Biology subjects

Fehl, C.

Publications and source records attributed to Fehl, C..

2 recordsLinked to original sources

bioRxiv2022

Spatiotemporal proximity labeling tools to track GlcNAc sugar-modified functional protein hubs during cellular signaling

A fundamental mechanism that all eukaryotic cells use to adapt to their environment is dynamic protein modification with monosaccharide sugars. In humans, O-linked N-acetylglucosamine (O-GlcNAc) is rapidly added to and removed from diverse protein sites as a response to fluctuating nutrient levels, stressors, and signaling cues. Two aspects remain challenging for tracking functional O-GlcNAc events with chemical strategies: spatial control over subcellular locations and time control during labeling. The objective of this study was to create intracellular proximity labeling tools to identify functional changes in O-GlcNAc patterns with spatiotemporal control. We developed a labeling strategy based on the TurboID proximity labeling system for rapid protein biotin conjugation that we directed to O-GlcNAc protein modifications inside cells, a set of tools we called "GlycoID." Localized variants to the nucleus and cytosol, nuc-GlycoID and cyt-GlycoID, labeled O-GlcNAc proteins and their interactomes in subcellular space. Labeling during insulin as well as serum stimulation revealed functional changes in O-GlcNAc proteins as soon as 30 minutes of signaling. We demonstrated using proteomic analysis that the GlycoID strategy captured O-GlcNAcylated "activity hubs" consisting of O-GlcNAc proteins and their associated protein-protein interactions. The ability to follow changes in O-GlcNAc hubs during physiological events like insulin stimulation poises these tools to be used for determining mechanisms of glycobiological cell regulation. Our functional O-GlcNAc datasets in human cells will be a useful resource for O-GlcNAc-driven mechanisms. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/488185v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@dbfde0org.highwire.dtl.DTLVardef@f8a27eorg.highwire.dtl.DTLVardef@17430f5org.highwire.dtl.DTLVardef@13a3e30_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

Hyperglycemic O-GlcNAc transferase activity drives cancer stem cell induction in TNBC

Enhanced glucose metabolism is a feature of almost all cancers, but downstream functional effects of aberrant glucose flux are difficult to mechanistically determine. The objective of this study is to characterize a mechanism by which elevated glucose level drives a tumorigenic pathway in triple negative breast cancer (TNBC). We used chemical biology methods to track how a metabolite of glucose, N-acetylglucosamine (GlcNAc), is linked to the transcriptional regulatory protein tet-methylcytosine dioxygenase 1 (TET1) as an O-linked GlcNAc post translational modification (O-GlcNAc). In this work, we revealed that intracellular protein glycosylation by O-GlcNAc is driven by high glucose levels in TNBC models, including on TET1. A single enzyme, O-GlcNAc transferase (OGT), is responsible for catalyzing protein modification of O-GlcNAc. We showed that OGT activity is higher in TNBC cell lines compared to non-tumor breast cell lines and is associated with hyperglycemia. Furthermore, enhanced OGT activity activated a pathway for cancer stem-like cell (CSC) reprogramming in TNBC cells. In our model, O-GlcNAcylated TET1 upregulated expression of splicing factor TAR-DNA binding protein (TARDBP), which drives CSC induction as well as higher OGT levels. We show that this OGT-TET1-TARDBP axis "feeds-forward" in hyperglycemic conditions both in cell lines and diet-induced obese mice, which displayed higher blood glucose levels and tumor O-GlcNAc levels than lean littermates. This data converges on a novel pathway whereby hyperglycemia drives aberrant OGT activity, activating a pathway for CSC induction in TNBC. Our findings partially explain a key aspect of how obesity is associated with TNBC risk and negative outcomes. Statement of ImplicationThis work presents a novel mechanism to explain how obesity is a risk factor for triple-negative breast cancer via elevated sugar-transfer activity by O-GlcNAc transferase in hyperglycemia, leading to the induction of a cancer stem-like cells and revealing a targetable pathway in obesity-associated tumors.

cancer biology↗