Biology subjects

Ecclestone, G. B.

Publications and source records attributed to Ecclestone, G. B..

3 recordsLinked to original sources

bioRxiv2023

UCHL1-dependent control of Hypoxia-Inducible Factor Transcriptional Activity in Liver Disease.

Liver fibrosis is the excessive accumulation of extracellular matrix proteins that occurs in most types of chronic liver diseases. Fibrosis is associated with the activation of hepatic stellate cells (HSCs) which transdifferentiate into a myofibroblast like phenotype that is contractile, proliferative and profibrogenic. Hypoxia-inducible factor 1 (HIF1), an oxygen-sensitive transcription factor, is elevated during HSC activation and promotes the expression of profibrotic mediator HIF target genes. HIF activation during HSC activation can by either due to localised decreases in oxygen levels, or through oxygen-independent mechanisms that are not completely understood. Here we describe a role for the deubiquitinase UCHL1 in regulating HIF levels and activity during HSC activation and liver fibrosis. Increased HIF1 expression correlated with induction of UCHL1 mRNA and protein with HSC activation. Genetic deletion or chemical inhibition of UCHL1 impaired HIF activity through reduction of HIF1 levels. UCHL1 specifically cleaves the degradative ubiquitin chains from HIF1 leading to increased HIF1 levels, even in sufficiently oxygenated cells. Furthermore, our mechanistic studies have shown that UCHL1 elevates HIF activity through specific cleavage of degradative ubiquitin chains, elevates levels of pro-fibrotic gene expression and increases proliferation rates. These results demonstrate how small molecule inhibitors of DUBs can modulate the activity of HIF transcription factors in liver disease. Furthermore, inhibition of HIF activity via modulation of the ubiquitin-proteasomal degradation pathway may represent a therapeutic opportunity with other HIF-related pathologies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/523142v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1d82ee9org.highwire.dtl.DTLVardef@1eff21aorg.highwire.dtl.DTLVardef@1ffb0eforg.highwire.dtl.DTLVardef@1b93dc9_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

Disruption of HIF1A translational control attenuates the HIF-dependent hypoxic response and solid tumour formation in vivo

Adaptation to reduced oxygen availability is mediated by the hypoxia-inducible factor (HIF) family of transcription factors. The activity and availability of HIF proteins is primarily driven by the stability of the HIF alpha subunits. However, it is becoming increasingly apparent that preferential translation of HIF1 mRNA is also necessary for full activation of the HIF1-dependent hypoxic response. Consequently, the mechanisms controlling HIF1 translation are of equivalent importance to the proline hydroxylase-dependent degradation pathways. Here we investigate the role of the 5UTR of the HIF1 mRNA in controlling preferential translation of endogenous HIF1 in hypoxic cells. CRISPR/Cas9-mediated genetic deletion of the 5 UTR of HIF1 results in reduced HIF1 levels following hypoxia, without alteration in mRNA or protein stability. HIF1 mRNA lacking the 5UTR was efficiently translated in adequately oxygenated cells but this was inhibited during hypoxia, consistent with the global block on protein synthesis. The HIF1 translational defect observed in cells missing the 5UTR led to reduced viability in hypoxic conditions in vitro and an impaired ability to form solid tumours in murine xenografts. Prevention of preferential HIF1 translation limits the duration and intensity of the HIF-dependent hypoxic response and disrupts the formation of solid tumours. Together these results demonstrate the importance of translation control over HIF1 and suggest that strategies to inhibit preferential HIF1 protein translation in hypoxic cancer cells will be an effective strategy to limit the growth of solid hypoxic tumours.

cancer biology↗

bioRxiv2021

Fur4 mediated uracil-scavenging to screen for surface protein regulators

Cell surface membrane proteins perform diverse and critical functions and are spatially and temporally regulated by membrane trafficking pathways. Although perturbations in these pathways underlie many pathologies, our understanding of these pathways at a mechanistic level remains incomplete. Using yeast as a model, we have developed an assay that reports on the surface activity of the Fur4 uracil permease in uracil auxotroph strains grown in the presence of limited uracil. This assay was used to screen a haploid deletion library that identified mutants with both diminished and enhanced comparative growth in restricted uracil media. Factors identified, including various multi-subunit complexes, were enriched for membrane trafficking and transcriptional functions, in addition to various uncharacterised genes. Bioinformatic analysis of expression profiles from many strains lacking identified transcription factors required for efficient uracil-scavenging revealed they control expression of other uracil-scavenging factors, in addition to membrane trafficking genes essential for viability, and therefore not represented in the screen. Finally, we performed a secondary mating factor secretion screen to functionally categorise factors implicated in uracil-scavenging, most of which are conserved throughout evolution.

cell biology↗