Neuregulin protein regulation of synaptic proteins

• US 20070042433A1
• Filed: 03/26/2004
• Published: 02/22/2007
• Est. Priority Date: 03/26/2003
• Status: Abandoned Application

• Current Assignee: Washington University In St Louis
• Original Assignee: Washington University In St Louis
• Inventors: Bao, Jianxin
• Application Number:
  US10/550,673
• Publication Number:
  US 20070042433A1
• Time in Patent Office: Days
• US Class Current: 435/7.200
• CPC Class Codes: A01K 2217/052 inducing gain of function
• CPC Class Codes: C07K 14/4756 Neuregulins, i.e. p185erbB2 ligands, glial growth factor, heregulin, ARIA, neu differentiation factor
• CPC Class Codes: C07K 14/485 Epidermal growth factor [EGF], i.e. urogastrone
• CPC Class Codes: G01N 2333/4756 Neuregulins, i.e. p185erbB2 ligands, glial growth factor, heregulin, ARIA, neu differentiation factor
• CPC Class Codes: G01N 2333/70567 Nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, nuclear orphan receptors
• 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 2500/04 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 2800/14 Disorders of ear, nose or throat
• CPC Class Codes: G01N 33/5035 on sub-cellular localization
• CPC Class Codes: G01N 33/5041 involving analysis of members of signalling pathways
• CPC Class Codes: G01N 33/6875 Nucleoproteins

Abstract

Methods are provided for discovery of new lead pharmaceuticals useful for addressing diseases and other problems of hearing. Compounds that modulate PSD-95 induction are discovered via use of the Nrg-1/Eos signaling pathway. Cell based assay systems are particularly described that identify modulation of binding of Nrg-ICD with an Eos binding site. Other features include the promotion and/or inhibition of Nrg-ICD translocation across nuclear membranes.

Human Nrg-ICD Polypeptide SequenceSEQ ID NO: 1KTKKQRKKLHDRLRQSLRSERNNVMNMANGPHHPNPPPDNVQLVNQYVSK NIISSERVVERETETSFSTSHYTSTTHHSMTVTQTPSHSWSNGHTESILS ESHSVLVSSSVENSRHTSPTGPRGRLNGIGGPREGNSFLRHARETPDSYR DSPHSERYVSAMTTPARMSPVDFHTPTSPKSPPSEMSPPVSSLTISIPSV AVSPFMDEERPLLLVTPPRLREKYDNHLQQFNSFHNNPTHESNSLPPSPL RIVEDEEYETTQEYEPAQEPPKKLTNSRRVKRTKPNGHISSRVEVDSDTS SQSTSSESETEDERTGEDTPFLSIQNPMATSLEPAAAYRLAENRTNPANR FSTPEELQARLSSVIANQDPIAV

Claims

1-34. -34. (canceled)

35. A method for decreasing the expression of PSD-95 protein in a cell, the method comprising contacting the cell with an agent that inhibits the interaction of Nrg-1 with Eos, the inhibition of interaction resulting in a decrease in PSD-95 promoter activity in the cell.

36. The method of claim 35, wherein the agent is a nucleotide sequence encoding a polypeptide that blocks interaction between Nrg-1 and Eos.

37. The method of claim 36, wherein the nucleotide sequence encodes a polypeptide having the amino acid sequence of SEQ ID NO. 8.

38. The method of claim 35, wherein the cell is a mammalian cell.

39. The method of claim 38, wherein the cell is a neuron.

40. A method for identifying a compound that modulates the Nrg-1/Eos signaling pathway in a cell, the method comprising:

(a) combining one or more test compounds with at least one or more agents that participate in the pathway;

(b) determining the amount of PSD-95 protein expressed in the cell in the presence of the test compound; and

(c) comparing the amount of PSD-95 protein expressed in the cell in the presence of the test compound with the amount of PSD-95 protein expressed in the cell in the absence of the test compound, wherein a change in the amount of PSD-95 expression in the presence of the test compound is indicative of a compound that modulates the pathway.

41. The method of claim 40, wherein the agent that participates in the signaling pathway is a nucleotide sequence encoding Nrg-ICD or a portion thereof, the nucleotide sequence hybridizing to a nucleotide sequence having SEQ ID. No:

1 in 5×

SSC at 42°

C.

42. The method of claim 41, wherein the test compound modulates the pathway by binding to Nrg-ICD.

43. The method of claim 41, wherein the test compound modulates the pathway by inhibiting the binding of Nrg-ICD to a binding site on Eos.

44. The method of claim of claim 41, wherein the test compound modulates translocation of Nrg-ICD into the nucleus of the cell.

45. The method of claim 44, wherein the Nrg-ICD is produced transgenically within the cell and the Nrg-ICD further comprises a conjugate of a polypeptide that is at least 90% homologous with SEQ ID NO. 1 and a detectable label.

46. The method of claim 45, wherein the Nrg-ICD further comprises a nuclear localization sequence selected from the group consisting of SEQ ID NO. 3 and SEQ ID. NO. 4.

47. The method of claim 46, wherein the detectable label is selected from the group consisting of green fluorescent protein, a chemilumiphore, an antigenic peptide sequence and a regulatory marker.

48. The method of claim 47, wherein the cell is a neuron.

49. The method of claim 44, wherein the Nrg-ICD further comprises a nuclear localization sequence comprising SEQ ID NO. 2.

50. The method of claim 49, wherein the detectable label is a regulatory marker selected from the group consisting of a promoter and an enhancer.

51. The method of claim 50, wherein the cell is a neuron.

52. A method for identifying a compound that modulates proteolysis of Nrg-1 to form Nrg-ICD, the method comprising:

(a) incubating a cellular membrane form of Nrg-1 in the presence of the compound; and

(b) detecting the formation of a carboxylic end portion of Nrg-1 that is less than approximately 60 kilodaltons in size.

53. The method of claim 52, wherein the cellular form of Nrg-1 is an intact cell and the carboxylic end portion of Nrg-1 is approximately 35 kilodaltons in size.

54. The method of claim 52, wherein the carboxylic end portion is detected by an immunologically reactive water soluble peptide.