S-SULFONATION OF SERUM TRANSTHYRETIN IN SSA/PTMS OF TTR IN AMYLOID FIBRILS

淀粉样原纤维中 TTR 的 SSA/PTMS 中血清运甲状腺素蛋白的 S-磺化

• 批准号: 7722963
• 负责人: Lawreen H. Connors
• 金额: $ 0.67万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7722963
• 关键词: Affect; Age; Alkanesulfonates; Amino Acid Substitution; Amino Acids; Amyloid; Amyloid Fibrils; Amyloidosis; Analytical Chemistry; Autopsy; Binding; Biochemical; Cardiac; Cardiomyopathies; Carrier Proteins; Complex; Computer Retrieval of Information on Scientific Projects Database; Condition; Cysteine; Deposition; Development; Diagnosis; Dimerization; Disease; Funding; Glutathione; Grant; Heart; Heterogeneity; Hormones; Individual; Institution; Link; Manuscripts; Methodology; Modification; Organ; Pathogenesis; Patients; Plasma; Play; Prealbumin; Proteins; Publishing; Purpose; Relative (related person); Research; Research Personnel; Resources; Retinol Binding Proteins; Role; Serum; Source; Structure; Sulfhydryl Compounds; Testing; Thyroxine; Tissues; United States National Institutes of Health; Variant; Vitamin A; adduct; cysteinylglycine; fibrillogenesis; immunoglobulin deposition disease; programs; protein aggregation

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. Transthyretin (TTR) is a plasma carrier protein consisting of 127 amino acid residues. TTR normally exists as a tetramer and binds the hormone thyroxine and the retinol-binding protein-vitamin A complex. Amino acid substitutions in TTR affect the stability of the tetramer and cause the protein to form intermediates that self-associate into amyloid fibrils. Familial transthyretin amyloidosis (ATTR) is associated with the deposition of the TTR variants as amyloid fibrils in tissues and organs. TTR is also associated with senile systemic amyloidosis (SSA), also referred to as senile cardiac amyloidosis (SCA). SSA is a nonhereditary disorder that affects about 25% of individuals over 80 years old. In SSA, the amyloid fibrils are usually composed of wild type TTR and its fragments and are found mainly in the heart. We have found that approximately 85-95% of serum TTR is post-translationally modified by S-sulfonation and S-thiolation (conjugation with cysteine, cysteinyl glycine, and glutathione). S-Sulfonation of TTR has been shown to increase amyloid forming potential and possibly plays a role in protein aggregation and fibrillogenesis. Furthermore, the extent of thiol conjugation of proteins has been shown to increase with age. The purpose of this study is to evaluate the serum levels of S-sulfonated TTR in SSA patients seen at the BUSM Amyloid Treatment and Research Program, in an attempt to link a biochemical structural feature to disease pathogenesis. We have hypothesized that increased S-sulfonation of transthyretin (TTR) at Cys10 and interactions with accessory proteins promote fibrillogenesis, and therefore act as effectors of senile systemic amyloidosis (SSA). To test this hypothesis, we have analyzed and compared the relative abundance of S-sulfonated TTR isolated from the sera of patients with SSA, primary amyloidosis (AL) with cardiomyopathy, and non-amyloid controls. The results do not indicate that there is a direct link between TTR S-sulfonation and development of SSA. To examine the possibility that the profile of cysteine adducts in serum varies from that in the amyloid deposits, cardiac fibrils were extracted from autopsy tissues obtained on two separate cases of clinically diagnosed SSA. We developed methodology to isolate TTR from these tissues under non-reducing conditions in order to establish the degree of heterogeneity with respect to mass. We observed both truncation, dimerization and Cys-10 modification in the TTR fibrils. Top-down MS analysis enabled assignment of structures to truncated forms of the protein. A manuscript describing these results was recently published in Analytical Chemistry.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 甲状腺素运载蛋白 (TTR) 是一种血浆载体蛋白，由 127 个氨基酸残基组成。 TTR 通常以四聚体形式存在，并结合激素甲状腺素和视黄醇结合蛋白-维生素 A 复合物。 TTR 中的氨基酸取代会影响四聚体的稳定性，并导致蛋白质形成自缔合成淀粉样原纤维的中间体。家族性转甲状腺素蛋白淀粉样变性 (ATTR) 与 TTR 变体以淀粉样原纤维形式沉积在组织和器官中有关。 TTR 还与老年系统性淀粉样变性 (SSA) 相关，也称为老年性心脏淀粉样变性 (SCA)。 SSA 是一种非遗传性疾病，影响约 25% 的 80 岁以上老年人。在SSA中，淀粉样原纤维通常由野生型TTR及其片段组成，主要存在于心脏中。我们发现大约 85-95% 的血清 TTR 通过 S-磺化和 S-硫醇化（与半胱氨酸、半胱氨酰甘氨酸和谷胱甘肽结合）进行翻译后修饰。 TTR 的 S-磺化已被证明可以增加淀粉样蛋白形成的潜力，并可能在蛋白质聚集和纤维形成中发挥作用。此外，蛋白质的硫醇结合程度已被证明随着年龄的增长而增加。本研究的目的是评估 BUSM 淀粉样蛋白治疗和研究计划中看到的 SSA 患者的 S-磺化 TTR 血清水平，试图将生化结构特征与疾病发病机制联系起来。我们假设，甲状腺素运载蛋白 (TTR) 在 Cys10 处的 S-磺化作用增加以及与辅助蛋白的相互作用促进原纤维形成，因此可作为老年系统性淀粉样变性 (SSA) 的效应因子。 为了检验这一假设，我们分析并比较了从 SSA、原发性淀粉样变性 (AL) 心肌病患者和非淀粉样蛋白对照患者的血清中分离出的 S-磺化 TTR 的相对丰度。结果并未表明 TTR S-磺化与 SSA 的发展之间存在直接联系。为了检查血清中半胱氨酸加合物的情况与淀粉样蛋白沉积物中半胱氨酸加合物的情况不同的可能性，从临床诊断的两例SSA的尸检组织中提取了心脏原纤维。 我们开发了在非还原条件下从这些组织中分离 TTR 的方法，以确定质量的异质性程度。 我们观察到 TTR 原纤维中的截短、二聚化和 Cys-10 修饰。自上而下的 MS 分析能够将结构分配给蛋白质的截短形式。描述这些结果的手稿最近发表在《分析化学》上。