scholarly journals Antibody toolkit reveals N-terminally ubiquitinated substrates of UBE2W

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Christopher W. Davies ◽  
Simon E. Vidal ◽  
Lilian Phu ◽  
Jawahar Sudhamsu ◽  
Trent B. Hinkle ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Monoclonal Antibodies ◽  
Molecular Basis ◽  
Selective Recognition ◽  
Tryptic Peptides ◽  
Ubiquitin Conjugating Enzyme ◽  
Endogenous Substrates ◽  
Conjugating Enzyme

AbstractThe ubiquitin conjugating enzyme UBE2W catalyzes non-canonical ubiquitination on the N-termini of proteins, although its substrate repertoire remains unclear. To identify endogenous N-terminally-ubiquitinated substrates, we discover four monoclonal antibodies that selectively recognize tryptic peptides with an N-terminal diglycine remnant, corresponding to sites of N-terminal ubiquitination. Importantly, these antibodies do not recognize isopeptide-linked diglycine (ubiquitin) modifications on lysine. We solve the structure of one such antibody bound to a Gly-Gly-Met peptide to reveal the molecular basis for its selective recognition. We use these antibodies in conjunction with mass spectrometry proteomics to map N-terminal ubiquitination sites on endogenous substrates of UBE2W. These substrates include UCHL1 and UCHL5, where N-terminal ubiquitination distinctly alters deubiquitinase (DUB) activity. This work describes an antibody toolkit for enrichment and global profiling of endogenous N-terminal ubiquitination sites, while revealing functionally relevant substrates of UBE2W.

scholarly journals Molecular Basis of Lys-63-linked Polyubiquitination Inhibition by the Interaction between Human Deubiquitinating Enzyme OTUB1 and Ubiquitin-conjugating Enzyme UBC13

2012 ◽  
Vol 287 (31) ◽  
pp. 25860-25868 ◽  
Author(s):  
Yusuke Sato ◽  
Atsushi Yamagata ◽  
Sakurako Goto-Ito ◽  
Keiko Kubota ◽  
Rikako Miyamoto ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Deubiquitinating Enzyme ◽  
Ubiquitin Conjugating Enzyme ◽  
Conjugating Enzyme

scholarly journals SILAC proteomics implicates the ubiquitin conjugating enzyme UBE2D in SOCS1-mediated downmodulation of the MET receptor in hepatocytes

2020 ◽  
Author(s):  
Madanraj Appiya Santharam ◽  
Akhil Shukla ◽  
Awais Ullan Ihsan ◽  
Maryse Cloutier ◽  
Dominique Levesque ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Growth Factor ◽  
Sh2 Domain ◽  
Mass Spectrometry Analysis ◽  
Cytokine Signaling ◽  
Spectrometry Analysis ◽  
Mitochondrial Ribosome ◽  
Ubiquitin Conjugating Enzyme ◽  
The Impact ◽  
Conjugating Enzyme

AbstractSuppressor of Cytokine Signaling 1 (SOCS1) functions as a tumor suppressor in hepatocellular carcinoma (HCC) and many other types of cancers. SOCS1 mediates its functions by inhibiting tyrosine kinases, promoting ubiquitination and proteasomal degradation of signal transducing proteins, and by modulating transcription factors. Here, we studied the impact of SOCS1 on the hepatocyte proteome using Stable Isotopic Labelling of Amino acids in Cell culture (SILAC)-based mass spectrometry on the Hepa1-6 murine HCC cell line stably expressing wildtype SOCS1 or a mutant SOCS1 with impaired SH2 domain. As SOCS1 regulates the hepatocyte growth factor (HGF) receptor MET, the SILAC-labelled cells were stimulated or not with HGF. Following mass spectrometry analysis, differentially modulated proteins were identified, quantified and analyzed for pathway enrichment. Of the 3440 proteins identified in Hepa-SOCS1 cells at steady state, 181 proteins were significantly modulated compared to control cells. The SH2 domain mutation and HGF increased the number of differentially modulated proteins. Protein interaction network analysis revealed enrichment of SOCS1-modulated proteins within multiprotein complexes such as ubiquitin conjugating enzymes, proteasome, mRNA spliceosome, mRNA exosome and mitochondrial ribosome. These findings suggest that SOCS1, induced by cytokines, growth factors and diverse other stimuli, may dynamically modulate of large macromolecular regulatory complexes to help maintain cellular homeostasis. Notably, the expression of UBE2D ubiquitin conjugating enzyme, which is implicated in the control of growth factor receptor tyrosine kinase signaling, was found to be regulated by SOCS1.

A Conserved Asparagine in a Ubiquitin Conjugating Enzyme Positions the Substrate for Nucleophilic Attack

2018 ◽  
Author(s):  
Walker M. Jones ◽  
Aaron G. Davis ◽  
R. Hunter Wilson ◽  
Katherine L. Elliott ◽  
Isaiah Sumner
Keyword(s):  
Molecular Dynamics ◽  
Quantum Mechanics ◽  
Molecular Mechanics ◽  
Reaction Rates ◽  
Nucleophilic Attack ◽  
Classical Molecular Dynamics ◽  
Ubiquitin Conjugating Enzyme ◽  
Conjugating Enzyme

We present classical molecular dynamics (MD), Born-Oppenheimer molecular dynamics (BOMD), and hybrid quantum mechanics/molecular mechanics (QM/MM) data. MD was performed using the GPU accelerated pmemd module of the AMBER14MD package. BOMD was performed using CP2K version 2.6. The reaction rates in BOMD were accelerated using the Metadynamics method. QM/MM was performed using ONIOM in the Gaussian09 suite of programs. Relevant input files for BOMD and QM/MM are available.

scholarly journals Development of a New Highly Selective Monoclonal Antibody against Preferentially Expressed Antigen in Melanoma (PRAME) and Identification of the Target Epitope by Bio-Layer Interferometry

2021 ◽  
Vol 22 (6) ◽  
pp. 3166
Author(s):  
Jwala Priyadarsini Sivaccumar ◽  
Antonio Leonardi ◽  
Emanuela Iaccarino ◽  
Giusy Corvino ◽  
Luca Sanguigno ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Western Blotting ◽  
Cancer Biomarkers ◽  
Trypsin Digestion ◽  
Protein Fragments ◽  
Target Epitope ◽  
Potential Activity ◽  
Antibody Epitopes

Background: Monoclonal antibodies (mAbs) against cancer biomarkers are key reagents in diagnosis and therapy. One such relevant biomarker is a preferentially expressed antigen in melanoma (PRAME) that is selectively expressed in many tumors. Knowing mAb’s epitope is of utmost importance for understanding the potential activity and therapeutic prospective of the reagents. Methods: We generated a mAb against PRAME immunizing mice with PRAME fragment 161–415; the affinity of the antibody for the protein was evaluated by ELISA and SPR, and its ability to detect the protein in cells was probed by cytofluorimetry and Western blotting experiments. The antibody epitope was identified immobilizing the mAb on bio-layer interferometry (BLI) sensor chip, capturing protein fragments obtained following trypsin digestion and performing mass spectrometry analyses. Results: A mAb against PRAME with an affinity of 35 pM was obtained and characterized. Its epitope on PRAME was localized on residues 202–212, taking advantage of the low volumes and lack of fluidics underlying the BLI settings. Conclusions: The new anti-PRAME mAb recognizes the folded protein on the surface of cell membranes suggesting that the antibody’s epitope is well exposed. BLI sensor chips can be used to identify antibody epitopes.

scholarly journals Targeting neddylation E2s: a novel therapeutic strategy in cancer

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Yi-Chao Zheng ◽  
Yan-Jia Guo ◽  
Bo Wang ◽  
Chong Wang ◽  
M. A. A. Mamun ◽  
...  
Keyword(s):  
Posttranslational Modification ◽  
Therapeutic Strategy ◽  
Neural Precursor Cell ◽  
Catalytic Core Domain ◽  
Catalytic Core ◽  
Core Domain ◽  
E3 Ligases ◽  
Ubiquitin Conjugating Enzyme ◽  
E2 Enzymes ◽  
Conjugating Enzyme

AbstractUbiquitin-conjugating enzyme E2 M (UBE2M) and ubiquitin-conjugating enzyme E2 F (UBE2F) are the two NEDD8-conjugating enzymes of the neddylation pathway that take part in posttranslational modification and change the activity of target proteins. The activity of E2 enzymes requires both a 26-residue N-terminal docking peptide and a conserved E2 catalytic core domain, which is the basis for the transfer of neural precursor cell-expressed developmentally downregulated 8 (NEDD8). By recruiting E3 ligases and targeting cullin and non-cullin substrates, UBE2M and UBE2F play diverse biological roles. Currently, there are several inhibitors that target the UBE2M-defective in cullin neddylation protein 1 (DCN1) interaction to treat cancer. As described above, this review provides insights into the mechanism of UBE2M and UBE2F and emphasizes these two E2 enzymes as appealing therapeutic targets for the treatment of cancers.

scholarly journals Cell Cycle-Dependent Expression of Mammalian E2-C Regulated by the Anaphase-Promoting Complex/Cyclosome

2000 ◽  
Vol 11 (8) ◽  
pp. 2821-2831 ◽  
Author(s):  
Atsushi Yamanaka ◽  
Shigetsugu Hatakeyama ◽  
Kin-ichiro Kominami ◽  
Masatoshi Kitagawa ◽  
Masaki Matsumoto ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Substrate Specificity ◽  
Critical Role ◽  
Anaphase Promoting Complex ◽  
Polyubiquitin Chain ◽  
M Phase ◽  
Ubiquitin Conjugating Enzyme ◽  
Recognition Motifs ◽  
Cycle Dependent ◽  
Conjugating Enzyme

Progression through mitosis requires the precisely timed ubiquitin-dependent degradation of specific substrates. E2-C is a ubiquitin-conjugating enzyme that plays a critical role with anaphase-promoting complex/cyclosome (APC/C) in progression of and exit from M phase. Here we report that mammalian E2-C is expressed in late G2/M phase and is degraded as cells exit from M phase. The mammalian E2-C shows an autoubiquitinating activity leading to covalent conjugation to itself with several ubiquitins. The ubiquitination of E2-C is strongly enhanced by APC/C, resulting in the formation of a polyubiquitin chain. The polyubiquitination of mammalian E2-C occurs only when cells exit from M phase. Furthermore, mammalian E2-C contains two putative destruction boxes that are believed to act as recognition motifs for APC/C. The mutation of this motif reduced the polyubiquitination of mammalian E2-C, resulting in its stabilization. These results suggest that mammalian E2-C is itself a substrate of the APC/C-dependent proteolysis machinery, and that the periodic expression of mammalian E2-C may be a novel autoregulatory system for the control of the APC/C activity and its substrate specificity.

scholarly journals Unraveling the molecular basis of subunit specificity in P pilus assembly by mass spectrometry

2008 ◽  
Vol 105 (35) ◽  
pp. 12873-12878 ◽  
Author(s):  
R. J. Rose ◽  
D. Verger ◽  
T. Daviter ◽  
H. Remaut ◽  
E. Paci ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Molecular Basis ◽  
Pilus Assembly
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