VMAT-2 N-TERMINAL PEPTIDE RESEARCH

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 来自VMAT-2 N端的一种肽被证明可以抑制5-羟色胺向嵌入全长蛋白的囊泡中的运输。具有扰乱序列的多肽不具有这种活性。该肽序列包含两个磷酸化位点；二磷酸化的多肽也不能抑制转运。这些结果表明，蛋白质的N末端可能在调节其功能中起重要作用，其活性的开启和关闭取决于磷酸化状态。关于天然序列多肽的初步数据表明，在30℃和pH 2.8的磷酸化位点区域有部分或原螺旋。建议的实验旨在收集在测定pH为7.8的情况下未磷酸化和二磷酸化的多肽的数据，以供进一步的结构计算和比较 VMAT-2的N末端被认为在蛋白质运输单胺类神经递质(如5-羟色胺)的能力中具有调节作用。来自蛋白质N端的20个残基的多肽抑制了氚标记的5-羟色胺进入嵌入VMAT-2的囊泡的运输。该多肽含有两个可能的磷酸化位点的丝氨酸；二磷酸化的多肽不能抑制5-羟色胺在小泡内的聚集。具有扰乱序列的多肽也不能抑制转运。对天然序列的非磷酸化多肽的初步核磁共振工作表明，α-螺旋结构趋势可能是观察到的生物活性的原因。将在要求的时间内进行的工作包括对二磷酸化和杂乱序列的多肽进行结构测定，以便进行比较，看看这一结构假设是否成立。 VMAT-2胞浆N末端含有5个带正电荷的残基(4个Arg，1个Lys)，是单胺转运的分子内介体/激活因子。用VMAT-2的N端肽处理转染人VMAT-2的CoS-7囊泡，可抑制囊泡内~3H-5-羟色胺的积聚。在其序列中的两个潜在的磷酸化位点上的任何一个位置的磷酸化导致该肽不能抑制~3H-5-羟色胺在小泡中的积累。磷酸化位点丝氨酸残基突变为丙氨酸或天冬氨酸，分别增强和减少了5-羟色胺的转运。关于多肽的初步结构数据表明，在含有丝氨酸残基的多肽区域有α-螺旋趋势。由于初步核磁共振工作的条件与功能分析所用的条件不同，在与功能分析相匹配的条件下进行进一步的核磁共振工作将允许更好地表征结构，并更好地了解序列特异性磷酸化可能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.