scholarly journals Technical Report: Targeted Proteomic Analysis Reveals Enrichment of Atypical Ubiquitin Chains in Contractile Murine Tissues

2020 ◽  
Author(s):  
Tiaan Heunis ◽  
Frederic Lamoliatte ◽  
José Luis Marín-Rubio ◽  
Abeer Dannora ◽  
Matthias Trost
Keyword(s):  
High Throughput Screening ◽  
Absolute Quantification ◽  
Polyubiquitin Chain ◽  
Post Translational Modification ◽  
Ubiquitin Chain ◽  
Form Complex ◽  
Murine Bone Marrow ◽  
Parallel Reaction Monitoring ◽  
Mouse Tissues ◽  
Total Pool

SummaryUbiquitylation is an elaborate post-translational modification involved in all biological processes. Its pleotropic effect is driven by the ability to form complex polyubiquitin chain architectures that can influence biological functions. In this study, we optimised sample preparation and chromatographic separation of Ubiquitin peptides for Absolute Quantification by Parallel Reaction Monitoring (Ub-AQUA-PRM). Using this refined Ub-AQUA-PRM assay, we were able to quantify all ubiquitin chain types in 10-minute LC-MS/MS runs. We used this method to determine the ubiquitin chain-linkage composition in murine bone marrow-derived macrophages and different mouse tissues. We could show tissue-specific differences in ubiquitin levels in murine tissues, with polyubiquitin chain types contributing a small proportion to the total pool of ubiquitin. Interestingly, we observed enrichment of atypical (K33) ubiquitin chains in heart and muscle. Our approach enabled high-throughput screening of ubiquitin chain-linkage composition in different murine tissues and highlighted a possible role for atypical ubiquitylation in contractile tissues.

scholarly journals Isoleucine 44 Hydrophobic Patch Controls Toxicity of Unanchored, Linear Ubiquitin Chains through NF-κB Signaling

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1519 ◽  
Author(s):  
Jessica R. Blount ◽  
Kozeta Libohova ◽  
Gustavo M. Silva ◽  
Sokol V. Todi
Keyword(s):  
Drosophila Melanogaster ◽  
Fruit Fly ◽  
Post Translational Modification ◽  
Ubiquitin Chain ◽  
Interaction Site ◽  
Cellular Processes ◽  
Protein Adduct ◽  
Linear Chains ◽  
Linear Poly ◽  
Free Ubiquitin

Ubiquitination is a post-translational modification that regulates cellular processes by altering the interactions of proteins to which ubiquitin, a small protein adduct, is conjugated. Ubiquitination yields various products, including mono- and poly-ubiquitinated substrates, as well as unanchored poly-ubiquitin chains whose accumulation is considered toxic. We previously showed that transgenic, unanchored poly-ubiquitin is not problematic in Drosophila melanogaster. In the fruit fly, free chains exist in various lengths and topologies and are degraded by the proteasome; they are also conjugated onto other proteins as one unit, eliminating them from the free ubiquitin chain pool. Here, to further explore the notion of unanchored chain toxicity, we examined when free poly-ubiquitin might become problematic. We found that unanchored chains can be highly toxic if they resemble linear poly-ubiquitin that cannot be modified into other topologies. These species upregulate NF-κB signaling, and modulation of the levels of NF-κB components reduces toxicity. In additional studies, we show that toxicity from untethered, linear chains is regulated by isoleucine 44, which anchors a key interaction site for ubiquitin. We conclude that free ubiquitin chains can be toxic, but only in uncommon circumstances, such as when the ability of cells to modify and regulate them is markedly restricted.

scholarly journals iTRAQ-Based Comparative Proteomics Analysis of Urolithiasis Rats Induced by Ethylene Glycol

2020 ◽  
Vol 2020 ◽  
pp. 1-10
Author(s):  
Yanan Cao ◽  
Bin Duan ◽  
Xiaowei Gao ◽  
E. Wang ◽  
Zhitao Dong
Keyword(s):  
Ethylene Glycol ◽  
Kidney Stones ◽  
Enrichment Analysis ◽  
Absolute Quantification ◽  
Rat Models ◽  
Protein Protein Interaction ◽  
Parallel Reaction Monitoring ◽  
High Prevalence ◽  
Isobaric Tags ◽  
And Control

Nephrolithiasis is a frequent chronic urological condition with a high prevalence and recurrence rate. Proteomics studies on urolithiasis rat models are highly important in characterizing the pathophysiology of kidney stones and identifying potential approaches for preventing and treating kidney stones. The isobaric tags for relative and absolute quantification (iTRAQ) were performed to identify differentially expressed proteins (DEPs) in the kidney between urolithiasis rats and control rats. The results showed that 127 DEPs (85 upregulated and 42 downregulated) were identified in urolithiasis and control rats. The functions of DEPs were predicted by Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, and protein–protein interaction (PPI) network analysis. The expression of four upregulated proteins (Tagln, Akr1c9, Spp1, and Fbn1) and four downregulated proteins (Hbb, Epb42, Hmgcs2, and Ca1) were validated by parallel reaction monitoring (PRM). Proteomics studies of ethylene glycol-induced urolithiasis rat models using iTRAQ and PRM helped to elucidate the molecular mechanism governing nephrolithiasis and to identify candidate proteins for the treatment of kidney stones.

scholarly journals Microfluidic Technologies for cfDNA Isolation and Analysis

Micromachines ◽  
2019 ◽  
Vol 10 (10) ◽  
pp. 672 ◽  
Author(s):  
Xu ◽  
Qiao ◽  
Tu
Keyword(s):  
High Throughput Screening ◽  
Absolute Quantification ◽  
Sample Volume ◽  
Tumor Burden ◽  
Precision Oncology ◽  
Sequencing Library ◽  
Potential Applications ◽  
Liquid Transfer ◽  
High Degree ◽  
Micrometer Scale

Cell-free DNA (cfDNA), which promotes precision oncology, has received extensive concern because of its abilities to inform genomic mutations, tumor burden and drug resistance. The absolute quantification of cfDNA concentration has been proved as an independent prognostic biomarker of overall survival. However, the properties of low abundance and high fragmentation hinder the isolation and further analysis of cfDNA. Microfluidic technologies and lab-on-a-chip (LOC) devices provide an opportunity to deal with cfDNA sample at a micrometer scale, which reduces required sample volume and makes rapid isolation possible. Microfluidic platform also allow for high degree of automation and high-throughput screening without liquid transfer, where rapid and precise examination and quantification could be performed at the same time. Microfluidic technologies applied in cfDNA isolation and analysis are limited and remains to be further explored. This paper reviewed the existing and potential applications of microfluidic technologies in collection and enrichment of cfDNA, quantification, mutation detection and sequencing library construction, followed by discussion of future perspectives.

scholarly journals High-Throughput Screening for Linear Ubiquitin Chain Assembly Complex (LUBAC) Selective Inhibitors Using Homogenous Time-Resolved Fluorescence (HTRF)-Based Assay System

2018 ◽  
Vol 23 (10) ◽  
pp. 1018-1029 ◽  
Author(s):  
Ken Katsuya ◽  
Yuji Hori ◽  
Daisuke Oikawa ◽  
Tomohisa Yamamoto ◽  
Kayo Umetani ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Inflammatory Responses ◽  
Mass Spectrometry Analysis ◽  
Regulatory Subunit ◽  
Luciferase Reporter ◽  
Time Resolved Fluorescence ◽  
Ubiquitin Chain ◽  
Time Resolved ◽  
Gene Assay

The nuclear factor κB (NF-κB) pathway is critical for regulating immune and inflammatory responses, and uncontrolled NF-κB activation is closely associated with various inflammatory diseases and malignant tumors. The Met1-linked linear ubiquitin chain, which is generated by linear ubiquitin chain assembly complex (LUBAC), is important for regulating NF-κB activation. This process occurs through the linear ubiquitination of NF-κB essential modulator, a regulatory subunit of the canonical inhibitor of the NF-κB kinase complex. In this study, we have established a robust and efficient high-throughput screening (HTS) platform to explore LUBAC inhibitors, which may be used as tool compounds to elucidate the pathophysiological role of LUBAC. The HTS platform consisted of both cell-free and cell-based assays: (1) cell-free LUBAC-mediated linear ubiquitination assay using homogenous time-resolved fluorescence technology and (2) cell-based LUBAC assay using the NF-κB luciferase reporter gene assay. By using the HTS platform, we performed a high-throughput chemical library screen and identified several hit compounds with selectivity against a counterassay. Liquid chromatography–mass spectrometry analysis revealed that these compounds contain a chemically reactive lactone structure, which is transformed to give reactive α,β-unsaturated carbonyl compounds. Further investigation revealed that the reactive group of these compounds is essential for the inhibition of LUBAC activity.

scholarly journals Novel Approach for Characterizing Ubiquitin E3 Ligase Function

2010 ◽  
Vol 15 (10) ◽  
pp. 1220-1228 ◽  
Author(s):  
Jeffrey G. Marblestone ◽  
K. G. Suresh Kumar ◽  
Michael J. Eddins ◽  
Craig A. Leach ◽  
David E. Sterner ◽  
...  
Keyword(s):  
Drug Discovery ◽  
High Throughput Screening ◽  
E3 Ligase ◽  
Cost Effective ◽  
Ubiquitin Proteasome System ◽  
Dependent Manner ◽  
Ubiquitin Chain ◽  
E3 Ligases ◽  
Effective Manner ◽  
Wide Range

The ubiquitin-proteasome system is central to the regulation of numerous cellular events, and dysregulation may lead to disease pathogenesis. E3 ubiquitin ligases typically function in concert with E1 and E2 enzymes to recruit specific substrates, thereby coordinating their ubiquitylation and subsequent proteasomal degradation or cellular activity. E3 ligases have been implicated in a wide range of pathologies, and monitoring their activity in a rapid and cost-effective manner would be advantageous in drug discovery. The relative lack of high-throughput screening (HTS)–compliant E3 ligase assays has significantly hindered the discovery of E3 inhibitors. Herein, the authors describe a novel HTS-compliant E3 ligase assay platform that takes advantage of a ubiquitin binding domain’s inherent affinity for polyubiquitin chains, permitting the analysis of ubiquitin chain formation in an E3 ligase-dependent manner. This assay has been used successfully with members of both the RING and HECT families, demonstrating the platform’s broad utility for analyzing a wide range of E3 ligases. The utility of the assay platform is demonstrated by the identification of inhibitors of the E3 ligase CARP2. As the number of E3 ligases associated with various disease states increases, the ability to quantitate the activity of these enzymes in an expeditious manner becomes imperative in drug discovery.

scholarly journals Crosstalk and ultrasensitivity in protein degradation pathways

2020 ◽  
Vol 16 (12) ◽  
pp. e1008492
Author(s):  
Abhishek Mallela ◽  
Maulik K. Nariya ◽  
Eric J. Deeds
Keyword(s):  
Protein Turnover ◽  
E3 Ligase ◽  
Biochemical Networks ◽  
Protein Homeostasis ◽  
Polyubiquitin Chain ◽  
Post Translational Modification ◽  
E3 Ligases ◽  
Protein Levels ◽  
Synthesis And Degradation

Protein turnover is vital to cellular homeostasis. Many proteins are degraded efficiently only after they have been post-translationally “tagged” with a polyubiquitin chain. Ubiquitylation is a form of Post-Translational Modification (PTM): addition of a ubiquitin to the chain is catalyzed by E3 ligases, and removal of ubiquitin is catalyzed by a De-UBiquitylating enzyme (DUB). Nearly four decades ago, Goldbeter and Koshland discovered that reversible PTM cycles function like on-off switches when the substrates are at saturating concentrations. Although this finding has had profound implications for the understanding of switch-like behavior in biochemical networks, the general behavior of PTM cycles subject to synthesis and degradation has not been studied. Using a mathematical modeling approach, we found that simply introducing protein turnover to a standard modification cycle has profound effects, including significantly reducing the switch-like nature of the response. Our findings suggest that many classic results on PTM cycles may not hold in vivo where protein turnover is ubiquitous. We also found that proteins sharing an E3 ligase can have closely related changes in their expression levels. These results imply that it may be difficult to interpret experimental results obtained from either overexpressing or knocking down protein levels, since changes in protein expression can be coupled via E3 ligase crosstalk. Understanding crosstalk and competition for E3 ligases will be key in ultimately developing a global picture of protein homeostasis.

Absolute quantification of dystrophin protein in human muscle biopsies using parallel reaction monitoring (PRM)

2019 ◽  
Vol 55 (2) ◽  
pp. e4437 ◽  
Author(s):  
Emily H. Canessa ◽  
Mansi V. Goswami ◽  
Tchilabalo D. Alayi ◽  
Eric P. Hoffman ◽  
Yetrib Hathout
Keyword(s):  
Absolute Quantification ◽  
Human Muscle ◽  
Reaction Monitoring ◽  
Parallel Reaction ◽  
Parallel Reaction Monitoring ◽  
Dystrophin Protein ◽  
Muscle Biopsies
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