Performance of a phosphoflow assay to determine phosphorylation of S6 ribosomal protein as a pharmacodynamic read out for mTOR inhibition

Abdel-Kahaar, Emaad; Kabakchiev, Mariana; Hartmann, Beate; Wieland, Eberhard; Shipkova, Maria

doi:10.1016/j.clinbiochem.2016.06.012

Cited by 5 publications

(4 citation statements)

References 24 publications

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“…54 Rapamycin functions by inhibiting the mTORC1 complex (comprising mTOR and Raptor), which blocks phosphorylation of S6K (ribosomal protein S6 kinase) and subsequent phosphorylation of S6 (ribosomal protein S6), as shown in Figure 5A. 55 We first validated that aRap could be used for light-controlled inhibition of S6 phosphorylation. HeLa cells were treated with 5 nM of rapamycin or aRap, followed by the presence or absence of 405 nm irradiation.…”

Section: ■ Results and Discussionmentioning

confidence: 89%

“…Lastly, we tested whether aRap could be used for optical control of mTOR signaling in both cells and zebrafish embryos . Rapamycin functions by inhibiting the mTORC1 complex (comprising mTOR and Raptor), which blocks phosphorylation of S6K (ribosomal protein S6 kinase) and subsequent phosphorylation of S6 (ribosomal protein S6), as shown in Figure A . We first validated that aRap could be used for light-controlled inhibition of S6 phosphorylation.…”

Section: Resultsmentioning

confidence: 99%

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Blue Light Activated Rapamycin for Optical Control of Protein Dimerization in Cells and Zebrafish Embryos

et al. 2021

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Rapamycin-induced dimerization of FKBP and FRB is the most commonly utilized chemically induced protein dimerization system. It has been extensively used to conditionally control protein localization, split-enzyme activity, and protein−protein interactions in general by simply fusing FKBP and FRB to proteins of interest. We have developed a new aminonitrobiphenylethyl caging group and applied it to the generation of a caged rapamycin analog that can be photoactivated using blue light. Importantly, the caged rapamycin analog shows minimal background activity with regard to protein dimerization and can be directly interfaced with a wide range of established (and often commercially available) FKBP/FRB systems. We have successfully demonstrated its applicability to the optical control of enzymatic function, protein stability, and protein subcellular localization. Further, we also showcased its applicability toward optical regulation of cell signaling, specifically mTOR signaling, in cells and aquatic embryos.

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Section: ■ Results and Discussionmentioning

confidence: 89%

Section: Resultsmentioning

confidence: 99%

Blue Light Activated Rapamycin for Optical Control of Protein Dimerization in Cells and Zebrafish Embryos

et al. 2021

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“…For precision medicine, PTM signatures may either be identified in disease models and then validated in clinical samples or directly identified using clinical samples. Although, for example, specific changes in phosphorylation and glycosylation are increasingly reported as potential markers of disease, and many PTM assays have been developed so far (for instance reviewed in [23][24][25]), only few are really routinely used for diagnostic purposes to date, due to a variety of reasons [26]. We are still in the early days of PTM-specific research and detection and many potential PTM biomarkers and the corresponding assays still need to be validated extensively.…”

Section: Can Ptms Be Used As Biomarkers?mentioning

confidence: 99%

Detecting post-translational modification signatures as potential biomarkers in clinical mass spectrometry

Mnatsakanyan

Shema

Basik

et al. 2018

Expert Review of Proteomics

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Numerous diseases are caused by changes in post-translational modifications (PTMs). Therefore, the number of clinical proteomics studies that include the analysis of PTMs is increasing. Combining complementary information-for example changes in protein abundance, PTM levels, with the genome and transcriptome (proteogenomics)-holds great promise for discovering important drivers and markers of disease, as variations in copy number, expression levels, or mutations without spatial/functional/isoform information is often insufficient or even misleading. Areas covered: We discuss general considerations, requirements, pitfalls, and future perspectives in applying PTM-centric proteomics to clinical samples. This includes samples obtained from a human subject, for instance (i) bodily fluids such as plasma, urine, or cerebrospinal fluid, (ii) primary cells such as reproductive cells, blood cells, and (iii) tissue samples/biopsies. Expert commentary: PTM-centric discovery proteomics can substantially contribute to the understanding of disease mechanisms by identifying signatures with potential diagnostic or even therapeutic relevance but may require coordinated efforts of interdisciplinary and eventually multi-national consortia, such as initiated in the cancer moonshot program. Additionally, robust and standardized mass spectrometry (MS) assays-particularly targeted MS, MALDI imaging, and immuno-MALDI-may be transferred to the clinic to improve patient stratification for precision medicine, and guide therapies.

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“…Blocking mTOR affects the activity of p70S6K and the function of 4E-BP1, leading to inhibition of protein synthesis [ 68 ]. Through inhibition of p70S6K, rapamycin also blocks the translation of 5′-TOP (5′-terminal oligopyrimidine tract) mRNAs to suppress mRNA translation and protein synthesis [ 69 ]. In addition to affecting p70S6K, 4E-BP1 is a translation inhibitor, which is phosphorylated and inactivated in response to a growth signal [ 70 ].…”

Section: Inhibition Of Mtor Suppresses Er Stress and Attenuates Rementioning

confidence: 99%

Neuroprotective Strategy in Retinal Degeneration: Suppressing ER Stress-Induced Cell Death via Inhibition of the mTOR Signal

Fan

Sun

Liu

et al. 2017

IJMS

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The retina is a specialized sensory organ, which is essential for light detection and visual formation in the human eye. Inherited retinal degenerations are a heterogeneous group of eye diseases that can eventually cause permanent vision loss. UPR (unfolded protein response) and ER (endoplasmic reticulum) stress plays an important role in the pathological mechanism of retinal degenerative diseases. mTOR (the mammalian target of rapamycin) kinase, as a signaling hub, controls many cellular processes, covering protein synthesis, RNA translation, ER stress, and apoptosis. Here, the hypothesis that inhibition of mTOR signaling suppresses ER stress-induced cell death in retinal degenerative disorders is discussed. This review surveys knowledge of the influence of mTOR signaling on ER stress arising from misfolded proteins and genetic mutations in retinal degenerative diseases and highlights potential neuroprotective strategies for treatment and therapeutic implications.

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Performance of a phosphoflow assay to determine phosphorylation of S6 ribosomal protein as a pharmacodynamic read out for mTOR inhibition

Cited by 5 publications

References 24 publications

Blue Light Activated Rapamycin for Optical Control of Protein Dimerization in Cells and Zebrafish Embryos

Blue Light Activated Rapamycin for Optical Control of Protein Dimerization in Cells and Zebrafish Embryos

Detecting post-translational modification signatures as potential biomarkers in clinical mass spectrometry

Neuroprotective Strategy in Retinal Degeneration: Suppressing ER Stress-Induced Cell Death via Inhibition of the mTOR Signal

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