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Unraveling mitotic protein networks by 3D multiplexed epitope drug screening.

ABSTRACT: Three-dimensional protein localization intricately determines the functional coordination of cellular processes. The complex spatial context of protein landscape has been assessed by multiplexed immunofluorescent staining or mass spectrometry, applied to 2D cell culture with limited physiological relevance or tissue sections. Here, we present 3D SPECS, an automated technology for 3D Spatial characterization of Protein Expression Changes by microscopic Screening. This workflow comprises iterative antibody staining, high-content 3D imaging, and machine learning for detection of mitoses. This is followed by mapping of spatial protein localization into a spherical, cellular coordinate system, a basis for model-based prediction of spatially resolved affinities of proteins. As a proof-of-concept, we mapped twelve epitopes in 3D-cultured spheroids and investigated the network effects of twelve mitotic cancer drugs. Our approach reveals novel insights into spindle fragility and chromatin stress, and predicts unknown interactions between proteins in specific mitotic pathways. 3D SPECS's ability to map potential drug targets by multiplexed immunofluorescence in 3D cell culture combined with our automated high-content assay will inspire future functional protein expression and drug assays.

SUBMITTER: Maier LJ

PROVIDER: S-EPMC6088390 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Unraveling mitotic protein networks by 3D multiplexed epitope drug screening.

Maier Lorenz J LJ Kallenberger Stefan M SM Jechow Katharina K Waschow Marcel M Eils Roland R Conrad Christian C

Molecular systems biology 20180813 8

Three-dimensional protein localization intricately determines the functional coordination of cellular processes. The complex spatial context of protein landscape has been assessed by multiplexed immunofluorescent staining or mass spectrometry, applied to 2D cell culture with limited physiological relevance or tissue sections. Here, we present 3D SPECS, an automated technology for 3D Spatial characterization of Protein Expression Changes by microscopic Screening. This workflow comprises iterative ...[more]

PMID: 30104419

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Project description:BackgroundA variety of approaches to understanding protein structure and function require production of recombinant protein. Mammalian based expression systems have advantages over bacterial systems for certain classes of protein but can be slower and more laborious. Thus the availability of a simple system for production and rapid screening of constructs or conditions for mammalian expression would be of great benefit. To this end we have coupled an efficient recombinant protein production system based on transient transfection in HEK-293 EBNA1 (HEK-293E) suspension cells with a dot blot method allowing pre-screening of proteins expressed in cells in a high throughput manner.ResultsA nested PCR approach was used to clone 21 extracellular domains of mouse receptors as CD4 fusions within a mammalian GATEWAY expression vector system. Following transient transfection, HEK-293E cells grown in 2 ml cultures in 24-deep well blocks showed similar growth kinetics, viability and recombinant protein expression profiles, to those grown in 50 ml shake flask cultures as judged by western blotting. Following optimisation, fluorescent dot blot analysis of transfection supernatants was shown to be a rapid method for analysing protein expression yielding similar results as western blot analysis. Addition of urea enhanced the binding of glycoproteins to a nitrocellulose membrane. A good correlation was observed between the results of a plate based small scale transient transfection dot blot pre-screen and successful purification of proteins expressed at the 50 ml scale.ConclusionThe combination of small scale multi-well plate culture and dot blotting described here will allow the multiplex analysis of different mammalian expression experiments enabling a faster identification of high yield expression constructs or conditions prior to large scale protein production. The methods for parallel GATEWAY cloning and expression of multiple constructs in cell culture will also be useful for applications such as the generation of receptor protein microarrays.

Project description:BackgroundFor treating a complex disease such as cancer, some effective means are needed to control biological networks that underlies the disease. The one-target one-drug paradigm has been the dominating drug discovery approach in the past decades. Compared to single target-based drugs, combination drug targets may overcome many limitations of single drug target and achieve a more effective and safer control of the disease. Most of existing combination drug targets are developed based on clinical experience or text-and-trial strategy, which cannot provide theoretical guidelines for designing and screening effective drug combinations. Therefore, systematic identification of multiple drug targets and optimal intervention strategy needs to be developed.ResultsWe developed a strategy to screen the synergistic combinations of two drug targets in disease networks based on the classification of single drug targets. The method tried to identify the sensitivity of single intervention and then the combination of multiple interventions that can restore the disease network to a desired normal state. In our strategy of screening drug target combinations, we first classified all drug targets into sensitive and insensitive single drug targets. Then, we identified the synergistic and antagonistic of drug target combinations, including the combinations of sensitive drug targets, the combinations of insensitive drug target and the combination of sensitive and insensitive targets. Finally, we applied our strategy to Arachidonic Acid (AA) metabolic network and found 18 pairs of synergistic drug target combinations, five of which have been proven to be viable by biological or medical experiments.ConclusionsDifferent from traditional methods for judging drug synergy and antagonism, we propose the framework of how to enhance the efficiency by perturbing two sensitive targets in a combinatorial way, how to decrease the drug dose and therefore its side effect and cost by perturbing combinatorially a main sensitive target and an auxiliary insensitive target, and how to perturb two insensitive targets to realize the transition from a disease state to a healthy one which cannot be realized by perturbing each insensitive target alone. Although the idea is mainly applied to an AA metabolic network, the strategy holds for more general molecular networks such as combinatorial regulation in gene regulatory networks.

Dataset Information

Unraveling mitotic protein networks by 3D multiplexed epitope drug screening.

Publications

Unraveling mitotic protein networks by 3D multiplexed epitope drug screening.

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