Analysis of the biochemical role of Lys‐11 in polyubiquitin chain formation using quantitative mass spectrometry

Jung, Jin Woo; Bae, Sung Jun; Kang, Gum–Yong; Kim, Kyun-Hwan; Yeo, Woon‐Seok; Park, Sang‐Hyun; Seol, Jae Hong; Yi, Eugene C.; Kim, Kwang Pyo

doi:10.1002/rcm.6447

Cited by 8 publications

(7 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Unlike previously described methods that relied on the use of radio-labeled ATP ( 32 P, 3 H) or/and radio-labeled Ub ( 125 I, 32 P) 21,24,27-29,70 , our method does not require radio-labeling of any of the components and therefore can be readily available in any biochemical laboratory having access to a mass spectrometer. Moreover, in contrast to the previous approaches our method quantifies Ub activation through direct measurement of the remaining amount of unactivated Ub and uses internal standards for absolute quantification.…”

Section: Discussionmentioning

confidence: 99%

Hydrophobic Patch of Ubiquitin is Important for its Optimal Activation by Ubiquitin Activating Enzyme E1

et al. 2017

View full text Add to dashboard Cite

Protein ubiquitination plays a role in essentially every process in eukaryotic cells. The attachment of ubiquitin (Ub) or Ub-like (UBL) proteins to target proteins is achieved by parallel but distinct cascades of enzymatic reactions involving three enzymes: E1, E2, and E3. The E1 enzyme functions at the apex of this pathway and plays a critical role in activating the C-terminus of ubiquitin or UBL, which is an essential step that triggers subsequent downstream transfer to their cognate E2s resulting in the fidelity of the Ub/UBL conjugation machinery. Despite the central role of the E1 enzyme in protein modification, a quantitative method to measure Ub/UBL activation by E1 is lacking. Here, we present a mass spectrometry-based assay to accurately measure the activation of Ub/UBL by E1 independent of the E2/E3 enzymes. Our method does not require radiolabeling of any components and therefore can be used in any biochemical laboratory having access to a mass spectrometer. This method allowed us to dissect the concerted process of E1-E2-catalyzed Ub conjugation in order to separately characterize the process of Ub activation and how it is affected by select mutations and other factors. We found that the hydrophobic patch of Ub is important for the optimal activation of Ub by E1. We further show that the blockers of the Ub-proteasome system such as ubistatin and fullerenol inhibit Ub activation by E1. Interestingly, our data indicate that the phosphorylation of Ub at the S65 position augments its activation by the E1 enzyme.

show abstract

Section: Discussionmentioning

confidence: 99%

Hydrophobic Patch of Ubiquitin is Important for its Optimal Activation by Ubiquitin Activating Enzyme E1

et al. 2017

View full text Add to dashboard Cite

show abstract

“…One advantage of tagged-UbLs is that they can be easily modified for specific goals, for example, mutations can be introduced to optimize MS identification [35,36] or to prevent formation of certain chain types [37,38]. In addition, UbL-modified proteins can be captured in a tissue-or temporal-specific manner in genetically tractable organisms [31,33,39].…”

Section: Expression Of Tagged-ub and Ub-like Proteinsmentioning

confidence: 99%

Using Ubiquitin Binders to Decipher the Ubiquitin Code

Mattern

Sutherland

Kadimisetty

et al. 2019

Trends in Biochemical Sciences

View full text Add to dashboard Cite

Post-translational modifications (PTMs) by ubiquitin (Ub) are versatile, highly dynamic, and involved in nearly all aspects of eukaryote biological function. The reversibility and heterogeneity of Ub chains attached to protein substrates have complicated their isolation, quantification, and characterization. Strategies have emerged to isolate endogenous ubiquitylated targets, including technologies based on the use of Ub-binding peptides, such as TUBEs (Tandem-repeated Ubiquitin-Binding Entities). TUBEs allow the identification and characterization of ubiquitin chains, novel substrates for deubiquitylases (DUBs) and Ub ligases (E3s). Here we review their impact on purification, analysis of pan or chain-selective polyubiquitylated proteins and underline the biological relevance of this information. Together with peptide aptamers and other Ub affinity-based approaches, TUBEs will contribute to unravelling the secrets of the Ub-code. The Complexity of the Ubiquitin CodeUbiquitination of proteins is a significant regulatory process that affects almost all cellular functions. Ubiquitin (Ub) is a small, compact, and highly conserved 76 amino acid protein. The C-terminus of Ub is attached by an isopeptide bond to the Ɛ-amino group of lysine residues on target proteins. After Ub is attached to a protein, this proximal Ub can act as a substrate for additional Ubs, which are conjugated to any of its seven lysine residues (K6, K11, K27, K29, K33, K48, and K63) or to its N-terminal methionine (M1) [1]. Protein ubiquitylation was first described as a signal for proteasomal degradation. The so called Ub-proteasome system (UPS) (see Glossary) uses mainly K48 and K11 polyubiquitylation as signals for substrate recognition [2,3]. The 26S proteasome include subunits that contain Ub-binding domains (UBDs) (Box 1) that participate in the binding of ubiquitin chains [4,5]. The binding and disassembly of many different types of Ub chains vary in both length and linkage specificity [6,7]. Although the presence of one Ub chain type is not by itself indicative of one function, accumulated evidence supports the existence of certain generic roles. For instance, proteasome inhibition leads to accumulation of ubiquitylated proteins containing all seven different types of Ub linkages except for K63 [8], strongly suggesting that K63 chains do not primarily target proteins to proteasomes. K63 Ub chains drive proteolysis primarily by the autophagylysosome system (ALS) [2] and might mediate DNA repair and other signaling pathways. Not much is known about the functions and mechanisms of more atypical linkages such Lys6, Lys27, Lys29, Lys33, and Met1 [9]. This Ub chain complexity known as the "Ub-code" includes mono-and multi-mono-Ub modifications, chains mixing multiple Ub linkages and other Ub-Like molecules (UbL). The biological relevance of most of these complex chains remains to be elucidated [10,11].

show abstract

“…Since keratin has an amino-acid composition different from collagen, the isotopic values may differ between the two. Previous studies have shown that for both δ 13 C and δ 15 N values from keratin need to be corrected for comparison with bone (O'Connell and Hedges 1999, O'Connell et al 2001, von Holstein et al 2013. Nail growth varies depending on the age of the individual, diet, climate, lifestyle, disease and between fingernails and toenails.…”

Section: Nail Keratinmentioning

confidence: 99%

Food and Cultural Traits in Coastal Northern Finnmark in the 14th–19th Centuries

Fjellström

Eriksson

Lidén

et al. 2019

Norwegian Archaeological Review

View full text Add to dashboard Cite

In this study, we used stable isotope analysis and radiocarbon dating to study diet, mobility and chronology in two late medieval/historical coastal populations in northern Norway. We have shown that the individuals buried at Kirkegårdsøya date between 1331 and 1953 cal AD and had a homogenous marine diet, whereas the individuals buried at Gullholmen had a more heterogeneous diet, consisting of both terrestrial and marine proteins and date between 1661 and 1953 cal AD. We have demonstrated that reindeer protein was not an important part of their diet, and also discussed the importance of correcting for the marine reservoir effect in populations with a coastal subsistence. Our interpretation is that individuals buried at Kirkegårdsøya primarily belonged to a Coastal Sámi community, although Norwegians with a similar diet (and likely comprising a minor population in the area) cannot be ruled out. The more varied diet and mobility at Gullholmen could, as predicted, indicate that these individuals may have had a more diverse cultural affinity.

show abstract

Analysis of the biochemical role of Lys‐11 in polyubiquitin chain formation using quantitative mass spectrometry

Cited by 8 publications

References 27 publications

Hydrophobic Patch of Ubiquitin is Important for its Optimal Activation by Ubiquitin Activating Enzyme E1

Hydrophobic Patch of Ubiquitin is Important for its Optimal Activation by Ubiquitin Activating Enzyme E1

Using Ubiquitin Binders to Decipher the Ubiquitin Code

Food and Cultural Traits in Coastal Northern Finnmark in the 14th–19th Centuries

Contact Info

Product

Resources

About