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SUMOylation Site Identification Via FRET Analysis
Author(s) -
Way George,
Xiong Zhehao,
Liao Jiayu
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.06085
Subject(s) - sumo protein , virus , lysine , computational biology , ubiquitin , biology , immunoprecipitation , virology , chemistry , microbiology and biotechnology , genetics , gene , amino acid
This year’s influenza is estimated to affect more than 20 million people in the United States alone. Although influenza has been extensively researched, it still remains a major public health threat every year. Current methods to cull the spread of the influenza virus are vaccination and anti‐influenza virus drugs. Although these methods can be effective, the infuenza’s capacity to mutate and undergo antigenic shift make these methods fall short. SUMOylation is a post‐translational modification that is important for cellular processes as well as protein function and stability. The dysregulation of SUMOylation has been observed in neurodegenerative diseases, many different types of cancers, and viral infections. SUMOylation has been shown to be an important host factor for the influenza A virus but the mechanism still needs to be better understood. Currently, the methodology to identify SUMOylated lysine residues relies on immunodetection after site‐directed mutagenesis. Although this method has yielded great success, it is prone to false positives due to a lack of sensitivity or the breakdown of the isopeptide bond from endogenous SENP. We have developed a highly‐sensitive FRET‐based methodology to identify the exact lysine residue that is targeted by SUMOylation in the NS1 protein of the influenza virus (Figure 1). Using our method, we found lysine 131 was SUMOylated, contrary to previous findings (Figure 2). Our method can be applied to other proteins and ubiquitin‐like modifications to identify important residues and evaluate posttranslational modifications as druggable targets for cancer and antiviral therapies.Development of in vitro FRET‐based SUMOylation assay of IAV NS1. A) FRET‐based SUMOylation assay of NS1. If a lysine residue is responsible for SUMOylation, the mutation of the lysine residue to alanine will result in a decrease of the FRET emission at 530 nm given a 414 nm excitation. B) The EmFRET RFU is specific to the SUMOylation of NS1, provided the requirements of SUMOylation (SAE1, E2, and ATP).Determination of SUMOylated lysine residues of NS1. A) Table of NS1 lysine mutants for the FRET assay. B) Systematic mutation of NS1 lysine residues to alanine.