As an important post-translational modification, ubiquitination mediates ∼80% of protein degradation in eukaryotes. The degree of protein ubiquitination is tightly determined by the delicate balance between specific ubiquitin ligase (E3)-mediated ubiquitination and deubiquitinase-mediated deubiquitination. In 2017, we developed UbiBrowser 1.0, which is an integrated database for predicted human proteome-wide E3–substrate interactions. Here, to meet the urgent requirement of proteome-wide E3/deubiquitinase–substrate interactions (ESIs/DSIs) in multiple organisms, we updated UbiBrowser to version 2.0 (http://ubibrowser.ncpsb.org.cn). Using an improved protocol, we collected 4068/967 known ESIs/DSIs by manual curation, and we predicted about 2.2 million highly confident ESIs/DSIs in 39 organisms, with >210-fold increase in total data volume. In addition, we made several new features in the updated version: (i) it allows exploring proteins’ upstream E3 ligases and deubiquitinases simultaneously; (ii) it has significantly increased species coverage; (iii) it presents a uniform confidence scoring system to rank predicted ESIs/DSIs. To facilitate the usage of UbiBrowser 2.0, we also redesigned the web interface for exploring these known and predicted ESIs/DSIs, and added functions of ‘Browse’, ‘Download’ and ‘Application Programming Interface’. We believe that UbiBrowser 2.0, as a discovery tool, will contribute to the study of protein ubiquitination and the development of drug targets for complex diseases.

ABSTRACT:An integrated sample pretreatment system, composed of a click maltose hydrophilic interaction chromatography (HILIC) column, a strong cation exchange (SCX) precolumn, and a PNGase F immobilized enzymatic reactor (IMER), was established for the simultaneous glycopeptide enrichment, sample buffer exchange, and online deglycosylation, by which the sample pretreatment for glycoproteome could be performed online automatically, beneficial to improve the efficiency and sensitivity of the N-linked glycosylation site identification. With such a system, the deglycosylated glycopeptide from the digests of avidin with the coexistence of 50 times (mass ratio) BSA could be selectively detected, and the detection limit as low as 5 fmol was achieved. Moreover, the sample pretreatment time was significantly shortened to ∼1 h. Such a system was further successfully applied for analyzing the digest of the soluble fraction extracted from rat brain. A total of 120 unique glycoprotein groups and 196 N-linked glycosylation sites were identified by nanoreversed phase liquid chromatographyÀelectrospray ionization-tandem mass spectrometry (nanoRPLC-ESI-MS/MS), with the injected digests amount as 6 μg. All these results demonstrate that the integrated system is of great promise for N-linked glycosylation site profiling and could be further online coupled with nanoHPLC-ESI-MS/MS to achieve high-throughput glycoproteome analysis. N -glycosylation is one of the most common and complex posttranslational modification of proteins and plays an important role in cellÀcell interaction, signal transduction, cancer immunology, and so forth. 1À4 The global mapping of the N-linked glycosylation site, which generally falls into the canonical N-!P-S/T (where !P denotes any amino acid except proline) sequence motif, is a prerequisite for fully understanding the biological functions of N-linked glycoproteins. Mass spectrometry (MS) coupled with chromatography has become an effective tool for analyzing N-linked glycoproteins and glycosylation sites in particular.5À9 However, the low abundance of glycopeptides relative to the large excess of nonglycosylated peptides as well as their low ionization efficiency often result in signal suppression when subjected to MS analysis.10 Therefore, the development of a fast and specific protocol to enrich glycopeptides prior to MS becomes indispensable.

The post-translational protein modification known as SUMOylation has conserved roles in the heat stress responses of various species. The functional connection between the global regulation of gene expression and chromatin-associated SUMOylation in plant cells is unknown. Here, we uncovered a genome-wide relationship between chromatin-associated SUMOylation and transcriptional switches in Arabidopsis thaliana grown at room temperature, exposed to heat stress, and exposed to heat stress followed by recovery. The small ubiquitin-like modifier (SUMO)-associated chromatin sites, characterized by whole-genome ChIP-seq, were generally associated with active chromatin markers. In response to heat stress, chromatin-associated SUMO signals increased at promoter-transcriptional start site regions and decreased in gene bodies. RNA-seq analysis supported the role of chromatin-associated SUMOylation in transcriptional activation during rapid responses to high temperature. Changes in SUMO signals on chromatin were associated with the upregulation of heat-responsive genes and the downregulation of growth-related genes. Disruption of the SUMO ligase gene SIZ1 abolished SUMO signals on chromatin and attenuated rapid transcriptional responses to heat stress. The SUMO signal peaks were enriched in DNA elements recognized by distinct groups of transcription factors under different temperature conditions. These observations provide evidence that chromatin-associated SUMOylation regulates the transcriptional switch between development and heat stress response in plant cells.

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