Internally tagged ubiquitin: a tool to identify linear polyubiquitin-modified proteins by mass spectrometry

Kliza, Katarzyna; Taumer, Christoph; Pinzuti, Irene; Franz‐Wachtel, Mirita; Kunzelmann, Simone; Stieglitz, Benjamin; Maček, Boris; Husnjak, Koraljka

doi:10.1038/nmeth.4228

Cited by 62 publications

(55 citation statements)

References 60 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Proteins were in‐gel digested by endoproteinase LysC (SILAC experiments) or trypsin and extracted peptides were desalted using C18 StageTips . LC‐MS/MS analyses were performed on an EASY‐nLC 1200 system (Thermo Fisher Scientific, Karlsruhe, Germany) coupled with an LTQ Orbitrap Elite mass spectrometer (Thermo Fisher Scientific) as described elsewhere with slight modifications: peptide mixtures were separated using a 127 segmented gradient of 10–33–50–90% of HPLC solvent B (80% acetonitrile in 0.1% formic acid) in HPLC solvent A (0.1% formic acid) at a flow rate of 200 nL·min −1 . In each scan cycle, the 15 most intense precursor ions were sequentially fragmented using collision‐induced dissociation.…”

Section: Methodsmentioning

confidence: 99%

APEX2‐mediated proximity labeling resolves protein networks in Saccharomyces cerevisiae cells

et al. 2019

Self Cite

View full text Add to dashboard Cite

Enzyme‐catalyzed proximity labeling (PL) with the engineered ascorbate peroxidase APEX2 is a novel approach to map organelle compartmentalization and protein networks in living cells. Current procedures developed for mammalian cells do not allow delivery of the cosubstrate, biotin‐phenol, into living yeast cells. Here, we present a new method based on semipermeabilized yeast cells. Combined with stable isotope labeling by amino acids in cell culture (SILAC), we demonstrate proteomic mapping of a membrane‐enclosed and a semiopen compartment, the mitochondrial matrix and the nucleus. APEX2 PL revealed nuclear proteins that were previously not identified by conventional techniques. One of these, the Yer156C protein, is highly conserved but of unknown function. Its human ortholog, melanocyte proliferating gene 1, is linked to developmental processes and dermatological diseases. A first characterization of the Yer156C neighborhood reveals an array of proteins linked to proteostasis and RNA binding. Thus, our approach establishes APEX2 PL as another powerful tool that complements the methods palette for the model system yeast.

show abstract

Section: Methodsmentioning

confidence: 99%

APEX2‐mediated proximity labeling resolves protein networks in Saccharomyces cerevisiae cells

et al. 2019

Self Cite

View full text Add to dashboard Cite

show abstract

“…The e-lysine (K) residues in the target serve as the typical Ub acceptors, forming isopeptide bonds with the C-terminal glycine carboxy group of ubiquitin (Okumoto et al, 2011). Ubiquitin also modifies itself at each of its seven lysine residues (K6, K11, K27, K29, K33, K48 and K63; Peng et al, 2003) or its N-terminal residues (Kliza et al, 2017). K48-linked poly-ub is the predominant (> 50%) linkage type functioning in directing targets to UPS for degradation (Hershko et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

Quantitative ubiquitylomics approach for characterizing the dynamic change and extensive modulation of ubiquitylation in rice seed germination

Damaris

et al. 2019

The Plant Journal

View full text Add to dashboard Cite

Summary During seed germination, cells embark on extensive post‐transcriptional and post‐translational modifications (PTM), providing a perfect platform to study these events in embryo rebooting from relative quiescenct to highly active state. PR‐619, a deubiquitylase inhibitor, delayed the rice seed germination and resulted in the accumulation of ubiquitylated proteins, which indicated the protein ubiquitylation is involved in this process. Using the K‐Ɛ‐GG antibody enrichment method integrated with high‐resolution mass spectrometry, a list of 2576 lysine ubiquitylated (Kub) sites in 1171 proteins was compiled for rice embryos at 0, 12 and 24 h after imbibition (HAI). Of these, the abundance of 1419 Kub sites in 777 proteins changed significantly. Most of them substantially increased within the first 12 HAI, which is similar to the dynamic state previously observed for protein phosphorylation, implying that the first 12 HAI are essential for subsequent switch during rice seed germination. We also quantitatively analyzed the embryo proteome in these samples. Generally, a specific protein’s abundance in the ubiquitylome was uncorrelated to that in the proteome. The differentially ubiquitinated proteins were greatly enriched in the categories of protein processing, DNA and RNA processing/regulation related, signaling, and transport. The DiGly footprint of the Kub sites was significantly reduced on K48, a linkage typically associated with proteasome‐mediated degradation. These observations suggest ubiquitylation may modulate the protein function more than providing 26S degradation signals in the early stage of rice seed germination. Revealing this comprehensive ubiquitylome greatly increases our understanding of this critical PTM during seed germination.

show abstract

“…Moreover, by applying an internally STREP II‐tagged ubiquitin (INT‐Ub.7KR) approach, Kliza et al. found that LUBAC‐mediated TRAF6 linear polyubiquitination is crucial for NF‐κB signaling …”

Section: Linear Polyubiquitination Plays Pivotal Roles In Inflammatormentioning

confidence: 99%

“…Recently, Kliza et al. utilized internally STREP II‐tagged ubiquitin (INT‐Ub.7KR) to confirm the regulatory role of linear polyubiquitination in the NF‐κB signaling pathway and to discover new linear ubiquitination targets . This internally tagged ubiquitin is a simple and powerful tool for studying the function of linear ubiquitin chains.…”

Section: Tools For Studying Atypical Polyubiquitinationmentioning

confidence: 99%

Emerging Roles and Research Tools of Atypical Ubiquitination

Huang

Zhang

2020

Proteomics

View full text Add to dashboard Cite

Ubiquitination is a posttranslational modification characterized by the covalent attachment of ubiquitin molecules to protein substrates. The ubiquitination modification process is reversible, dynamic, and involved in the regulation of various biological processes, such as autophagy, inflammatory responses, and DNA damage responses. The forms of ubiquitin modification are very diverse, incorporating either a single ubiquitin molecule or a complicated ubiquitin polymer, and different types of ubiquitination usually elicit corresponding cellular responses. The development of research tools and strategies has afforded more detailed insight into atypical ubiquitin signaling pathways that were previously poorly understood. Here, an update on the understanding of atypical ubiquitin chain signaling pathways is provided and the recent development of representative research tools for ubiquitin systems is discussed. In addition, the future challenges in ubiquitin research are reflected on and summarized.

show abstract

Internally tagged ubiquitin: a tool to identify linear polyubiquitin-modified proteins by mass spectrometry

Cited by 62 publications

References 60 publications

APEX2‐mediated proximity labeling resolves protein networks in Saccharomyces cerevisiae cells

APEX2‐mediated proximity labeling resolves protein networks in Saccharomyces cerevisiae cells

Quantitative ubiquitylomics approach for characterizing the dynamic change and extensive modulation of ubiquitylation in rice seed germination

Emerging Roles and Research Tools of Atypical Ubiquitination

Contact Info

Product

Resources

About