Synthesis and Assembly of Lipoprotein(a)

脂蛋白（a）（Lp（a））是存在于人类血液中的脂肪/蛋白质颗粒。它与低密度脂蛋白（LDL，也称为“坏胆固醇”）有关，但含有一种称为载脂蛋白（a）（apo（a））的额外蛋白质成分。Lp（a）水平升高是心血管疾病如心脏病发作和中风的危险因素。虽然已知Lp（a）的水平由颗粒的生物合成速率（而不是其从血流中的清除率）控制，但Lp（a）生物合成的确切机制在很大程度上仍然未知或有争议。特别是，我们希望检查Lp（a）从LDL和载脂蛋白（a）组装的位点，载脂蛋白（a）组装的脂蛋白颗粒的确切性质，催化Lp（a）组装的酶的身份和特征，以及载脂蛋白（a）连接的LDL蛋白组分（称为apoB 100）上特定氨基酸的身份。当Lp（a）组装时，apo（a）首先与apoB 100进行非共价（瞬时）接触，然后在两种蛋白质之间形成特定的共价（永久）键，称为二硫键。我们和其他人已经报道了载脂蛋白（a）和低密度脂蛋白的组装发生在细胞外，可能在细胞表面。使用不同的方法，其他人间接地表明组装可能发生在细胞内。为了解决这一争议，我们将在细胞内寻找apo（a）和apoB 100之间的非共价或共价复合物。我们还将比较apo（a）和apoB 100的分泌速率，apoB 100是LDL或Lp（a）的目标。甚至有人提出，apo（a）与一种不同于LDL的颗粒组装，但仍然含有apoB 100。因此，我们将测量载脂蛋白（a）是否与不同尺寸和密度的含apoB 100的颗粒更有效地组装。作为这些研究的一部分，我们将确定载脂蛋白（a）的大小是否影响组装速率;载脂蛋白（a）在不同个体之间的大小可能存在显着差异，小的载脂蛋白（a）大小与较高的Lp（a）水平相关。我们有一些关于催化apo（a）和apoB 100之间二硫键形成的酶的性质的初步证据，我们称之为Lp（a）氧化酶，我们将尝试纯化氧化酶或确定几种已知氧化酶中的一种是否可以进行此反应。最后，我们将直接鉴定apoB 100中与apo（a）形成二硫键的氨基酸（半胱氨酸），方法是将Lp（a）切割成几个大片段，鉴定含有apo（a）和apoB 100片段的片段（以及连接它们的二硫键），然后使用称为质谱的技术表征片段的序列。我们的研究将解决长期存在的问题，并在我们理解Lp（a）生物合成控制的生化机制方面开辟令人兴奋的新领域。

Lipoprotein(a) (Lp(a)) is a fat/protein particle present in human blood. It is related to low density lipoprotein (LDL, also known as the "bad cholesterol") but contains an additional protein component termed apolipoprotein(a) (apo(a)). Elevated levels of Lp(a) are a risk factor for the development of cardiovascular diseases such as heart attacks and strokes. While it is known that the levels of Lp(a) are controlled by the rate of the biosynthesis of the particle (rather than its clearance from the bloodstream), the exact mechanisms of Lp(a) biosynthesis remain largely unknown or controversial. In particular, we wish to examine the site of Lp(a) assembly from LDL and apo(a), the exact nature of the lipoprotein particle that apo(a) assembles with, the identity and characteristics of the enzyme that catalyzes Lp(a) assembly, and the identity of the specific amino acid on the protein component of LDL (known as apoB100) that apo(a) links to. When Lp(a) is assembled, apo(a) first makes noncovalent (transient) contacts with apoB100, and then a specific covalent (permanent) bond is formed between the two proteins called a disulfide bond. We and others have reported that assembly of apo(a) and LDL takes place outside the cell, possibly on the cell surface. Using different methodology, others have indirectly shown that assembly may happen inside the cell. To resolve this controversy, we will look for complexes between apo(a) and apoB100 inside the cell that are either noncovalent or covalent. We will also compare the rates of secretion of apo(a) and apoB100 that is destined for either LDL or Lp(a). It has even been suggested that apo(a) assembles with a particle distinct from LDL, yet one that still contains apoB100. Therefore, we will measure if apo(a) is assembled more or less efficiently with apoB100-containing particles of different size and density. As part of these studies, we will determine if the size of apo(a) influences the rate of assembly; apo(a) can differ remarkably in size between different individuals and small apo(a) sizes are associated with higher Lp(a) levels. We have some preliminary evidence about the nature of the enzyme that catalyzes disulfide bond formation between apo(a) and apoB100, which we call Lp(a) oxidase, and we will attempt to either purify the oxidase or determine if one of several known oxidases can perform this reaction. Finally, we will directly identify the amino acid (cysteine) in apoB100 that forms a disulfide bond with apo(a) by cleaving Lp(a) into several large pieces, identifying the piece that contains both apo(a) and apoB100 segments (and thus the disulfide bond that joins them), and then characterizing the sequence of the segments using a technique called mass spectrometry. Our research will both address longstanding questions as well as break exciting new ground in our understanding what biochemical mechanisms underlie control of the biosynthesis of Lp(a).