Regulation of autophagy pathway phosphorylation and uses thereof

Abstract

The invention relates to polypeptides and proteins known to function in the autophagy pathway that have novel phosphorylation sites. The invention also relates to antibodies specific to these polypeptides and proteins that are phosphorylated or not phosphorylated at novel phosphorylated sites. The invention also relates to methods of producing these antibodies and use of these antibodies in the treatment of diseases related to autophagocytosis.

1 . An isolated autophagy peptide comprising an amino acid sequence selected from the group consisting of sequences set forth in Table 1, wherein the x residue is nonphosphorylated or phosphorylated serine, threonine, or tyrosine, and with the proviso that the peptide is not a full-length autophagy protein. 
     
     
         2 . An isolated autophagy peptide of  claim 1  wherein the peptide does not comprise a sequence set forth in Table 2. 
     
     
         3 . The isolated autophagy peptide of  claim 1 , which comprises an amino acid sequence selected from the group consisting of the sequences set forth in Table 3. 
     
     
         4 . The isolated autophagy peptide of  claim 1 , which comprises an amino acid sequence selected from the group consisting of sequences set forth in Table 1. 
     
     
         5 . The isolated autophagy peptide of  claim 4 , wherein the x residue is nonphosphorylated serine, threonine, or tyrosine. 
     
     
         6 . The isolated autophagy peptide of  claim 1 , wherein the x residue is phosphorylated serine, threonine, or tyrosine. 
     
     
         7 . A method for producing an antibody to an autophagy polypeptide, which method comprises:
 (a) introducing an isolated autophagy peptide of  claim 1  to a mammal in an amount sufficient to produce an antibody to said autophagy peptide; and   (b) recovering said antibody from said mammal.   
     
     
         8 . The method of  claim 7 , wherein the x residue in the isolated autophagy peptide is nonphosphorylated serine, threonine, or tyrosine and the method is used to produce an antibody to a nonphosphorylated autophagy polypeptide. 
     
     
         9 . The method of  claim 7 , wherein the x residue in the isolated autophagy peptide is phosphorylated serine, threonine, or tyrosine and the method is used to produce an antibody to a phosphorylated autophagy polypeptide. 
     
     
         10 . A method for detecting an autophagy protein or fragment comprising an amino acid sequence set forth in Table 4, wherein the x residue is phosphorylated or nonphosphorylated serine, threonine, or tyrosine in a sample, which method comprises:
 (a) contacting a sample containing or suspected of containing an autophagy protein or fragment comprising the amino acid sequence set forth in Table 4, wherein the x residue is phosphorylated or nonphosphorylated serine, threonine, or tyrosine, with an isolated antibody that specifically binds to an epitope that comprises the amino acid residue x in the amino acid sequence set forth in Table 4, wherein the x residue is phosphorylated or nonphosphorylated serine, threonine, or tyrosine; and   (b) assessing a complex formed between said autophagy protein or fragment, if present in said sample, and said antibody, to determine the presence, absence and/or amount of said autophagy protein or fragment in said sample.   
     
     
         11 . The method of  claim 10 , wherein the epitope comprises the amino acid residue x in the amino acid sequence set forth in Table 4 and an amino acid residue of the autophagy protein that is outside the amino acid sequence set forth in Table 4. 
     
     
         12 . The method of  claim 10 , wherein the epitope comprises the amino acid residue x in the amino acid sequence set forth in Table 4 and amino acid residues of the autophagy protein that are outside the amino acid sequence set forth in Table 4. 
     
     
         13 . The method of  claim 10 , wherein the epitope is comprised in the amino acid sequence set forth in Table 4. 
     
     
         14 . The method of  claim 10 , wherein the epitope is comprised in an amino acid sequence set forth in Table 3. 
     
     
         15 . The method of  claim 10 , wherein the epitope is comprised in an amino acid sequence selected from the sequence set forth in Table 1. 
     
     
         16 . The method of  claim 10 , wherein the isolated antibody specifically binds to the epitope that comprises the x residue in the amino acid sequence set forth in Table 4, wherein the x residue is nonphosphorylated serine, threonine, or tyrosine. 
     
     
         17 . The method of  claim 10 , wherein the isolated antibody specifically binds to the epitope that comprises the x residue in the amino acid sequence set forth in Table 4, wherein the x residue is phosphorylated serine, threonine, or tyrosine. 
     
     
         18 . The method of  claim 10 , wherein the isolated antibody specifically binds to an epitope comprised in the amino acid sequence set forth in Table 4, wherein the x residue is nonphosphorylated serine, threonine, or tyrosine, but does not specifically bind to an epitope comprised in the amino acid sequence set forth in Table 4 wherein the x residue is phosphorylated serine, threonine, or tyrosine. 
     
     
         19 . The method of  claim 10 , wherein the isolated antibody specifically binds to an epitope comprised in the amino acid sequence set forth in Table 4, wherein the x residue is phosphorylated serine, threonine, or tyrosine, but does not specifically bind to an epitope comprised in the amino acid sequence set forth in Table 4 wherein the x residue is nonphosphorylated serine, threonine, or tyrosine.