A Toolkit of Protein-Fragment Complementation Assays for Studying and Dissecting Large-Scale and Dynamic Protein–Protein Interactions in Living Cells

Michnick, Stephen W.; Ear, Po Hien; Landry, Christian R.; Malleshaiah, Mohan; Messier, Vincent

doi:10.1016/s0076-6879(10)70014-8

Cited by 48 publications

(32 citation statements)

References 44 publications

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“…We initially used the split Venus system (19), where one-half of the protein was expressed in the cytosol and the other half was targeted to the ER through in-frame fusion with the H2-K b signal sequence ( Fig. 1 A and B and Fig.…”

Section: Resultsmentioning

confidence: 99%

Deglycosylation-dependent fluorescent proteins provide unique tools for the study of ER-associated degradation

Grotzke

Lu²,

Cresswell

2013

Proc. Natl. Acad. Sci. U.S.A.

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Endoplasmic reticulum-associated degradation (ERAD) is a constitutive process that identifies misfolded proteins in the ER and shuttles them to the cytosol, where they can be degraded by the proteasome. The accumulation of misfolded proteins can be catastrophic at both the cellular and organismal level. Although the players involved and mechanistic details of ERAD are being characterized, much remains to be learned. Because of the complexity of the pathway, experimental studies generally require labor-intensive biochemical techniques. Here, we report the development of a system to analyze ERAD based on mutants of split or intact Venus fluorescent protein for which fluorescence depends on enzymatic deglycosylation. We have generated variants that only become fluorescent when they are first glycosylated in the ER and subsequently deglycosylated after retrotranslocation into the cytosol. The E3 ubiquitin ligase HMG-coA reductase degradation 1 homolog (Hrd1) and, consistent with the demonstrated glycosylation/deglycosylation requirement, the cytosolic deglycosylating enzyme peptide:N′glycanase are both required for fluorescence. Furthermore, although these deglycosylation-dependent fluorescent proteins are themselves ERAD substrates, they can also be fused to additional ERAD substrates to interrogate substrate-specific pathways. To validate the system we performed a genomewide siRNA screen that successfully identified known ERAD factors such as Hrd1; homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1 (HERP); sel-1 suppressor of lin-12-like (SEL1L); and p97. These tools should greatly facilitate the identification of ERAD components and investigation of the mechanisms involved in this critical pathway.

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Section: Resultsmentioning

confidence: 99%

Deglycosylation-dependent fluorescent proteins provide unique tools for the study of ER-associated degradation

Grotzke

Lu²,

Cresswell

2013

Proc. Natl. Acad. Sci. U.S.A.

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“…Through direct monomeric binding to the Nav channel C-terminal tail, FGF14 forms a complex with the channel that is required for proper gating, expression and trafficking of the channel to the AIS as well as the control of neuronal excitability (Ali et al, 2014, 2016; Alshammari et al, 2016a; Laezza et al, 2007, 2009; Lou et al, 2005; Wang et al, 2000, 2002; Xiao et al, 2007; Goetz et al, 2009). Using the bioluminescence-based luciferase complementation assay (LCA) (Michnick et al, 2010; Remy & Michnick, 2006; Shavkunov et al, 2012; Villalobos et al, 2007, 2008), a PPI assay that quantitatively measures the complementation of reconstituted FGF14 and Nav1.6 channel complexes, we previously identified glycogen synthase kinase 3 (GSK–3), a multifunctional kinase dysregulated in Alzheimer's, depression and schizophrenia (Emamian, 2012; Jope et al, 2007; Jope & Roh, 2006; Liu et al, 2013; Budni et al, 2012; Scala et al, 2015; Maqbool et al, 2016; Morris & Berk, 2016; Provensi et al, 2016; Avila et al, 2010), as a key modulator of the FGF14:Nav1.6 complex. Inhibition of GSK–3 reduces the assembly of the FGF14:Nav channel complex, modifies FGF14-dependent regulation of Na + currents, and induces dissociation and subcellular redistribution of the native FGF14:Nav channel complex (Shavkunov et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

PPARgamma agonists rescue increased phosphorylation of FGF14 at S226 in the Tg2576 mouse model of Alzheimer's disease

Hsu

Wildburger

Haidacher

et al. 2017

Experimental Neurology

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Background Cognitive impairment in humans with Alzheimer's disease (AD) and in animal models of Aβ-pathology can be ameliorated by treatments with the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARγ) agonists, such as rosiglitazone (RSG). Previously, we demonstrated that in the Tg2576 animal model of AD, RSG treatment rescued cognitive deficits and reduced aberrant activity of granule neurons in the dentate gyrus (DG), an area critical for memory formation. Methods We used a combination of mass spectrometry, confocal imaging, electrophysiology and split-luciferase assay and in vitro phosphorylation and Ingenuity Pathway Analysis. Results Using an unbiased, quantitative nano-LC-MS/MS screening, we searched for potential molecular targets of the RSG-dependent rescue of DG granule neurons. We found that S226 phosphorylation of fibroblast growth factor 14 (FGF14), an accessory protein of the voltage-gated Na+ (Nav) channels required for neuronal firing, was reduced in Tg2576 mice upon treatment with RSG. Using confocal microscopy, we confirmed that the Tg2576 condition decreased PanNav channels at the AIS of the DG, and that RSG treatment of Tg2576 mice reversed the reduction in PanNav channels. Analysis from previously published data sets identified correlative changes in action potential kinetics in RSG-treated T2576 compared to untreated and wildtype controls. In vitro phosphorylation and mass spectrometry confirmed that the multifunctional kinase GSK–3β, a downstream target of insulin signaling highly implicated in AD, phosphorylated FGF14 at S226. Assembly of the FGF14:Nav1.6 channel complex and functional regulation of Nav1.6-mediated currents by FGF14 was impaired by a phosphosilent S226A mutation. Bioinformatics pathway analysis of mass spectrometry and biochemistry data revealed a highly interconnected network encompassing PPARγ, FGF14, SCN8A (Nav 1.6), and the kinases GSK–3 β, casein kinase 2β, and ERK1/2. Conclusions These results identify FGF14 as a potential PPARγ-sensitive target controlling Aβ-induced dysfunctions of neuronal activity in the DG underlying memory loss in early AD.

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“…These approaches identify groups of proteins that are directly or indirectly associated, generally in a stable manner, and are extremely powerful to identify protein complexes. The third approach is based on protein-fragment complementation assays (PCAs) 11,21 . This approach provides an intermediate level of resolution between the two former approaches, as it allows detecting direct and proximal associations between proteins.…”

Section: Introductionmentioning

confidence: 99%

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Rochette¹,

Diss²,

Filteau³

et al. 2015

JoVE

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Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein's function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

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A Toolkit of Protein-Fragment Complementation Assays for Studying and Dissecting Large-Scale and Dynamic Protein–Protein Interactions in Living Cells

Cited by 48 publications

References 44 publications

Deglycosylation-dependent fluorescent proteins provide unique tools for the study of ER-associated degradation

Deglycosylation-dependent fluorescent proteins provide unique tools for the study of ER-associated degradation

PPARgamma agonists rescue increased phosphorylation of FGF14 at S226 in the Tg2576 mouse model of Alzheimer's disease

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

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