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

生物体系拥有高效的降解途径来处理由细胞代谢和环境产生的活性氧物种（ROS）导致的蛋白质和DNA的氧化损伤。然而，聚集和交联现象亦可能发生，进而引发一系列问题，包括细胞调控的破坏、突变，乃至细胞死亡。关于蛋白质聚集发生的机制以及涉及的宏分子种类，目前的理解尚显不足。在本研究中，我们提供了关于核糖体中蛋白质与RNA之间新型聚集体的证据。在研究酵母蛋白质组中由氧化应激诱导的效果时，注意到核糖体蛋白被广泛氧化。在用过氧化氢应激酵母细胞培养后，发现在酵母核糖体中的蛋白质中有百分之八十六被碳酰化。此外，根据其在RPC分离过程中的共洗脱模式，许多这些蛋白质似乎存在交联。鉴于它们之间并未直接接触，除非是通过将它们隔开的核糖体中的RNA，否则这种交联现象并不明朗。这一推测通过一系列多步骤过程得到证实，第一步是通过缩醛形成对细胞裂解物中的所有碳酰化蛋白进行生物素肟化处理。随后，通过氰硼酸钠对缩醛进行还原，利用亲和层析从细胞裂解物中筛选出生物素化的蛋白。由此捕获的氧化蛋白随后再次通过硼酸亲和层析筛选，以捕获含有邻二醇的蛋白质。这包括与含有2‘,3‘-羟基基团的核糖残基的RNA片段交联的蛋白质。一些糖蛋白也会通过此过程被筛选出来。对通过此过程捕获的蛋白质衍生的肽段进行液相色谱-质谱-质谱（LC/MS/MS）分析，并结合MASCOT搜索，结果鉴定出37种似乎与RNA交联的核糖体蛋白。蛋白质与核糖体RNA的聚集现象尚未有报道。此现象对细胞可能产生的影响是降低蛋白质的合成能力。

关键词：蛋白质组学、氧化应激、过氧化氢、酵母、生物素肟、亲和层析、硼酸亲和层析、蛋白质裂解、蛋白质交联、核糖体蛋白、蛋白质-RNA交联、串联质谱、蛋白质鉴定