PROTEOMIC APPROACH TO THE STUDY OF SYSTEMIC AMYLOIDOSIS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 在系统性淀粉样变性中，淀粉样蛋白的广泛沉积会导致严重的器官功能障碍。有必要区分不同形式的淀粉样蛋白，以设计适当的治疗策略。我们开发了一种蛋白质组学方法，利用二维聚丙烯酰胺凝胶电泳、基质辅助激光解吸/电离质谱仪和多肽质量指纹图谱直接表征腹部皮下脂肪中的淀粉样沉积，这些脂肪是通过细针抽吸获得的，这些患者被诊断患有免疫球蛋白轻链或甲状腺激素样变性。在对照组和患者之间以及两种类型的患者之间，脂肪组织的二维凝胶蛋白质组有显著的差异，患者标本中存在明显的额外斑点，这些斑点可以被归类为全长和截断形式的淀粉样蛋白。在转甲状腺素杂合子突变的患者中，从相同的蛋白质点分离出大致相同数量的野生型多肽和含有淀粉样变性转甲状腺素变异体的多肽，这表明这两种物种都合并到了沉淀物中。此外，在患者样本中出现了与淀粉样蛋白无关的新斑点；其中一些被鉴定为血清淀粉样蛋白P和载脂蛋白E的异构体，这两种蛋白质先前已被描述为与淀粉样蛋白沉积有关。最后，观察到驻留脂肪蛋白的正常表达模式的变化，如α-晶体蛋白、过氧化还蛋白6和醛酮还原酶I的下调，与淀粉样蛋白的存在明显相关，尽管它们的水平与淀粉样蛋白沉积的级别没有严格的相关性。这种蛋白质组学方法不仅提供了一种检测淀粉样变患者腹部皮下脂肪沉积并明确分型的方法，而且还可能通过识别与淀粉样变渗透相关的新蛋白质或蛋白质翻译后修饰，对疾病的机制产生新的见解。在本年度，编写了一项提案的初步结果，并与其他中心一起提交了提案，提议扩大该项目，但尚未获得资金。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In systemic amyloidoses, widespread deposition of protein as amyloid causes severe organ dysfunction. It is necessary to discriminate among the different forms of amyloid to design an appropriate therapeutic strategy. We developed a proteomics methodology utilizing two-dimensional polyacrylamide gel electrophoresis followed by matrix-assisted laser desorption/ionization mass spectrometry and peptide mass fingerprinting to directly characterize amyloid deposits in abdominal subcutaneous fat obtained by fine needle aspiration from patients diagnosed as having amyloidoses typed as immunoglobulin light chain or transthyretin. Striking differences in the two-dimensional gel proteomes of adipose tissue were observed between controls and patients and between the two types of patients with distinct, additional spots present in the patient specimens that could be assigned as the amyloidogenic proteins in full-length and truncated forms. In patients heterozygotic for transthyretin mutations, wild-type peptides and peptides containing amyloidogenic transthyretin variants were isolated in roughly equal amounts from the same protein spots, indicative of incorporation of both species into the deposits. Furthermore novel spots unrelated to the amyloidogenic proteins appeared in patient samples; some of these were identified as isoforms of serum amyloid P and apolipoprotein E, proteins that have been described previously to be associated with amyloid deposits. Finally changes in the normal expression pattern of resident adipose proteins, such as down-regulation of alphaB-crystallin, peroxiredoxin 6, and aldo-keto reductase I, were observed in apparent association with the presence of amyloid, although their levels did not strictly correlate with the grade of amyloid deposition. This proteomics approach not only provides a way to detect and unambiguously type the deposits in abdominal subcutaneous fat aspirates from patients with amyloidoses but it may also have the capability to generate new insights into the mechanism of the diseases by identifying novel proteins or protein post-translational modifications associated with amyloid infiltration. During the current year, preliminary results for a proposal were prepared and the proposal was submitted, together with other centers, that proposed an expansion of this project, but it has not yet been funded.