Proteome-wide identification and functional analysis of ubiquitinated proteins in peach leaves

Abstract

Ubiquitination is a critical post-translational modification machinery that governs a wide range of cellular functions by regulating protein homeostasis. Identification of ubiquitinated proteins and lysine residues can help researchers better understand the physiological roles of ubiquitin modification in different biological systems. In this study, we report the first comprehensive analysis of the peach ubiquitome by liquid chromatography-tandem mass spectrometry-based diglycine remnant affinity proteomics. Our systematic profiling revealed a total of 544 ubiquitination sites on a total of 352 protein substrates. Protein annotation and functional analysis suggested that ubiquitination is involved in modulating a variety of essential cellular and physiological processes in peach, including but not limited to carbon metabolism, histone assembly, translation and vesicular trafficking. Our results could facilitate future studies on how ubiquitination regulates the agricultural traits of different peach cultivars and other crop species.Peach is a highly popular and well-recognized fruit first cultivated in China during ancient times. It is considered an excellent source of vitamin C, niacin, potassium and dietary fiber. As a result, the recent global trend of healthy eating has greatly stimulated the demand for peach and the related food products. As of 2014, the combined area of peach orchards in China is estimated at around 7,260 square kilometers, with a total production of 12.4 million tons 1 . The long history of peach cultivation has resulted in hundreds of distinct cultivars. Peach growers have spent decades in a continuous quest to enhance the quality of different peach cultivars through crossbreeding. Recently, biotechnology-facilitated engineering has shown enormous promise in rapidly generating new peach varieties with desirable agricultural traits. Still, further accomplishments would depend on improved understanding of the diverse molecular pathways in peach and their connections to its physiology.Ubiquitination is one of the most critical post-translational modification mechanisms with a wide range of cellular functions that center on the control of protein load 2,3 . Ubiquitin is a small but abundantly present protein consisting of 76 amino acids with a C-terminal diglycine tail. The ubiquitination pathway is essentially an enzymatic cascade involving the cooperative action of three enzymes, including the E1, E2 and E3 ligases 2,4 . During this process, the C-terminal carboxyl group of ubiquitin is first adenylated by E1 in the presence of ATP and then forms a thioester linkage with the catalytic cysteine residue of the ligase 5 . The E1 then binds a second ubiquitin with its adenylation domain and recruits its cognate E2.This triggers a transthioesterification reaction where the E1-conjugated ubiquitin unit is transferred to the catalytic cysteine of the E2 6 . Finally, an E3 is involved in the transfer of ubiquitin from E2 to the ε-amino group of one or more lysine residues in the protein substrate 7...

Decreased ubiquitin modifying enzyme A20 associated with hyper-responsiveness to ovalbumin challenge following intrauterine growth restriction

“…The ubiquitinated protein identifications were performed according to the previous studies [ 15 , 16 ]. The lung tissues of newborn rats were used for protein extraction and in-solution digestion.…”