Proteomic identification of binding partners for the brain metabolite lanthionine ketimine (LK) and documentation of LK effects on microglia and motoneuron cell cultures.
Proteomic identification of binding partners for the brain metabolite lanthionine ketimine (LK) and documentation of LK effects on microglia and motoneuron cell cultures.
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DOI：
10.1523/jneurosci.5247-09.2010
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发表时间：
2010-02-24
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通讯作者：
Post J
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作者：
Hensley K;Christov A;Kamat S;Zhang XC;Jackson KW;Snow S;Post J
羊毛硫基酮亚胺（LK）是哺乳动物中枢神经系统中存在的一类知之甚少的硫醚。以前的工作已经表明LK与突触体膜蛋白的高亲和力相互作用，但既没有LK结合配偶体也没有特定的生物活性的报道。在这项研究中，LK是化学合成的，并用作亲和剂从哺乳动物脑裂解物中捕获结合伴侣。液相色谱-电喷雾电离-质谱LC-ESI-MS/MS法分析经质谱分离的LK结合蛋白质，鉴定了参与轴突重塑或囊泡运输以及包括阿尔茨海默病和精神分裂症在内的疾病的多肽：塌陷蛋白反应介体蛋白-2/二氢嘧啶酶样蛋白-2（CRMP 2/DRP 2/DPYSL 2）;髓鞘碱性蛋白（MBP）;和突触融合蛋白结合蛋白-1（STXBP 1/Munc-18）。还确定了最近发现的谷胱甘肽（GSH）结合蛋白，羊毛硫氨酸合成酶样蛋白-1（LanCL 1）。通过使用脑裂解物的免疫沉淀研究来探测LK：CRMP 2相互作用的功能结果，其中发现LK增加CRMP 2与其配偶体神经纤维蛋白-1（NF 1）的共沉淀，但减少CRMP 2与β-微管蛋白的共沉淀。NSC-34运动神经元样细胞的功能研究表明，细胞可渗透的LK-酯，LKE，是无毒的，并保护免受过氧化氢的氧化挑战。LKE处理的NSC-34细胞以血清浓度依赖性方式显著增加神经突数量和长度，与CRMP 2相互作用一致。最后，LKE拮抗炎症原对EOC-20小胶质细胞的激活。结果进行了讨论，参考可能的生化起源，旁分泌功能，神经学意义和药理潜力的lanthionyl化合物。
Lanthionine ketimine (LK) represents a poorly-understood class of thioethers present in mammalian central nervous system. Previous work has indicated high-affinity interaction of LK with synaptosomal membrane protein(s) but neither LK binding partners nor specific bioactivities have been reported. In this study LK was chemically synthesized and used as an affinity agent to capture binding partners from mammalian brain lysate. Liquid chromatography with electrospray ionization-mass spectrometry (LC-ESI-MS/MS) of electrophoretically-separated, LK-bound proteins identified polypeptides implicated in axon remodeling or vesicle trafficking, and diseases including Alzheimer’s disease and schizophrenia: Collapsin response mediator protein-2/dihydropyrimidinase-like protein-2 (CRMP2/DRP2/DPYSL2); myelin basic protein (MBP); and syntaxin-binding protein-1 (STXBP1/Munc-18). Also identified was the recently discovered glutathione (GSH)-binding protein, lanthionine synthetase-like protein-1 (LanCL1). Functional consequences of LK:CRMP2 interactions were probed through immunoprecipitation studies using brain lysate wherein LK was found to increase CRMP2 co-precipitation with its partner neurofibromin-1 (NF1), but decreased CRMP2 co-precipitation with β-tubulin. Functional studies of NSC-34 motor neuron-like cells indicated that a cell-permeable LK-ester, LKE, was non-toxic and protective against oxidative challenge with H2O2. LKE-treated NSC-34 cells significantly increased neurite number and length in a serum concentration-dependent fashion, consistent with a CRMP2 interaction. Finally, LKE antagonized the activation of EOC-20 microglia by inflammogens. The results are discussed with reference to possible biochemical origins, paracrine functions, neurological significance and pharmacological potential of lanthionyl compounds.