Diversified Application of Barcoded PLATO (PLATO-BC) Platform for Identification of Protein Interactions

Weili Kong,Netty Santoso,Guillaume Fiches,Stephen J Elledge,Jun Qi,Qikai Xu,Jianwen Que,Jian Zhu,Mamie Z Li,Wei Zhang,Tsuyoshi Hayashi,Shuai Liu

doi:10.1016/j.gpb.2018.12.010

Abstract

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.

Highlights

Proteins are the workhorses that control many biological processes
At 72 h post transfection, the medium was removed and the cells were infected with Zika virus (ZIKV) at $ 0.2 to 0.3 multiplicity of infection (MOI) in 40 ll complete media
Knockdown of C19orf53 or PARD3 in MAGI cells significantly enhanced ZIKV replication (Figure 5F). These results suggest that the parallel analysis of translated ORFs (PLATO)-BC enables the identification of novel host proteins that interact with ZIKV NS5 protein, and these host proteins may interfere with ZIKV viral replication

Summary

Introduction

Proteins are the workhorses that control many biological processes. They recognize or are recognized by other molecules through physical interactions to achieve proper biological functions. A variety of proteomic tools have been developed to allow the massive identification of protein interactions for a ‘‘bait” molecule [1,2]. Identity of protein/peptide can be directly analyzed by mass spectrometers or by pre-arrayed protein microarrays. Such direct detection of protein/peptide signal usually requires enormous efforts. Protein/peptidecorresponding nucleotide signal is relatively easy to identify, due to the recent advances of high-throughput sequencing technologies. To ‘‘pair” protein/peptide with its coding sequence, a vector is usually needed to express and display the protein/peptide. The commonly used vectors include phages, yeasts, and ribosomes [3]

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Discussion

Conclusion