Biology subjects

Hawkins, E. D.

Publications and source records attributed to Hawkins, E. D..

3 recordsLinked to original sources

bioRxiv2023

Divergent roles for caspase-8 and MLKL in high-fat diet induced obesity and NAFLD in mice

Cell death frequently occurs in the pathogenesis of obesity and non-alcoholic fatty liver disease (NAFLD). However, the exact contribution of core cell death machinery to disease manifestations remains ill-defined. Here, we show via the direct comparison of mice genetically deficient in apoptotic caspase-8 in myeloid cells, or the essential necroptotic regulators, Receptor-interacting protein kinase-3 (RIPK3) and Mixed lineage kinase domain-like (MLKL), that RIPK3-caspase-8 signaling regulates macrophage inflammatory responses and drives adipose tissue inflammation and NAFLD upon high-fat diet feeding. In contrast, MLKL, divergent to RIPK3, contributes to both obesity and NAFLD in a manner largely independent of inflammation. We also uncover that MLKL regulates the expression of molecules involved in lipid uptake, transport and metabolism and, congruent with this, we discover a shift in the hepatic lipidome upon MLKL deletion. Collectively, these findings highlight MLKL as an attractive therapeutic target to combat the growing obesity pandemic and metabolic disease.

immunology↗

bioRxiv2022

RNA decay defines the therapeutic response to transcriptional perturbation in cancer

Therapeutic targeting of dysregulated transcriptional programs has arisen as a promising strategy for the treatment of leukaemias. The therapeutic response to small molecule inhibitors of Bromodomain-Containing Proteins (BRD), such as BRD2 and BRD4, P300/cAMP-response element binding protein (CBP) and Cyclin Dependent Kinases (CDKs), is generally attributed to the selective disruption of oncogenic gene expression networks driven by enhancers, super-enhancers (SEs) and lineage-specific transcription factors (TFs), including the c-MYC oncogene. Using technologies such as thiol (SH)-linked alkylation for the metabolic sequencing of RNA sequencing (SLAM-seq) to profile messenger RNA (mRNA) decay and production rates, we demonstrate that gene intrinsic properties largely govern the selectivity associated with transcriptional inhibition, where total mRNA response signatures are dominated with genes that have short transcript half-lives, including those regulated by SEs and oncogenic TFs. Further highlighting that gene sensitivities only occur in the context of short transcript half-lives, stabilisation of the c-MYC transcript through changes in the 3 UTR rendered it insensitive to transcriptional targeting. However, this was not sufficient to rescue c-MYC target gene transcription and anti-leukaemia effects following transcriptional inhibition. Importantly, long-lived mRNAs encoding essential genes that evade transcriptional targeting can be rendered sensitive via modulation of mRNA decay kinetics through inhibition of the RNA Binding Protein (RBP), ELAV Like RNA binding protein 1 (ELAVL1)/ Human Antigen R (HuR). Taken together, these data demonstrate that mRNA decay shapes the therapeutic response to transcriptional perturbation and can be modulated for novel therapeutic outcomes using transcriptional agents in leukaemia.

cancer biology↗

bioRxiv2020

A toolbox for imaging RIPK1, RIPK3 and MLKL in mouse and human cells

Necroptosis is a lytic, inflammatory cell death pathway that is dysregulated in many human pathologies. The pathway is executed by a core machinery comprising the RIPK1 and RIPK3 kinases, which assemble into necrosomes in the cytoplasm, and the terminal effector pseudokinase, MLKL. RIPK3-mediated phosphorylation of MLKL induces oligomerization and translocation to the plasma membrane where MLKL accumulates as hotspots and perturbs the lipid bilayer to cause death. The precise choreography of events in the pathway, where they occur within cells, and pathway differences between species, are of immense interest. However, they have been poorly characterized due to a dearth of validated antibodies for microscopy studies. Here, we describe a toolbox of antibodies for immunofluorescent detection of the core necroptosis effectors, RIPK1, RIPK3 and MLKL, and their phosphorylated forms, in human and mouse cells. By comparing reactivity with endogenous proteins in wild-type cells and knockout controls in basal and necroptosis-inducing conditions, we characterise the specificity of frequently-used commercial and recently-developed antibodies for detection of necroptosis signaling events. Importantly, our findings demonstrate that not all frequently-used antibodies are suitable for monitoring necroptosis by immunofluorescence microscopy, and methanol-is preferable to paraformaldehyde-fixation for robust detection of specific RIPK1, RIPK3 and MLKL signals.

cell biology↗