Detection of Protein–Protein Interactions and Posttranslational Modifications Using the Proximity Ligation Assay: Application to the Study of the SUMO Pathway

2016 ◽  
pp. 279-290 ◽  
Author(s):  
Marko Ristic ◽  
Frédérique Brockly ◽  
Marc Piechaczyk ◽  
Guillaume Bossis
Keyword(s):  
Protein Interactions ◽  
Posttranslational Modifications ◽  
Proximity Ligation Assay ◽  
Protein Protein Interactions ◽  
Proximity Ligation ◽  
Sumo Pathway

The “In Situ” Proximity Ligation Assay to Probe Protein–Protein Interactions in Intact Tissues

2014 ◽  
pp. 397-405 ◽  
Author(s):  
Arianna Bellucci ◽  
Chiara Fiorentini ◽  
Michela Zaltieri ◽  
Cristina Missale ◽  
PierFranco Spano
Keyword(s):  
Protein Interactions ◽  
Proximity Ligation Assay ◽  
Protein Protein Interactions ◽  
Proximity Ligation

Abstract 1976: Systematic identification of protein-protein interactions by Proximity Ligation Assay

2011 ◽  
Author(s):  
Tzu-Chi Chen ◽  
Kuan-Ting Lin ◽  
Yu-Lun Kuo ◽  
Pei-Ying Lee ◽  
Chun-Houh Chen ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Proximity Ligation Assay ◽  
Protein Protein Interactions ◽  
Proximity Ligation ◽  
Systematic Identification

scholarly journals Proximity Ligation Assay for Detecting Protein‐Protein Interactions and Protein Modifications in Cells and Tissues in Situ

2020 ◽  
Vol 89 (1) ◽  
Author(s):  
Marihan Hegazy ◽  
Eran Cohen‐Barak ◽  
Jennifer L. Koetsier ◽  
Nicole A. Najor ◽  
Constadina Arvanitis ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Proximity Ligation Assay ◽  
Protein Protein Interactions ◽  
Protein Modifications ◽  
Proximity Ligation ◽  
In Cells

scholarly journals Proximity ligation assay to study protein–protein interactions of proteins on two different cells

BioTechniques ◽  
2018 ◽  
Vol 65 (3) ◽  
pp. 149-157 ◽  
Author(s):  
Rushikesh Sable ◽  
Nithya Jambunathan ◽  
Sitanshu Singh ◽  
Sandeep Pallerla ◽  
Konstantin G Kousoulas ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Proximity Ligation Assay ◽  
Protein Protein Interactions ◽  
Proximity Ligation

scholarly journals Glyceraldehyde-3-Phosphate Dehydrogenase and Seven in Absentia Homolog -1 Interactions in Mammalian Cells Visualized Using Proximity Ligation Assay

2016 ◽  
Author(s):  
Harkewal Singh ◽  
Christopher Melm
Keyword(s):  
Cell Lines ◽  
Protein Interactions ◽  
Genetic Modification ◽  
Rolling Circle Amplification ◽  
Target Protein ◽  
Proximity Ligation Assay ◽  
Protein Protein Interactions ◽  
Proximity Ligation ◽  
Mammalian Cell Lines ◽  
Seven In Absentia

AbstractProteins seldom function in isolation and thus protein-protein interactions are critical in understanding the molecular basis of diseases and health (1, 2). There are several well established techniques that are used to investigate protein-protein interactions(3). Most of the methods require some form of genetic modification of the target protein and thus always adds extra steps. However, Proximity Ligation Assay(4-6) (PLA) aka Duolink® is one such method that requires no genetic modification of the target protein and probes protein-protein interactions in fixed live cells and tissues. Briefly, PLA requires the use of primary antibodies specific to the proteins of interest. Once the sample (fixed cells or tissues) is incubated with species specific primary antibodies, secondary antibodies that are conjugated with oligonucleotides (also known as PLUS and MINUS probes respectively) and connecter oligonucleotides are added. This complex is ligated if the two PLUS and MINUS probes are within 40nm of each other. The resulting nucleic acid is amplified using rolling circle amplification and then probed with appropriate fluorescent probes. If the two proteins are interacting, one could visualize the interaction as a single red foci (for example Far Red Detection) using fluorescent microscopy. Here, we used PLA to probe protein-protein interactions between Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and seven in absentia homolog -1 (Siah-1) – an E3 ubiquitin ligase. We first use PLA to show that GAPDH and Siah-1 proteins exist endogenously in the cytosol of multiple mammalian cell lines. Our data suggest the use of DU145 and T98G cell lines to show translocation of the GAPDH-Siah- 1 complex. Next, we used common nitrosylation agents(7, 8) (S-nitrosoglutathione-GSNO and S-Nitroso-N-acetyl-DL-pencillamine–SNAP) in different concentrations and observed that GAPDH and Siah-1 interact presumably due to the nitrosylation of the former, which is consistent with previous studies(9, 10). Interestingly, no interactions were observed between the two proteins in the absence of GSNO or SNAP indicating that nitrosylation might be critical for GAPDH-Siah1 interactions. Our results suggest that GAPDH-Siah-1 interactions originate in the cytosol and migrate to the nucleus under the conditions tested. We quantify the PLA signal using Duolink® Image Tool and observe a clear enhancement of GAPDH-Siah-1 PLA signal upon treating the cells with GSNO or SNAP. Next, we used R-(-)-Deprenyl (deprenyl), a known inhibitor of GAPDH4, and show that it abrogates GAPDH-Siah-1 PLA complex under the conditions tested. Finally, our data suggest that PLA can detect and quantify the GAPDH-Siah1 complex; a well-known protein-protein interaction implicated in neurodegeneration(9-11) and thus could be a method of choice for similar applications.

Using an in Situ Proximity Ligation Assay to Systematically Profile Endogenous Protein–Protein Interactions in a Pathway Network

2014 ◽  
Vol 13 (12) ◽  
pp. 5339-5346 ◽  
Author(s):  
Tzu-Chi Chen ◽  
Kuan-Ting Lin ◽  
Chun-Houh Chen ◽  
Sheng-An Lee ◽  
Pei-Ying Lee ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Proximity Ligation Assay ◽  
Protein Protein Interactions ◽  
Pathway Network ◽  
Proximity Ligation ◽  
Endogenous Protein

scholarly journals The Role of Posttranslational Modifications in DNA Repair

2020 ◽  
Vol 2020 ◽  
pp. 1-13
Author(s):  
Miaomiao Bai ◽  
Dongdong Ti ◽  
Qian Mei ◽  
Jiejie Liu ◽  
Xin Yan ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Protein Interactions ◽  
Posttranslational Modifications ◽  
Genome Stability ◽  
Protein Localization ◽  
Complex Structure ◽  
Protein Protein Interactions ◽  
Regulate Protein

The human body is a complex structure of cells, which are exposed to many types of stress. Cells must utilize various mechanisms to protect their DNA from damage caused by metabolic and external sources to maintain genomic integrity and homeostasis and to prevent the development of cancer. DNA damage inevitably occurs regardless of physiological or abnormal conditions. In response to DNA damage, signaling pathways are activated to repair the damaged DNA or to induce cell apoptosis. During the process, posttranslational modifications (PTMs) can be used to modulate enzymatic activities and regulate protein stability, protein localization, and protein-protein interactions. Thus, PTMs in DNA repair should be studied. In this review, we will focus on the current understanding of the phosphorylation, poly(ADP-ribosyl)ation, ubiquitination, SUMOylation, acetylation, and methylation of six typical PTMs and summarize PTMs of the key proteins in DNA repair, providing important insight into the role of PTMs in the maintenance of genome stability and contributing to reveal new and selective therapeutic approaches to target cancers.

Sign in / Sign up

Export Citation Format

Share Document