VMAT-2 N-TERMINAL PEPTIDE RESEARCH

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 已显示衍生自VMAT-2的N-末端的肽抑制5-羟色胺转运到包埋有全长蛋白质的囊泡中。具有乱序序列的肽不具有这种活性。肽序列含有两个磷酸化位点;二磷酸化肽也不能抑制转运。这些结果表明，N-末端的蛋白质可能是重要的，在调节其功能，根据磷酸化状态的活动被打开和关闭。关于天然序列肽的初步数据表明在30 ℃和pH 2.8下在磷酸化位点区域中存在部分螺旋或原螺旋。所提出的实验旨在收集在测定pH为7.8时未磷酸化和二磷酸化肽的数据，用于进一步的结构计算和比较 VMAT-2的N-末端被认为在蛋白质转运单胺神经递质如5-羟色胺的能力中具有调节作用。一个20个残基的肽来自N-末端的蛋白质抑制氚标记的5-羟色胺的运输到囊泡嵌入VMAT-2。该肽含有两个丝氨酸，这是可能的磷酸化位点;二磷酸化肽不能抑制囊泡内的5-羟色胺积累。具有乱序序列的肽也不能抑制转运。对天然序列未磷酸化肽的初步NMR研究表明α-螺旋结构趋势可能是观察到的生物活性的原因。在要求的时间内进行的工作包括二磷酸化和乱序肽的结构测定，以进行比较，确定该结构假设是否成立。 VMAT-2的20个氨基酸胞质N-末端含有5个带正电荷的残基（4个Arg，1个Lys），正在研究其作为单胺转运的分子内介体/使能者。用VMAT-2的N-末端肽处理人VMAT-2转染的cos-7囊泡抑制囊泡内3 H-5-羟色胺的积累。肽在其序列内的两个潜在磷酸化位点中的任一个处的磷酸化导致肽不能抑制3 H-5-羟色胺在囊泡内的积累。磷酸化位点丝氨酸残基突变为丙氨酸或天冬氨酸分别增强和减少5-羟色胺的转运。肽的初步结构数据表明在含有丝氨酸残基的肽区域中存在α-螺旋趋势。由于进行初步NMR工作的条件与用于功能测定的条件不同，因此在与功能测定的条件匹配的条件下进行的进一步NMR工作将允许更好地表征结构并更好地理解序列特异性磷酸化如何影响肽的可能调节活性。

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.