Luminescence- and fluorescence-based complementation assays to screen for GPCR oligomerization : current state of the art

G protein-coupled receptors (GPCRs) have the propensity to form homo- and heterodimers. Dysfunction of these dimers has been associated with multiple diseases, e.g., pre-eclampsia, schizophrenia, and depression, among others. Over the past two decades, considerable efforts have been made towards the development of screening assays for studying these GPCR dimer complexes in living cells. As a first step, a robust in vitro assay in an overexpression system is essential to identify and characterize specific GPCR-GPCR interactions, followed by methodologies to demonstrate association at endogenous levels and eventually in vivo. This review focuses on protein complementation assays (PCAs) which have been utilized to study GPCR oligomerization. These approaches are typically fluorescence- and luminescence-based, making identification and localization of protein-protein interactions feasible. The GPCRs of interest are fused to complementary fluorescent or luminescent fragments that, upon GPCR di- or oligomerization, may reconstitute to a functional reporter, of which the activity can be measured. Various protein complementation assays have the disadvantage that the interaction between the reconstituted split fragments is irreversible, which can lead to false positive read-outs. Reversible systems offer several advantages, as they do not only allow to follow the kinetics of GPCR-GPCR interactions, but also allow evaluation of receptor complex modulation by ligands (either agonists or antagonists). Protein complementation assays may be used for high throughput screenings as well, which is highly relevant given the growing interest and effort to identify small molecule drugs that could potentially target disease-relevant dimers. In addition to providing an overview on how PCAs have allowed to gain better insights into GPCR-GPCR interactions, this review also aims at providing practical guidance on how to perform PCA-based assays

Personalized medicine : the impact on chemistry

An effective strategy for personalized medicine requires a major conceptual change in the development and application of therapeutics. In this article, we argue that further advances in this field should be made with reference to another conceptual shift, that of network pharmacology. We examine the intersection of personalized medicine and network pharmacology to identify strategies for the development of personalized therapies that are fully informed by network pharmacology concepts. This provides a framework for discussion of the impact personalized medicine will have on chemistry in terms of drug discovery, formulation and delivery, the adaptations and changes in ideology required and the contribution chemistry is already making. New ways of conceptualizing chemistry’s relationship with medicine will lead to new approaches to drug discovery and hold promise of delivering safer and more effective therapies

Combining induced protease fragment assembly and microarray analysis to monitor signaling in living cells.

Die Fähigkeit Signalkaskaden zu vermessen ist für das Verständnis komplexer biologischer Prozesse essentiell. Bis jetzt versorgt uns die DNA Microarray Technologie mit umfassenden Daten, deren Auflösung jedoch auf der Ebene der Genexpression endet. Diese Informationen reichen nicht aus um die vorgeschalteten regulatorischen Mechanismen der Genexpression zu verstehen. Die meisten proteomischen Technologien hängen von in vitro synthetisierten Peptiden ab oder benötigen weitere biochemische Manipulationen. Für die Charakterisierung und Beobachtung einzelner Bestandteile von Signalkaskaden in lebenden Zellen sind Hochdurchsatz-Verfahren notwendig. In der vorliegenden Arbeit wird ein experimentelles Verfahren namens EXTassay beschrieben, dass eine quantitative und parallele Messung multipler Signal-Ereignisse ermöglicht, die der mRNA Expression vorgelagert sind. EXTassays vereinen verschiedene zelluläre Assays, die an die Reporter Gen Expression gekoppelt sind. Um Multiplexing zu erreichen wurde eine komplexe und optimierte Bibliotek kurzer expressed oligonucleotide tags (EXTs) generiert. Jedes einzelne EXT ersetzt hierbei ein klassisches Reportergen und dient als eindeutiger Identifikator für einen definierten zellulären Assay. Es können verschiedene EXTs, die entweder in einer Zelle oder in einer Zellpopulation exprimiert sein können, über Microarray Hybridisierung analysiert werden. In dieser Arbeit wurden Protokolle für das verlässliche Auslesen von Microarrays für EXTs optimiert. Weiterhin wurden EXT-basierte Assays verwendet, um die durch Neuregulin-1 induzierte Dimerisierung und Aktivierung von Rezeptortyrosinkinasen der ErbB Familie zu untersuchen. Für die quantitative Messung von Rezeptordimeriserung und phosphorylationsabhängige Kopplung an Interaktionspartner wurden Protein-Komplementations-Assays der TEV Protease, split-TEV Assays, verwendet. Hierzu wurde jeder Assay an eindeutige EXT-Reporter gekoppelt. Zusätzlich wurde die Aktivierung von 30 verschiedenen EXT-gekoppelten cis-regulatorischen Elementen erfaßt, um so einen Einblick in die nachfolgende Aspekte der Signalverarbeitung zu erhalten. Alle Assays wurden mit eindeutigen EXTs durchgeführt und mittels Microarray analysiert. Die simultane Analyse dreier verschiedener und regulierter Rezeptor Komplexe (ErbB2/2, 2/3, 2/4) zeigte, dass EXT-basierte Assays geeignet sind rezeptor-spezifische Signalereignisse zu unterscheiden. EXTassays sind daher geeignet quantitative Profile aktivierter Signalkaskaden in Zellen erstellen zu können.The ability to monitor multiple signaling events simultaneously in living cells is essential to better understand complex biological processes. So far, DNA-microarray technologies provide global scale data mainly restricted to the level of gene expression. This information is not sufficient to understand the upstream regulatory mechanisms that lead to gene expression changes. Most proteomic technologies also provide large scale measurement but usually depend on in vitro synthesized peptides or require biochemical manipulations. High throughput technologies are required for functional characterization and monitoring of signaling components in living cells. Here, an experimental approach is presented termed EXTassay that enables quantitative and parallel measurements of various signaling events upstream of mRNA expression. EXTassay incorporates various cellular assays that are coupled to reporter gene expression. To achieve multiplexing, we have generated a complex and optimized library of short expressed oligonucleotide tags (EXTs). Each unique EXT can replace a classical reporter gene and serves as a unique identifier for tracking and quantification of a defined cellular assay. Multiple EXT-reporters expressed in the same cell or cell population can be isolated and analyzed by custom microarray hybridization. We have established protocols and optimized the microarray readout for reliable EXT quantification. We applied the EXTassay to analyze the neuregulin 1 induced ErbB receptor tyrosine kinase signaling in PC-12 cells. We used transcriptionally coupled split TEV protein complementation assays to monitor ErbB receptor dimerization and phosphorylation dependent interaction with downstream signaling proteins. In addition, we employed 30 different cis-regulatory elements to assess the downstream signaling. All assays were coupled to unique EXTs and analyzed by microarrays. By analyzing three different receptor complexes (ErbB 2/2, 2/3 and 2/4), we were able to measure receptor specific differential signaling effects and demonstrate that EXTassays can be applied for the quantitative profiling of activated signaling pathways

The mgrB gene as a key target for acquired resistance to colistin in Klebsiella pneumoniae

Objectives Alterations in the PhoPQ two-component regulatory system may be associated with colistin resistance in Klebsiella pneumoniae. MgrB is a small transmembrane protein produced upon activation of the PhoPQ signalling system, and acts as a negative regulator on this system. We investigated the role of the MgrB protein as a source of colistin resistance in a series of K. pneumoniae. Methods Colistin-resistant K. pneumoniae isolates were recovered from hospitalized patients worldwide (France, Turkey, Colombia and South Africa). The mgrB gene was amplified and sequenced. A wild-type mgrB gene was cloned and the corresponding recombinant plasmid was used for complementation assays. Clonal diversity was evaluated by MLST and Diversilab analysis. Results Of 47 colistin-resistant isolates, 12 were identified as having a mutated mgrB gene. Five clonally unrelated isolates had an mgrB gene truncated by an IS5-like IS, while one clone also harboured an insertional inactivation at the exact same position of the mgrB gene, but with ISKpn13. Another clone harboured an insertional inactivation due to ISKpn14 at another location of the mgrB gene. Two clonally related isolates harboured an IS (IS10R) in the promoter region of mgrB. Finally, three clonally unrelated isolates harboured substitutions leading to anticipated stop codon in the MgrB protein. Complementation assays with a wild-type MgrB protein restored full susceptibility to colistin for all colistin-resistant isolates identified with qualitative or quantitative MgrB modifications. Conclusion The inactivation or down-regulation of the mgrB gene was shown to be a source of colistin resistance in K. pneumoniae. Interestingly, identical genetic events were identified among clonally unrelated isolate

The mgrB gene as a key target for acquired resistance to colistin in Klebsiella pneumoniae

Objectives Alterations in the PhoPQ two-component regulatory system may be associated with colistin resistance in Klebsiella pneumoniae. MgrB is a small transmembrane protein produced upon activation of the PhoPQ signalling system, and acts as a negative regulator on this system. We investigated the role of the MgrB protein as a source of colistin resistance in a series of K. pneumoniae.Methods Colistin-resistant K. pneumoniae isolates were recovered from hospitalized patients worldwide (France, Turkey, Colombia and South Africa). The mgrB gene was amplified and sequenced. A wild-type mgrB gene was cloned and the corresponding recombinant plasmid was used for complementation assays. Clonal diversity was evaluated by MLST and Diversilab analysis.Results Of 47 colistin-resistant isolates, 12 were identified as having a mutated mgrB gene. Five clonally unrelated isolates had an mgrB gene truncated by an IS5-like IS, while one clone also harboured an insertional inactivation at the exact same position of the mgrB gene, but with ISKpn13. Another clone harboured an insertional inactivation due to ISKpn14 at another location of the mgrB gene. Two clonally related isolates harboured an IS (IS10R) in the promoter region of mgrB. Finally, three clonally unrelated isolates harboured substitutions leading to anticipated stop codon in the MgrB protein. Complementation assays with a wild-type MgrB protein restored full susceptibility to colistin for all colistin-resistant isolates identified with qualitative or quantitative MgrB modifications.Conclusion The inactivation or down-regulation of the mgrB gene was shown to be a source of colistin resistance in K. pneumoniae. Interestingly, identical genetic events were identified among clonally unrelated isolates

InSiDDe: A server for designing artificial disordered proteins

InSiDDe (In Silico Disorder Design) is a program for the in silico design of intrinsically disordered proteins of desired length and disorder probability. The latter is assessed using IUPred and spans values ranging from 0.55 to 0.95 with 0.05 increments. One to ten artificial sequences per query, each made of 50 to 200 residues, can be generated by InSiDDe. We describe the rationale used to set up InSiDDe and show that an artificial sequence of 100 residues with an IUPred score of 0.6 designed by InSiDDe could be recombinantly expressed in E. coli at high levels without degradation when fused to a natural molecular recognition element (MoRE). In addition, the artificial fusion protein exhibited the expected behavior in terms of binding modulation of the specific partner recognized by the MoRE. To the best of our knowledge, InSiDDe is the first publicly available software for the design of intrinsically disordered protein (IDP) sequences. InSiDDE is publicly available online

Virus-host interactome: Putting the accent on how it changes

[EN] Viral infections are extremely complex processes that could only be well understood by precisely characterizing the interaction networks between the virus and the host components. In recent years, much effort has gone in this directionwith the aimof unveiling themolecular basis of viral pathology. These networks are mostly formed by viral and host proteins, and are expected to be dynamic bothwith time and space (i.e., with the progression of infection, as well as with the virus and host genotypes; what we call plastodynamic). This largely overlooked spatio-temporal evolution urgently calls for a change both in the conceptual paradigms and experimental techniques used so far to characterize virus-host interactions. More generally, molecular plasticity and temporal dynamics are unavoidable components of themechanisms that underlie any complex disease; components whose understandingwill eventually enhance our ability to modulate those networkswith the aimof improving disease treatments.This work is supported by the grants BFU2015-66894-P (to G.R.), BI02014-54269-R (to J-A.D.) and BFU2015-65037-P (to S.F.E.) from the Ministerio de Economia, Industria y Competitividad, and by the grant PROMETEOII/2014/021 from the Generalitat Valenciana (to S.F.E. and J-A.D.).Rodrigo Tarrega, G.; Daros Arnau, JA.; Elena Fito, SF. (2017). Virus-host interactome: Putting the accent on how it changes. Journal of Proteomics. 156:1-4. https://doi.org/10.1016/j.jprot.2016.12.007S1415

Split-DHFR as a protein complementation assay for localisation and interactions of yeast mitochondrial proteins

Protein complementation assays (PCAs) utilising two fragments of a reporter protein – fused to two potentially interacting proteins of interest – are a common method of analysing protein-protein interactions (PPIs). This approach, using split dihydrofolate reductase (DHFR) as a reporter protein, has been previously carried out for cytosolic Saccharomyces cerevisiae proteins. The focus of this study was to establish a split-DHFR assay specifically for use in analysing yeast mitochondrial PPIs in the intermembrane space (IMS), which has not been done before. A strategy to overcome the problem endogenous DHFR activity had to be developed using a modified strain of S. cerevisiae for the specific application here. Further, plasmids containing two positive control proteins, Tim9 and Tim10 (two well-known interacting proteins of the IMS) were cloned for transformation into yeast strain BY4741. Several other plasmids bearing various control proteins were designed and some of them cloned, although we required more time to have the full set of tools to establish the assay