Purified and isolated protein zero related (PZR) and therapeutic and screening method using same
AssigneeVanderbilt University
InventorZhizhuang Zhao
Life of this patent
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Abstract
Isolated and purified proteins and nucleic acids including a novel member of the immunoglobulin super-family characterized as having SHP-2 binding activity and cell signaling activity and called protein zero related or PZR, and cDNA encoding the same. Recombinant host cells, recombinant nucleic acids and recombinant proteins are also disclosed, along with methods of producing each. Isolated and purified antibodies to PZR, and methods of producing the same, are also disclosed. PZR is characterized as having SHP-2 binding activity and cell signaling activity and thus, therapeutic methods involving these activities are also disclosed.
Claims79
An isolated and purified biologically active mammalian PZR polypeptide, or fragment or analog or derivative thereof.
An isolated and purified nucleic acid molecule that encodes a biologically active PZR polypeptide, or fragment thereof.
An antibody produced by the method of claim 25.
An assay kit for detecting the presence, in a biological sample, of an antibody immunoreactive with a PZR polypeptide, the kit comprising a first container containing a PZR polypeptide that immunoreacts with the antibody, with the polypeptide present in an amount sufficient to perform at least one assay.
A method of screening candidate substances for an ability to modulate PZR biological activity, the method comprising:
- (a) establishing replicate test and control samples that comprise a biologically active PZR polypeptide;
- (b) administering a candidate substance to test sample but not the control sample;
- (c) measuring the biological activity of PZR in the test and the control samples;
- and
- (d) determining that the candidate substance modulates PZR biological activity if the biological activity of PZR measured for the test sample is greater or less than the biological activity of PZR level measured for the control sample.
A method for identifying a compound that modulates tyrosine kinase or tyrosine phosphatase biological activity, the method comprising:
- (a) contacting a cell lysate of a target cell that has been exposed to a candidate substance with an anchoring molecule that is specific for a PZR protein substrate which has been dephosphorylated as a result of signal transduction in the target cell;
- (b) detecting phosphotyrosine residues on the PZR protein substrate that is bound to the anchoring molecule;
- (c) determining that the candidate substance modulates tyrosine kinase or tyrosine phosphatase biological activity if the amount of phosphotyrosine residues on the bound PZR protein substrate derived from the target cell lysate is greater or less than an amount of phosphotyrosine residues on a bound PZR protein substrate derived from a control target cell.
A method for determining the presence or amount of a protein tyrosine phosphatase enzymatic activity in a sample, the method comprising:
- (a) incubating a sample suspected of containing a protein tyrosine phosphatase with a phosphorylated PZR peptide under conditions and for a time sufficient to permit dephosphorylation of the phosphorylated PZR peptide by a protein tyrosine phosphatase in the sample, wherein the phosphorus atom of the phosphorylated PZR peptide is detectably labeled;
- (b) separating non-dephosphorylated PZR peptide from free detectably labeled phosphorus released by dephosphorylation of the phosphorylated PZR peptide;
- and
- (c) detecting the presence or amount of detectable labeled phosphorus released by dephosphorylation of the phosphorylated PZR peptide to thereby determine the presence or amount of protein tyrosine phosphatase enzymatic activity in the sample.
A method for determining the presence or amount of a protein tyrosine kinase enzymatic activity in a sample, the method comprising:
- (a) incubating a sample suspected of containing a protein tyrosine kinase with a dephosphorylated PZR peptide and a predetermined amount of detectably labeled phosphorus atom under conditions and for a time sufficient to permit phosphorylation of the dephosphorylated PZR peptide by a protein tyrosine kinase in the sample;
- (b) separating phosphorylated PZR peptide from free detectably labeled phosphorus not incorporated onto the phosphorylated PZR peptide;
- and
- (c) detecting the presence or amount of detectably labeled phosphorus incorporated onto the phosphorylated PZR peptide to thereby determine the presence or amount of protein tyrosine kinase enzymatic activity in the sample.
A method of modulating cell signaling in a warm-blooded vertebrate, the method comprising administering to the warm-blooded vertebrate a PZR biological activity-modulating amount of a PZR biological activity-modulating composition;
- and modulating cell signaling in the warm-blooded vertebrate through the modulating of PZR biological activity with the composition.
A method for modulating cell signaling in a warm-blooded vertebrate, the method comprising introducing to a cell in the warm-blooded vertebrate a construct comprising a nucleic acid sequence encoding a PZR gene product operatively linked to a promoter, wherein production of the PZR gene product in the cell results in modulation of cell signaling.
An isolated and purified biologically active PZR ectodomain polypeptide, or fragment or analog or derivative thereof.
Dependencies are inferred from the claim text.