VMAT-2 N-TERMINAL PEPTIDE RESEARCH

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A peptide derived from the N-terminus of VMAT-2 has been shown to inhibit transport of serotonin into vesicles embedded with the full-length protein. A peptide with a scrambled sequence does not have this activity. The peptide sequence contains two phosphorylation sites; the diphosphorylated peptide is also incapable of inhibiting transport. These results suggest that the N-terminus of the protein may be important in regulating its function, with the activity being switched on and off according to the phosphorylation state. Preliminary data on the native-sequence peptide suggest a partial or proto- helix in the region of the phosphorylation sites at 30 C and pH 2.8. The proposed experiments aim to collect data on the unphosphorylated and diphosphorylated peptides at the assay pH of 7.8 for further structural calculations and comparisons The N-terminus of VMAT-2 is believed to have a regulatory role in the ability of the protein to transport monoamine neurotransmitters such as serotonin. A 20-residue peptide derived from the N-terminus of the protein inhibited transport of tritium-labelled serotonin into vesicles embedded with VMAT-2. The peptide contains two serines which are possible phosphorylation sites; the diphosphorylated peptide was unable to inhibit serotonin accumulation within the vesicles. A peptide with a scrambled sequence was also unable to inhibit transport. Preliminary NMR work on the native-sequence, unphosphorylated peptide suggests alpha-helical structural tendencies which may be responsible for the observed biological activity. The work to be carried out in the requested time involves structural determination of the diphosphorylated and scrambled-sequence peptides, for comparison to see if this structural hypothesis is tenable. The 20 amino acid cytosolic N-terminus of VMAT-2, which contains five positively charged residues (4 Arg, 1 Lys), is being examined as an intramolecular mediator/enabler of monoamine transport. Treatment of human VMAT-2 transfected cos-7 vesicles with an N-terminal peptide of VMAT-2 inhibited accumulation of 3H-serotonin within the vesicles. Phosphorylation of the peptide at either of two potential phosphorylation sites within its sequence cause the peptide to fail to inhibit accumulation of 3H-serotonin within the vesicles. Mutation of the phosphorylation site serine residues to alanine or aspartate enhanced and diminished transport of serotonin, respectively. Preliminary structural data on the peptide suggest alpha-helical tendencies in the region of the peptide containing the serine residues. As the conditions under which the preliminary NMR work was done differ from the conditions used for the functional assay, further NMR work at conditions matching those of the functional assay will allow better characterization of the structure and better understanding of how sequence-specific phosphorylation might affect the possible regulatory activity of the peptide.