Identification of brain proteins that interact with 2-methylnorharman. An analog of the parkinsonian-inducing toxin, MPP+.
N-Methylated beta-carbolines, including 2-methylnorharman, are structural and functional analogs of the parkinsonian-inducing toxin, MPP+. We are investigating N-methylated beta-carbolines, including 2-methylnorharman, as possible etiologic factors in the pathogenesis of Parkinson's disease. The cellular targets of N-methylated beta-carboline-mediated cytotoxicity are unknown; therefore, we used the T7Select Phage Display System in a novel approach to identify brain proteins that bind to 2-methylnorharman. We incubated (biopanned) immobilized 2-methylnorharman with a phage display cDNA library that expressed a library of human brain proteins on the surface of bacteriophage T7. We washed off unbound phage, amplified the phage that were bound to 2-methylnorharman, and enriched for toxin-interacting phage by repeating the biopanning and amplification steps. The cDNA sequences from the toxin-interacting phage were used to derive the amino acid sequences of the phage-displayed proteins. Five of the six 2-methylnorharman-interacting proteins may have relevance to Parkinson's disease: alpha-tubulin, paraoxonase, dorfin, fatty acid binding protein, and platelet-activating factor acetylhydrolase. Dorfin has sequence homology with parkin, which is interesting because mutations in the parkin gene associate with early-onset Parkinson's disease. Our findings are the basis for future studies aimed at determining whether 2-methylnorharman affects the function of these specific proteins in vitro and in vivo.
Gearhart
Debra A
DA
Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta, GA 30912-2000, USA. dgearhar@mail.meg.edu
Toole
Patricia F
PF
Warren Beach
J
J
D015655:1-Methyl-4-phenylpyridinium / Q000737:chemistry*; D000595:Amino Acid Sequence; D001923:Brain Chemistry; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003101:Collodion; D004279:DNA, Viral / Q000096:biosynthesis / Q000235:genetics; D006247:Harmine / Q000031:analogs & derivatives* / Q000737:chemistry*; D006801:Humans; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D009498:Neurotoxins / Q000737:chemistry*; D019151:Peptide Library; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D004279:DNA, Viral; D007202:Indicators and Reagents; D009419:Nerve Tissue Proteins; D009498:Neurotoxins; D019151:Peptide Library; D011994:Recombinant Proteins; D006247:Harmine; C062099:2-methylnorharman; D003101:Collodion; D015655:1-Methyl-4-phenylpyridinium