Proteomics in neurotoxicology

神经毒理学中的蛋白质组学

• 批准号: 7304029
• 负责人: SANFORD P MARKEY
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF MENTAL HEALTH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7304029
• 关键词: Proteomics; neurotoxicology

We are applying proteomic methodology to unresolved problems in neuropathologic diseases. Methods to identify protein biomarkers of neuropsychiatric disorders, such as the obsessive compulsive syndrome that follows streptococcal infections (PANDAS) in pediatric patients, were tested. A combination of immunoaffinity strategies was used to separate proteins from patient sera. Mass spectral patterns of proteins were compared to determine whether there were statistically significant characteristics of patient state and the disease trait. We prepared several sub-proteome fractions and applied chemometric analyses to test whether MALDI/TOF measurements of the intact proteins produce disease characteristic profiles. There was no significant difference among the spectra related to disease state by principle components analysis. Using an alternative sample preparation (Conconavin A affinity), plasma and serum glycoproteomes associated with PANDAS, Sydenham chorea patients and controls were screened for differentially expressed proteins using SELDI-TOF-MS, 1D and 2D-gels. The absence of demonstrable differential proteomes from IVIgG, IgG or glycoproteomes precluded further analyses. A second strategy is to isolate proteins implicated from genomic studies as being associated with schizophrenia. We tested dysbindin antibody immunoaffinity, but were unsuccessful in capturing peptides related to dysbindin. Consequently, we are exploring the expression of affinity tag labeled dysbindin in an in vitro translation system. We will then explore transient or stable expression transfection of mammalian cells. In collaborative studies with NHGRI, the mitochondrial electron transport complex-I proteomics are being characterized. The hypothesis that deficiency in this complex results in Parkinson Disease is being tested. Preliminary work suggested phosphorylation dependent differences in proteins interacting with the C-terminus of alpha-synuclein. Of particular interest is the interaction of only the non-phosphorylated aS form with mitochondrial electron transport chain proteins. This data suggests that the function and localization of alpha-synuclein might be regulated by kinases in the synapse and that phosphorylation may be an important factor in protein aggregation and Lewy body formation. Although overexpression of alpha-synuclein induces signs of neurodegeneration, alpha-synuclein knockout mice show little to no obvious phenotype. Therefore, our proposal to identify proteins interacting with the alpha-synuclein tail under a variety of conditions will be of importance to understanding the function and localization of alpha-synuclein, possible physiological pathways of regulation and insight into its role in Parkinson Disease. Collaborative studies with NINDS have focused on defining the composition of the post-synaptic density complex. PSD-95, a specialized scaffold protein with multiple protein interaction domains, forms the backbone of an extensive postsynaptic protein complex that organizes receptors and signal transduction molecules at the synaptic contact zone. Large, detergent-insoluble PSD-95-based postsynaptic complexes can be affinity-purified from conventional postsynaptic density (PSD) fractions using magnetic beads coated with a PSD-95 antibody. Purified PSD-95 complexes were analyzed by mass spectrometry. In order to assess enrichment/depletion of proteins upon affinity purification, the relative abundances of proteins in the parent PSD fraction and the isolated PSD-95 complexes were evaluated, based on cumulative ion current intensities of corresponding peptides. The affinity-purified preparation was largely depleted of presynaptic proteins, spectrin, intermediate filaments and other contaminants prominent in the parent PSD fraction. A group of 26 proteins were identified as major components in the PSD-95 complex on the basis of abundance ranking and enrichment upon affinity purification. This group of constituent proteins includes, in addition to the specialized scaffolds and NMDA receptors, an abundance of AMPA receptors, as well as certain proteins that were not previously acknowledged as major elements of the postsynaptic complex. Two of these, BRAG1 (O60275) and BRAG2b (Q6DN90), are of the same family of brefeldin A-resistant regulators of the small G-protein Arf; another is a hypothetical protein (Q8BZM2) with two SAM domains. The properties of these new major components suggest that they may serve pivotal functions in spine structure and plasticity, including reorganization of the actin cytoskeleton, and insertion and retrieval of proteins to and from the plasma membrane. In collaborative studies with NYU, we are performing proteome analyses of several affinity purified protein extracts from HeLa cells expressing the amino terminus of the Huntington?s Disease protein with different numbers of glutamines (the wild type and disease states). The objective of this project is to test the hypothesis that there is transcriptional dysregulation associated with Huntington?s Disease. The first objective is to identify and compare proteins that co-purify with Huntintin (Htt) and Huntintin mutant (HttMut) from nuclear and cytoplasmic fractions. The retroviral vector pOZ-N was used to establish several HeLa cell lines that express FLAG/HA epitope-tagged Htt N-terminus in order to purify Htt-containing protein complexes. Htt amino-terminal constructs that express the 171 or 590 amino acids with 25, 46, or 97 Qs have been made. The 25Q constructs are representative of the wild- type protein. The 46 Q represents an expansion near the pathogenic threshold, and the 97 Q represents the fully expanded form. The Htt-FLAG/HA gene is under the control of the MMLV LTR promoter and the tagged proteins are expressed at levels only slightly higher than that of endogenous Htt. Immunopurification conditions were optimized to maximize positive identifications while keeping nonspecific identifications at a minimum, using small scale coimmuno-precipitations of endogenous proteins to investigate previously reported Htt interactions. A collaborative study with NIDCR is to identify relevant proteins involved in TRPC3 function and regulation in the rat brain. Mammalian transient receptor potential canonical (TRPC) family of cation channels consists of seven members (TRPC1-TRPC7) that are activated in response to agonist-stimulated PIP2 hydrolysis in a variety of tissues. Mass spectrometric analyses have been completed, and significant identified proteins confirmed by Western blot.

我们正在将蛋白质组学方法应用于神经病理疾病中未解决的问题。测试了鉴定神经精神疾病的蛋白质生物标志物，例如儿科患者链球菌感染（PANDAS）的强迫症综合征。免疫亲和力策略的结合用于将蛋白质与患者血清分开。比较蛋白质的质谱模式，以确定患者状态和疾病特征的统计学特征是否具有统计学意义。我们制备了几个亚蛋白蛋白分析，并应用化学​​计量分析以测试完整蛋白质的MALDI/TOF测量是否产生疾病特征。通过原理成分分析，与疾病状态有关的光谱之间没有显着差异。使用替代样品制剂（辅助A），使用SELDI-TOF-MS，1D和2D-凝胶，筛选了与PANDAS，Sydenham Chorea Chorea患者和对照组相关的血浆和血清糖蛋白酶，Sydenham Chorea患者和对照组。 IVIGG，IgG或糖蛋白酶没有明显的差异蛋白质组缺乏进一步的分析。 第二种策略是分离与精神分裂症有关的基因组研究所涉及的蛋白质。我们测试了胞霉素抗体免疫亲和力，但在捕获与dysbindin相关的肽方面没有成功。因此，我们正在探索在体外翻译系统中标记为dysbindin的亲和力标签的表达。然后，我们将探索哺乳动物细胞的瞬时或稳定表达转染。 在与NHGRI的协作研究中，线粒体电子传输复合物I蛋白质组学的表征。这一复合物缺乏导致帕金森氏病的假设正在测试中。初步工作表明，蛋白质与α-突触核蛋白的C末端相互作用的磷酸化依赖性差异。特别令人感兴趣的是仅非磷酸化作为形式与线粒体电子传输链蛋白的相互作用。该数据表明，α-突触核蛋白的功能和定位可能受到突触中的激酶的调节，并且磷酸化可能是蛋白质聚集和路易体形成的重要因素。尽管α-突触核蛋白的过表达诱导了神经退行性的迹象，但α-核蛋白敲除小鼠几乎没有明显的表型。因此，我们鉴定在各种疾病下与α-突触核蛋白尾巴相互作用的蛋白质的提议对于理解α-核蛋白的功能和定位至关重要，这可能是调节的生理途径以及对其在帕金森病中的作用的洞察力。 与Ninds的协作研究重点是定义后突触密度复合物的组成。 PSD-95是一种具有多种蛋白质相互作用结构域的专门支架蛋白，形成了广泛的突触后蛋白质复合物的骨干，该蛋白质复合物在突触接触区组织受体和信号转导分子。大型，洗涤剂不溶的PSD-95基于突触的后复合物可以使用涂有PSD-95抗体的磁珠来与常规的突触后密度（PSD）分数相关。通过质谱法分析了纯化的PSD-95复合物。为了评估亲和力纯化后蛋白质的富集/消耗，根据相应肽的累积离子电流强度，评估了父pSD馏分中蛋白质的相对丰度和分离的PSD-95复合物。亲和力纯化的制剂在很大程度上耗尽了突触前蛋白，光谱，中间丝和其他污染物在母体PSD级分中突出的污染物。基于亲和力纯化的丰度排名和富集，将一组26个蛋白质确定为PSD-95复合物中的主要成分。除了专门的支架和NMDA受体外，这组组成蛋白还包括大量的AMPA受体，以及某些以前未被认为是突触后复合物的主要元素的蛋白质。其中两个，即Brag1（O60275）和Brag2b（Q6DN90），是小型G蛋白ARF的Brefeldin A-抗性调节剂的家族；另一个是具有两个SAM结构域的假设蛋白（Q8BZM2）。这些新的主要成分的特性表明，它们可以在脊柱结构和可塑性中起关键功能，包括重组肌动蛋白细胞骨架，以及蛋白质与质膜的插入和检索。 在与纽约大学的协作研究中，我们正在对表达亨廷顿疾病蛋白的氨基末端的几种亲和力纯化蛋白提取物进行蛋白质组分析，这些蛋白质具有不同数量的谷氨酰胺（野生型和疾病状态）。该项目的目的是检验与亨廷顿病有关的转录失调的假设。第一个目标是鉴定和比较与核和细胞质级分的Huntintin（HTT）和Huntintin突变体（HTTMUT）共纯化的蛋白质。逆转录病毒载体POZ-N用于建立几种表达FLAG/HA表位标记的HTT N末端的HELA细胞系，以净化含HTT的蛋白质复合物。已经制作了25、46或97 QS的171或590氨基酸的HTT氨基末端构建体。 25Q构建体是野生型蛋白质的代表。 46 Q表示致病阈值附近的膨胀，97 Q表示完全膨胀的形式。 HTT-FLAG/HA基因在MMLV LTR启动子的控制之下，标记的蛋白的水平仅高于内源性HTT的水平。优化了免疫疗法条件，以最大程度地鉴定阳性，同时使用小规模的内源性蛋白质共沉淀来调查先前报道的HTT相互作用。 与NIDCR的协作研究是确定与大鼠大脑中有关TRPC3功能的相关蛋白质和调节。哺乳动物瞬态受体潜在的典型（TRPC）阳离子通道家族由七个成员（TRPC1-TRPC7）组成，这些成员因激动剂刺激的PIP2水解在多种组织中被激活。质谱分析已经完成，并通过Western印迹证实了明显的蛋白质。