scholarly journals Improving the Stability of Protein–Protein Interaction Assay FlimPIA Using a Thermostabilized Firefly Luciferase

2021 ◽  
Vol 9 ◽  
Author(s):  
Yuki Ohmuro-Matsuyama ◽  
Keiko Gomi ◽  
Takuya Shimoda ◽  
Hideki Yamaji ◽  
Hiroshi Ueda
Keyword(s):  
Protein Interaction ◽  
Firefly Luciferase ◽  
Second Step ◽  
Photinus Pyralis ◽  
Luminescence Signal ◽  
Key Technology ◽  
Protein Protein Interaction ◽  
Unique Protein ◽  
The Stability ◽  
Low Activity

The protein–protein interaction assay is a key technology in various fields, being applicable in drug screening as well as in diagnosis and inspection, wherein the stability of assays is important. In a previous study, we developed a unique protein–protein interaction assay “FlimPIA” based on the functional complementation of mutant firefly luciferases (Fluc). The catalytic step of Fluc was divided into two half steps: D-luciferin was adenylated in the first step, while adenylated luciferin was oxidized in the second step. We constructed two mutants of Fluc from Photinus pyralis (Ppy); one mutant named Donor is defective in the second half reaction, while the other mutant named Acceptor exhibited low activity in the first half reaction. To date, Ppy has been used in the system; however, its thermostability is low. In this study, to improve the stability of the system, we applied Fluc from thermostabilized Luciola lateralis to FlimPIA. We screened suitable mutants as probes for FlimPIA and obtained Acceptor and Donor candidates. We detected the interaction of FKBP12-FRB with FlimPIA using these candidates. Furthermore, after the incubation of the probes at 37°C for 1 h, the luminescence signal of the new system was 2.4-fold higher than that of the previous system, showing significant improvement in the stability of the assay.

scholarly journals Delineation of a unique protein-protein interaction site on the surface of the estrogen receptor

2005 ◽  
Vol 102 (10) ◽  
pp. 3593-3598 ◽  
Author(s):  
E. H. Kong ◽  
N. Heldring ◽  
J.-A. Gustafsson ◽  
E. Treuter ◽  
R. E. Hubbard ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Protein Interaction ◽  
Interaction Site ◽  
Protein Protein Interaction ◽  
Unique Protein

scholarly journals A conserved acetylation switch enables pharmacological control of tubby-like protein stability

2020 ◽  
pp. jbc.RA120.015839 ◽  
Author(s):  
Evan M Kerek ◽  
Kevin H Yoon ◽  
Shu Y Luo ◽  
Jerry Chen ◽  
Robert Valencia ◽  
...  
Keyword(s):  
Protein Interaction ◽  
Developmental Disorders ◽  
Intracellular Transport ◽  
Interaction Network ◽  
Membrane Receptors ◽  
Aberrant Expression ◽  
Protein Protein Interaction ◽  
Protein Levels ◽  
Lysine Residues ◽  
The Stability

Tubby-like proteins (TULPs) are characterized by a conserved C-terminal domain that binds phosphoinositides. Collectively, mammalian TULP1-4 proteins play essential roles in intracellular transport, cell differentiation, signaling, and motility. Yet, little is known about how the function of these proteins is regulated in cells. Here, we present the protein-protein interaction network of TULP3, a protein that is responsible for the trafficking of G-protein coupled receptors to cilia, and whose aberrant expression is associated with severe developmental disorders and polycystic kidney disease. We identify several protein interaction nodes linked to TULP3 that include enzymes involved in acetylation and ubiquitination. We show that acetylation of two key lysine residues on TULP3 by p300 increases TULP3 protein abundance, and that deacetylation of these sites by HDAC1 decreases protein levels. Furthermore, we show that one of these sites is ubiquitinated in the absence of acetylation, and that acetylation inversely correlates with ubiquitination of TULP3. This mechanism is evidently conserved across species and is active in zebrafish during development. Finally, we identify this same regulatory module in TULP1, TULP2, and TULP4, and demonstrate that the stability of these proteins is similarly modulated by an acetylation switch. This study unveils a signaling pathway that links nuclear enzymes to ciliary membrane receptors via TULP3, describes a dynamic mechanism for the regulation of all tubby-like proteins, and explores how to exploit it pharmacologically using drugs.

Ultra sensitive firefly luciferase‐based protein‐protein interaction assay (FlimPIA) attained by hinge region engineering and optimized reaction conditions

2015 ◽  
Vol 11 (1) ◽  
pp. 91-99 ◽  
Author(s):  
Makoto Kurihara ◽  
Yuki Ohmuro‐Matsuyama ◽  
Keiichi Ayabe ◽  
Takahiro Yamashita ◽  
Hideki Yamaji ◽  
...  
Keyword(s):  
Protein Interaction ◽  
Firefly Luciferase ◽  
Hinge Region ◽  
Reaction Conditions ◽  
Protein Protein Interaction

What factors determine the stability of a weak protein–protein interaction in a charged aqueous droplet?

2017 ◽  
Vol 19 (47) ◽  
pp. 31965-31981 ◽  
Author(s):  
Myong In Oh ◽  
Styliani Consta
Keyword(s):  
Mass Spectrometry ◽  
Electrospray Ionization ◽  
Protein Interaction ◽  
Bulk Solution ◽  
Ionization Mass ◽  
Critical Question ◽  
Gaseous State ◽  
Protein Protein Interaction ◽  
The Stability ◽  
Aqueous Droplet

Maintaining the interface of a weak transient protein complex transferred from bulk solution to the gaseous state via evaporating droplets is a critical question in the detection of the complex association (dissociation) constant by using electrospray ionization mass spectrometry (ESI-MS).

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