CHARACTERIZATION OF HUNTINGTON AND PARKINSON AGGREGATES BY MASS SPECTROMETRY

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 多聚谷氨酰胺病是由致病突变蛋白中多聚谷氨酰胺链扩张引起的遗传性神经退行性疾病。多聚谷氨酰胺疾病的一个统一的病理特征是在受影响的神经元中存在显微镜可识别的细胞内包涵体。 已经提出，扩展的多聚谷氨酰胺束形成的聚集体参与导致细胞死亡的不适当的蛋白质-蛋白质相互作用。迄今为止，仍然没有可靠的方法来分离高度纯化的聚集体，这些聚集体通常在形态上是异质的。我们开发了一种新的纯化方法，并进行了蛋白质组学程序，通过质谱分析聚集体相互作用蛋白。MS提供了一个强大的和直接的分析聚集蛋白在微量，并将帮助我们了解聚集体的多聚谷氨酰胺疾病的诞生。 我们获得了MS数据，表明聚集体中存在几种预期的蛋白质，以及其他不容易预测的蛋白质。免疫化学方法证实了这些聚集相互作用的蛋白质;这些结果已被发表。后来的实验提供了酵母细胞从亨廷顿蛋白的某些同种型形成攻击体的证据;这是攻击体可以在酵母中形成的第一个迹象。描述了侵略性基因组形成所需的蛋白质的鉴定并提出了我们的侵略性基因组形成新模型的手稿已发表在FASEB杂志上。 异常蛋白质聚集体可引起细胞毒性，导致各种其他病理，包括帕金森病（PD）。 Synphilin-1是这些包涵体的主要成分之一，并且与PD的发病机制有关。 Synphilin-1的表达导致细胞质聚集体，其在蛋白酶体抑制后被转运以形成侵袭体。Synphilin-1功能结构域的缺失揭示了未知的信号传导蛋白必须与Synphilin-1相关联以触发攻击体形成[N. Zaarur等人，J Biol Chem，2008，283，27575-27584]。 谢尔曼小组最近报道了这些未知蛋白候选物的鉴定和定量，以及它们相关功能和途径的研究。在目前的研究中，构建了在N-和C-末端分别携带His-标签和GFP-标签的Synphilin-1。 制备含有该重组突触素-1的质粒并转染到HEK 293 T细胞中。从用蛋白酶体抑制剂处理的细胞或幼稚细胞中纯化亲合蛋白-1和相关蛋白。纯化的亲合蛋白-1蛋白复合物通过胰蛋白酶在溶液中消化，通过数据依赖性纳米毛细管反相LC-MS/MS使用LTQ-Orbitrap MS（Thermo Scientific）一式三份进行分析。使用Progenesis LCMS（非线性动力学）进行无标记定量以确定相对蛋白质表达水平。 通过使用MASCOT（Matrix Science）搜索Swiss-Prot人蛋白质数据库来鉴定蛋白质。为了解决非特异性相互作用物，将载体转染的HEK 293 T细胞相同地处理并作为阴性对照平行分析。对显著调节的蛋白质（p<0.05，倍数变化>2）进行网络、途径和功能分析，使用Intimity途径分析（Intimity）来确定相互作用的配偶体候选物及其参与途径和细胞功能。 我们确定，Synphinlin-1复合物被高效分离。 对通过无标记定量（图1）确定的显著调节蛋白（p<0.05，倍数变化>2）进行网络、途径和功能分析。网络分析揭示了潜在的蛋白候选进一步Western印迹验证。途径分析揭示了参与蛋白质泛素化途径的调节蛋白，这对异常蛋白质聚集体的降解至关重要。功能分析表明，调控蛋白主要影响蛋白质合成和RNA转录后修饰，从而影响异常蛋白的形成。这些结果表明，Synphilin-1及其相互作用物参与蛋白质合成和降解，以调节蛋白质聚集体和侵袭体的形成。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Polyglutamine diseases are inherited neurodegenerative diseases caused by the expansion of polyglutamine tract in the disease-causing mutant proteins. A unifying pathological feature of polyglutamine disorders is the presence of microscopically discernible intracellular inclusions in the affected neurons. It has been proposed that expanded polyglutamine tract formed-aggregates participate in inappropriate protein-protein interactions that lead to cell death. To date, there is still no reliable method to isolate highly purified aggregates which are usually morphologically heterogeneous. We developed a new purification method and the proteomics procedure was carried out to analyze the aggregate-interacting proteins by mass spectrometry. MS provides a powerful and direct analysis of aggregate proteins in tiny quantities and will help us understand the nativity of aggregates in the polyglutamine disorders. We obtained MS data that indicates the presence of several expected proteins in the aggregate and others that would not have been easily predicted. Immunochemical approaches confirmed these aggregate interacting proteins; these results have been published. later experiments provided evidence that the yeast cells form aggresomes from some isoforms of the Huntington proteins; this was the first indication that aggresomes can form in yeast. The manuscript describing the identification of proteins that are required for aggresome formation and presenting our new model for aggresome formation has been published in FASEB journal. Abnormal protein aggregates can cause cell toxicity leading to various other pathologies, including Parkinson disease (PD). Synphilin-1 is one of the major components of these inclusions, and is implicated in the pathogenesis of PD. Expression of Synphilin-1 led to cytoplasmic aggregates, which were transported to form aggresomes upon proteasome inhibition. Deletion of Synphilin-1 functional domains revealed that unknown signaling proteins must be associated with Synphilin-1 to trigger aggresome formation [N. Zaarur, et al., J Biol Chem, 2008, 283, 27575-27584]. The Sherman group has recently reported identification and quantification of these unknown protein candidates, and investigation of their relevant functions and pathways. in the current studies, Synphilin-1 was constructed to carry a His-tag and a GFP-tag at the N- and C- termini, respectively. A plasmid containing this recombinant synphinlin-1 was produced and transfected into HEK293T cells. Synphilin-1 and associated proteins were purified from cells treated with proteasome inhibitor or na¿ve cells. Purified synphilin-1 protein complexes were digested by trypsin in solution, analyzed by data-dependent nano-capillary reversed-phase LC-MS/MS using an LTQ-Orbitrap MS (Thermo Scientific) in triplicate. Label-free quantification to determine relative protein expression levels was performed using Progenesis LCMS (Nonlinear Dynamics). Proteins were identified by searching against the Swiss-Prot human protein database using MASCOT (Matrix Science). To address nonspecific interactors, vector transfected HEK293T cells were treated identically and analyzed in parallel as a negative control. Significantly regulated proteins (p<0.05, fold change>2) were subjected to network, pathway and function analyses using Ingenuity pathway analysis (Ingenuity) to determine interacting partner candidates and their involvement in pathways and cellular functions. We determined that the Synphinlin-1 complex was isolated with high efficiency. Network, pathway, and function analyses of significant regulated proteins (p<0.05, fold change>2) determined by label-free quantification (Figure 1) were performed. Network analysis uncovered potential protein candidates for further Western blot validations. Pathway analysis revealed regulated proteins involved in the protein ubiquitination pathway, which is critical for degradation of abnormal protein aggregates. Functional analysis showed that regulated proteins mainly affected protein synthesis and RNA post-transcriptional modification, which impact on formation of abnormal proteins. These results indicate that Synphilin-1 and its interactors are involved in both protein synthesis and degradation to regulate formation of protein aggregates and aggresomes.