Chemistry: analytical and immunological testing – Biospecific ligand binding assay
Reexamination Certificate
1999-02-18
2002-12-17
Stucker, Jeffrey (Department: 1648)
Chemistry: analytical and immunological testing
Biospecific ligand binding assay
C435S004000, C435S007100, C435S007600, C435S007720, C435S007930, C435S007940, C435S007950, C530S300000, C530S350000
Reexamination Certificate
active
06495376
ABSTRACT:
BACKGROUND OF THE INVENTION
Homeostasis of the organism depends upon interactions between protein-interacting modules and ligands to activate and deactivate cell signaling pathways for biological processes such as cell proliferation, cell death and protein degradation. Protein-interacting modules are conserved regions of amino acids that bind specific sequences in target proteins or position enzymes in close proximity to their substrates. For example, src homology domain 2 (SH2) binds phosphotyrosine residues on target cells to mediate receptor activation and receptor-ligand binding (Pawson, T., et al.,
Science
278:2075 (1997)). An example are WW-domains which are highly conserved regions of approximately 40 amino acids residues with two invariant tryptophans (W) in a triple stranded &bgr; sheet (Sudol, M.
Prog. Biophys. Mol. Biol.
65:113 (1996); Rotin, D.
Curr. Topics Microbiol. Immunol.
228:115 (1998)). Although the WW-domains of certain polypeptides have been implicated in protein-protein interactions by binding to proline rich sequences, many of their ligands do not contain proline rich sequences. (Sudol, M.
Prog. Biophys. Mol. Biol.
65:113 (1996); Staub, O. et al.,
Structure
4:495 (1996), Rotin, D.,
Curr. Top. Microbiol. Immunol.
228:115 (1998)). Therefore, the role of WW-domain-containing proteins in mediating cell signaling events in biological processes is not known. However, due to their potential importance in cellular processes, it is important to elucidate a clearer understanding of the role of WW-domains in protein-protein interactions and cell signaling.
SUMMARY OF THE INVENTION
The present invention is based upon the discovery that WW-domains are phosphoserine or phosphothreonine binding modules. As further described herein, the present invention is also based upon the discovery that the WW-domain itself is phosphorylated, and that phosphorylation/dephosphorylation of the WW-domain polypeptide regulates the interaction of the WW-domain polypeptide with its phosphorylated ligand. As a result of this discovery, methods and compositions are available to modulate protein-protein interactions, e.g., the interaction between a signaling or regulatory polypeptide and its phosphorylated ligands.
The invention relates to methods of modulating protein-protein interactions comprising modulating the binding of WW-domain polypeptides with phosphorylated ligands. In one embodiment the binding interaction between the WW-domain containing polypeptide and phosphorylated ligand is inhibited. In another embodiment the binding interaction of the WW-domain containing polypeptide and phosphorylated ligand is enhanced. As used herein, a phosphorylated ligand is a molecule (e.g., protein, peptide, peptide mimetic or small organic molecule) containing a phosphoserine or phosphothreonine that binds to a WW-domain containing polypeptide. For example, ligands specifically encompassed by the present invention include tau protein, amyloid precursor protein, Cdc25C, Cdc27, Plk1, NIMA, Myt1, Rab4, Wee1, Mos, Sox3, Xbr1b, MP75 (E-MAP-115), MP110 (Cdc5), MP68, and MP30. WW-domain containing polypeptides specifically encompassed by the present invention include Pin1, NEDD4, YAP, FE65, formin binding protein, dystrophin, utropin, Ess1p/Ptf1p, Rsp5, Pub1, Dodo, Msb1, ORF1, YKB2, DP71, C38D4.5, P9659.21, Yo61, Yfx1, ZK1248.15, KO15c11, CD45AP, FBP11, FBP21, FBP23, FBP28 and FBP30.
Also encompassed by the present invention are molecules which mimic a WW-domain, referred to herein as WW-domain mimic molecules or pseudo-WW-domain molecules. Such molecules possess structural similarity with the WW-domains described herein or contain the consensus sequence LxxGWtx
6
Gtx(Y/F)(Y/F)h(N/D)Hx(T/S)tT(T/S)tWxtPt SEQ ID NO: 40 (where x=any amino acid, t=turn like or polar residue, and h=hydrophobic amino acid as described by Rotin, D.,
Curr. Top. Microbiol. Immunol.
228:115-133 (1998) the teachings of which are incorporated herein by reference in their entirety). For example, a WW-domain can contained the consensus sequence LP
x
GWE
xxxxxxx
G
xx
YY
x
NH
x
T
xx
T
x
W
xx
P SEQ ID NO: 41, where x=any amino acid. The WW-domain mimic molecules are amino acid sequences, peptides, peptide mimetics, or polypeptides. The WW-domain mimic molecules are capable of interacting with, or binding to, phosphoserine/phosphothreonine ligands, thus modulating the activity of the phosphorylated ligand.
Also encompassed by the present invention are phosphorylated ligand sequences, referred to herein as phosphorylated ligand mimics, or phosphorylated pseudo-ligands. Phosphorylated ligand mimics are amino acid sequences, peptides, peptide mimetics, or polypeptides that contain a phosphoserine or phosphothreonine residue(s) and are of sufficient length and share sufficient amino acid identity with the ligand that the ligand mimics and interacts with, or binds to, the WW-domain containing polypeptide and thus modulates the activity of the WW-domain containing polypeptide.
A method of modulating the activity of a phosphorylated ligand or ligand mimic for a WW-domain, or a WW-domain containing polypeptide, comprises providing a WW-domain or WW-domain mimic which interacts with the ligand, wherein the activity of the phosphorylated ligand, ligand mimic, WW-domain polypeptide or WW-domain mimic is modulated (e.g., inhibited or enhanced). The activity can be binding activity between the ligand and WW-domain; enzymatic/regulatory activity of the WW-domain polypeptide; or both. For example, the prolyl-peptidyl cis-trans isomerase activity of Pin1 or ubiquitin ligase activity of Nedd4 can increase following binding of the WW-domain to a phosphorylated ligand.
Another aspect of the invention relates to regulating cell growth comprising mediating the binding of the WW-domain of Pin1 to a mitotic regulatory protein. The WW-domain can bind to a phosphorylated ligand (e.g., NIMA) resulting in cell proliferation. Cell proliferation can be regulated by regulating the phosphorylation state of the WW-domain. Dephosphorylation of the WW-domain of Pin1 leads to binding of the WW-domain to a phosphorylated ligand resulting in cell proliferation. Likewise, phosphorylation of the WW-domain inhibits binding to phosphorylated ligands resulting in cell death.
The invention also encompasses methods of regulating neurodegenerative diseases by modulating the interaction of a WW-domain and a ligand in cells (e.g., neurons, glial cells, Schwann cells) of the central (e.g., brain and spinal cord) and peripheral nervous system and any cells associated with the central or peripheral nervous systems (e.g., skeletal muscle). The interaction between the WW-domain and a neural cellular target can inhibit, halt, prevent or reverse neural degeneration by, for example, interfering with neuronal cell death (e.g., apoptosis, necrosis) or restoring neuronal function.
A further aspect of the invention encompasses a method of regulating the function of phosphorylated ligands of WW-domain containing polypeptides comprising mediating the binding of the ligand to the WW-domain. Specifically encompassed by the invention is a method of regulating the activity of hyperphosphorylated tau protein in Alzheimer's disease comprising enhancing the binding of the WW-domain of Pin1 to the phosphorylated threonine 231 of tau whereby the binding of the WW-domain to tau results in binding of tau to microtubules leading to microtubule assembly. Another method of the invention relates to a method of regulating the interaction between the WW-domain of dystrophin and phosphorylated ligands.
The present invention further relates to a method of identifying a substance that modulates the interaction of a WW-domain containing polypeptide and a ligand, wherein the ligand is a phosphoserine or phosphothreonine ligand comprising contacting the WW-domain containing polypeptide with one, or more, test substances; maintaining the test substances and the WW-domain containing polypeptide under conditions suitable for interaction; and determining the interaction betwee
Lu Kun Ping
Zhou Xiao Zhen
Beth Israel Deaconess Medical Center
Hanley, Esq. Elizabeth A.
Kanik, Esq. Cynthia L.
Lahive & Cockfield LLP
Stucker Jeffrey
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