Novel Goodpasture antigen-binding protein isoforms and protein misfolded-mediated disorders

A substantially purified processed GPBP polypeptide derived from a precursor polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and/or SEQ ID NO:8 wherein the substantially purified polypeptide is reactive with an antibody selective for one or more epitopes within one or more of the GPBP isoforms disclosed herein, wherein the substantially purified processed GPBP polypeptide is selected from the group consisting of:

• (a) a 60 Da GPBP with a molecular weight of approximately 60 kDa in denaturing gel electrophoresis, wherein the 60 kDa GPBP is present in lysosomes, cytoplasm, microsomes, and mitochondria in liver tissue, wherein the 60 kDa GPBP is membrane-associated or soluble in the lysosomes in liver tissue;
• (b) a 4447 kDa GPBP with a molecular weight of approximately 4447 kDa in denaturing gel electrophoresis, wherein the 44-47 kDa GPBP is present in lysosomes in liver tissue, wherein the 4447 kDa GPBP is predominately formed through a leupeptin-sensitive proteolysis in liver tissue;
• and
• (c) a 32 kDa GPBP with a molecular weight of approximately 32 kDa in denaturing gel electrophoresis, wherein the 32 kDa GPBP is present in cytoplasm, mitochondria, microsomes, and lysosomes in liver tissue, and wherein the 32 kDa GPBP is formed through a leupeptin-insensitive proteolysis in liver lysosomes.

A method for identifying candidate compounds to treat an autoimmune condition comprising one or more of the following techniques:

• (a) incubating a target polypeptide selected from the group consisting of α3(IV) NC1 domain polypeptide and MBP, and functional equivalents thereof with a GPBP isoform comprising an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:SEQ ID NO:26, and SEQ ID NO:28 or a GPBP isoform selected from the group consisting of 60 kDa GPBP, 4447 kDa GPBP, and 32 kDa GPBP, in the presence of ATP in the presence or absence of one or more test compounds under conditions that promote phosphorylation of the target polypeptide by the GPBP isoform in the absence of the one or more test compounds;
• detecting phosphorylation of the target polypeptide;
• and identifying test compounds that reduce phosphorylation of the target polypeptide relative to phosphorylation of the target polypeptide in the absence of the one or more test compounds, wherein such compounds are candidate compounds for treating an autoimmune condition;
• (b) incubating a GPBP isoform comprising an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:SEQ ID NO:26, and SEQ ID NO:28, or a GPBP isoform selected from the group consisting of 60 kDa GPBP, 4447 kDa GPBP, and 32 kDa GPBP with ATP in the presence or absence of one or more test compounds under conditions that promote autophosphorylation of the GPBP isoform in the absence of the one or more test compounds;
• detecting autophosphorylation of the GPBP isoform;
• and identifying test compounds that reduce autophosphorylation of the GPBP isoform protein relative to autophosphorylation of the GPBP isoform in the absence of the one or more test compounds, wherein such compounds are candidate compounds for treating an autoimmune condition;
• (c) incubating a target polypeptide selected from the group consisting of α3(IV) NC1 domain polypeptide and MBP, and functional equivalents thereof;
• and a GPBP isoform comprising an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:SEQ ID NO:26, and SEQ ID NO:28, or a GPBP isoform selected from the group consisting of 60 kDa GPBP, 4447 kDa GPBP, and 32 kDa GPBP;
• in the presence or absence of one or more test compounds, under conditions that promote conformational isomerization of the target polypeptide catalyzed by the GPBP isoform in the absence of the one or more test compounds, detecting conformational isomerization of the target protein;
• and identifying test compounds that reduce conformational isomerization of the target protein relative to conformational isomerization of the target protein in the absence of the one or more test compounds, wherein such compounds are candidate compounds to treat an autoimmune condition;
• and
• (d) incubating a GPBP isoform comprising an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:SEQ ID NO:26, and SEQ ID NO:28, or a GPBP isoform selected from the group consisting of 60 kDa GPBP, 44-47 kDa GPBP, and 32 kDa GPBP with a target polypeptide selected from the group consisting of α3(IV) NC1 domain polypeptide and MBP, and functional equivalents thereof, in the presence of one or more test compounds, under conditions that promote formation of an interaction between the GPBP isoform and the target polypeptide in the absence of test compounds and identifying test compounds that inhibit the interaction, wherein such compounds are candidate compounds to treat an autoimmune condition.

A method for identifying candidate compounds to treat a protein deposit-mediated disorder comprising one or more of the following techniques:

• (a) incubating a target polypeptide selected from the group consisting of α3(IV) NC1 domain polypeptide, MBP, and prion protein, and functional equivalents thereof with a GPBP protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:SEQ ID NO:26, and SEQ ID NO:28 or a GPBP isoform selected from the group consisting of 60 kDa GPBP, 44-47 kDa GPBP, and 32 kDa GPBP, in the presence of ATP in the presence or absence of one or more test compounds under conditions that promote phosphorylation of the target polypeptide by the GPBP in the absence of the one or more test compounds;
• detecting phosphorylation of the target polypeptide;
• and identifying test compounds that reduce phosphorylation of the target polypeptide relative to phosphorylation of the target polypeptide in the absence of the one or more test compounds, wherein such compounds are candidate compounds for treating a protein deposit-mediated disorder;
• (b) incubating a GPBP isoform comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:SEQ ID NO:26, and SEQ ID NO:28 or a GPBP isoform selected from the group consisting of 60 kDa GPBP, 44-47 kDa GPBP, and 32 kDa GPBP, with ATP in the presence or absence of one or more test compounds under conditions that promote autophosphorylation of the GPBP isoform in the absence of the one or more test compounds;
• detecting autophosphorylation of the GPBP isoform;
• and identifying test compounds that reduce autophosphorylation of the GPBP isoform relative to autophosphorylation of the GPBP isoform in the absence of the one or more test compounds, wherein such compounds are candidate compounds for treating a protein deposit-mediated disorder;
• (c) incubating a target polypeptide selected from the group consisting of α3, MBP, and prion protein, and functional equivalents thereof;
• and a GPBP isoform comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:SEQ ID NO:26, and SEQ ID NO:28 or a GPBP isoform selected from the group consisting of 60 kDa GPBP, 44-47 kDa GPBP, and 32 kDa GPBP;
• in the presence or absence of one or more test compounds, under conditions that promote conformational isomerization of the target polypeptide catalyzed by the GPBP isoform in the absence of the one or more test compounds, detecting conformational isomerization of the target protein;
• and identifying test compounds that reduce conformational isomerization of the target protein relative to conformational isomerization of the target protein in the absence of the one or more test compounds, wherein such compounds are candidate compounds to treat a protein deposit-mediated disorder;
• and
• (d) incubating a GPBP isoform comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:SEQ ID NO:26, and SEQ ID NO:28 or a GPBP isoform selected from the group consisting of 60 kDa GPBP, 4447 kDa GPBP, and 32 kDa GPBP with a target polypeptide selected from the group consisting of α3, MBP, prion protein, Aβ1-42, and functional equivalents thereof, in the presence of one or more test compounds, under conditions that promote formation of an interaction between the GPBP isoform and the target polypeptide in the absence of test compounds and identifying test compounds that inhibit the interaction, wherein such compounds are candidate compounds to treat a protein deposit-mediated disorder.

An isolated polypeptide consisting of X1-SHCIX2-X3

• wherein X1 is 0-10 amino acids of the sequence ATTAGILATL (SEQ ID NO:41);
• X2 is E or Q;
• and
• X2 is 0-10 amino acids of the sequence LMVKREDSWQ (SEQ ID NO:42)