In Vitro Protein Ubiquitination Assay

2011 ◽  
pp. 163-172 ◽  
Author(s):  
Qingzhen Zhao ◽  
Lijing Liu ◽  
Qi Xie
Keyword(s):  
Protein Ubiquitination ◽  
Ubiquitination Assay ◽  
Vitro Protein

scholarly journals miR-101-3p Contributes to α-Synuclein Aggregation in Neural Cells through the miR-101-3p/SKP1/PLK2 Pathway

2021 ◽  
Vol 2021 ◽  
pp. 1-8
Author(s):  
Min Zhang ◽  
Wei Liu ◽  
Qingan Zhang ◽  
Hongfeng Hu
Keyword(s):  
Substantia Nigra ◽  
Target Gene ◽  
Luciferase Activity ◽  
Substantia Nigra Pars Compacta ◽  
Activity Assay ◽  
Protein Ubiquitination ◽  
Ubiquitination Assay ◽  
Luciferase Activity Assay ◽  
Vitro Protein

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by progressive neuronal loss in different brain regions, including the dopaminergic (DA) neurons of the substantia nigra pars compacta (SNc). The aggregation of α-synuclein (α-Syn) plays an essential role in the progression of PD-related neuron toxicity. In this study, bioinformatic analysis was used to confirm differentially expressed genes between patients with PD and healthy donors. Immunofluorescence was used to study the aggregation of α-Syn. Flow cytometry was used to confirm the apoptosis of neurons. Western blot was used to investigate the underlying mechanism. Coimmunoprecipitation (co-IP) was used to verify the interaction between proteins. Luciferase activity assay was used to confirm the target gene of miRNA. In vitro protein ubiquitination assay was used to ascertain the role of S-phase kinase-associated protein 1 (SKP1) on the ubiquitination processes of polo-like kinase 2 (PLK2). The result indicated that miR-101-3p was overexpressed in the substantia nigra of the postmortem brains of patients with PD. The underlying role was investigated in the SH-SY5Y cell line. The overexpression of α-Syn did not result in toxicity or aggregation. However, the co-overexpression of miR-101-3p and α-Syn promoted aggregation and neuron toxicity. Luciferase activity assay indicated that SKP1 is a target gene of miR-101-3p. The co-IP experiment confirmed that SKP1 could directly interact with PLK2. In vitro protein ubiquitination assay confirmed that SKP1 could promote the ubiquitination and subsequent protein degradation of PLK2. We also observed that the cotransfection of short hairpin RNA that targets PLK2 and α-Syn overexpression plasmid results in the endoplasmic reticulum stress of neurons. Our results collectively provide evidence that miR-101-3p contributes to α-Syn aggregation in neurons through the miR-101-3p/SKP1/PLK2 pathway.

In vitro Protein Ubiquitination Assays

BIO-PROTOCOL ◽  
2013 ◽  
Vol 3 (19) ◽  
Author(s):  
Qingzhen Zhao ◽  
Qi Xie
Keyword(s):  
Protein Ubiquitination ◽  
Vitro Protein

scholarly journals A Conserved LIR Motif in Connexins Mediates Ubiquitin-Independent Binding to LC3/GABARAP Proteins

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 902 ◽  
Author(s):  
Steve Catarino ◽  
Teresa M Ribeiro-Rodrigues ◽  
Rita Sá Ferreira ◽  
José Ramalho ◽  
Christine Abert ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Protein Interaction ◽  
Alternative Mechanism ◽  
Direct Communication ◽  
Protein Protein Interaction ◽  
Amino Terminal ◽  
Protein Ubiquitination ◽  
Lir Motif ◽  
Vitro Protein

Gap junctions (GJ) are specialized cell-cell contacts formed by connexins (Cxs), which provide direct communication between adjacent cells. Cx43 ubiquitination has been suggested to induce the internalization of GJs, as well as the recruitment of the autophagy receptor p62 to mediate binding to LC3B and degradation by macroautophagy. In this report, we describe a functional LC3 interacting region (LIR), present in the amino terminal of most Cx protein family members, which can mediate the autophagy degradation of Cx43 without the need of ubiquitin. Mutation of the LIR motif on Cx37, Cx43, Cx46 and Cx50 impairs interaction with LC3B and GABARAP without compromising protein ubiquitination. Through in vitro protein-protein interaction assays, we demonstrate that this LIR motif is required for the binding of Cx43 to LC3B and GABARAP. Overall, our findings describe an alternative mechanism whereby Cxs interact with LC3/GABARAP proteins, envisioning a new model for the autophagy degradation of connexins.

scholarly journals A general in vitro assay to study enzymatic activities of the ubiquitin system

2019 ◽  
Author(s):  
Yukun Zuo ◽  
Boon Keat Chong ◽  
Kun Jiang ◽  
Daniel Finley ◽  
David Klenerman ◽  
...  
Keyword(s):  
Reaction Rates ◽  
Vitro Assay ◽  
Protein Ubiquitination ◽  
Ubiquitination Assay ◽  
Ubiquitin System ◽  
Wide Range ◽  
Machine Learning Approach ◽  
Fret Efficiency ◽  
Kinetics Of

AbstractThe ubiquitin (Ub) system regulates a wide range of cellular signaling pathways. Several hundred E1, E2 and E3 enzymes are together responsible for protein ubiquitination, thereby controlling cellular activities. Due to the numerous enzymes and processes involved, studies on ubiquitination activities have been challenging. We here report a novel FRET-based assay to study the in vitro kinetics of ubiquitination. FRET is established between binding of fluorophore-labeled Ub to eGFP-tagged ZnUBP, a domain that exclusively binds unconjugated Ub. We name this assay the Free Ub Sensor System (FUSS). Using Uba1, UbcH5 and CHIP as model E1, E2 and E3 enzymes, respectively, we demonstrate that ubiquitination results in decreasing FRET efficiency, from which reaction rates can be determined. Further treatment with USP21, a deubiquitinase, leads to increased FRET efficiency, confirming the reversibility of the assay. We subsequently use this assay to show that increasing the concentration of CHIP or UbcH5 but not Uba1 enhances ubiquitination rates, and develop a novel machine learning approach to model ubiquitination. The overall ubiquitination activity is also increased upon incubation with tau, a substrate of CHIP. Our data together demonstrate the versatile applications of a novel ubiquitination assay that does not require labeling of E1, E2, E3 or substrates, and is thus likely compatible with any E1-E2-E3 combinations.

Applicability of Instability Index for In vitro Protein Stability Prediction

2019 ◽  
Vol 26 (5) ◽  
pp. 339-347 ◽  
Author(s):  
Dilani G. Gamage ◽  
Ajith Gunaratne ◽  
Gopal R. Periyannan ◽  
Timothy G. Russell
Keyword(s):  
Protein Stability ◽  
Caulobacter Crescentus ◽  
Effect Of Temperature ◽  
Instability Index ◽  
Dipeptide Composition ◽  
Experimental Conditions ◽  
The Stability ◽  
Vitro Protein

Background: The dipeptide composition-based Instability Index (II) is one of the protein primary structure-dependent methods available for in vivo protein stability predictions. As per this method, proteins with II value below 40 are stable proteins. Intracellular protein stability principles guided the original development of the II method. However, the use of the II method for in vitro protein stability predictions raises questions about the validity of applying the II method under experimental conditions that are different from the in vivo setting. Objective: The aim of this study is to experimentally test the validity of the use of II as an in vitro protein stability predictor. Methods: A representative protein CCM (CCM - Caulobacter crescentus metalloprotein) that rapidly degrades under in vitro conditions was used to probe the dipeptide sequence-dependent degradation properties of CCM by generating CCM mutants to represent stable and unstable II values. A comparative degradation analysis was carried out under in vitro conditions using wildtype CCM, CCM mutants and two other candidate proteins: metallo-β-lactamase L1 and α -S1- casein representing stable, borderline stable/unstable, and unstable proteins as per the II predictions. The effect of temperature and a protein stabilizing agent on CCM degradation was also tested. Results: Data support the dipeptide composition-dependent protein stability/instability in wt-CCM and mutants as predicted by the II method under in vitro conditions. However, the II failed to accurately represent the stability of other tested proteins. Data indicate the influence of protein environmental factors on the autoproteolysis of proteins. Conclusion: Broader application of the II method for the prediction of protein stability under in vitro conditions is questionable as the stability of the protein may be dependent not only on the intrinsic nature of the protein but also on the conditions of the protein milieu.

scholarly journals Oncogenic Potential of the Dual-Function Protein MEX3A

Biology ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 415
Author(s):  
Marcell Lederer ◽  
Simon Müller ◽  
Markus Glaß ◽  
Nadine Bley ◽  
Christian Ihling ◽  
...  
Keyword(s):  
Poor Prognosis ◽  
Rna Binding ◽  
Dual Function ◽  
Self Renewal ◽  
Oncogenic Potential ◽  
Protein Ubiquitination ◽  
Domain Composition ◽  
New Gene ◽  
Ligase Function

MEX3A belongs to the MEX3 (Muscle EXcess) protein family consisting of four members (MEX3A-D) in humans. Characteristic for MEX3 proteins is their domain structure with 2 HNRNPK homology (KH) domains mediating RNA binding and a C-terminal really interesting new gene (RING) domain that harbors E3 ligase function. In agreement with their domain composition, MEX3 proteins were reported to modulate both RNA fate and protein ubiquitination. MEX3 paralogs exhibit an oncofetal expression pattern, they are severely downregulated postnatally, and re-expression is observed in various malignancies. Enforced expression of MEX3 proteins in various cancers correlates with poor prognosis, emphasizing their oncogenic potential. The latter is supported by MEX3A’s impact on proliferation, self-renewal as well as migration of tumor cells in vitro and tumor growth in xenograft studies.

scholarly journals In Vivo and In Vitro Protein Ligation by Naturally Occurring and Engineered Split DnaE Inteins

PLoS ONE ◽  
2009 ◽  
Vol 4 (4) ◽  
pp. e5185 ◽  
Author(s):  
A. Sesilja Aranko ◽  
Sara Züger ◽  
Edith Buchinger ◽  
Hideo Iwaï
Keyword(s):  
Protein Ligation ◽  
Naturally Occurring ◽  
Vitro Protein

In Vitro Protein Degradation of 38 Sainfoin Accessions and Its Relationship to Tannin Content by Different Assays

2012 ◽  
Vol 60 (20) ◽  
pp. 5071-5075 ◽  
Author(s):  
Martin M. Lorenz ◽  
Christine Hayot Carbonero ◽  
Lydia Smith ◽  
Peter Udén
Keyword(s):  
Protein Degradation ◽  
Tannin Content ◽  
Vitro Protein
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