PROTEIN CYSTEINE POST-TRANSLATIONAL MODIFICATION IN AMYLOIDOSIS

淀粉样变性中的蛋白质半胱氨酸翻译后修饰

• 批准号: 8365496
• 负责人: Catherine E. Costello
• 金额: $ 0.46万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-08-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8365496
• 关键词: Amino Acid Substitution; Amyloid; Amyloid Fibrils; Amyloidosis; Binding; Biology; Blood capillaries; Carrier Proteins; Centrifugation; Chemicals; Collaborations; Collection; Cysteine; Data; Databases; Deposition; Digestion; Funding; Grant; High Pressure Liquid Chromatography; Immunoglobulin G; Light; Light-Chain Immunoglobulins; Liver; Manuals; Mass Spectrum Analysis; Medicine; Methods; Modification; Mutation; National Center for Research Resources; Organ; Patients; Phase; Plasma; Play; Post-Translational Protein Processing; Prealbumin; Principal Investigator; Proteins; Proteomics; Research; Research Infrastructure; Resources; Retinol Binding Proteins; Role; Science; Serum; Serum Proteins; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; System; Thyroxine; Time; Tissues; United States National Institutes of Health; Variant; capillary; clinical Diagnosis; clinical application; clinically significant; cost; effective therapy; interest; liver transplantation; nano; overexpression; patient population; programs; tool

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. Post-translational modifications at cysteine may be involved in making certain proteins amyloidogenic. We are investigating the correlation of such PTMs on two types of proteins that have great interest for the amyloid diseases that are especially central to the patient population seen at the BUSM Amyloid Treatment and Research Center, the serum protein transthyretin and the overexpressed immunoglobulin light chains. Transthyretin (TTR) is a 13.7-kDa transport protein which is synthesized predominantly by the liver. In plasma, tetrameric TTR binds retinol-binding protein and thyroxine. Amino acid substitutions in TTR, and/or post-translational modifications are hypothesized to destabilize the tetramer and cause the TTR to form an intermediate that self associates into amyloid fibrils. Familial transthyretin amyloidosis (ATTR), is associated with the deposition of the TTR variants as amyloid fibrils in various tissues and organs. More than 90 TTR variants have been identified, with the majority being amyloidogenic. Senile systemic amyloidosis is associated with deposition of the wt protein and becomes common (>25%) in patinets over 80 yr. Since the only effective treatment of ATTR is liver transplantation, the correct clinical diagnosis is critical. The MS Resource, in collaboration with the Amyloid Treatment and Research Program, has characterized a number of TTR variants of clinical significance. We are now exploring the use of QoTOF MS/MS., LTQ-Orbitrap MS/MS and FTMS/MS with emphasis on on-line information dependent acquisition (IDA) nano LC MS/MS for the characterization of TTR via automated database searching. TTR is immunoprecipitated from the serum of patients and purified by centrifugation and reversed phase HPLC. Proteolytic digestions are performed and the digests are first analyzed by MALDI-TOFMS. On-line capillary and nanoLC-MS with information dependent acquisition (IDA) MS/MS is performed on the QStar and LTQ-Orbitrap systems. Data from IDA-LC-MS/MS are analyzed against Mascot (Matrix Science) and user programmed PRO-ID (ABI) databases. Mass spectrometry (MS) has played an important role in the clinical diagnosis of ATTR. Since manual collection and interpretation of ESI/MALDI/MS/MS data is time consuming and inefficient, use of proteomics developed MS/MS methods, such as IDA-LC-MS/MS with automated database searching, offers advantages to the clinical applications of mass spectrometry. New separation methods are being evaluated to see whether they may offer advantages. Conclusive variant identification is obtained in cases where the mutation has been pre-programmed into the database.and automated assignment of PTMs such as modifications at cysteine can be handled using the preprogrammed database. The same approach is being developed for cysteine-modified IgG light chains. Chemical tools are also being developed to aid in determination of unusual modifications that may accompany cysteinylation.

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 半胱氨酸的翻译后修饰可能与某些蛋白质的淀粉样变性有关。我们正在研究这种PTM与两种蛋白质的相关性，这两种蛋白质对BUSM淀粉样疾病治疗和研究中心看到的患者群体特别重要的淀粉样疾病非常感兴趣，这两种蛋白质是血清蛋白转甲状腺素和过表达的免疫球蛋白轻链。转甲状腺素(TTR)是一种13.7 kDa的转运蛋白，主要由肝脏合成。在血浆中，四聚体TTR结合视黄醇结合蛋白和甲状腺激素。假设TTR中的氨基酸替换和/或翻译后修饰会破坏四聚体的稳定，并导致TTR形成一种自结合成淀粉样纤维的中间体。家族性甲状腺激素性淀粉样变性(ATTR)是一种以淀粉样纤维形式存在于各种组织和器官中的遗传性遗传病。目前已鉴定出90多种TTR变异体，其中大多数是淀粉样变性的。老年性系统性淀粉样变性与wt蛋白沉积有关，并在80岁以上的患者中变得常见(&gt；25%)。由于唯一有效的治疗方法是肝移植，因此正确的临床诊断至关重要。MS资源与淀粉样蛋白治疗和研究计划合作，已经确定了一些具有临床意义的TTR变体的特征。我们目前正在探索QOTOF MS/MS、LTQ-Orbitrap MS/MS和FTMS/MS的使用，重点是在线信息依赖获取(IDA)纳米LC MS/MS，通过自动数据库搜索来表征TTR.TTR是从患者血清中免疫沉淀并通过离心法和反相高效液相色谱法纯化而成。进行蛋白质消化，并首先用MALDI-TOFMS分析消化产物。在QStar和LTQ-Orbitrap系统上进行了在线毛细管和带有信息依赖采集(IDA)的纳米LC-MS/MS联用。将来自IDA-LC-MS/MS的数据对照Mascot(矩阵科学)和用户编程PRO-ID(ABI)数据库进行分析。质谱仪(MS)在临床诊断中发挥了重要作用。由于人工收集和解释ESI/MALDI/MS/MS数据费时且效率低下，使用蛋白质组学开发的MS/MS方法，如具有自动数据库搜索的IDA-LC-MS/MS，为质谱学的临床应用提供了优势。正在对新的分离方法进行评估，看看它们是否会带来优势。在突变已被预先编程到数据库中的情况下，获得确凿的变异识别。并且可以使用预先编程的数据库来处理PTM的自动分配，例如半胱氨酸的修饰。同样的方法正在被开发用于半胱氨酸修饰的免疫球蛋白轻链。化学工具也正在开发，以帮助确定可能伴随半胱氨酸化的不寻常的修饰。