Protein- and Peptide-Derived Cofactors

描述（由申请人提供）：已发现许多医学相关的哺乳动物蛋白含有衍生自氨基酸侧链的新型氧化还原辅因子。这些辅因子是在蛋白质的铜胺氧化酶家族中发现的2，4，5三羟基苯丙氨酰醌（TPQ）和存在于赖氨酰氧化酶的活性位点中的赖氨酸酪氨酰醌（LTQ）。在真菌和原核生物中也描述了另外两种酪氨酸衍生的辅因子。这些是半乳糖氧化酶中的蛋白质结合的半胱氨酸酪氨酰自由基（CTR）和由多种细菌产生的肽衍生的吡咯喹啉醌（PQQ）。这个建议的一个主要目标是阐明这些酪氨酸衍生的辅因子中的每一个的生物发生的途径，并确定在何种程度上共同的策略已经（或没有）被采用，以实现非常不同的最终辅因子结构。在两种情况下（TPQ和CTR），已经提出的证据表明，生物发生仅通过添加O2和Cu 2+的前体蛋白。这可能也适用于LTQ，而PQQ的生产需要许多基因产物。拟议的实验策略，研究生物合成涉及前体蛋白或多肽的表达（连同辅助蛋白的表达的情况下，PQQ），其次是应用动力学和光谱检测和表征生物合成中间体。 辅因子的产生对细胞生理的影响可能是非常显著的。在赖氨酰氧化酶的情况下，已知许多疾病状态与酶活性相关，所述酶活性将受铜可用性和辅因子产生的速率和程度影响。哺乳动物铜胺氧化酶的细胞作用现在才变得明显。描述了解决位于内皮细胞和脂肪细胞外表面上的铜胺氧化酶的作用的实验。实验方案将集中在表征的内皮酶，我们最近在昆虫细胞中表达的高产量和使用的脂肪细胞系遵循铜胺氧化酶活性对细胞功能的影响。

DESCRIPTION (provided by applicant): A number of medically relevant mammalian proteins have been found to contain novel redox cofactors that are derived from amino acid side chains. These cofactors are 2,4,5 trihydroxyphenylalanyl quinone (TPQ), discovered in the copper amine oxidase family of proteins, and the lysine tyrosyl quinone (LTQ), present in the active site of lysyl oxidases. Two other tyrosine-derived cofactors have also been described in fungi and prokaryotes. These are the protein bound cysteine tyrosyl radical (CTR) in galactose oxidase and the peptide derived pyrroloquinoline quinone (PQQ) produced by a variety of bacteria. A major goal of this proposal is to elucidate the pathway for the biogenesis of each of these tyrosine-derived cofactors and to determine the degree to which common strategies have (or have not) been employed to achieve the very different final cofactor structures. In two instances (TPQ and CTR), evidence has been presented that biogenesis occurs solely by the addition of 02 and Cu 2+ to precursor protein. This is likely to also be true for LTQ, while PQQ production requires a number of gene products. The proposed experimental strategy for studying biogenesis involves the expression of precursor protein or polypeptide (together with the expression of accessory proteins in the case of PQQ), followed by the application of kinetics and spectroscopy to detect and characterize biogenesis intermediates. The impact of cofactor production on cellular physiology may be very significant. In the case of the lysyl oxidases, it is known that many disease states correlate with enzyme activity that will be affected by copper availability and the rate and extent of cofactor production. The cellular roles of the mammalian copper amine oxidases are only now becoming apparent. Experiments are described that address the role of the copper amine oxidase located on the outer surface of the endothelium and the adipocyte. Experimental protocols will focus on the characterization of the endothelial enzyme that we have recently expressed in high yield in insect cells and the use of an adipocyte cell line to follow the impact of copper amine oxidase activity on cellular function.