Current methodologies in protein ubiquitination characterization: from ubiquitinated protein to ubiquitin chain architecture

Abstract

Ubiquitination is a versatile post-translational modification (PTM), which regulates diverse fundamental features of protein substrates, including stability, activity, and localization. Unsurprisingly, dysregulation of the complex interaction between ubiquitination and deubiquitination leads to many pathologies, such as cancer and neurodegenerative diseases. The versatility of ubiquitination is a result of the complexity of ubiquitin (Ub) conjugates, ranging from a single Ub monomer to Ub polymers with different length and linkage types. To further understand the molecular mechanism of ubiquitination signaling, innovative strategies are needed to characterize the ubiquitination sites, the linkage type, and the length of Ub chain. With advances in chemical biology tools, computational methodologies, and mass spectrometry, protein ubiquitination sites and their Ub chain architecture have been extensively revealed. The obtained information on protein ubiquitination helps to crack the molecular mechanism of ubiquitination in numerous pathologies. In this review, we summarize the recent advances in protein ubiquitination analysis to gain updated knowledge in this field. In addition, the current and future challenges and barriers are also reviewed and discussed.

Does the Ubiquitination Degradation Pathway Really Reach inside of the Chloroplast? A Re-Evaluation of Mass Spectrometry-Based Assignments of Ubiquitination

“…Chain position, linkage type, and topology determine the fate of a ubiquitylated substrate. The linkage polyubiquitination leading to proteasomal degradation typically occurs on K48-linked chains. , The determination of the nature of the substrate ubiquitination is technically challenging. ,, Figure D of Sun et al showed a linkage analysis indicating that 32% was from ubiquitin K48 (no further information was provided on this analysis). It was stated that “because K48 was the most abundant linkage type, much of the chloroplast ubiquitinome can be assumed to be primed for proteasomal degradation.” However, there are two issues with this analysis: (i) we identified 5500 proteins in these samples of which only ∼25% was chloroplast-localized (Table ) and (ii) off-target lysine di-carbamidomethylation is confounding this analysis.…”

“…Based on our own analysis presented here, the most recent reviews on “ubiquitinomics”, ,,,,,, and the primary literature, we recommend the following for the MS-based determination of possible in vivo ubiquitination of intra-chloroplast proteins of viable and intact chloroplasts: Ensure that the plants are not under significant chloroplast stress (e.g., due to high light treatment) as this will result in (a) rapid release of chloroplast proteins into the cytosol as demonstrated by confocal microscopy of plants expressing chloroplast-targeted green fluorescent protein (GFP) in the flu mutant , and (b) ATG-dependent and independent autophagy of swollen or otherwise damaged chloroplasts or selected chloroplast content and delivery and degradation in the vacuole. − ,− Use low concentration of appropriate (e.g., CAA but not IAA) alkylating agents (e.g., 20 mM) for in-solution digests and ensure removal or quenching of alkylating agent prior to addition of trypsin (or other proteases) to avoid N-terminal peptide and lysine alkylation. An alternative method to block the cysteine sulfhydryl group without the introduction of any potentially confounding acetamide at all is by treatment with S-methyl methanethiosulfonate (MMTS) with a +45.9877 modification or acrylamide resulting in a +71.037 modification .…”

“…One could also consider omitting sample alkylation since this will avoid di-carbamidomethylation of lysines (and other amino acid residues) while still obtaining relatively high PSM match rates Include appropriate negative affinity-enrichment controls to determine the true false discovery rate of the K-ε-GG footprint (or K + 114) and that affinity enrichment for-K-ε-GG containing peptides is highly selective. Use parallel alternative methods to enrich for peptides with ubiquitination; there are several independent methods now available. ,,, Perform an open search on the acquired MS data to ensure that the mass modifications in the data are consistent with the intended sample handling. For the analysis of MS data, include only the most relevant (variable) modifications (ideally based on open searches) and site scoring algorithms to reduce false discovery rate of ubiquitination (in particular +57.022 and +114.0429 Da on lysine, cysteine, and peptide N terminus). The sequence database used for MS data analysis should include all proteins of the target organism (e.g., Arabidopsis predicted protein sequences in Araport11 or TAIR10) as well as additional proteins that may be in the sample (including contaminants such as keratin, trypsin, and E. coli proteins) not just proteins of interest. …”

“…Furthermore, there are conflicting reports about whether trypsin cleaves the C-terminal of modified lysines, and therefore, assigning ubiquitination at a C-terminal K of a peptide needs to be treated with care , but see ref . Nevertheless, we can conclude that MS is a very powerful tool to detect ubiquitination and indeed several large scale ubiquitination studies have been published for Arabidopsis − as well as many non-plant species. ,− However, as in all large-scale “omics” studies, even low false discovery rates can result in many false positive observations especially for PTMs, and a critical evaluation of the MS results is therefore needed. Because the original raw MS data for many protein MS-based publications are available through ProteomeXchange (), it is possible to re-evaluate reported results by re-searching these raw MS data. , However, because many of these data sets are large and because of the need for specific expertise, many readers are not in the position to explore the raw data by themselves.…”

From seeds to trees: how E2 enzymes grow ubiquitin chains

“…The ubiquitin system is most well-known for its role in managing protein degradation [ 1 ], however ubiquitin transfer (ubiquitylation) has extensive non-proteolytic roles including the recruitment of signalling complexes [ 2 ], and regulating the cellular localisation of proteins [ 3 ]. Proteins can be modified by single ubiquitin moieties or by polyubiquitin chains of eight different linkages [ 4 ]. Attachment of ubiquitin involves a cascade of three families of proteins.…”