Biology subjects

Kenneth, N. S.

Publications and source records attributed to Kenneth, N. S..

7 recordsLinked to original sources

bioRxiv2026

Phosphorylation at S345 Converts HIF-2α from a Transcription Factor to an RNA binding protein

Oxygen sensing and adaptation are vital for metazoan survival. At the cellular level the response to hypoxia is characterised by a switch in transcriptional programming, primarily mediated by hypoxia-inducible factors (HIFs). HIFs comprise an obligatory heterodimer between an oxygen-sensitive HIF- subunit and an oxygen-insensitive HIF-1{beta} subunit, which together regulate gene expression in response to hypoxia. Among the three HIF- proteins in vertebrates (HIF-1, HIF-2, and HIF-3), post-translational modifications (PTMs) play a crucial role in modulating their stability, localisation, and activity. While many modifications have been investigated on HIF-1, our knowledge of HIF-2 regulation is very limited. Here, we investigated the function of the recently identified HIF-2 S345 phosphorylation site and demonstrate that this disrupts the interaction between HIF-2 and HIF-1{beta}, thereby silencing the HIF-2 transcriptional response. Additionally, our findings suggest that this modification redirects HIF-2 from its normal transcription factor function towards a role in mRNA fate control, as indicated by mass spectrometry interactome analysis and RNA-immunoprecipitation. Our discoveries highlight a new regulatory mechanism of HIF-2 activity, where S345 phosphorylation impedes HIF-2/HIF-1{beta} heterodimer formation, creating a switch in HIF-2 function. As HIF-2 is the only HIF- isoform with a clinically approved inhibitor, these insights are crucial for advancing therapeutic strategies targeting hypoxia signalling.

molecular biology↗

bioRxiv2025

NF-κB is a Central Regulator of Hypoxia-Induced Gene Expression

Hypoxia is both a physiological and pathological signal in cells. Changes in gene expression play a critical role in the cellular response to hypoxia, enabling cells to adapt to reduced oxygen availability. These changes are primarily mediated by the HIF family of transcription factors, however other transcription factors such as NF-{kappa}B, are also activated by hypoxia. Although NF-{kappa}B is known to be activated by hypoxia, the extent to which NF-{kappa}B contributes to the hypoxic response remains poorly understood. Here, we analysed hypoxia-induced, NF-{kappa}B-dependent gene expression, to define the NF-{kappa}B-dependent hypoxic signature. Our analysis reveals that most genes downregulated by hypoxia require NF-{kappa}B for their repression. We show that although the NF-{kappa}B-mediated hypoxic response may vary between cell types, a core subset of hypoxia-inducible genes requires NF-{kappa}B across multiple cell backgrounds. We demonstrate that NF-{kappa}B is critical for reactive oxygen species (ROS) generation and regulation of genes involved in oxidative phosphorylation under hypoxia. This work highlights NF-{kappa}Bs central role in the hypoxia response and offering new insights into gene expression regulation by hypoxia and NF-{kappa}B.

molecular biology↗

bioRxiv2024

RAF inhibitors activate the integrated stress response by direct activation of GCN2

Paradoxical RAF activation by chemical RAF inhibitors (RAFi) is a well-understood on-target biological and clinical response. In this study, we show that a range of RAFi drive ERK1/2-independent activation of the Unfolded Protein Response (UPR), including expression of ATF4 and CHOP, that required the translation initiation factor eIF2. RAFi-induced ATF4 and CHOP expression was not reversed by inhibition of PERK, a known upstream activator of the eIF2-dependent Integrated Stress Response (ISR). Rather, we found that RAFi exposure activated GCN2, an alternate eIF2 kinase, leading to eIF2-dependent (and ERK1/2-independent) ATF4 and CHOP expression. The GCN2 kinase inhibitor A-92, GCN2 RNAi, GCN2 knock-out or ISRIB (an eIF2 antagonist) all reversed RAFi-induced expression of ATF4 and CHOP indicating that RAFi require GCN2 to activate the ISR. RAFi also activated full-length recombinant GCN2 in vitro and in cells, generating a characteristic bell-shaped concentration-response curve, reminiscent of RAFi-driven paradoxical activation of WT RAF dimers. Activation of the ISR by RAFi was abolished by GCN2 kinase dead mutations and M802A or M802G gatekeeper mutations, suggesting that RAFi bind directly to the GCN2 kinase domain; this was supported by mechanistic structural models of RAFi interaction with GCN2. Since the ISR is a critical pathway for determining cell survival or death, our observations may be relevant to the clinical use of RAFi, where paradoxical GCN2 activation may be a previously unappreciated off-target effect that may modulate tumour cell responses.

biochemistry↗

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↗

bioRxiv2020

Autophagy promotes cell and organismal survival by maintaining NAD(H) pools

Autophagy is an essential catabolic process that promotes clearance of surplus or damaged intracellular components1. As a recycling process, autophagy is also important for the maintenance of cellular metabolites during periods of starvation2. Loss of autophagy is sufficient to cause cell death in animal models and is likely to contribute to tissue degeneration in a number of human diseases including neurodegenerative and lysosomal storage disorders3-7. However, it remains unclear which of the many cellular functions of autophagy primarily underlies its role in cell survival. Here we have identified a critical role of autophagy in the maintenance of nicotinamide adenine dinucleotide (NAD+/NADH) levels. In respiring cells, loss of autophagy caused NAD(H) depletion resulting in mitochondrial membrane depolarisation and cell death. We also found that maintenance of NAD(H) is an evolutionary conserved function of autophagy from yeast to human cells. Importantly, cell death and reduced viability of autophagy-deficient animal models can be partially reversed by supplementation with an NAD(H) precursor. Our study provides a mechanistic link between autophagy and NAD(H) metabolism and suggests that boosting NAD(H) levels may be an effective intervention strategy to prevent cell death and tissue degeneration in human diseases associated with autophagy dysfunction.

cell biology↗

bioRxiv2018

Regulation of checkpoint kinase signalling and tumorigenesis by the NF-κB regulated gene, CLSPN

Inhibition of the tumour promoting activities of NF-{kappa}B by cell signalling pathways has been proposed as a natural mechanism to limit the development of cancer. However, there has been a lack of evidence for these effects in vivo. Here we report that RelAT505A mice, where a CHK1 targeted Thr505 phosphosite is mutated to alanine, display earlier onset of MYC driven lymphoma than wild type littermates. We describe a positive feedback loop in which the NF-{kappa}B subunits RelA and c-Rel, in a manner dependent upon RelA Thr505 phosphorylation, drive the expression of the ATR checkpoint kinase regulator Claspin in response to DNA replication stress in cancer cells. This in turn is required for maintenance of CHK1 activity. Loss of a single allele of the Clspn gene in mice is sufficient to drive earlier tumorigenesis and low levels of CLSPN mRNA expression are associated with worse survival in some forms of human cancer. We propose that loss of this pathway early in tumorigenesis promotes cancer development through increased genomic instability. However, in malignant cancer cells it can help promote their addiction to the checkpoint kinase signalling required for the maintenance of genomic integrity. Importantly, disruption of this pathway leads to resistance of cells to treatment with CHK1 inhibitors. Claspin expression could therefore act as a biomarker for responsiveness of patients to CHK1 inhibitors and provide a potential pathway for the development of tumour resistance.

cancer biology↗