A5011709620- CRISPR and Genetic Engineering
- Endoplasmic Reticulum Stress and Disease
- Advanced biosensing and bioanalysis techniques
- RNA regulation and disease
- Cellular transport and secretion
- Crystallography and molecular interactions
- X-ray Diffraction in Crystallography
- RNA and protein synthesis mechanisms
- CAR-T cell therapy research
- Crystallization and Solubility Studies
- Transgenic Plants and Applications
- interferon and immune responses
- Fungal and yeast genetics research
- Bacterial Genetics and Biotechnology
- Virus-based gene therapy research
- Lipid Membrane Structure and Behavior
- RNA Interference and Gene Delivery
- Plant Virus Research Studies
- Adenosine and Purinergic Signaling
- Single-cell and spatial transcriptomics
- Toxin Mechanisms and Immunotoxins
- Autophagy in Disease and Therapy
- Ion channel regulation and function
- Innovative Microfluidic and Catalytic Techniques Innovation
- Glycosylation and Glycoproteins Research
Horizon Discovery Group (United Kingdom)
2015-2021
Benedict College
2018
University of Cambridge
2012-2013
NIHR Cambridge Biomedical Research Centre
2012-2013
National Institute for Health Research
2012
University of Manchester
2007-2012
IRE1 couples endoplasmic reticulum unfolded protein load to RNA cleavage events that culminate in the sequence-specific splicing of Xbp1 mRNA and regulated degradation diverse membrane-bound mRNAs. We report on identification a small molecule inhibitor attains its selectivity by forming an unusually stable Schiff base with lysine 907 endonuclease domain, explained solvent inaccessibility imine bond enzyme-inhibitor complex. The (abbreviated 4μ8C) blocks substrate access active site...
Interfering with disulfide bond formation impedes protein folding and promotes endoplasmic reticulum (ER) stress. Due to limitations in measurement techniques, the relationships of altered thiol redox ER stress have been difficult assess. We report that fluorescent lifetime measurements circumvented crippling dimness an ER-tuned redox-responsive probe (roGFPiE), faithfully tracking activity major ER-localized isomerase, PDI. In vivo imaging by time-correlated single-photon counting (TCSPC)...
Production and trafficking of proteins entering the secretory pathway eukaryotic cells is coordinated at endoplasmic reticulum (ER) in a process that begins with protein translocation via membrane-embedded ER translocon. The same complex also responsible for co-translational integration membrane orchestrates polypeptide modifications are often essential function. We now show previously identified inhibitor ER-associated degradation (ERAD) eeyarestatin 1 (ESI) potent translocation. have...
Abstract Pooled CRISPR–Cas9 knock out screens provide a valuable addition to the methods available for novel drug target identification and validation. However, where gene editing is targeted amplified loci, resulting multiple DNA cleavage events can be cause of false positive hit identification. The generation nuclease deficient versions Cas9 has enabled development two additional techniques – CRISPR interference (CRISPRi) activation (CRISPRa) that enable repression or overexpression,...
Despite their usefulness as fluorophores and synthetic precursors, efficient reliable routes to coumarin-8-carbaldehydes are lacking. We describe here a high-yielding continuous flow synthesis that requires no manual intermediate purification or work-up, giving access multigram quantities of the aldehyde product.
Abstract Components of the type II CRISPR–Cas complex in bacteria have been used successfully eukaryotic cells to facilitate rapid and accurate cell line engineering, animal model generation functional genomic screens. Such developments are providing new opportunities for drug target identification validation, particularly with application pooled genetic screening. As is a relatively screening tool, it important assess its functionality number different lines analyse potential improvements...
A site-specific cross-linking approach was used to study the integration of TM (transmembrane) segments 4–7 polytopic membrane protein, opsin, at ER (endoplasmic reticulum). We found that although TM4 exits translocon rapidly, 5, 6 and 7 are all retained until opsin biosynthesis is terminated. Furthermore, artificial extension nascent chain not sufficient release C-terminal region from translocon, substitution native segment with a more hydrophobic results in its rapid lateral exit into...
Discovery of small-molecule degraders that activate ubiquitin ligase–mediated ubiquitination and degradation targeted oncoproteins in cancer cells has been an elusive therapeutic strategy. Here, we report a cell–based drug screen the NCI drug-like compounds library enabled identification small ubiquitin-related modifier 1 (SUMO1). Structure-activity relationship studies analogs hit compound CPD1 led to lead HB007 with improved properties anticancer potency vitro vivo. A genome-scale...
The membrane integration of polytopic proteins is coordinated at the endoplasmic reticulum (ER) by conserved Sec61 translocon, which facilitates lateral release transmembrane (TM) segments into lipid phase during polypeptide translocation. Here we use a site-specific crosslinking strategy to study new model protein and show that TM P2X2 receptor are retained complex for entire duration biosynthetic process. This extremely prolonged association implicates in regulation process, both vitro...
The discovery of CRISPR-Cas9 systems has fueled a rapid expansion gene editing adoption and impacted pharmaceutical biotechnology research substantially. Here, is used at an industrial scale to identify validate new biological targets for precision medicines, with functional genomic screening having increasingly important role. Functional strategies provide crucial link between observed phenomena the genes that influence drive those phenomena. Although such studies are not new, use in this...
The presence of two basic amino acids strategically located within a single spanning transmembrane region has previously been shown to act as signal for the endoplasmic reticulum associated degradation (ERAD) several polypeptides. In contrast, functionality this degron motif context polytopic membrane protein not established. Using opsin model system, we have investigated consequences inserting in first its seven (TM) spans. Whilst these residue reduce binding targeting factor, recognition...
Summary The integration of transmembrane (TM)-spanning regions many channels and ion transporters is potentially compromised by the presence polar charged residues required for biological function. Although two TMs ATP-gated channel subunit P2X2 each contain charged/polar amino acids, we found that TM efficiently membrane inserted when it analysed in isolation, uncovered no evidence cooperativity between these during integration. However, using minimal N-glycosylation distance mapping, find...
The endoplasmic reticulum (ER) is a major site of protein synthesis in eukaryotes. Newly synthesized proteins are monitored by process quality control, which removes misfolded or unassembled polypeptides from the ER for degradation proteasome. This requires retrotranslocation lumen into cytosol via pathway that, some substrates, involves members recently discovered Derlin family. Derlin-1 isoform present as dimer ER, and we now show that its dimerization modulated stress. Three distinct...
Functional genomic screening with CRISPR has provided a powerful and precise new way to interrogate the phenotypic consequences of gene manipulation in high-throughput, unbiased analyses. However, some experimental paradigms prove especially challenging require carefully appropriately adapted approaches. In particular, negative selection (or sensitivity) screening, often most experimentally desirable modality remained challenge drug discovery. Here we assess whether our new, modular...
The response of S. pombe, also known as fission yeast, to misfolded proteins involves mechanisms that have not been observed in other species.
Abstract The RNA-guided CRISPR-Cas9 gene editing technology can be adapted to provide a loss-of-function genetic screening platform. Knockout (KO) uses sequence-specific disruption and circumvents many of the problems encountered by RNAi technologies. In particular, complete ablation expression allows opportunity examine cellular physiological effects resulting from loss targets rather than partial knockdown target, which might not have phenotypic effect. Our screens are conducted with...
Abstract Some cancers lack clearly druggable driver mutations and might also have too few neo-antigen generating to provoke a tumor-specific immune response, even in the presence of antibodies targeting checkpoint mechanisms. Synthetic lethal/non-oncogene addiction represents an alternative approach therapy such cancers, as exemplified by recent approval olaparib. RNA interference has identified several putative synthetic lethal targets, but few, if any, these withstood vigorous validation....