User Guide. Page content. You are here : Catalogue Display. Dietary modulation of cell signaling pathways. Bookmark Catalogue Record Item Information Catalogue Record Catalogue Information Catalogue Record Physical Details xxii, p. Series Oxidative stress and disease ; no. Related Works Catalogue Record Add Title to Basket Catalogue Record Branch Status Due Date Res. Download Title Catalogue Record Reserve Title Catalogue Record Top Titles Author Title 1. Climate new leaders, new approaches 1 2. Find more information about Crossref citation counts.
The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric. Find more information on the Altmetric Attention Score and how the score is calculated. The complicated and entangled cell signaling network is dynamically regulated by a wide array of enzymes such as kinases.
It remains desirable but challenging to specifically modulate individual, endogenous kinases within a cell, particularly in a spatial—temporally controlled fashion. Current strategies toward regulating the intracellular functions of a kinase of interest either lack specificity or require genetic engineering that may perturb its physiological activity.
Herein, we harnessed a bacterial effector OspF for optical and chemical modulation of the endogenous mitogen-activated protein kinase MAPK cascade in living cells and mice. The phospho-lyase OspF provided high specificity and spatial resolution toward the desired kinase such as the extracellular signal-regulated kinase ERK , while the genetically encoded bioorthogonal decaging strategy enabled its temporal activation in living systems.
Finally, our spatially and chemically controlled OspF c was further used to precisely tune immune responses in T cells. Together, our bioorthogonal engineering strategy on bacterial effectors offers a general tool to modulate cell signaling with high specificity and spatial—temporal resolution.
The catalytic center of OspF is highlighted. The in situ rescued OspF can specifically remove the phosphate group on phosphothreonine at residue on ERK and residue on p38, respectively. The resulting dehydrobutyrine at these sites can no longer be rephosphorylated, leading to permanently abrogated ERK and p38 activity. Figure 1. OspF activity was detected by phosphorylation of ERK and p38 using specific antibodies.
Cell Signaling and Stress Responses
OspF-WT and actin were used as controls. Cells with no ONBK supplementation were used as a control. The relative luminescence activity is proportional to the endogenous phosphorylation level of ERK. Cells transfected with empty vector or OspF-WT were used as controls.
Figure 2. Data were analyzed by FlowJo software.hemimaborr.ga
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The nucleus was stained with Hoechst Overlay of fluorescence image and DIC image is shown, and all fluorescence images are shown in Figure S No obvious variation on p90RSK phosphorylation was detected. Data shown in parts E and G are representative of at least three independent experiments. Figure 3. Chemical rescue of OspF c in living cells and mice.
Cells expressing OspF-WT or transfected with empty vector were used as controls, and actin was used as loading controls. B Dual-luciferase analysis of activation of OspF c in living cells. C Schematic flow showing the chemical rescue of OspF c in living mice. Mice were fed for another 24 h for luciferase expression, and luciferin was injected 10 min before bioluminescence imaging. OspF c by Me 2 -Tz treatment would attenuate ERK activity and thus reduce luciferase expression and bioluminescence signal.
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D Representative images of rescued OspF c activity as measured by bioluminescence after Me 2 -Tz treatment. An obvious decrease of bioluminescence was observed only in the leg injected with cells harboring OspF c after Me 2 -Tz treatment. Figure 4. Precise tuning of T cell activation and immune responses by subcellular-targeted, chemically controlled OspF c. A Stimulation on T cells will induce immune responses and cytokine release, while overstimulation on T cells can induce cytokine release syndrome.
OspF activity was detected by phosphorylation of ERK using specific antibodies, and actin was used as loading controls. Detailed experimental procedures for plasmid construction and cell biological experiments and additional figures including structures, mass spectrometry, immunofluorescent staining, flow cytometry quantification, immunoblotting analyses, substrate specificity, temporal modulation, subcellular fractionation, optical rescue, densitometric analysis, time-dependent alternation, and cytotoxicity study PDF.
We thank Dr.
Cong Li and Dr. Rongfeng Zhu for constructive discussions and proofreading of the manuscript. This article references 52 other publications.
More by Jingyi Zhao. More by Yanjun Liu. More by Feng Lin. More by Weixia Wang. More by Shaojun Yang. More by Yun Ge. More by Peng R. Cite this: ACS Cent. ACS AuthorChoice. Article Views Altmetric -.
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PDF 4 MB. Abstract High Resolution Image. Eukaryotic cells have evolved a diverse repertoire of enzymes for catalyzing potent chemical modifications on proteins that dictate diverse signaling events. Diverse effector proteins have been evolved by bacteria to modulate signaling pathways inside host cells with high specificity. However, since the constitutively active form of OspF will irreversibly inhibit ERK and p38 activity and thus permanently turn off the MAPK signaling cascade, temporally controlled activation of OspF is highly desired. Previous methods relying on controlled expression of OspF protein have a poor temporal resolution, 23 and are exceedingly difficult for applications in live animals.
We envisioned that our recently developed genetically encoded bioorthogonal decaging strategy would enable precise and temporally controlled activation of OspF under living conditions. Herein, by bioorthogonal engineering and targeting of the bacterial effector OspF to different subcellular compartments e. Scheme 1.
High Resolution Image.
Dietary Modulation of Cell Signaling Pathways
Results and Discussion. We started by engineering an optically controlled OspF in living cells based on the genetic code expansion system. In contrast, no bioluminescence variation was observed in the same batch of cells without light treatment Figure 1 C. Taken together, our engineered photoactivatable OspF can modulate the endogenous MAPK signaling in living cells with high temporal resolution. ERK is known to shuttle between the nucleus and cytoplasm and phosphorylate a series of substrates with different physiological outputs.
Meanwhile, phosphorylated ERK remains in the cytoplasm to phosphorylate over 50 substrates and mediate biological processes such as negative feedback regulation. As an oncogenic transcriptional factor, the amplification of c-MYC has been shown as a hallmark in cancer and malignant tumor generation. No alternation of c-MYC phosphorylation and protein abundance was observed within 30 min, and a decreased phosphorylation was detected at 45 min Figure S14B.
Taken together, our engineered OspF can modulate endogenous ERK functions with high spatial—temporal resolution. Meanwhile, small-molecule-mediated chemical-rescue strategies possess higher penetration capability in deep tissue and are thus more compatible for kinase signaling modulation at the tissue and animal level. The addition of the decaging reagent Me 2 -Tz would regenerate free lysine at K with its phospho-lyase activity rescued Scheme 1 B and Figure S1. We also performed a time course study to show the stability of OspF c and its effect on endogenous MAPK signaling after chemical decaging.
Immunoblotting results showed that an obviously attenuated phosphorylation level of ERK and p38 was observed within 10 min after Me 2 -Tz treatment and can sustain for over 60 min Figure S Meanwhile, no apparent change of OspF c abundance was detected even 60 min after chemical decaging Figure S We then employed the aforementioned luciferase reporter, SRE-luc, to monitor OspF c activity in living cells.
In contrast, no bioluminescence variation was observed in the same batch of cells without Me 2 -Tz treatment Figure 3 B. We next pursued the chemical rescue of OspF c in living mice. A previously developed xenograft model was used to demonstrate the chemical activation of OspF c in vivo. As a control, a noncleavable lysine analogue, CbzK benzyloxycarbonyl caged lysine , was incorporated into OspF at the same residue K to generate an inactivated counterpart OspF-KCbzK.
Bioluminescence imaging showed that the lyase activity of OspF c can be effectively rescued with Me 2 -Tz treatment, as made evident by the significantly attenuated bioluminescence signal in mice upon Me 2 -Tz addition Figure 3 D and Figure S In contrast. Therefore, our chemical-decaging strategy on OspF can be applied to living animals. We further expanded our chemically rescued OspF c to tune immune responses.