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In-situ generation of differential sensors that fingerprint kinases and the cellular response to their expression.

ABSTRACT: Mitogen-activated protein (MAP) kinases are responsible for many cellular functions, and their malfunction manifests itself in several human diseases. Usually, monitoring the phosphorylation states of MAP kinases in vitro requires the preparation and purification of the proteins or Western blotting. Herein, we report an array sensing approach for the differentiation of MAP kinases and their phosphorylated counterparts in vitro. This technique utilizes a library of differential receptors created in situ containing peptides known for affinity to MAP kinases, and a Zn(II)-dipicolylamine complex that binds phosphate groups on proteins. An indicator-displacement assay signals the binding of the individual receptors to the kinases, while chemometrics is used to create a fingerprint for the kinases and their state of activity. For example, linear discriminant analysis correctly identified kinase activity with a classification accuracy of 97.5% in vitro, while the cellular response to kinase expression was classified with 100% accuracy.

SUBMITTER: Zamora-Olivares D

PROVIDER: S-EPMC3846390 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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In-situ generation of differential sensors that fingerprint kinases and the cellular response to their expression.

Zamora-Olivares Diana D Kaoud Tamer S TS Dalby Kevin N KN Anslyn Eric V EV

Journal of the American Chemical Society 20130918 39

Mitogen-activated protein (MAP) kinases are responsible for many cellular functions, and their malfunction manifests itself in several human diseases. Usually, monitoring the phosphorylation states of MAP kinases in vitro requires the preparation and purification of the proteins or Western blotting. Herein, we report an array sensing approach for the differentiation of MAP kinases and their phosphorylated counterparts in vitro. This technique utilizes a library of differential receptors created ...[more]

PMID: 23991633

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Project description:BackgroundA cellular stress response (CSR) is triggered upon recombinant protein synthesis which acts as a global feedback regulator of protein expression. To remove this key regulatory bottleneck, we had previously proposed that genes that are up-regulated post induction could be part of the signaling pathways which activate the CSR. Knocking out some of these genes which were non-essential and belonged to the bottom of the E. coli regulatory network had provided higher expression of GFP and L-asparaginase.ResultsWe chose the best performing double knockout E. coli BW25113?elaA?cysW and demonstrated its ability to enhance the expression of the toxic Rubella E1 glycoprotein by 2.5-fold by tagging it with sfGFP at the C-terminal end to better quantify expression levels. Transcriptomic analysis of this hyper-expressing mutant showed that a significantly lower proportion of genes got down-regulated post induction, which included genes for transcription, translation, protein folding and sorting, ribosome biogenesis, carbon metabolism, amino acid and ATP synthesis. This down-regulation which is a typical feature of the CSR was clearly blocked in the double knockout strain leading to its enhanced expression capability. Finally, we supplemented the expression of substrate uptake genes glpK and glpD whose down-regulation was not prevented in the double knockout, thus ameliorating almost all the negative effects of the CSR and obtained a further doubling in recombinant protein yields.ConclusionThe study validated the hypothesis that these up-regulated genes act as signaling messengers which activate the CSR and thus, despite having no casual connection with recombinant protein synthesis, can improve cellular health and protein expression capabilities. Combining gene knockouts with supplementing the expression of key down-regulated genes can counter the harmful effects of CSR and help in the design of a truly superior host platform for recombinant protein expression.

Project description:BackgroundSkull diversity in the neotropical leaf-nosed bats (Phyllostomidae) evolved through a heterochronic process called peramorphosis, with underlying causes varying by subfamily. The nectar-eating (subfamily Glossophaginae) and blood-eating (subfamily Desmondontinae) groups originate from insect-eating ancestors and generate their uniquely shaped faces and skulls by extending the ancestral ontogenetic program, appending new developmental stages and demonstrating peramorphosis by hypermorphosis. However, the fruit-eating phyllostomids (subfamilies Carollinae and Stenodermatinae) adjust their craniofacial development by speeding up certain developmental processes, displaying peramorphosis by acceleration. We hypothesized that these two forms of peramorphosis detected by our morphometric studies could be explained by differential growth and investigated cell proliferation during craniofacial morphogenesis.ResultsWe obtained cranial tissues from four wild-caught bat species representing a range of facial diversity and labeled mitotic cells using immunohistochemistry. During craniofacial development, all bats display a conserved spatiotemporal distribution of proliferative cells with distinguishable zones of elevated mitosis. These areas were identified as modules by the spatial distribution analysis. Ancestral state reconstruction of proliferation rates and patterns in the facial module between species provided support, and a degree of explanation, for the developmental mechanisms underlying the two models of peramorphosis. In the long-faced species, Glossophaga soricina, whose facial shape evolved by hypermorphosis, cell proliferation rate is maintained at lower levels and for a longer period of time compared to the outgroup species Miniopterus natalensis. In both species of studied short-faced fruit bats, Carollia perspicillata and Artibeus jamaicensis, which evolved under the acceleration model, cell proliferation rate is increased compared to the outgroup.ConclusionsThis is the first study which links differential cellular proliferation and developmental modularity with heterochronic developmental changes, leading to the evolution of adaptive cranial diversity in an important group of mammals.

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In-situ generation of differential sensors that fingerprint kinases and the cellular response to their expression.

Publications

In-situ generation of differential sensors that fingerprint kinases and the cellular response to their expression.

Similar Datasets

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