Proteomics in neurotoxicology

我们正在将蛋白质组学方法应用于神经病理疾病中尚未解决的问题。在用于治疗情绪障碍的药物对突触后密度的结构及其重塑、精神分裂症的基因组研究所涉及的蛋白质复合体以及伴随长期抑郁(LTD)的突触蛋白变化的研究方面继续取得进展。 钙是一种普遍存在的基本信号成分，被细胞用来调节一系列关键的细胞功能。瞬时受体电位(Trp)超家族离子通道参与钙信号转导机制，调节细胞的关键感觉功能以及其他过程，如分泌、增殖、神经元引导、细胞死亡和发育。我们已经鉴定了TRPC的蛋白质组，并在TRPC1和TRPC3蛋白质组中观察到许多相同的蛋白质(约50%)。此外，当我们分析常见蛋白质之间的功能相似性时，很明显存在非常不对称的分布。两者之间的功能比较表明，某些功能基团有更大的重叠，例如参与内吞作用的蛋白质。已提出存在TRPC1/TRPC3混合通道，与此观察一致，TRPC3与大鼠脑提取液中的TRPC1-IP免疫共沉淀。在某些功能区，TRPC1和TRPC3可能以异源二聚体的形式共存，从而共享相同的蛋白质伙伴。相反，在结合伙伴中几乎没有重叠的功能区可能表明TRPC1和TRPC3的独立作用。需要进一步的研究来阐明TRPC1和TRPC3独立相互作用的复合体，而不是同时存在的复合体。 ErbB4和Dystrobrevin结合蛋白1(Dybindin)蛋白信号复合体都被精神分裂症的基因组研究所涉及，而且在大脑中仍然很难分析。我们正在继续一个项目，以识别、表征和验证与这两种蛋白质相关的复合体。ERBB4是一种受体酪氨酸蛋白激酶，我们已经使用抗体将其与磁珠共价连接。为该项目开发的使用珠状消化程序的比较LC/MS/MS蛋白质组学研究未能提供回收ErbB4和进行质谱分析所需的浓缩因子。然而，新的多肽靶向策略被计划使用来自合成基因的标记蛋白，以允许更好地跟踪该复合体的浓缩和回收。 蛋白酶对蛋白质的降解/修饰涉及许多生理过程，如细胞凋亡。Caspase-3是一种半胱氨酸蛋白酶，在细胞凋亡中被称为刽子手酶。最近的研究表明，caspase-3在突触中也发挥着重要的非凋亡功能。在海马神经元中，caspase-3活性是NMDA受体依赖的长期抑制(LTD)所特需的，但不是长期增强所必需的。更重要的是，LTD中CASAPSE-3的激活促进AMPA受体内吞而不是细胞死亡。为了了解caspase-3激活导致LTD的分子机制，我们应用了一种已发表的方法，选择性地捕获NMDA受体激活和诱导凋亡时蛋白水解酶裂解产生的突现多肽。简而言之，利用工程酶枯草杆菌酶，用生物素化的多肽标记新形成的蛋白质N-末端，这些蛋白质是由蛋白酶裂解产生的。用固定化亲和素对标记蛋白进行富集化，并进行质谱分析。比较了对照(未处理的神经元)、NMDA处理和星形孢菌素处理的样品的底物降解穹顶。目前，我们正在重点研究具有caspase裂解位点的底物，并在LTD中测试它们的功能。

We are applying proteomic methodology to unresolved problems in neuropathologic diseases. Progress continues in studies on the structure of the postsynaptic density and its remodeling by drugs used in the treatment of mood disorders, on protein complexes implicated by genomic studies of schizophrenia, and synaptic protein changes accompanying long term depression (LTD). Calcium is a ubiquitous and fundamental signaling component that is utilized by cells to regulate a diverse range of critical cellular functions. The transient receptor potential (TRP) superfamily of ion channels are involved in calcium signaling mechanisms which regulate critical sensory functions in cells as well as other processes such as secretion, proliferation, neuronal guidance, cell death, and development. We have characterized the proteome of TRPCs and observed many of the same proteins in both the TRPC1 and TRPC3 proteome (approximately 50%). Moreover, when we analyzed the functional similarities among common proteins, a very asymmetrical distribution was evident. The functional comparison between the two reveals a greater overlap in certain functional groups, e.g. protein involved in endocytosis. Hybrid TRPC1/TRPC3 channels have been proposed to exist, and, consistent with this observation, TRPC3 co-immunoprecipitates with TRPC1-IP from rat brain extracts. In certain functional areas TRPC1 and TRPC3 may co-exist as a heterodimer and thus share the same protein partners. In contrast, the functional areas where little overlap in binding partners is observed could indicate independent roles for TRPC1 and TRPC3. Further studies will be needed to elucidate the complexes where TRPC1 and TRPC3 interact independently as opposed to complexes where both are present. The ErbB4 and Dystrobrevin binding protein 1 (dysbindin) protein signaling complexes have both been implicated by genomic studies on schizophrenia and have remained difficult to analyze in brain. We are continuing a project to identify, characterize and validate complexes associated with both proteins. ErbB4 is a receptor tyrosine-protein kinase, and we have used antibodies to covalently link it to magnetic beads. Comparative LC/MS/MS proteomic studies using on-bead digestion procedures developed for this project have failed to provide the enrichment factor required for ErbB4 recovery and mass spectrometric detection. However, new peptide targeting strategies are planned using labeled proteins from synthetic genes to allow better tracking of enrichment and recovery of this complex. Protein degradation/modification by proteases involves many physiological processes such as apoptosis. Caspase-3 is a cysteine protease best known as an executioner protease in apoptosis. Recent studies suggest that caspase-3 plays an important non-apoptotic function in synapses as well. In hippocampal neurons, caspase-3 activity is specifically required for NMDA receptor-dependent long-term depression (LTD), but not for long-term potentiation. More importantly, activation of casapse-3 in LTD promotes AMPA receptor endocytosis instead of cell death. To understand the molecular mechanism by which caspase-3 activation leads to LTD, we applied a published method developed to selectively capture emergent peptides derived from proteolytic cleavage upon NMDA receptor activation and apoptosis induction. Briefly, the newly formed free, unblocked protein N-termini produced from protease cleavage events are tagged with a biotinylated peptide using an engineered enzyme subtiligase. The labeled proteins are enriched by immobilized avidin and analyzed by mass spectrometry. The substrate degradomes of control (untreated neurons), NMDA treated, and staurosporine treated samples are compared. Currently, we are focusing on the substrates with putative caspase-cleavage sites and testing their functions in LTD.