Publications by authors named "Katherine Newling"

10 Publications

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Essential roles for deubiquitination in Leishmania life cycle progression.

PLoS Pathog 2020 06 16;16(6):e1008455. Epub 2020 Jun 16.

York Biomedical Research Institute and Department of Biology, University of York, United Kingdom.

The parasitic protozoan Leishmania requires proteasomal, autophagic and lysosomal proteolytic pathways to enact the extensive cellular remodelling that occurs during its life cycle. The proteasome is essential for parasite proliferation, yet little is known about the requirement for ubiquitination/deubiquitination processes in growth and differentiation. Activity-based protein profiling of L. mexicana C12, C19 and C65 deubiquitinating cysteine peptidases (DUBs) revealed DUB activity remains relatively constant during differentiation of procyclic promastigote to amastigote. However, when life cycle phenotyping (bar-seq) was performed on a pool including 15 barcoded DUB null mutants created in promastigotes using CRISPR-Cas9, significant loss of fitness was observed during differentiation and intracellular infection. DUBs 4, 7, and 13 are required for successful transformation from metacyclic promastigote to amastigote and DUBs 3, 5, 6, 8, 10, 11 and 14 are required for normal amastigote proliferation in mice. DUBs 1, 2, 12 and 16 are essential for promastigote viability and the essential role of DUB2 in establishing infection was demonstrated using DiCre inducible gene deletion in vitro and in vivo. DUB2 is found in the nucleus and interacts with nuclear proteins associated with transcription/chromatin dynamics, mRNA splicing and mRNA capping. DUB2 has broad linkage specificity, cleaving all the di-ubiquitin chains except for Lys27 and Met1. Our study demonstrates the crucial role that DUBs play in differentiation and intracellular survival of Leishmania and that amastigotes are exquisitely sensitive to disruption of ubiquitination homeostasis.
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http://dx.doi.org/10.1371/journal.ppat.1008455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7319358PMC
June 2020

Photoactivated cell-killing involving a low molecular weight, donor-acceptor diphenylacetylene.

Chem Sci 2019 May 21;10(17):4673-4683. Epub 2019 Mar 21.

Department of Biosciences , Durham University , South Road , Durham, DH1 3LE , UK.

Photoactivation of photosensitisers can be utilised to elicit the production of ROS, for potential therapeutic applications, including the destruction of diseased tissues and tumours. A novel class of photosensitiser, exemplified by , has been designed possessing a modular, low molecular weight and 'drug-like' structure which is bioavailable and can be photoactivated by UV-A/405 nm or corresponding two-photon absorption of near-IR (800 nm) light, resulting in powerful cytotoxic activity, ostensibly through the production of ROS in a cellular environment. A variety of cellular assays confirmed ROS formation and cytotoxic activity was exemplified irradiation and subsequent targeted destruction of specific areas of a zebrafish embryo.
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http://dx.doi.org/10.1039/c9sc00199aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6495688PMC
May 2019

The mRNA-bound Proteome of : Novel Genetic Insight into an Ancient Parasite.

Mol Cell Proteomics 2019 07 4;18(7):1271-1284. Epub 2019 Apr 4.

From the ‡Centre for Immunology and Infection,. Electronic address:

parasite infections, termed the leishmaniases, cause significant global infectious disease burden. The lifecycle of the parasite embodies three main stages that require precise coordination of gene regulation to survive environmental shifts between sandfly and mammalian hosts. Constitutive transcription in kinetoplastid parasites means that gene regulation is overwhelmingly reliant on post-transcriptional mechanisms, yet strikingly few -regulators are known. Using optimized crosslinking and deep, quantified mass spectrometry, we present a comprehensive analysis of 1400 mRNA binding proteins (mRBPs) and whole cell proteomes from the three main lifecycle stages. Supporting the validity, although the crosslinked RBPome is magnitudes more enriched, the protein identities of the crosslinked and non-crosslinked RBPomes were nearly identical. Moreover, multiple candidate RBPs were endogenously tagged and found to associate with discrete mRNA target pools in a stage-specific manner. Results indicate that in parasites, mRNA levels are not a strong predictor of the whole cell expression or RNA binding potential of encoded proteins. Evidence includes a low correlation between transcript and corresponding protein expression and stage-specific variation in protein expression RNA binding potential. Unsurprisingly, RNA binding protein enrichment correlates strongly with relative replication efficiency of the specific lifecycle stage. Our study is the first to quantitatively define and compare the mRBPome of multiple stages in kinetoplastid parasites. It provides novel, in-depth insight into the -regulatory mRNA:Protein (mRNP) complexes that drive parasite lifecycle progression.
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http://dx.doi.org/10.1074/mcp.RA118.001307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6601212PMC
July 2019

miR-132 suppresses transcription of ribosomal proteins to promote protective Th1 immunity.

EMBO Rep 2019 04 4;20(4). Epub 2019 Mar 4.

Centre for Immunology and Infection and York Biomedical Research Institute, Hull York Medical School and Department of Biology, University of York, York, UK

Determining the mechanisms that distinguish protective immunity from pathological chronic inflammation remains a fundamental challenge. miR-132 has been shown to play largely immunoregulatory roles in immunity; however, its role in CD4 T cell function is poorly understood. Here, we show that CD4 T cells express high levels of miR-132 and that T cell activation leads to miR-132 up-regulation. The transcriptomic hallmark of splenic CD4 T cells lacking the miR-132/212 cluster during chronic infection is an increase in mRNA levels of ribosomal protein (RP) genes. BTAF1, a co-factor of B-TFIID and novel miR-132/212-3p target, and p300 contribute towards miR-132/212-mediated regulation of RP transcription. Following infection with CD4 T cells display enhanced expression of IL-10 and decreased IFNγ. This is associated with reduced hepatosplenomegaly and enhanced pathogen load. The enhanced IL-10 expression in Th1 cells is recapitulated following treatment with phenylephrine, a drug reported to promote ribosome synthesis. Our results uncover that miR-132/212-mediated regulation of RP expression is critical for optimal CD4 T cell activation and protective immunity against pathogens.
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http://dx.doi.org/10.15252/embr.201846620DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6446204PMC
April 2019

Fluorescent Retinoic Acid Analogues as Probes for Biochemical and Intracellular Characterization of Retinoid Signaling Pathways.

ACS Chem Biol 2019 03 13;14(3):369-377. Epub 2019 Feb 13.

Department of Biosciences , Durham University , South Road , Durham DH1 3LE , U.K.

Retinoids, such as all- trans-retinoic acid (ATRA), are endogenous signaling molecules derived from vitamin A that influence a variety of cellular processes through mediation of transcription events in the cell nucleus. Because of these wide-ranging and powerful biological activities, retinoids have emerged as therapeutic candidates of enormous potential. However, their use has been limited, to date, due to a lack of understanding of the complex and intricate signaling pathways that they control. We have designed and synthesized a family of synthetic retinoids that exhibit strong, intrinsic, solvatochromatic fluorescence as multifunctional tools to interrogate these important biological activities. We utilized the unique photophysical characteristics of these fluorescent retinoids to develop a novel in vitro fluorometric binding assay to characterize and quantify their binding to their cellular targets, including cellular retinoid binding protein II (CRABPII). The dihydroquinoline retinoid, DC360, exhibited particularly strong binding ( K = 34.0 ± 2.5 nM), and we further used X-ray crystallography to determine the structure of the DC360-CRABPII complex to 1.8 Å, which showed that DC360 occupies the known hydrophobic retinoid binding pocket. Finally, we used confocal fluorescence microscopy to image the cellular behavior of the compounds in cultured human epithelial cells, highlighting a fascinating nuclear localization, and used RNA sequencing to confirm that the compounds regulate cellular processes similar to those of ATRA. We anticipate that the unique properties of these fluorescent retinoids can now be used to cast new light on the vital and highly complex retinoid signaling pathway.
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http://dx.doi.org/10.1021/acschembio.8b00916DOI Listing
March 2019

Maintenance of epigenetic landscape requires CIZ1 and is corrupted in differentiated fibroblasts in long-term culture.

Nat Commun 2019 01 28;10(1):460. Epub 2019 Jan 28.

Department of Biology, University of York, York, YO10 5DD, UK.

The inactive X chromosome (Xi) serves as a model for establishment and maintenance of repressed chromatin and the function of polycomb repressive complexes (PRC1/2). Here we show that Xi transiently relocates from the nuclear periphery towards the interior during its replication, in a process dependent on CIZ1. Compromised relocation of Xi in CIZ1-null primary mouse embryonic fibroblasts is accompanied by loss of PRC-mediated H2AK119Ub1 and H3K27me3, increased solubility of PRC2 catalytic subunit EZH2, and genome-wide deregulation of polycomb-regulated genes. Xi position in S phase is also corrupted in cells adapted to long-term culture (WT or CIZ1-null), and also accompanied by specific changes in EZH2 and its targets. The data are consistent with the idea that chromatin relocation during S phase contributes to maintenance of epigenetic landscape in primary cells, and that elevated soluble EZH2 is part of an error-prone mechanism by which modifying enzyme meets template when chromatin relocation is compromised.
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http://dx.doi.org/10.1038/s41467-018-08072-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6484225PMC
January 2019

Epidermal growth factor can signal via β-catenin to control proliferation of mesenchymal stem cells independently of canonical Wnt signalling.

Cell Signal 2019 01 1;53:256-268. Epub 2018 Oct 1.

Department of Biology, University of York, York YO10 5DD, United Kingdom. Electronic address:

Bone marrow mesenchymal stem/stromal cells (MSCs) maintain bone homeostasis and repair through the ability to expand in response to mitotic stimuli and differentiate into skeletal lineages. Signalling mechanisms that enable precise control of MSC function remain unclear. Here we report that by initially examining differences in signalling pathway expression profiles of individual MSC clones, we identified a previously unrecognised signalling mechanism regulated by epidermal growth factor (EGF) in primary human MSCs. We demonstrate that EGF is able to activate β-catenin, a key component of the canonical Wnt signalling pathway. EGF is able to induce nuclear translocation of β-catenin in human MSCs but does not drive expression of Wnt target genes or T cell factor (TCF) activity in MSC reporter cell lines. Using an efficient Design of Experiments (DoE) statistical analysis, with different combinations and concentrations of EGF and Wnt ligands, we were able to confirm that EGF does not influence the Wnt/β-catenin pathway in MSCs. We show that the effects of EGF on MSCs are temporally regulated to initiate early "classical" EGF signalling mechanisms (e.g via mitogen activated protein kinase) with delayed activation of β-catenin. By RNA-sequencing, we identified gene sets that were exclusively regulated by the EGF/β-catenin pathway, which were distinct from classical EGF-regulated genes. However, subsets of classical EGF gene targets were significantly influenced by EGF/β-catenin activation. These signalling pathways cooperate to enable EGF-mediated proliferation of MSCs by alleviating the suppression of cell cycle pathways induced by classical EGF signalling.
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http://dx.doi.org/10.1016/j.cellsig.2018.09.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6293317PMC
January 2019

Microexon gene transcriptional profiles and evolution provide insights into blood processing by the Schistosoma japonicum esophagus.

PLoS Negl Trop Dis 2018 02 12;12(2):e0006235. Epub 2018 Feb 12.

Departamento de Ciências Biológicas, Universidade Federal de Ouro Preto, Campus Morro do Cruzeiro, Ouro Preto, Minas Gerais, Brasil.

Background: Adult schistosomes have a well-developed alimentary tract comprising an oral sucker around the mouth, a short esophagus and a blind ending gut. The esophagus is not simply a muscular tube for conducting blood from the mouth to gut but is divided into compartments, surrounded by anterior and posterior glands, where processing of ingested blood is initiated. Self-cure of rhesus macaques from a Schistosoma japonicum infection appears to operate by blocking the secretory functions of these glands so that the worms cease feeding and slowly starve to death. Here we use subtractive RNASeq to characterise the genes encoding the principal secretory products of S. japonicum esophageal glands, preparatory to evaluating their relevance as targets of the self-cure process.

Methodology/principal Findings: The heads and a small portion of the rear end of male and female S. japonicum worms were separately enriched by microdissection, for mRNA isolation and library construction. The sequence reads were then assembled de novo using Trinity and those genes enriched more than eightfold in the head preparation were subjected to detailed bioinformatics analysis. Of the 62 genes selected from the male heads, more than one third comprised MEGs encoding secreted or membrane-anchored proteins. Database searching using conserved motifs revealed that the MEG-4 and MEG-8/9 families had counterparts in the bird schistosome Trichobilharzia regenti, indicating an ancient association with blood processing. A second group of MEGs, including a MEG-26 family, encoded short peptides with amphipathic properties that most likely interact with ingested host cell membranes to destabilise them. A number of lysosomal hydrolases, two protease inhibitors, a secreted VAL and a putative natterin complete the line-up. There was surprisingly little difference between expression patterns in males and females despite the latter processing much more blood.

Significance/conclusions: The mixture of approximately 40 proteins specifically secreted by the esophageal glands is responsible for initiating blood processing in the adult worm esophagus. They comprise the potential targets for the self-cure process in the rhesus macaque, and thus represent a completely new cohort of secreted proteins that can be investigated as vaccine candidates.
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http://dx.doi.org/10.1371/journal.pntd.0006235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5825161PMC
February 2018

Motion and actions in language: semantic representations in occipito-temporal cortex.

Brain Lang 2013 Apr 28;125(1):94-105. Epub 2013 Feb 28.

Department of Psychology, University of York, Heslington YO10 5DD, UK.

Understanding verbs typically activates posterior temporal regions and, in some circumstances, motion perception area V5. However, the nature and role of this activation remains unclear: does language alone indeed activate V5? And are posterior temporal representations modality-specific motion representations, or supra-modal motion-independent event representations? Here, we address these issues by investigating human and object motion sentences compared to corresponding state descriptions. We adopted the blank screen paradigm, which is known to encourage visual imagery, and used a localizer to identify V5 and temporal structures responding to motion. Analyses in each individual brain suggested that language modulated activity in the posterior temporal lobe but not within V5 in most participants. Moreover, posterior temporal structures strongly responded to both motion sentences and human static sentences. These results suggest that descriptive language alone need not recruit V5 and instead engages more schematic event representations in temporal cortex encoding animacy and motion.
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http://dx.doi.org/10.1016/j.bandl.2013.01.008DOI Listing
April 2013

Image-invariant responses in face-selective regions do not explain the perceptual advantage for familiar face recognition.

Cereb Cortex 2013 Feb 17;23(2):370-7. Epub 2012 Feb 17.

Department of Psychology, York Neuroimaging Centre, University of York, York YO10 5DD, UK.

The ability to recognize familiar faces across different viewing conditions contrasts with the inherent difficulty in the perception of unfamiliar faces across similar image manipulations. It is widely believed that this difference in perception and recognition is based on the neural representation for familiar faces being less sensitive to changes in the image than it is for unfamiliar faces. Here, we used an functional magnetic resonance-adaptation paradigm to investigate image invariance in face-selective regions of the human brain. We found clear evidence for a degree of image-invariant adaptation to facial identity in face-selective regions, such as the fusiform face area. However, contrary to the predictions of models of face processing, comparable levels of image invariance were evident for both familiar and unfamiliar faces. This suggests that the marked differences in the perception of familiar and unfamiliar faces may not depend on differences in the way multiple images are represented in core face-selective regions of the human brain.
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http://dx.doi.org/10.1093/cercor/bhs024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3539454PMC
February 2013