Bibliographic Details
| Title: |
Components of the presenilin-complex |
| Document Number: |
20050288212 |
| Publication Date: |
December 29, 2005 |
| Appl. No: |
10/519238 |
| Application Filed: |
June 25, 2003 |
| Abstract: |
The present invention is based on a novel direct interaction between a Presenilin and a novel protein identified herein and named Sambiasin-1, a homolog thereof named Sambiasin-2, as well as a protein complex further comprising a Nicastrin. Also comprised are uses of said components and complexes, as well as methods for use of the protein and the complex, inter alia, screening, diagnosis and therapy, as well as methods of preparing the complexes. |
| Inventors: |
Hale, Richard (Prestwood, GB); Rowley, Adele (St Albans, GB) |
| Assignees: |
CELLZOME AG (Heidelberg, DE) |
| Claim: |
1. A protein complex comprising (a) a first protein, or a functionally active fragment or functionally active derivative thereof, which first protein is selected from the group consisting of: (i) Sambiasin-1 (SEQ ID No: 1) or a functionally active derivative thereof, or a functionally active fragment thereof, or a homolog thereof, or a variant of Sambiasin-1 encoded by a nucleic acid that hybridizes to the Sambiasin-1 nucleic acid or its complement under low stringency conditions, (b) and at least one second protein, or a functionally active fragment or functionally active derivative thereof, which second protein is selected from the group consisting of: (i) Presenilin-1 (SEQ ID No: 2), or a functionally active derivative thereof, or a functionally active fragment thereof, or a homolog thereof, or a variant of Presenilin-1 encoded by a nucleic acid that hybridizes to the Presenilin-1 nucleic acid or its complement under low stringency conditions, (ii) Nicastrin (SEQ ID No: 3), or a functionally active derivative thereof, or a functionally active fragment thereof, or a homolog thereof, or a variant of Nicastrin encoded by a nucleic acid that hybridizes to the Nicastrin nucleic acid or its complement under low stringency conditions, wherein said first protein and said second protein are members of a native cellular complex, and wherein said low stringency conditions comprise hybridization in a buffer comprising 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 ug/ml denatured salmon sperm DNA, and 10% (wt/vol) dextran sulfate for 18-20 hours at 40° C., washing in a buffer consisting of 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS for 1.5 hours at 55° C., and washing in a buffer consisting of 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS for 1.5 hours at 60° C. |
| Claim: |
2. A protein complex comprising Sambiasin-1 (SEQ ID No: 1) or Sambiasin-2 (SEQ ID No: 4) and Presenilin-1 (SEQ ID No: 2) or Presenilin-2 (SEQ ID No: 5). |
| Claim: |
3. A protein complex according to claim 1 further comprising Nicastrin (SEQ ID No: 3) |
| Claim: |
4. A protein complex according to claim 1 comprising Sambiasin-1 (SEQ ID No: 1) and Presenilin-1 (SEQ ID No: 2) and Nicastrin (SEQ ID No: 3). |
| Claim: |
5. The complex of claim 1 comprising a functionally active derivative of any of the proteins of said complex, wherein the functionally active derivative is a fusion protein comprising said protein fused to an amino acid sequence different from said protein. |
| Claim: |
6. The complex of claim 5 wherein the functionally active derivative is a fusion protein comprising said protein fused to an affinity tag or label. |
| Claim: |
7. The complex of claim 1 comprising a fragment of any of the proteins of said complex, which fragment binds to another protein component of said complex. |
| Claim: |
8. The complex of claim 1 that is involved in the gamma-secretase activity. |
| Claim: |
9. A protein comprising the amino acid sequence of SEQ ID No: 1, or a functionally active derivative thereof, or a functionally active fragment thereof, or a homolog thereof, or a variant of Sambiasin-1 encoded by a nucleic acid that hybridizes to the Sambiasin-1 nucleic acid or its complement under low stringency conditions, wherein said low stringency conditions comprise hybridization in a buffer comprising 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 ug/ml denatured salmon sperm DNA, and 10% (wt/vol) dextran sulfate for 18-20 hours at 40° C., washing in a buffer consisting of 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS for 1.5 hours at 55° C., and washing in a buffer consisting of 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS for 1.5 hours at 60° C., with the proviso that the protein does not have the amino acid sequence according to SEQ ID No: 6. |
| Claim: |
10. Protein comprising the amino acid sequence of SEQ ID No: 1. |
| Claim: |
11. A nucleic acid encoding a protein according to claim 9. |
| Claim: |
12. A construct comprising (a) a nucleic acid according to claim 11 and at least one further nucleic acid which is normally not associated with said nucleic acid, or (b) at least two separate nucleic acid sequences each encoding a different protein, which protein is selected from the group consisting of Sambiasin-1 (SEQ ID No: 1), Presenilin-1 (SEQ ID No: 2), and Nicastrin (SEQ ID No: 3), or a functionally active fragment or a functionally active derivative thereof. |
| Claim: |
13. A host cell containing one or more constructs according to claim 12. |
| Claim: |
14. An antibody or a fragment of said antibody containing the binding domain thereof, which binds the complex of claim 1, and which does not bind the first protein when uncomplexed or the second protein when uncomplexed. |
| Claim: |
15. A kit comprising in one or more containers the complex of claim 1 optionally together with reagents and working instructions. |
| Claim: |
16. A kit according to claim 15 for the diagnosis or prognosis of a disease or a disease risk, wherein the disease is selected from the group consisting of neurodegenerative diseases and developmental disorders caused by defects in the Notch pathway. |
| Claim: |
17. An array comprising the complex according to claim 1 attached to a solid carrier. |
| Claim: |
18. A method for processing the physiological substrates of the complex of claim 1 comprising the step of bringing into contact the complex with said substrate, such that said substrate is processed. |
| Claim: |
19. A pharmaceutical composition comprising the protein complex of claim 1 and a pharmaceutically acceptable carrier. |
| Claim: |
20. A pharmaceutical composition according to claim 19 for the treatment of diseases and disorders, wherein the disease or disorder is selected from the group consisting of neurodegenerative diseases and developmental disorders caused by defects in the Notch pathway. |
| Claim: |
21. A method for screening for a molecule that binds to the complex of claim 1, comprising the following steps: (a) exposing said complex, or a cell or organism containing same, to one or more candidate molecules; and (b) determining whether said candidate molecule is bound to the complex. |
| Claim: |
22. A method for screening for a molecule that modulates the function, activity, composition or formation of the complex of claim 1 comprising the steps of: (a) exposing said complex, or a cell or organism containing said complex to one or more candidate molecules; and (b) determining the amount of, activity of, protein components of, and/or intracellular localization of, said complex and/or the transcription level of a gene dependent on the complex and/or the abundance and/or activity of a protein or protein complex dependent on the function of the complex and/or product of a gene dependent on the complex in the presence of the one or more candidate molecules, wherein a change in said amount, activity, protein components or intracellular localization relative to said amount, activity, protein components and/or intracellular localization and/or a change in the transcription level of a gene dependent on the complex and/or the abundance and/or activity of a protein or protein complex dependent on the function of the complex and/or product of a gene dependent on the complex in the absence of said candidate molecules indicates that the molecule modulates function, activity or composition of said complex. |
| Claim: |
23. The method of claim 22 wherein the amount of said complex is determined. |
| Claim: |
24. The method of claim 22, wherein the activity of said complex is determined. |
| Claim: |
25. The method of claim 24, wherein said determining step comprises isolating from the cell or organism said complex to produce said isolated complex and contacting said isolated complex in the presence or absence of a candidate molecule with a physiological substrate of the complex and determine whether said substrate is processed. |
| Claim: |
26. The method of claim 22, wherein the amount of the individual protein components of said complex are determined. |
| Claim: |
27. The method of claim 26, wherein said determining step comprises determining whether (i) Sambiasin-1 (SEQ ID No: 1) or a functionally active derivative thereof, or a functionally active fragment thereof, or a homolog thereof, or a variant of Sambiasin-1 encoded by a nucleic acid that hybridizes to the Sambiasin-1 nucleic acid or its complement under low stringency conditions, and/or (ii) Presenilin-1 (SEQ ID No: 2), or a functionally active derivative thereof, or a functionally active fragment thereof, or a homolog thereof, or a variant of Presenilin-1 encoded by a nucleic acid that hybridizes to the Presenilin-1 nucleic acid or its complement under low stringency conditions, and/or (iii) Nicastrin (SEQ ID No: 3), or a functionally active derivative thereof, or a functionally active fragment thereof, or a homolog thereof, or a variant of Nicastrin encoded by a nucleic acid that hybridizes to the Nicastrin nucleic acid or its complement under low stringency conditions, are present in the complex and wherein said low stringency conditions comprise hybridization in a buffer comprising 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 ug/ml denatured salmon sperm DNA, and 10% (wt/vol) dextran sulfate for 18-20 hours at 40° C., washing in a buffer consisting of 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS for 1.5 hours at 55° C., and washing in a buffer consisting of 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS for 1.5 hours at 60° C. |
| Claim: |
28. The method of claim 22, wherein said method is a method of screening for a drug for treatment or prevention of a disease or disorder, wherein the disease or disorder is selected from the group consisting of neurodegenerative diseases and developmental disorders caused by defects in the Notch pathway. |
| Claim: |
29. A method of manufacturing a medicament comprising the use of a molecule that modulates the amount of, activity of, or the protein components of the complex of claim 1 to manufacture a medicament for the treatment or prevention of a disease or disorder, wherein the disease or disorder is selected from the group consisting of neurodegenerative diseases and developmental disorders caused by defects in the Notch pathway. |
| Claim: |
30. A method for the production of a pharmaceutical composition comprising carrying out the method of claim 22 to identify a molecule that modulates the function, activity, composition or formation of said complex, and further comprising mixing the identified molecule with a pharmaceutically acceptable carrier. |
| Claim: |
31. A method for diagnosing or screening for the presence of a disease or disorder or a predisposition for developing a disease or disorder in a subject, which disease or disorder is characterized by an aberrant amount of, activity of, component composition of, or intracellular localization of the complex of claim 1 comprising determining the amount of, activity of, protein components of, and/or intracellular localization of, said complex and/or the transcription level of a gene dependent on the complex and/or the abundance and/or activity of a protein or protein complex dependent on the function of the complex and/or product of a gene dependent on the complex in a comparative sample derived from a subject, wherein a difference in said amount, activity, or protein components of, said complex in an analogous sample from a subject not having the disease or disorder or predisposition indicates the presence in the subject of the disease or disorder or predisposition in the subject. |
| Claim: |
32. The method of claim 31, wherein the amount of said complex is determined. |
| Claim: |
33. The method of claim 31, wherein the activity of said complex is determined. |
| Claim: |
34. The method of claim 33, wherein said determining step comprises isolating from the cell or organism said complex to produce said isolated complex and contacting said isolated complex in the presence or absence of a candidate molecule with a physiological substrate of the complex, and determine whether said substrate is processed. |
| Claim: |
35. The method of claim 31, wherein the amount of the individual protein components of said complex are determined. |
| Claim: |
36. The method of claim 35, wherein said determining step comprises determining whether (i) Sambiasin-1 (SEQ ID No: 1) or a functionally active derivative thereof, or a functionally active fragment thereof, or a homolog thereof, or a variant of Sambiasin-1 encoded by a nucleic acid that hybridizes to the Sambiasin-1 nucleic acid or its complement under low stringency conditions, and/or (ii) Presenilin-1 (SEQ ID No: 2), or a functionally active derivative thereof, or a functionally active fragment thereof, or a homolog thereof, or a variant of Presenilin-1 encoded by a nucleic acid that hybridizes to the Presenilin-1 nucleic acid or its complement under low stringency conditions, and/or (iii) Nicastrin (SEQ ID No: 3), or a functionally active derivative thereof, or a functionally active fragment thereof, or a homolog thereof, or a variant of Nicastrin encoded by a nucleic acid that hybridizes to the Nicastrin nucleic acid or its complement under low stringency conditions, are present in the complex and wherein said low stringency conditions comprise hybridization in a buffer comprising 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 ug/ml denatured salmon sperm DNA, and 10% (wt/vol) dextran sulfate for 18-20 hours at 40° C., washing in a buffer consisting of 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS for 1.5 hours at 55° C., and washing in a buffer consisting of 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS for 1.5 hours at 60° C. |
| Claim: |
37. The method of claim 31, wherein the disease or disorder is selected from the group consisting of neurodegenerative diseases and developmental disorders caused by defects in the Notch pathway. |
| Claim: |
38. A method for treating or preventing a disease or disorder characterized by an aberrant amount of, activity of, component composition of or intracellular localization of, the complex of claim 1 comprising administering to a subject in need of such treatment or prevention a therapeutically effective amount of one or more molecules that modulate the amount of, activity of, or protein components of, said complex. |
| Claim: |
39. The method according to claim 38, wherein said disease or disorder involves decreased levels of the amount or activity of said complex. |
| Claim: |
40. The method according to claim 39, wherein said disease or disorder involves increased levels of the amount or activity of said complex. |
| Claim: |
41. (canceled) |
| Claim: |
42. An antibody or a fragment of said antibody containing the binding domain thereof, which binds to the protein of claim 9. |
| Claim: |
43. A kit comprising in one or more containers the protein of claim 9, optionally together with reagents and working instructions. |
| Claim: |
44. The kit of claim 15, which further comprises a protein comprising the amino acid sequence of SEQ ID No: 1, or a functionally active derivative thereof, or a functionally active fragment thereof, or a homolog thereof |
| Claim: |
45. The kit of claim 15, which further comprises an antibody or a fragment of said antibody containing the binding domain thereof, which binds the complex. |
| Claim: |
46. An array comprising the protein of claim 9 attached to a solid carrier. |
| Claim: |
47. An array comprising at least one antibody according to claim 14 attached to a solid carrier. |
| Claim: |
48. A pharmaceutical composition comprising the protein of claim 9 and a pharmaceutically acceptable carrier. |
| Claim: |
49. A method for screening for a molecule that binds to the protein of claim 9 comprising the following steps: (a) exposing said protein, or a cell or organism containing same, to one or more candidate molecules; and (b) determining whether said candidate molecule is bound to the protein. |
| Current U.S. Class: |
514002/000 |
| Accession Number: |
edspap.20050288212 |
| Database: |
USPTO Patent Applications |
