Proteins usually associate with other molecules physically to execute their functions. Identifying these interactions is important for the functional analysis of proteins. Previously, we reported the parallel analysis of translated ORFs (PLATO) to couple ribosome display of full-length ORFs with affinity enrichment of mRNA/protein/ribosome complexes for the “bait” molecules, followed by the deep sequencing analysis of mRNA. However, the sample processing, from extraction of precipitated mRNA to generation of DNA libraries, includes numerous steps, which is tedious and may cause the loss of materials. Barcoded PLATO (PLATO-BC), an improved platform was further developed to test its application for protein interaction discovery. In this report, we tested the antisera-antigen interaction using serum samples from patients with inclusion body myositis (IBM). Tripartite motif containing 21 (TRIM21) was identified as a potentially new IBM autoantigen. We also expanded the application of PLATO-BC to identify protein interactions for JQ1, single ubiquitin peptide, and NS5 protein of Zika virus. From PLATO-BC analyses, we identified new protein interactions for these “bait” molecules. We demonstrate that Ewing sarcoma breakpoint region 1 (EWSR1) binds to JQ1 and their interactions may interrupt the EWSR1 association with acetylated histone H4. RIO kinase 3 (RIOK3), a newly identified ubiquitin-binding protein, is preferentially associated with K63-ubiquitin chain. We also find that Zika NS5 protein interacts with two previously unreported host proteins, par-3 family cell polarity regulator (PARD3) and chromosome 19 open reading frame 53 (C19orf53), whose attenuated expression benefits the replication of Zika virus. These results further demonstrate that PLATO-BC is capable of identifying novel protein interactions for various types of “bait” molecules.
蛋白质通常需要通过与其他分子（抗体，小分子药物，肽段或者蛋白质）互作来发挥其功能，鉴定蛋白质与这些分子的相互作用有助于我们来理解该蛋白质的功能。之前， 我们建立了一种PLATO方法用来鉴定这些相互作用，该方法采用核糖体展示全长基因的开放阅读框（ORF）来形成一种mRNA/蛋白质/核糖体复合体，通过与诱饵分子共同孵育，pulldown与其相互作用的分子，通过二代测序mRNA序列可以鉴定出与某一特定诱饵分子作用的特异性蛋白质。但是该方法在样品制备上存在一些弊端，包括从mRNA的提取到DNA 文库的制备，其步骤繁琐，耗时较长，并且会导致一些基因的丢失。我们通过给每个基因添加一段特有的条形码，建立了一种优化的Barcoded PLATO 。本研究中我们通过测定四种不同的诱饵分子（包涵体肌炎病人的血清抗体、BRD4抑制剂JQ1、泛素肽和ZIKV病毒的非结构蛋白NS5）来评估该方法的实用性。研究结果表明，新的Barcoded PLATO不仅可以鉴定先前报道的自身抗原，同时也鉴定出了一种新的未被报道的自身抗原TRIM21。JQ1可以与癌症相关的蛋白ESWR1结合, 同时JQ1还可以阻断EWSR1与乙酰化的组蛋白H4间的相互作用。一种新的泛素结合蛋白RIOK3在本研究中发现，进一步研究证明RIOK3优先结合K63泛素链。PARD3 和C19orf53不仅可以与ZIKV 病毒NS5结合，还可以抑制ZIKV病毒的复制。总之，这些结果证实barcoded PLATO 是一种可以用来鉴定多种诱饵分子相互作用的方法。





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