scholarly journals GqPCR-stimulated dephosphorylation of AKT is induced by an IGBP1-mediated PP2A switch

2022 ◽  
Vol 20 (1) ◽  
Author(s):  
Guy Nadel ◽  
Zhong Yao ◽  
Ehud Wainstein ◽  
Izel Cohen ◽  
Ido Ben-Ami ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Intracellular Signaling ◽  
Regulatory Subunit ◽  
Akt Pathway ◽  
Resting Cells ◽  
Protein Protein Interactions ◽  
Cellular Processes ◽  
Proximity Ligation ◽  
Main Regulator ◽  
G Protein Coupled

Abstract Background G protein-coupled receptors (GPCRs) usually regulate cellular processes via activation of intracellular signaling pathways. However, we have previously shown that in several cell lines, GqPCRs induce immediate inactivation of the AKT pathway, which leads to JNK-dependent apoptosis. This apoptosis-inducing AKT inactivation is essential for physiological functions of several GqPCRs, including those for PGF2α and GnRH. Methods Here we used kinase activity assays of PI3K and followed phosphorylation state of proteins using specific antibodies. In addition, we used coimmunoprecipitation and proximity ligation assays to follow protein–protein interactions. Apoptosis was detected by TUNEL assay and PARP1 cleavage. Results We identified the mechanism that allows the unique stimulated inactivation of AKT and show that the main regulator of this process is the phosphatase PP2A, operating with the non-canonical regulatory subunit IGBP1. In resting cells, an IGBP1-PP2Ac dimer binds to PI3K, dephosphorylates the inhibitory pSer608-p85 of PI3K and thus maintains its high basal activity. Upon GqPCR activation, the PP2Ac-IGBP1 dimer detaches from PI3K and thus allows the inhibitory dephosphorylation. At this stage, the free PP2Ac together with IGBP1 and PP2Aa binds to AKT, causing its dephosphorylation and inactivation. Conclusion Our results show a stimulated shift of PP2Ac from PI3K to AKT termed “PP2A switch” that represses the PI3K/AKT pathway, providing a unique mechanism of GPCR-stimulated dephosphorylation.

scholarly journals Mechanism of the G-protein mimetic nanobody binding to a muscarinic G-protein-coupled receptor

2018 ◽  
Vol 115 (12) ◽  
pp. 3036-3041 ◽  
Author(s):  
Yinglong Miao ◽  
J. Andrew McCammon
Keyword(s):  
Protein Binding ◽  
G Protein ◽  
Protein Interactions ◽  
Intracellular Signaling ◽  
Md Simulations ◽  
Binding Pocket ◽  
Signaling Proteins ◽  
Protein Protein Interactions ◽  
Intracellular Loops ◽  
G Protein Coupled

Protein–protein binding is key in cellular signaling processes. Molecular dynamics (MD) simulations of protein–protein binding, however, are challenging due to limited timescales. In particular, binding of the medically important G-protein-coupled receptors (GPCRs) with intracellular signaling proteins has not been simulated with MD to date. Here, we report a successful simulation of the binding of a G-protein mimetic nanobody to the M2 muscarinic GPCR using the robust Gaussian accelerated MD (GaMD) method. Through long-timescale GaMD simulations over 4,500 ns, the nanobody was observed to bind the receptor intracellular G-protein-coupling site, with a minimum rmsd of 2.48 Å in the nanobody core domain compared with the X-ray structure. Binding of the nanobody allosterically closed the orthosteric ligand-binding pocket, being consistent with the recent experimental finding. In the absence of nanobody binding, the receptor orthosteric pocket sampled open and fully open conformations. The GaMD simulations revealed two low-energy intermediate states during nanobody binding to the M2 receptor. The flexible receptor intracellular loops contribute remarkable electrostatic, polar, and hydrophobic residue interactions in recognition and binding of the nanobody. These simulations provided important insights into the mechanism of GPCR–nanobody binding and demonstrated the applicability of GaMD in modeling dynamic protein–protein interactions.

Structure, Function, Involvement in Diseases and Targeting of 14-3-3 Proteins: An Update

2018 ◽  
Vol 25 (1) ◽  
pp. 5-21 ◽  
Author(s):  
Ylenia Cau ◽  
Daniela Valensin ◽  
Mattia Mori ◽  
Sara Draghi ◽  
Maurizio Botta
Keyword(s):  
Protein Interactions ◽  
Molecular Mechanisms ◽  
Physiological Role ◽  
Specific Protein ◽  
Protein Protein Interactions ◽  
Cellular Processes ◽  
Protein Partners ◽  
High Sequence Homology ◽  
Small Molecule Modulators

14-3-3 is a class of proteins able to interact with a multitude of targets by establishing protein-protein interactions (PPIs). They are usually found in all eukaryotes with a conserved secondary structure and high sequence homology among species. 14-3-3 proteins are involved in many physiological and pathological cellular processes either by triggering or interfering with the activity of specific protein partners. In the last years, the scientific community has collected many evidences on the role played by seven human 14-3-3 isoforms in cancer or neurodegenerative diseases. Indeed, these proteins regulate the molecular mechanisms associated to these diseases by interacting with (i) oncogenic and (ii) pro-apoptotic proteins and (iii) with proteins involved in Parkinson and Alzheimer diseases. The discovery of small molecule modulators of 14-3-3 PPIs could facilitate complete understanding of the physiological role of these proteins, and might offer valuable therapeutic approaches for these critical pathological states.

scholarly journals Multiple Interactions within the AKAP220 Signaling Complex Contribute to Protein Phosphatase 1 Regulation

2001 ◽  
Vol 276 (15) ◽  
pp. 12128-12134 ◽  
Author(s):  
Robynn V. Schillace ◽  
James W. Voltz ◽  
Alistair T. R. Sim ◽  
Shirish Shenolikar ◽  
John D. Scott
Keyword(s):  
Intermolecular Interactions ◽  
Protein Interactions ◽  
Protein Phosphatase ◽  
Competitive Inhibitor ◽  
Regulatory Subunit ◽  
Protein Protein Interactions ◽  
Signaling Complex ◽  
Activity Measurements ◽  
Anchoring Protein ◽  
Pka Regulatory Subunit

The phosphorylation status of cellular proteins is controlled by the opposing actions of protein kinases and phosphatases. Compartmentalization of these enzymes is critical for spatial and temporal control of these phosphorylation/dephosphorylation events. We previously reported that a 220-kDa A-kinase anchoring protein (AKAP220) coordinates the location of the cAMP-dependent protein kinase (PKA) and the type 1 protein phosphatase catalytic subunit (PP1c) (Schillace, R. V., and Scott, J. D. (1999)Curr. Biol.9, 321–324). We now demonstrate that an AKAP220 fragment is a competitive inhibitor of PP1c activity (Ki= 2.9 ± 0.7 μm). Mapping studies and activity measurements indicate that several protein-protein interactions act synergistically to inhibit PP1. A consensus targeting motif, between residues 1195 and 1198 (Lys-Val-Gln-Phe), binds but does not affect enzyme activity, whereas determinants between residues 1711 and 1901 inhibit the phosphatase. Analysis of truncated PP1c and chimeric PP1/2A catalytic subunits suggests that AKAP220 inhibits the phosphatase in a manner distinct from all known PP1 inhibitors and toxins. Intermolecular interactions within the AKAP220 signaling complex further contribute to PP1 inhibition as addition of the PKA regulatory subunit (RII) enhances phosphatase inhibition. These experiments indicate that regulation of PP1 activity by AKAP220 involves a complex network of intra- and intermolecular interactions.

scholarly journals Heterodimerization Between the Lutropin and Follitropin Receptors is Associated With an Attenuation of Hormone-Dependent Signaling

Endocrinology ◽  
2013 ◽  
Vol 154 (10) ◽  
pp. 3925-3930 ◽  
Author(s):  
Xiuyan Feng ◽  
Meilin Zhang ◽  
Rongbin Guan ◽  
Deborah L. Segaloff
Keyword(s):  
Protein Interactions ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
G Protein Coupled Receptors ◽  
Fsh Receptor ◽  
Bioluminescence Resonance Energy Transfer ◽  
Protein Protein Interactions ◽  
Lh Receptor ◽  
G Protein Coupled ◽  
Stimulation Of

The LH receptor (LHR) and FSH receptor (FSHR) are each G protein-coupled receptors that play critical roles in reproductive endocrinology. Each of these receptors has previously been shown to self-associate into homodimers and oligomers shortly after their biosynthesis. As shown herein using bioluminescence resonance energy transfer to detect protein-protein interactions, our data show that the LHR and FSHR, when coexpressed in the same cells, specifically heterodimerize with each other. Further experiments confirm that at least a portion of the cellular LHR/FSHR heterodimers are present on the cell surface and are functional. We then sought to ascertain what effects, if any, heterodimerization between the LHR and FSHR might have on signaling. It was observed that when the LHR was expressed under conditions promoting the heterodimerization with FSHR, LH or human chorionic gonadotropin (hCG) stimulation of Gs was attenuated. Conversely, when the FSHR was expressed under conditions promoting heterodimerization with the LHR, FSH-stimulated Gs activation was attenuated. These results demonstrate that the coexpression of the LHR and FSHR enables heterodimerizaton between the 2 gonadotropin receptors and results in an attenuation of signaling through each receptor.

Visual detection of binary, ternary, and quaternary protein interactions in fission yeast by Pil1 co-tethering assay

2021 ◽  
Author(s):  
Zhong-Qiu Yu ◽  
Xiao-Man Liu ◽  
Dan Zhao ◽  
Dan-Dan Xu ◽  
Li-Lin Du
Keyword(s):  
Fission Yeast ◽  
Protein Interactions ◽  
Visual Detection ◽  
Protein Protein Interactions ◽  
Phosphatidylinositol 3 Kinase ◽  
Basic Form ◽  
Bait Protein ◽  
Cellular Processes ◽  
Prey Protein ◽  
Versatile Tool

Protein-protein interactions are vital for executing nearly all cellular processes. To facilitate the detection of protein-protein interactions in living cells of the fission yeast Schizosaccharomyces pombe, here we present an efficient and convenient method termed the Pil1 co-tethering assay. In its basic form, we tether a bait protein to mCherry-tagged Pil1, which forms cortical filamentary structures, and examine whether a GFP-tagged prey protein colocalizes with the bait. We demonstrate that this assay is capable of detecting pairwise protein-protein interactions of cytosolic proteins and nuclear proteins. Furthermore, we show that this assay can be used for detecting not only binary protein-protein interactions, but also ternary and quaternary protein-protein interactions. Using this assay, we systematically characterized the protein-protein interactions in the Atg1 complex and in the phosphatidylinositol 3-kinase (PtdIns3K) complexes and found that Atg38 is incorporated into the PtdIns3K complex I via an Atg38-Vps34 interaction. Our data show that this assay is a useful and versatile tool and should be added to the routine toolbox of fission yeast researchers.

scholarly journals Protein–protein interactions in bacteria: a promising and challenging avenue towards the discovery of new antibiotics

2018 ◽  
Vol 14 ◽  
pp. 2881-2896 ◽  
Author(s):  
Laura Carro
Keyword(s):  
Protein Interactions ◽  
Bacterial Infections ◽  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
Protein Protein Interactions ◽  
Lead Discovery ◽  
Antibiotic Development ◽  
Cellular Processes ◽  
Ppi Networks ◽  
New Antibiotics

Antibiotics are potent pharmacological weapons against bacterial infections; however, the growing antibiotic resistance of microorganisms is compromising the efficacy of the currently available pharmacotherapies. Even though antimicrobial resistance is not a new problem, antibiotic development has failed to match the growth of resistant pathogens and hence, it is highly critical to discover new anti-infective drugs with novel mechanisms of action which will help reducing the burden of multidrug-resistant microorganisms. Protein–protein interactions (PPIs) are involved in a myriad of vital cellular processes and have become an attractive target to treat diseases. Therefore, targeting PPI networks in bacteria may offer a new and unconventional point of intervention to develop novel anti-infective drugs which can combat the ever-increasing rate of multidrug-resistant bacteria. This review describes the progress achieved towards the discovery of molecules that disrupt PPI systems in bacteria for which inhibitors have been identified and whose targets could represent an alternative lead discovery strategy to obtain new anti-infective molecules.

scholarly journals Assay Systems for Profiling Deubiquitinating Activity

2020 ◽  
Vol 21 (16) ◽  
pp. 5638
Author(s):  
Jinhong Cho ◽  
Jinyoung Park ◽  
Eunice EunKyeong Kim ◽  
Eun Joo Song
Keyword(s):  
Protein Degradation ◽  
Protein Interactions ◽  
Live Cells ◽  
Deubiquitinating Enzyme ◽  
Protein Protein Interactions ◽  
Deubiquitinating Enzymes ◽  
Cell Lysates ◽  
Cellular Processes

Deubiquitinating enzymes regulate various cellular processes, particularly protein degradation, localization, and protein–protein interactions. The dysregulation of deubiquitinating enzyme (DUB) activity has been linked to several diseases; however, the function of many DUBs has not been identified. Therefore, the development of methods to assess DUB activity is important to identify novel DUBs, characterize DUB selectivity, and profile dynamic DUB substrates. Here, we review various methods of evaluating DUB activity using cell lysates or purified DUBs, as well as the types of probes used in these methods. In addition, we introduce some techniques that can deliver DUB probes into the cells and cell-permeable activity-based probes to directly visualize and quantify DUB activity in live cells. This review could contribute to the development of DUB inhibitors by providing important information on the characteristics and applications of various probes used to evaluate and detect DUB activity in vitro and in vivo.

Deep learning based prediction of reversible HAT/HDAC-specific lysine acetylation

2019 ◽  
Vol 21 (5) ◽  
pp. 1798-1805 ◽  
Author(s):  
Kai Yu ◽  
Qingfeng Zhang ◽  
Zekun Liu ◽  
Yimeng Du ◽  
Xinjiao Gao ◽  
...  
Keyword(s):  
Deep Learning ◽  
Protein Interactions ◽  
Web Server ◽  
Data Sets ◽  
Lysine Acetylation ◽  
Protein Acetylation ◽  
Protein Protein Interactions ◽  
Cellular Processes ◽  
Protein Lysine Acetylation ◽  
Specific Regulation

Abstract Protein lysine acetylation regulation is an important molecular mechanism for regulating cellular processes and plays critical physiological and pathological roles in cancers and diseases. Although massive acetylation sites have been identified through experimental identification and high-throughput proteomics techniques, their enzyme-specific regulation remains largely unknown. Here, we developed the deep learning-based protein lysine acetylation modification prediction (Deep-PLA) software for histone acetyltransferase (HAT)/histone deacetylase (HDAC)-specific acetylation prediction based on deep learning. Experimentally identified substrates and sites of several HATs and HDACs were curated from the literature to generate enzyme-specific data sets. We integrated various protein sequence features with deep neural network and optimized the hyperparameters with particle swarm optimization, which achieved satisfactory performance. Through comparisons based on cross-validations and testing data sets, the model outperformed previous studies. Meanwhile, we found that protein–protein interactions could enrich enzyme-specific acetylation regulatory relations and visualized this information in the Deep-PLA web server. Furthermore, a cross-cancer analysis of acetylation-associated mutations revealed that acetylation regulation was intensively disrupted by mutations in cancers and heavily implicated in the regulation of cancer signaling. These prediction and analysis results might provide helpful information to reveal the regulatory mechanism of protein acetylation in various biological processes to promote the research on prognosis and treatment of cancers. Therefore, the Deep-PLA predictor and protein acetylation interaction networks could provide helpful information for studying the regulation of protein acetylation. The web server of Deep-PLA could be accessed at http://deeppla.cancerbio.info.

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