Protein-RNA cross-linking in the ribosomes of yeast under oxidative stress
Protein-RNA cross-linking in the ribosomes of yeast under oxidative stress
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DOI：
10.1021/pr060337l
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发表时间：
2006-12-01
影响因子：
4.4
通讯作者：
Regnier, Fred
中科院分区：
文献类型：
--
作者：
Mirzaei, Hamid;Regnier, Fred
生命系统具有有效的降解途径来处理来自细胞代谢和环境的活性氧（ROS）氧化损伤蛋白质和DNA的事实。但是聚集和交联也会发生，导致一系列问题，包括细胞调节的破坏，突变，甚至细胞死亡。蛋白质聚集发生的机制和所涉及的大分子种类知之甚少。在这里报道的研究中，为核糖体中蛋白质和RNA之间的新型聚集体提供了证据。在研究酵母蛋白质组中诱导的氧化应激的影响时，注意到核糖体蛋白被广泛氧化。酵母核糖体中86%的蛋白质被发现在用过氧化氢对酵母细胞培养物施加压力后被羰基化。此外，许多这些蛋白质似乎是交联的基础上，他们的共洗脱模式在RPC分离。由于它们没有直接接触，因此不清楚这是如何发生的，除非是通过核糖体中将它们分开的RNA。这在多步过程中得到证实，第一步是通过形成席夫碱，用生物素酰肼衍生化细胞裂解物中的所有羰基化蛋白质。在用氰基硼氢化钠还原席夫碱之后，用亲和素亲和层析从细胞裂解物中选择生物素化蛋白。然后使用硼酸盐亲和色谱法再次选择由此捕获的氧化蛋白质以捕获含邻位二醇的蛋白质。这将包括与含有具有2 '，3'-羟基的核糖残基的RNA片段交联的蛋白质。一些糖蛋白也将通过该过程被选择。LC/MS/MS分析胰蛋白酶肽，这些肽来源于通过该过程捕获的蛋白质，沿着MASCOT搜索，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的聚集以前没有报道过。这种现象的可能影响细胞是减少蛋白质合成能力。
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.