scholarly journals The Small Molecule 2-Azido-2-deoxy-glucose Is a Metabolic Chemical Reporter of O-GlcNAc Modifications in Mammalian Cells, Revealing an Unexpected Promiscuity of O-GlcNAc Transferase

2017 ◽  
Vol 12 (3) ◽  
pp. 787-794 ◽  
Author(s):  
Balyn W. Zaro ◽  
Anna R. Batt ◽  
Kelly N. Chuh ◽  
Marisol X. Navarro ◽  
Matthew R. Pratt
Keyword(s):  
Small Molecule ◽  
Mammalian Cells ◽  
Chemical Reporter ◽  
Glcnac Transferase

scholarly journals Small-molecule inducible transcriptional control in mammalian cells

2020 ◽  
Vol 40 (8) ◽  
pp. 1131-1150
Author(s):  
Aarti Doshi ◽  
Fatemeh Sadeghi ◽  
Navin Varadarajan ◽  
Patrick C. Cirino
Keyword(s):  
Small Molecule ◽  
Mammalian Cells ◽  
Transcriptional Control

scholarly journals The nutrient sensor OGT regulates Hipk stability and tumorigenic-like activities in Drosophila

2020 ◽  
Vol 117 (4) ◽  
pp. 2004-2013 ◽  
Author(s):  
Kenneth Kin Lam Wong ◽  
Ta-Wei Liu ◽  
Jessica M. Parker ◽  
Donald A. R. Sinclair ◽  
Yi-Yun Chen ◽  
...  
Keyword(s):  
Protein Stability ◽  
Mammalian Cells ◽  
Normal Diet ◽  
Cancer Proliferation ◽  
Hexosamine Biosynthetic Pathway ◽  
Interacting Protein ◽  
Molecular Sensors ◽  
Cellular Behaviors ◽  
Glcnac Transferase ◽  
Necessary And Sufficient

Environmental cues such as nutrients alter cellular behaviors by acting on a wide array of molecular sensors inside cells. Of emerging interest is the link observed between effects of dietary sugars on cancer proliferation. Here, we identify the requirements of hexosamine biosynthetic pathway (HBP) and O-GlcNAc transferase (OGT) for Drosophila homeodomain-interacting protein kinase (Hipk)-induced growth abnormalities in response to a high sugar diet. On a normal diet, OGT is both necessary and sufficient for inducing Hipk-mediated tumor-like growth. We further show that OGT maintains Hipk protein stability by blocking its proteasomal degradation and that Hipk is O-GlcNAcylated by OGT. In mammalian cells, human HIPK2 proteins accumulate posttranscriptionally upon OGT overexpression. Mass spectrometry analyses reveal that HIPK2 is at least O-GlcNAc modified at S852, T1009, and S1147 residues. Mutations of these residues reduce HIPK2 O-GlcNAcylation and stability. Together, our data demonstrate a conserved role of OGT in positively regulating the protein stability of HIPKs (fly Hipk and human HIPK2), which likely permits the nutritional responsiveness of HIPKs.

Identification of novel small molecule enhancers of protein production by cultured mammalian cells

2008 ◽  
Vol 100 (6) ◽  
pp. 1193-1204 ◽  
Author(s):  
Martin J. Allen ◽  
James P. Boyce ◽  
Michael T. Trentalange ◽  
David L. Treiber ◽  
Brian Rasmussen ◽  
...  
Keyword(s):  
Small Molecule ◽  
Mammalian Cells ◽  
Protein Production ◽  
Small Molecule Enhancers

scholarly journals A rational blueprint for the design of chemically-controlled protein switches

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sailan Shui ◽  
Pablo Gainza ◽  
Leo Scheller ◽  
Che Yang ◽  
Yoichi Kurumida ◽  
...  
Keyword(s):  
Small Molecule ◽  
Protein Design ◽  
Protein Interactions ◽  
Mammalian Cells ◽  
Chemical Space ◽  
Domain Architecture ◽  
Receptor Protein ◽  
Drug Side Effects ◽  
Protein Protein Interactions ◽  
Drug Receptors

AbstractSmall-molecule responsive protein switches are crucial components to control synthetic cellular activities. However, the repertoire of small-molecule protein switches is insufficient for many applications, including those in the translational spaces, where properties such as safety, immunogenicity, drug half-life, and drug side-effects are critical. Here, we present a computational protein design strategy to repurpose drug-inhibited protein-protein interactions as OFF- and ON-switches. The designed binders and drug-receptors form chemically-disruptable heterodimers (CDH) which dissociate in the presence of small molecules. To design ON-switches, we converted the CDHs into a multi-domain architecture which we refer to as activation by inhibitor release switches (AIR) that incorporate a rationally designed drug-insensitive receptor protein. CDHs and AIRs showed excellent performance as drug responsive switches to control combinations of synthetic circuits in mammalian cells. This approach effectively expands the chemical space and logic responses in living cells and provides a blueprint to develop new ON- and OFF-switches.

scholarly journals The Metabolic Chemical Reporter Ac46AzGal Could Incorporate Intracellular Protein Modification in the Form of UDP-6AzGlc Mediated by OGT and Enzymes in the Leloir Pathway

2021 ◽  
Vol 9 ◽  
Author(s):  
Jiajia Wang ◽  
Biao Dou ◽  
Lu Zheng ◽  
Wei Cao ◽  
Peiyu Dong ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Protein Modification ◽  
Time Dependent ◽  
Dependent Manner ◽  
Intracellular Protein ◽  
Galactose Metabolism ◽  
Naturally Occurring ◽  
Leloir Pathway ◽  
Chemical Reporter ◽  
Complex Glycans

Galactose is a naturally occurring monosaccharide used to build complex glycans that has not been targeted for labeling as a metabolic reporter. Here, we characterize the cellular modification of proteins by using Ac46AzGal in a dose- and time-dependent manner. It is noted that a vast majority of this labeling of Ac46AzGal occurs intracellularly in a range of mammalian cells. We also provided evidence that this labeling is dependent on not only the enzymes of OGT responsible for O-GlcNAcylation but also the enzymes of GALT and GALE in the Leloir pathway. Notably, we discover that Ac46AzGal is not the direct substrate of OGT, and the labeling results may attribute to UDP-6AzGlc after epimerization of UDP-6AzGal via GALE. Together, these discoveries support the conclusion that Ac46AzGal as an analogue of galactose could metabolically label intracellular O-glycosylation modification, raising the possibility of characterization with impaired functions of the galactose metabolism in the Leloir pathway under certain conditions, such as galactosemias.

scholarly journals A general strategy to construct small molecule biosensors in eukaryotes

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Justin Feng ◽  
Benjamin W Jester ◽  
Christine E Tinberg ◽  
Daniel J Mandell ◽  
Mauricio S Antunes ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Ligand Binding ◽  
Small Molecules ◽  
Small Molecule ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Dynamic Range ◽  
Plant Cells ◽  
General Strategy ◽  
Target Molecule

Biosensors for small molecules can be used in applications that range from metabolic engineering to orthogonal control of transcription. Here, we produce biosensors based on a ligand-binding domain (LBD) by using a method that, in principle, can be applied to any target molecule. The LBD is fused to either a fluorescent protein or a transcriptional activator and is destabilized by mutation such that the fusion accumulates only in cells containing the target ligand. We illustrate the power of this method by developing biosensors for digoxin and progesterone. Addition of ligand to yeast, mammalian, or plant cells expressing a biosensor activates transcription with a dynamic range of up to ~100-fold. We use the biosensors to improve the biotransformation of pregnenolone to progesterone in yeast and to regulate CRISPR activity in mammalian cells. This work provides a general methodology to develop biosensors for a broad range of molecules in eukaryotes.

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