Biology subjects

Arrowsmith, C.

Publications and source records attributed to Arrowsmith, C..

23 records · Page 2Linked to original sources

bioRxiv2020

LSD1 represses a neonatal/reparative gene program in adult intestinal epithelium

Intestinal epithelial homeostasis is maintained by adult intestinal stem cells, which, alongside Paneth cells, appear after birth in the neonatal period. We aimed to identify new regulators of neonatal intestinal epithelial development by testing a small library of epigenetic modifier inhibitors in Paneth cell-skewed organoid cultures. We found that Lysine-specific demethylase 1A (Kdm1a/Lsd1) is absolutely required for Paneth cell differentiation. Lsd1-deficient crypts, devoid of Paneth cells, are still able to form organoids without a requirement of exogenous or endogenous Wnt. Mechanistically, we find that LSD1 represses genes that are normally expressed in fetal and neonatal epithelium. This gene profile is similar to what is seen in repairing epithelium, and indeed, we find that Lsd1-deficient epithelium has superior regenerative capacities after irradiation injury. In summary, we found an important regulator of neonatal intestinal development and identified a druggable target to reprogram intestinal epithelium towards a reparative state.

developmental biology↗

bioRxiv2019

Identification and Characterization of the First Fragment Hits for SETDB1 Tudor Domain

SET domain bifurcated protein 1 (SETDB1) is a human histone-lysine methyltransferase, which is amplified in human cancers and was shown to be crucial in the growth of non-small and small cell lung carcinoma. In addition to its catalytic domain, SETDB1 harbors a unique tandem tudor domain which recognizes histone sequences containing both methylated and acetylated lysines, and likely contributes to its localization on chromatin. Using X-ray crystallography and NMR spectroscopy fragment screening approaches, we have identified the first small molecule fragment hits that bind to histone peptide binding groove of the TTD of SETDB1. Herein, we describe the binding modes of these fragments and analogues and the biophysical characterization of key compounds. These confirmed small molecule fragments will inform the development of potent antagonists of SETDB1 interaction with histones.

pharmacology and toxicology↗

bioRxiv2018

Design and characterisation of mutant and wild-type huntingtin proteins produced from a toolkit of scalable eukaryotic expression systems

The gene mutated in Huntingtons disease (HD) patients encodes the 348 kDa huntingtin (HTT) protein. The pathogenic HD CAG-expansion mutation causes a polyglutamine (polyQ) tract at the N-terminus of the HTT protein to expand above a critical threshold of ~35 glutamine residues. The effect of HD mutations on HTT is not well understood, in part due to difficulties in carrying out biochemical, biophysical and structural studies of this large protein. To facilitate such studies, we have generated expression constructs for the scalable production of HTT in multiple eukaryotic expression systems. Our set of HTT expression clones comprises both N and C-terminally FLAG-tagged HTT constructs with polyQ lengths representative of the general population, HD patients, juvenile HD patients as well as the more extreme polyQ expansions used in some HD tissue and animal models. These reagents yield milligram quantities of pure recombinant HTT protein, including many of the previously mapped posttranslational modifications. We have characterised both apo and HTT-HAP40 complex samples produced using this HD resource, demonstrating that this toolkit can be used to generate physiologically meaningful complexes of HTT. We demonstrate how these resources can produce sufficient material for protein-intensive experiments such as small angle X-ray scattering (SAXS), providing biochemical insight into HTT protein structure. The work outlined in this manuscript and the tools generated, lay a foundation for further biochemical and structural work on the HTT protein and its functional interactions with other biomolecules.

biochemistry↗

bioRxiv2018

Metabolic adaptations underlie epigenetic vulnerabilities in chemoresistant breast cancer.

Cancer cell survival upon cytotoxic drug exposure leads to changes in cell identity, dictated by the epigenome. Several metabolites serve as substrates or co-factors to chromatin-modifying enzymes, suggesting that metabolic changes can underlie change in cell fate. Here, we show that progression of triple-negative breast cancer (TNBC) to taxane-resistance is characterized by altered methionine metabolism and S-adenosylmethionine (SAM) availability, giving rise to DNA hypomethylation in regions enriched for transposable elements (TE). Compensatory redistribution of H3K27me3 forming Large Organized Chromatin domains of lysine (K) modification (LOCK) prevents expression of TE in taxane-resistant cells. Pharmacological inhibition of EZH2, the H3K27me3 methyltransferase, alleviates TE repression, leading to the accumulation of dsRNA and activation of the interferon viral mimicry-response, specifically inhibiting the growth of taxane-resistant TNBC. Together, our work delineates a role for metabolic adaptations in redefining the epigenome of taxane-resistant TNBC cells and underlies an epigenetic vulnerability toward pharmacological inhibition of EZH2.

genomics↗

bioRxiv2018

Identification and Structure-Activity Relationship of HDAC6 Zinc-finger Ubiquitin Binding Domain Inhibitors

HDAC6 plays a central role in the recruitment of protein aggregates for lysosomal degradation, and is a promising target for combination therapy with proteasome inhibitors in multiple myeloma. Pharmacologically displacing ubiquitin from the zinc-finger ubiquitin-binding domain (ZnF-UBD) of HDAC6 is an underexplored alternative to catalytic inhibition. Here, we present the discovery of a HDAC6 ZnF-UBD-focused chemical series and its progression from virtual screening hits to low micromolar inhibitors. A carboxylate mimicking the C-terminal extremity of ubiquitin, and an extended aromatic system stacking with W1182 and R1155 are necessary for activity. One of the compounds induced a conformational remodeling of the binding site where the primary binding pocket opens-up onto a ligand-able secondary pocket that may be exploited to increase potency. The preliminary structure-activity relationship accompanied by nine crystal structures should enable further optimization into a chemical probe to investigate the merit of targeting the ZnF-UBD of HDAC6 in multiple myeloma and other diseases.

biophysics↗