Apolipoprotein conformation in amyloid and cardiovascular diseases

摘要 脂蛋白代谢异常是影响>40%的美国人的肥胖症的标志。肥胖及其并发症 包括2型糖尿病（T2 D）、心血管、淀粉样蛋白和严重病毒性疾病在内的并发症是主要的 过早死亡的原因。这项研究的最终目标是帮助诊断和治疗这些疾病。这 该项目解决了肥胖症中脂蛋白功能性的关键未解决问题。高、低和非常低- 密度脂蛋白（HDL、LDL和VLDL）是由脂质和载脂蛋白组成的不同纳米颗粒， 直接影响血浆脂质代谢。这个过程非常复杂，许多细节都是未知的。我们目前 重点是脂蛋白和蛋白质与硫酸乙酰肝素（HS）的相互作用，这可以触发心血管疾病， 血管和淀粉样疾病。我们将确定载脂蛋白-HS相互作用的分子机制 和脂蛋白功能的改变。我们强大的方法，它集成了广泛的生物化学- 科学、生物物理和计算工具，使我们能够假定令人信服的新假设， 三个互补的具体目标。目标1将确定脂蛋白功能如何在肥胖中变化- 治疗前后。将基于以下方法分离来自健康和病态肥胖受试者的脂蛋白： 将测定肝素结合亲和力以及它们的生物化学和功能性质。我们的新假设 在强有力的试点研究的支持下，将进行测试：脂蛋白脂肪酶作用于循环时形成的小颗粒， 标记VLDL提供了新的肥胖生物标志物和用于治疗的读数。目标2将定义生物化学 和肥胖症中血清淀粉样蛋白A（SAA）从HDL向LDL转移的功能细节。我们的初步研究表明 这种小蛋白的转移诱导LDL中促动脉粥样硬化的变化，并提出两种假设， 测试：i）肥胖，SAA升高和动脉粥样硬化之间的新机制联系，和ii）新的有益的 SAA在肥胖症中作为脂质清除剂的作用。目的3将确定HS和肝素如何影响SAA错误折叠， 淀粉样蛋白A（AA）淀粉样变性，肥胖和慢性炎症的危及生命的并发症。SAA结合至 HS是AA淀粉样变性的治疗靶点，但缺乏分子细节。我们的试点研究表明：i） SAA与HS结合启动淀粉样蛋白沉积的细胞外途径; ii）HS结合中的周期性阴离子阵列 使淀粉样蛋白中的碱性残基堆积并稳定淀粉样蛋白核。为了测试这些有趣的新想法，我们将 结合联合收割机生物化学、生物物理、氢氘交换质谱和计算方法， 在晶体和原纤维中具有SAA原子结构的Ods。结果：这项研究将确定新的机制 肥胖和动脉粥样硬化之间的联系;验证SAA作为脂质清除剂的假设重要作用;澄清 它在淀粉样蛋白中的错误折叠途径;并帮助开发新的诊断工具和治疗肥胖及相关疾病的方法。 紊乱这项研究的更广泛的影响是，它将：i）帮助理解和调节 肥胖、T2 D和严重的COVID-19疾病，以及ii）揭示HS-淀粉样蛋白间 这些行动不仅可以严重影响AA，还可以影响阿尔茨海默氏症和其他主要淀粉样疾病。

Abstract Aberrant lipoprotein metabolism is a hallmark of obesity that affects >40% of Americans. Obesity and its com- plications, including type-2 diabetes (T2D), cardiovascular, amyloid and severe viral diseases, are the leading causes of premature death. The ultimate goal of this research is to help diagnose and treat these disorders. This project addresses critical unresolved questions in the lipoprotein functionality in obesity. High-, low- and very low- density lipoproteins (HDL, LDL and VLDL) are diverse nanoparticles comprised of lipids and apolipoproteins that direct lipid metabolism in plasma. This process is extremely complex and many details are unknown. Our current focus is on the interactions of lipoproteins and proteins with heparan sulfate (HS), which can trigger both cardio- vascular and amyloid diseases. We will determine the molecular mechanisms of apolipoprotein-HS interactions and altered lipoprotein functionality in obesity. Our powerful approach, which integrates a wide array of biochem- ical, biophysical and computational tools, enabled us to postulate compelling new hypotheses that will be tested in three complementary specific aims. Aim 1 will determine how lipoprotein functionality changes in obesity be- fore and after treatment. Lipoproteins from healthy and morbidly obese subjects will be isolated based on the heparin binding affinity, and their biochemical and functional properties will be determined. Our new hypothesis supported by strong pilot studies will be tested: small particles formed upon action of lipoprotein lipase on circu- lating VLDL provide novel biomarkers of obesity and readouts for treatment. Aim 2 will define the biochemical and functional details of serum amyloid A (SAA) transfer from HDL to LDL in obesity. Our pilot studies indicate that transfer of this small protein induces pro-atherogenic changes in LDL, and suggest two hypotheses that will be tested: i) new mechanistic links between obesity, elevated SAA and atherosclerosis, and ii) novel beneficial role of SAA as a lipid scavenger in obesity. Aim 3 will determine how HS and heparin affect SAA misfolding in amyloid A (AA) amyloidosis, a life-threatening complication of obesity and chronic inflammation. SAA binding to HS is a therapeutic target in AA amyloidosis but molecular details are lacking. Our pilot studies suggest that: i) SAA binding to HS initiates an extracellular pathway of amyloid deposition; ii) periodic anionic arrays in HS bind to stacks of basic residues in amyloid and stabilize amyloid nuclei. To test these intriguing new ideas, we will combine biochemical, biophysical, hydrogen-deuterium exchange mass spectrometry, and computational meth- ods with atomic structures of SAA in crystals and fibrils. Outcome: this research will identify new mechanistic links between obesity and atherosclerosis; verify the hypothetical vital role of SAA as a lipid scavenger; clarify its misfolding pathway in amyloid; and help develop new diagnostic tools and treatments for obesity and related disorders. Broader impact of this research is that it will: i) help understand and modulate the causal link between obesity, T2D and severe COVID-19 disease, and ii) uncover the molecular underpinnings of HS-amyloid inter- actions that can critically impact not only in AA but also Alzheimer's and other major amyloid diseases.