+ Site Statistics
References:
54,258,434
Abstracts:
29,560,870
PMIDs:
28,072,757
+ Search Articles
+ Subscribe to Site Feeds
Most Shared
PDF Full Text
+ PDF Full Text
Request PDF Full Text
+ Follow Us
Follow on Facebook
Follow on Twitter
Follow on LinkedIn
+ Translate
+ Recently Requested

Ubiquitin Chain Enrichment Middle-Down Mass Spectrometry (UbiChEM-MS) Reveals Cell-Cycle Dependent Formation of Lys11/Lys48 Branched Ubiquitin Chains



Ubiquitin Chain Enrichment Middle-Down Mass Spectrometry (UbiChEM-MS) Reveals Cell-Cycle Dependent Formation of Lys11/Lys48 Branched Ubiquitin Chains



Journal of Proteome Research 16(9): 3363-3369



The dynamics of cellular signaling events are tightly regulated by a diverse set of ubiquitin chains. Recent work has suggested that branched ubiquitin chains composed of Lys11 and Lys48 isopeptide linkages play a critical role in regulating cell cycle progression. Yet, endogenous Lys11/Lys48 branched chains could not be detected. By combining a Lys11 linkage specific antibody with high-resolution middle-down mass spectrometry (an approach termed UbiChEM-MS) we sought to identify endogenous Lys11/Lys48 branched ubiquitin chains in cells. Using asynchronous cells, we find that Lys11-linked branched chains can only be detected upon cotreatment with a proteasome and nonselective deubiquitinase inhibitor. Releasing cells from mitotic arrest results in a marked accumulation of Lys11/Lys48 branched chains in which branch points represent ∼3-4% of the total ubiquitin population. This report highlights the utility of UbiChEM-MS in characterizing the architecture of Lys11 Ub chains under various cellular conditions and corroborates the formation of Lys11/Lys48 branched chains during mitosis.

(PDF emailed within 0-6 h: $19.90)

Accession: 060434631

Download citation: RISBibTeXText

PMID: 28737031

DOI: 10.1021/acs.jproteome.7b00381


Related references

Ubiquitin Chain Enrichment Middle-Down Mass Spectrometry Enables Characterization of Branched Ubiquitin Chains in Cellulo. Analytical Chemistry 89(8): 4428-4434, 2017

Lys11- and Lys48-linked ubiquitin chains interact with p97 during endoplasmic-reticulum-associated degradation. Biochemical Journal 459(1): 205-216, 2014

Middle-down mass spectrometry enables characterization of branched ubiquitin chains. Biochemistry 53(30): 4979-4989, 2014

Immunoaffinity enrichment coupled to quantitative mass spectrometry reveals ubiquitin-mediated signaling events. Journal of Molecular Biology 427(11): 2121-2134, 2015

The ubiquitin-conjugating enzyme, UbcM2, is restricted to monoubiquitylation by a two-fold mechanism that involves backside residues of E2 and Lys48 of ubiquitin. Biochemistry 53(24): 4004-4014, 2014

Selective Binding of AIRAPL Tandem UIMs to Lys48-Linked Tri-Ubiquitin Chains. Structure 24(3): 412-422, 2016

Ubiquitin-dependent activation of NF-kappaB: K63-linked ubiquitin chains: a link to cancer?. Cancer Biology and Therapy 3(3): 286-288, 2004

Certain pairs of ubiquitin-conjugating enzymes (E2s) and ubiquitin-protein ligases (E3s) synthesize nondegradable forked ubiquitin chains containing all possible isopeptide linkages. Journal of Biological Chemistry 282(24): 17375-17386, 2007

Dimeric Ube2g2 simultaneously engages donor and acceptor ubiquitins to form Lys48-linked ubiquitin chains. Embo Journal 33(1): 46-61, 2014

Increased ubiquitin expression suppresses the cell cycle defect associated with the yeast ubiquitin conjugating enzyme, CDC34 (UBC3). Evidence for a noncovalent interaction between CDC34 and ubiquitin. Journal of Biological Chemistry 270(16): 9347-9352, 1995

Ubiquitin-specific Protease 11 (USP11) Deubiquitinates Hybrid Small Ubiquitin-like Modifier (SUMO)-Ubiquitin Chains to Counteract RING Finger Protein 4 (RNF4). Journal of Biological Chemistry 290(25): 15526-15537, 2016

Preparing to read the ubiquitin code: characterization of ubiquitin trimers by top-down mass spectrometry. Journal of Mass Spectrometry 51(4): 315-321, 2016

Using mass spectrometry to identify ubiquitin and ubiquitin-like protein conjugation sites. Proteomics 9(4): 922-934, 2009

Probing affinity and ubiquitin linkage selectivity of ubiquitin-binding domains using mass spectrometry. Journal of the American Chemical Society 134(14): 6416-6424, 2012

Definitive evidence for Ufd2-catalyzed elongation of the ubiquitin chain through Lys48 linkage. Biochemical and Biophysical Research Communications 320(3): 840-845, 2004