Abstract
Reversible phosphorylation of the transcription factor EB (TFEB) coordinates cellular responses to metabolic and other stresses. During nutrient replete and stressor-free conditions, phosphorylated TFEB is primarily localized to the cytoplasm. Stressor-mediated reduction of TFEB phosphorylation promotes its nuclear translocation and context-dependent transcriptional activity. In this study, we explored targeted dephosphorylation of TFEB as an approach to activate TFEB in the absence of nutrient deprivation or other cellular stress. Through an induction of proximity between TFEB and several phosphatases using the AdPhosphatase system, we demonstrate targeted dephosphorylation of TFEB in cells. Furthermore, by developing a heterobifunctional molecule BDPIC (bromoTAG-dTAG proximity-inducing chimera), we demonstrate targeted dephosphorylation of TFEB-dTAG through induced proximity to bromoTAG-PPP2CA. Targeted dephosphorylation of TFEB-dTAG by bromoTAG-PPP2CA with BDPIC at the endogenous levels is sufficient to induce nuclear translocation and some transcriptional activity of TFEB.
| Originalsprog | Engelsk |
|---|---|
| Artikelnummer | 110432 |
| Tidsskrift | iScience |
| Vol/bind | 27 |
| Udgave nummer | 8 |
| Antal sider | 24 |
| ISSN | 2589-0042 |
| DOI | |
| Status | Udgivet - 2024 |
Bibliografisk note
Funding Information:G.P.S. is funded by the UKRI Medical Research Council (MC_UU_00018/6) and is also supported by the pharmaceutical companies supporting the Division of Signal Transduction Therapy (Boehringer-Ingelheim, GlaxoSmithKline, Merck-Serono). G.S. is supported by UKRI MRC funds awarded to G.P.S. A.B. is supported by the UKRI BBSRC EASTBIO PhD studentship (grant BB/M010996/1) and UKRI MRC. We thank the members of G.P.S. lab for experimental advice and discussions during the study. We thank the MRC tissue culture facility staff (E. Allen, A. Muir, S. Dalglish, E. Webster and J. Stark), the staff at the DNA Sequencing services (School of Life Sciences, University of Dundee), the cloning, antibody, inhibitor and protein production teams within the MRC PPU Reagents & Services (University of Dundee) coordinated by J. Hastie and the staff at FACS service for their technical and reagents support. Graphic abstract was created with BioRender.com (license number BP26ZAGDQP). Novo Nordisk Foundation Center for Basic Metabolic Research is an independent Research Center based at the University of Copenhagen, Denmark, and partially funded by an un-conditional donation from the Novo Nordisk Foundation (Grant number NNF18CC0034900 and NNF23SA0084103). This work is supported by International Alliances between University of Copenhagen and University of Dundee using the funding from the Novo Nordisk Foundation (Grant number NNF18CC0034900 and NNF23SA0084103). J.-F.Z. performed most of the experiments, collected and analyzed data, and contributed to the writing of the manuscript. G.P.S. designed BDPIC and HDPIC compounds, and N.S. synthesized BDPIC, HDPIC, and PhosTAC7. G.S. performed and analyzed some cell-based experiments and all proteomic experiments. A.B. performed some experiments and analyzed data. T.J.M. N.T.W. and J.-F.Z. generated constructs used in this study. T.J.M. designed the strategies, and made constructs, for CRISPR/Cas9 genome editing experiments. F.N. characterized and validated specificity of phospho-specific TFEB antibodies. K.S. and G.P.S. conceived and supervised the project, analyzed data and contributed to the writing of the manuscript. University of Dundee is currently in the process of filing a patent application for the bivalent molecule BDPIC that was developed as part of this study. The authors declare no other competing interests.
Funding Information:
G.P.S. is funded by the UKRI Medical Research Council ( MC_UU_00018/6 ) and is also supported by the pharmaceutical companies supporting the Division of Signal Transduction Therapy (Boehringer-Ingelheim, GlaxoSmithKline, Merck-Serono). G.S. is supported by UKRI MRC funds awarded to G.P.S.. A.B. is supported by the UKRI BBSRC EASTBIO PhD studentship (grant BB/M010996/1 ) and UKRI MRC . We thank the members of G.P.S. lab for experimental advice and discussions during the study. We thank the MRC tissue culture facility staff (E. Allen, A. Muir, S. Dalglish, E. Webster and J. Stark), the staff at the DNA Sequencing services (School of Life Sciences, University of Dundee), the cloning, antibody, inhibitor and protein production teams within the MRC PPU Reagents & Services (University of Dundee) coordinated by J. Hastie and the staff at FACS service for their technical and reagents support. Graphic abstract was created with BioRender.com (license number BP26ZAGDQP ).
Funding Information:
Novo Nordisk Foundation Center for Basic Metabolic Research is an independent Research Center based at the University of Copenhagen , Denmark, and partially funded by an un-conditional donation from the Novo Nordisk Foundation (Grant number NNF18CC0034900 and NNF23SA0084103 ). This work is supported by International Alliances between University of Copenhagen and University of Dundee using the funding from the Novo Nordisk Foundation (Grant number NNF18CC0034900 and NNF23SA0084103 ).
Publisher Copyright:
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