:

providing atomic coordinates defining a three-dimensional structure of the protein complex of claim 5; and

designing or selecting compounds capable of modulating the interaction between the first and second proteins based on said atomic coordinates.

37. A method for providing antagonists of a protein-protein interaction, comprising:

providing atomic coordinates defining a three-dimensional structure of the protein complex of claim 5; and

designing or selecting compounds capable of interfering with the interaction between the first and second proteins based on said atomic coordinates.

38. An isolated antibody selectively immunoreactive with the protein complex of claim 5.

7. A fusion protein having a first polypeptide covalently linked to a second polypeptide, wherein said first polypeptide is BCL-XL or a homologue or fragment thereof, and wherein said second polypeptide is TCTP or a homologue or fragment thereof.

8. A nucleic acid encoding the fusion protein of claim 7.

11. A method for selecting modulators of an interaction between a first protein and a second protein,

(a) said first protein being selected from the group consisting of

(i) BCL-XL,

(ii) a BCL-XL homologue having an amino acid sequence at least 90% identical to that of BCL-XL and capable of interacting with TCTP,

(iii) a BCL-XL fragment capable of interacting with TCTP, and

(iv) a fusion protein containing BCL-XL, said BCL-XL homologue or said BCL-XL fragment;

and

(b) said second protein being selected from the group consisting of

(1) TCTP,

(2) a TCTP homologue having an amino acid sequence at least 90% identical to that of TCTP and capable of interacting with BCL-XL,

(3) a TCTP fragment capable of interacting with BCL-XL, and

(4) a fusion protein containing TCTP, said TCTP homologue or said TCTP fragment, said method comprising;

contacting said first protein with said second protein in the presence of a test compound; and

detecting the interaction between said first protein and said second protein.

12. The method of claim 11, wherein at least one of said first and second proteins is a fusion protein having a detectable tag.

13. The method of claim 11, wherein said contacting step is conducted in a substantially cell free environment.

14. The method of claim 11, wherein the interaction between said first protein and said second protein is determined in a host cell.

15. The method of claim 14, wherein said host cell is a yeast cell.

16. The method of claim 11, wherein said detecting step comprises measuring the amount of the protein complex formed by said first and second proteins.

17. The method of claim 11, further comprising a step of generating a data set defining one or more selected test compounds, said data set being embodied in a transmittable form.

19. The method of claim 18, further comprising a step of generating a data set defining one or more selected test compounds, said data set being embodied in a transmittable form.

20. A method for selecting modulators of an interaction between a first polypeptide and a second polypeptide, said first polypeptide being BCL-XL or a homologue or fragment thereof and said second polypeptide being TCTP or a homologue or fragment thereof, said method comprising:

providing in a host cell a first fusion protein having said first polypeptide, and a second fusion protein having said second polypeptide, wherein a DNA binding domain is fused to one of said first and second polypeptides while a transcription-activating domain is fused to the other of said first and second polypeptides;

providing in said host cell a reporter gene, wherein the transcription of the reporter gene is controlled by the interaction between the first polypeptide and the second polypeptide;

allowing said first and second fusion proteins to interact with each other within said host cell in the presence of a test compound; and

determining the expression of said reporter gene.

21. The method of claim 20, wherein said host cell is a yeast cell.

22. A method for selecting a compound capable of interfering with the interaction between a first protein and a second protein, wherein

(a) said first protein is selected from the group consisting of

(i) BCL-XL,

(ii) a BCL-XL homologue having an amino acid sequence at least 90% identical to that of BCL-XL and capable of interacting with TCTP,

(iii) a BCL-XL fragment capable of interacting with TCTP, and

(iv) a fusion protein containing BCL-XL, said BCL-XL homologue or said BCL-XL fragment;

and

(b) said second protein is selected from the group consisting of

(1) TCTP,

(2) a TCTP homologue having an amino acid sequence at least 90% identical to that of TCTP and capable of interacting with BCL-XL,

(3) a TCTP fragment capable of interacting with BCL-XL, and

(4) a fusion protein containing TCTP, said TCTP homologue or said TCTP fragment, said method comprising;

contacting said first protein with said second protein in the presence of a test compound and detecting the interaction between said first protein and said second protein; and

contacting said first protein with said second protein in the absence of said test compound and detecting the interaction between said first protein and said second protein.

23. The method of claim 22, wherein said contacting steps are conducted in a substantially cell free environment.

24. The method of claim 22, wherein said contacting steps are conducted in a host cell.

25. The method of claim 22, wherein the first protein is a fusion protein containing BCL-XL, said BCL-XL homologue or said BCL-XL fragment, and said second protein is a fusion protein containing TCTP, said TCTP homologue or said TCTP fragment.

26. The method of claim 22, further comprising a step of generating a data set defining one or more selected test compounds, said data set being embodied in a transmittable form.

29. A host cell comprising the expression vector of claim 28.

27. A composition comprising:

a first expression vector having a nucleic acid encoding a first protein; and

a second expression vector having a nucleic acid encoding a second protein, wherein;

(a) said first protein is selected from the group consisting of

(i) BCL-XL,

(ii) a BCL-XL homologue having an amino acid sequence at least 90% identical to that of BCL-XL and capable of interacting with TCTP,

(iii) a BCL-XL fragment capable of interacting with TCTP, and

(iv) a fusion protein containing BCL-XL, said BCL-XL homologue or said BCL-XL fragment; and

(b) said second protein is selected from the group consisting of

(1) TCTP,

(2) a TCTP homologue having an amino acid sequence at least 90% identical to that of TCTP and capable of interacting with BCL-XL,

(3) a TCTP fragment capable of interacting with BCL-XL, and

(4) a fusion protein containing TCTP, said TCTP homologue or said TCTP fragment.

28. An expression vector comprising:

(a) a first nucleic acid encoding a first protein selected from the group consisting of

(i) BCL-XL,

(ii) a BCL-XL homologue having an amino acid sequence at least 90% identical to that of BCL-XL and capable of interacting with TCTP,

(iii) a BCL-XL fragment capable of interacting with TCTP, and

(iv) a fusion protein containing BCL-XL, said BCL-XL homologue or said BCL-XL fragment; and

(b) a second nucleic acid encoding a second protein selected from the group consisting of

(1) TCTP,

(2) a TCTP homologue having an amino acid sequence at least 90% identical to that of TCTP and capable of interacting with BCL-XL,

(3) a TCTP fragment capable of interacting with BCL-XL, and

(4) a fusion protein containing TCTP, said TCTP homologue or said TCTP fragment.

30. A host cell comprising:

a first expression cassette having a nucleic acid encoding a first protein; and

a second expression cassette having a nucleic acid encoding a second protein, wherein;

(a) said first protein is selected from the group consisting of

(i) BCL-XL,

(ii) a BCL-XL homologue having an amino acid sequence at least 90% identical to that of BCL-XL and capable of interacting with TCTP,

(iii) a BCL-XL fragment capable of interacting with TCTP, and

(iv) a fusion protein containing BCL-XL, said BCL-XL homologue or said BCL-XL fragment; and

(b) said second protein is selected from the group consisting of

(1) TCTP,

(2) a TCTP homologue having an amino acid sequence at least 90% identical to that of TCTP and capable of interacting with BCL-XL,

(3) a TCTP fragment capable of interacting with BCL-XL, and

(4) a fusion protein containing TCTP, said TCTP homologue or said TCTP fragment.

31. The host cell of claim 30, wherein said host cell is a yeast cell.

32. The host cell of claim 30, wherein said first and second proteins are fusion proteins.

33. The host cell of claim 30, wherein one of said first and second nucleic acids is linked to a nucleic acid encoding a DNA binding domain, and the other of said first and second nucleic acids is linked to a nucleic acid encoding a transcription-activation domain, whereby two fusion proteins can be produced in said host cell.

34. The host cell of claim 30, further comprising a reporter gene, wherein the expression of the reporter gene is controlled by the interaction between the first protein and the second protein.

36. The method of claim 35, further comprising a step of generating a data set defining one or more selected test compounds, said data set being embodied in a transmittable form.