Protein−RNA Cross-Linking in the Ribosomes of Yeast under Oxidative Stress

Living systems have efficient degradative pathways for dealing with the fact that reactive oxygen species
(ROS) derived from cellular metabolism and the environment oxidatively damage proteins and DNA.
But aggregation and cross-linking can occur as well, leading to a series of problems including disruption
of cellular regulation, mutations, and even cell death. The mechanism(s) by which protein aggregation
occurs and the macromolecular species involved are poorly understood. In the study reported here,
evidence is provided for a new type of aggregate between proteins and RNA in ribosomes. While
studying the effect of oxidative stress induced in the yeast proteome it was noted that ribosomal proteins
were widely oxidized. Eighty six percent of the proteins in yeast ribosomes were found to be
carbonylated after stressing yeast cell cultures with hydrogen peroxide. Moreover, many of these
proteins appeared to be cross-linked based on their coelution patterns during RPC separation. Since
they were not in direct contact, it was not clear how this could occur unless it was through the RNA
separating them in the ribosome. This was confirmed in a multiple-step process, the first being
derivatization of all carbonylated proteins in cell lysates with biotin hydrazide through Schiff base
formation. Following reduction of Schiff bases with sodium cyanoborohydride, biotinylated proteins
were selected from cell lysates with avidin affinity chromatography. Oxidized proteins thus captured
were then selected again using boronate affinity chromatography to capture vicinal diol-containing
proteins. This would include proteins cross-linked to an RNA fragment containing a ribose residue
with 2‘,3‘-hydroxyl groups. Some glycoproteins would also be selected by this process. LC/MS/MS
analyses of tryptic peptides derived from proteins captured by this process along with MASCOT searches
resulted in the identification of 37 ribosomal proteins that appear to be cross-linked to RNA. Aggregation
of proteins with ribosomal RNA has not been previously reported. The probable impact of this
phenomenon cells is to diminish the protein synthesis capacity.
Keywords: Proteomics • oxidative stress • hydrogen peroxide • yeast • biotin hydrazide • avidin affinity
chromatography • boronate affinity chromatography • protein fragmentation • protein cross-linking • ribosomal
proteins • protein−RNA cross-linking • tandem mass spectrometry • protein identification

生命系统拥有高效的降解通路，以应对源自细胞代谢与外界环境的活性氧（reactive oxygen species, ROS）对蛋白质与DNA造成的氧化损伤。但与此同时，蛋白质聚集与交联现象也可能发生，进而引发一系列问题，包括细胞调控紊乱、基因突变乃至细胞死亡。目前人们对蛋白质聚集的发生机制及其所涉及的大分子物种仍知之甚少。
本研究为此提供了证据，证明核糖体中存在一类新型的蛋白质-RNA聚集体。在研究氧化应激对酵母蛋白质组的影响时，研究人员观察到核糖体蛋白发生了广泛的氧化修饰。在用过氧化氢处理酵母细胞培养物后，研究人员发现酵母核糖体中86%的蛋白质均发生了羰基化修饰。此外，基于反相色谱（reverse phase chromatography, RPC）分离过程中的共洗脱行为，研究人员发现此类蛋白质多发生了交联。由于这些蛋白质之间并无直接接触，研究人员一度难以解释该交联现象如何发生，除非是通过核糖体中分隔它们的RNA介导。
该猜想通过多步实验流程得到了验证：第一步是利用生物素酰肼（biotin hydrazide）通过希夫碱（Schiff base）形成反应，对细胞裂解液中所有发生羰基化修饰的蛋白质进行衍生化处理；随后利用氰基硼氢化钠（sodium cyanoborohydride）还原希夫碱，再通过抗生物素蛋白亲和色谱（avidin affinity chromatography）从细胞裂解液中富集被生物素标记的蛋白质。将上述捕获到的氧化修饰蛋白质再次通过硼酸亲和色谱（boronate affinity chromatography）进行富集，以捕获带有邻二醇基团的蛋白质，此类蛋白质包括与带有2',3'-羟基的核糖残基的RNA片段发生交联的蛋白质，该流程也会同时富集部分糖蛋白。
对通过该流程捕获的蛋白质所得到的胰蛋白酶肽段进行液相色谱-串联质谱（LC/MS/MS）分析，并结合MASCOT数据库检索，最终鉴定出37种与RNA发生交联的核糖体蛋白质。此前尚无关于蛋白质与核糖体RNA发生聚集的相关报道。该现象可能会降低细胞的蛋白质合成能力。
关键词：蛋白质组学 • 氧化应激 • 过氧化氢 • 酵母 • 生物素酰肼 • 抗生物素蛋白亲和色谱 • 硼酸亲和色谱 • 蛋白质片段化 • 蛋白质交联 • 核糖体蛋白质 • 蛋白质-RNA交联 • 串联质谱 • 蛋白质鉴定