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.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 半胱氨酸的翻译后修饰可能与制造某些蛋白质淀粉样蛋白发生有关。 我们正在研究这种PTM在两种类型的蛋白质上的相关性，这些蛋白质对淀粉样蛋白具有极大的兴趣，这对于在Busm淀粉样蛋白治疗和研究中心所见的患者群体，血清蛋白经胆管素和过表达过表达的免疫球蛋白轻链链中尤其重要。经硫代蛋白（TTR）是13.7-KDA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素。假设TTR中的氨基酸取代和/或翻译后修饰是为了破坏四聚体的稳定，并导致TTR形成一个中间体，该中间体会自我促进成淀粉样蛋白的原纤维。家族性经胆囊素淀粉样变性（ATTR）与TTR变体作为淀粉样蛋白原纤维的沉积有关。已经确定了90多种TTR变体，大多数是淀粉样蛋白生成的。老年全身性淀粉样变性与WT蛋白的沉积有关，并在80年以上的patinets中变得常见（> 25％）。由于对ATT的唯一有效治疗方法是肝移植，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，表征了许多具有临床意义的TTR变体。现在，我们正在探索Qotof MS/MS。，LTQ-Orbitrap MS/MS和FTMS/MS的使用，重点是在线信息依赖性获取（IDA）NANO LC MS/MS通过自动数据库搜索来表征TTR。 TTR从患者的血清中免疫沉淀，并通过离心和反向相HPLC纯化。进行蛋白水解消化，并首先通过MALDI-TOFMS分析消化。在QSTAR和LTQ-ORBITRAP系统上执行具有信息依赖性采集（IDA）MS/MS的在线毛细管和Nanolc-MS。 分析来自IDA-LC-MS/MS的数据针对Mascot（Matrix Science）和用户编程的Pro-ID（ABI）数据库进行分析。质谱法（MS）在ATTR的临床诊断中起着重要作用。由于对ESI/MALDI/MS/MS数据的手动收集和解释很耗时且效率低下，因此开发的蛋白质组学使用MS/MS方法（例如具有自动数据库搜索的IDA-LC-MS/MS），因此为质谱法的临床应用提供了优势。正在评估新的分离方法，以查看它们是否提供优势。在已预编程到数据库中的突变的情况下，可以获得最终的变体识别。可以使用预编程数据库来处理PTMS（例如Cysysteine）之类的PTM的自动分配。为半胱氨酸改性的IgG轻链开发了相同的方法。还开发了化学工具，以帮助确定可能伴随半胱氨酸化的异常修饰。