Biology subjects

Graham, S.

Publications and source records attributed to Graham, S..

48 records · Page 3Linked to original sources

bioRxiv2020

The dynamic interplay of host and viral enzymes in type III CRISPR-mediated cyclic nucleotide signalling

Cyclic nucleotide second messengers are increasingly implicated in prokaryotic anti-viral defence systems. Type III CRISPR systems synthesise cyclic oligoadenylate (cOA) upon detecting foreign RNA, activating ancillary nucleases that can be toxic to cells, necessitating mechanisms to remove cOA in systems that operate via immunity rather than abortive infection. Previously, we demonstrated that the Sulfolobus solfataricus type III-D CRISPR complex generates cyclic tetra-adenylate (cA4), activating the ribonuclease Csx1, and showed that subsequent RNA cleavage and dissociation acts as an "off-switch" for the cyclase activity (Rouillon et al., 2018). Subsequently, we identified the cellular ring nuclease Crn1, which slowly degrades cA4 to reset the system, and demonstrated that viruses can subvert type III CRISPR immunity by means of a potent anti-CRISPR ring nuclease variant. Here, we present a comprehensive analysis of the dynamic interplay between these enzymes, governing cyclic nucleotide levels and infection outcomes in virus-host conflict.

biochemistry↗

bioRxiv2019

Expression of inhibitory checkpoint ligands by Glioblastoma Multiforme cells and the implications of an enhanced stem cell-like phenotype

Glioblastoma Multiforme is a highly aggressive brain malignancy commonly refractory to classical and novel chemo-, radio- and immuno-therapies, with median survival times of ~15 months following diagnosis. Poor immunological responses exemplified by the down-regulation of T-cell activity, and upregulation of immunosuppressive cells within the tumour micro-environment have limited the effectiveness of immunotherapy in GBM to date. Here we show that GBM cells express a large repertoire of inhibitory checkpoint ligands. Furthermore, GBM cells with an enhanced stem cell-like phenotype exhibit heightened levels of inhibitory checkpoint ligands, compared to non-stem cell-like GBM cells. Understanding how GBM modulates an extensive repertoire of immune checkpoint ligands and the functional consequence on immune evasion are necessary to develop effective immuno-therapeutics.

cell biology↗

bioRxiv2019

Structure and mechanism of a Type III CRISPR defence DNA nuclease activated by cyclic oligoadenylate

The CRISPR system provides adaptive immunity against mobile genetic elements in prokaryotes. On binding invading RNA species, Type III CRISPR systems generate cyclic oligoadenylate (cOA) molecules which act as a second messenger, signalling infection and potentiating a powerful immune response by activating a range of downstream effector proteins that can lead to viral clearance, cell dormancy or death. Only one type of effector enzyme has been studied - the Csm6/Csx1 ribonuclease domain, and the mechanism of cOA activation is not understood at a molecular level. Here we describe the structure and mechanism of a novel cOA-activated CRISPR defence DNA endonuclease, Can1 (\"CRISPR ancillary nuclease 1\"). Can1 has a unique monomeric structure with two CRISPR associated Rossman fold (CARF) CARF domains and two DNA nuclease-like domains. The crystal structure of the enzyme has been captured in the activated state, with a cyclic tetra-adenylate (cA4) molecule bound at the core of the protein. cA4 binding reorganises the structure to license a metal-dependent DNA nuclease activity specific for nicking of supercoiled DNA. DNA nicking by Can1 is predicted to slow down viral replication kinetics by leading to the collapse of DNA replication forks.

biochemistry↗

bioRxiv2019

A viral ring nuclease anti-CRISPR subverts type III CRISPR immunity

The CRISPR system provides adaptive immunity against mobile genetic elements in bacteria and archaea. On detection of viral RNA, type III CRISPR systems generate a cyclic oligoadenylate (cOA) second messenger1-3, activating defence enzymes and sculpting a powerful antiviral response that can drive viruses to extinction4,5. Cyclic nucleotides are increasingly implicated as playing an important role in host-pathogen interactions6,7. Here, we identify a widespread new family of viral anti-CRISPR (Acr) enzymes that rapidly degrade cyclic tetra-adenylate (cA4). The viral ring nuclease (AcrIII-1) is the first Acr described for type III CRISPR systems and is widely distributed in archaeal and bacterial viruses, and proviruses. The enzyme uses a novel fold to bind cA4 specifically and utilizes a conserved active site to rapidly cleave the signalling molecule, allowing viruses to neutralise the type III CRISPR defence system. The AcrIII-1 family has a broad host range as it targets cA4 signalling molecules rather than specific CRISPR effector proteins. This study highlights the crucial role of cyclic nucleotide signalling in the conflict between viruses and their hosts.

biochemistry↗

bioRxiv2019

Cannabinoid Receptor 2 (CB2) Signals via G-alpha-s and Induces IL-6 and IL-10 Cytokine Secretion in Primary Human Leukocytes

Cannabinoid receptor 2 (CB2) is a promising therapeutic target for immunological modulation. There is, however, a deficit of knowledge regarding CB2 signaling and function in human primary immunocompetent cells. We applied an experimental paradigm which closely models the in situ state of human primary leukocytes (PBMC; peripheral blood mononuclear cells) to characterize activation of a number of signaling pathways in response to a CB2-selective ligand (HU308). We observed a \"lag\" phase of unchanged cAMP concentration prior to development of classically-expected Gi-mediated inhibition of cAMP synthesis. Application of G protein inhibitors revealed that this apparent lag was a result of counteraction of Gi effects by concurrent Gs activation. Monitoring downstream signaling events, activation of p38 was mediated by Gi whereas ERK1/2 and Akt phosphorylation were mediated by Gi-coupled {beta}{gamma}. Activation of CREB integrated multiple components; Gs and {beta}{gamma} mediated [~]85% of the response, while [~]15% was attributed to Gi. Responses to HU308 had an important functional outcome - secretion of interleukins 6 (IL-6) and 10 (IL-10). IL-2, IL-4, IL-12, IL-13, IL-17A, MIP-1, and TNF- were unaffected. IL-6/IL-10 induction had a similar G protein coupling profile to CREB activation. All response potencies were consistent with that expected for HU308 acting via CB2. Additionally, signaling and functional effects were completely blocked by a CB2-selective inverse agonist, giving additional evidence for CB2 involvement. This work expands the current paradigm regarding cannabinoid immunomodulation and reinforces the potential utility of CB2 ligands as immunomodulatory therapeutics.\n\nSignificance statementCannabinoid receptor 2 (CB2) is a G protein-coupled receptor which plays a complex role in immunomodulation and is a promising target in a range of disorders with immune system involvement. However, to date the majority of the studies in this field have been performed on cell lines, rodent models, or stimulated primary cells. Here we provide a detailed account of CB2-mediated signaling in primary human immune cells under conditions which closely mimic their in vivo state. We reveal a complex signaling system involving an unprecedented CB2 signaling pathway and leading to immunomodulatory functional outcomes. This work provides not only a critical foundation impacting CB2-targeted drug discovery, but reveals important wider considerations for GPCR signaling studies and model validity.\n\nTable of Contents Summary Figure\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC=\"FIGDIR/small/663831v2_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (22K):\norg.highwire.dtl.DTLVardef@599095org.highwire.dtl.DTLVardef@2331a9org.highwire.dtl.DTLVardef@917860org.highwire.dtl.DTLVardef@75e8f7_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2019

Cyclic oligoadenylate signalling mediates Mycobacterium tuberculosis CRISPR defence

The CRISPR system provides adaptive immunity against mobile genetic elements (MGE) in prokaryotes. In type III CRISPR systems, an effector complex programmed by CRISPR RNA detects invading RNA, triggering a multi-layered defence that includes target RNA cleavage, licencing of an HD DNA nuclease domain and synthesis of cyclic oligoadenylate (cOA) molecules. cOA activates the Csx1/Csm6 family of effectors, which degrade RNA non-specifically to enhance immunity. Type III systems are found in diverse archaea and bacteria, including the human pathogen Mycobacterium tuberculosis. Here, we report a comprehensive analysis of the in vitro and in vivo activities of the type III-A M. tuberculosis CRISPR system. We demonstrate that immunity against MGE is achieved predominantly via a cyclic hexa-adenylate (cA6) signalling pathway and the ribonuclease Csm6, rather than through DNA cleavage by the HD domain. Furthermore, we show that the mechanism can be reprogrammed to operate as a cyclic tetra-adenylate (cA4) system by replacing the effector protein. These observations demonstrate that M. tuberculosis has a fully-functioning CRISPR interference system that generates a range of cyclic and linear oligonucleotides of known and unknown functions, potentiating fundamental and applied studies.

biochemistry↗

bioRxiv2019

Mendelian Randomization Analysis Dissects the Relationship between NAFLD, T2D, and Obesity and Provides Implications to Precision Medicine

BackgroundNon-alcoholic fatty liver disease (NAFLD) is epidemiologically correlated with both type 2 diabetes (T2D) and obesity. However, the causal inter-relationships among the three diseases have not been completely investigated.\n\nAimWe aim to explore the causal relationships among the three diseases.\n\nDesign and methodsWe performed a genome-wide association study (GWAS) on fatty liver disease in [~]400,000 UK BioBank samples. Using this data as well as the largest-to-date publicly available summary-level GWAS data, we performed a two-sample bidirectional Mendelian Randomization (MR) analysis. This analysis tested the causal inter-relationship between NAFLD, T2D, and obesity, as well as the association between genetically driven NAFLD (with two well-established SNPs at the PNPLA3 and TM6SF2 loci) and glycemic and lipidemic traits, respectively. Transgenic mice expressing the human PNPLA3 I148I (TghPNPLA3-I148I) and PNPLA3 I148M (TghPNPLA3-I148M) isoforms were used to further validate the causal effects.\n\nResultsWe found that genetically instrumented hepatic steatosis significantly increased the risk for T2D (OR=1.3, 95% CI: [1.2, 1.4], p=8.3e-14) but not the intermediate glycemic phenotypes at the Bonferroni-adjusted level of significance (p<0.002). There was a moderate, but significant causal association between genetically driven hepatic steatosis and decreased risk for BMI ({beta}=- 0.027 SD, 95%CI: [-0.043, -0.01], p=1.3e-4), but an increased risk for WHRadjBMI (Waist-Hip Ratio adjusted for BMI) ({beta}=0.039 SD, 95%CI: [0.023, 0.054], p=8.2e-7), as well as a decreased level for total cholesterol ({beta}=-0.084 SD, 95%CI [-0.13, -0.036], p=6.8e-4), but not triglycerides ({beta}=0.02 SD, 95%CI [-0.023, 0.062], p=0.36). The reverse MR analyses suggested that genetically driven T2D (OR=1.1, 95% CI: [1.0, 1.2], p=1.7e-3), BMI (OR=2.3, 95% CI: [2.0, 2.7], p=1.4e-25) and WHRadjBMI (OR=1.5, 95% CI: [1.3, 1.8], p=1.1e-6) causally increase the NAFLD risk. In the animal study, as compared to the TghPNPLA3-I148I controls, the TghPNPLA3-I148M mice developed higher fasting glucose level and reduced glucose clearance. Meanwhile, the TghPNPLA3-I148M mice demonstrated a reduced body weight, increased central to peripheral fat ratio, decreased circulating total cholesterol as compared to the TghPNPLA3-I148I controls.\n\nConclusionThis large-scale bidirectional MR study suggests that lifelong, genetically driven NAFLD is a causal risk factor for T2D (hence potentially a \"NAFLD-driven T2D\" subtype) and central obesity (or \"NAFLD-driven obesity\" subtype), but protects against overall obesity; while genetically driven T2D, obesity, and central obesity also causally increase the risk of NAFLD, hence a \"metabolic NAFLD\". This causal relationship revealed new insights into disease subtypes and provided novel hypotheses for precision treatment or prevention for the three diseases.

epidemiology↗

bioRxiv2019

Validation of a CRISPR/cas9-based technology platform for examining specific immune gene functions in an experimental murine model of IBD

Inflammatory bowel diseases (IBD) are complex, multifactorial disorders characterized by chronic relapsing intestinal inflammation. Association studies have identified hundreds of genes that are linked to IBD and potentially regulate its pathology. The further dissection of the genetic network underlining IBD pathogenesis and pathophysiology is hindered by the limited capacity to investigate the role of each GWAS association through functional studies, including the generation of knockout animal models for each of the associated genes. The CRISPR/Cas9 system represents a cutting edge technology which has the potential to transform the field of IBD research by facilitating the introduction of genetic alterations in an efficient and effective manner. Using the CD40-mediated-colitis model, our results demonstrate the validity of a CRISPR/Cas9-based platform as a tool for the validation of target genes or interference strategies in experimental IBD. The utilization of this discovery strategy will allow for the timely in vivo validation of therapeutic targets as the rapidly emerge from current genetic and genomics efforts with human disease tissue. As such, the CRISPR/Cas9-based platform can significantly shorten the time span between target identification and generation of proof of principle experiments for drug discovery.

immunology↗

bioRxiv2019

A type III CRISPR ancillary ribonuclease degrades its cyclic oligoadenylate activator

Cyclic oligoadenylate (cOA) secondary messengers are generated by type III CRISPR systems in response to viral infection. cOA allosterically activates the CRISPR ancillary ribonucleases Csx1/Csm6, which degrade RNA non-specifically using a HEPN (Higher Eukaryotes and Prokaryotes, Nucleotide binding) active site. This provides effective immunity, but can also lead to growth arrest in infected cells, necessitating a means to deactivate the ribonuclease once viral infection has been cleared. In the crenarchaea, dedicated ring nucleases degrade cA4 (cOA consisting of 4 AMP units), but the equivalent enzyme has not been identified in bacteria. We demonstrate that, in Thermus thermophilus HB8, the uncharacterised protein TTHB144 is a cA4-activated HEPN ribonuclease that also degrades its activator. TTHB144 binds and degrades cA4 at an N-terminal CARF (CRISPR Associated Rossman Fold) domain. The two activities can be separated by site-directed mutagenesis. TTHB144 is thus the first example of a self-limiting CRISPR ribonuclease.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC=\"FIGDIR/small/582114_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (21K):\norg.highwire.dtl.DTLVardef@d4ac46org.highwire.dtl.DTLVardef@7a67b0org.highwire.dtl.DTLVardef@2d8c7aorg.highwire.dtl.DTLVardef@61b12a_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

biochemistry↗

bioRxiv2018

Disrupted reinforcement learning during post-error slowing in ADHD

ADHD is associated with altered dopamine regulated reinforcement learning on prediction errors. Despite evidence of categorically altered error processing in ADHD, neuroimaging advances have largely investigated models of normal reinforcement learning in greater detail. Further, although reinforcement leaning critically relies on ventral striatum exerting error magnitude related thresholding influences on substantia nigra (SN) and dorsal striatum, these thresholding influences have never been identified with neuroimaging. To identify such thresholding influences, we propose that error magnitude related activities must first be separated from opposite activities in overlapping neural regions during error detection. Here we separate error detection from magnitude related adjustment (post-error slowing) during inhibition errors in the stop signal task in typically developing (TD) and ADHD adolescents using fMRI. In TD, we predicted that: 1) deactivation of dorsal striatum on error detection interrupts ongoing processing, and should be proportional to right frontoparietal response phase activity that has been observed in the SST; 2) deactivation of ventral striatum on post-error slowing exerts thresholding influences on, and should be proportional to activity in dorsal striatum. In ADHD, we predicted that ventral striatum would instead correlate with heightened amygdala responses to errors. We found deactivation of dorsal striatum on error detection correlated with response-phase activity in both groups. In TD, post-error slowing deactivation of ventral striatum correlated with activation of dorsal striatum. In ADHD, ventral striatum correlated with heightened amygdala activity. Further, heightened activities in locus coeruleus (norepinephrine), raphe nucleus (serotonin) and medial septal nuclei (acetylcholine), which all compete for control of DA, and are altered in ADHD, exhibited altered correlations with SN. All correlations in TD were replicated in healthy adults. Results in TD are consistent with dopamine regulated reinforcement learning on post-error slowing. In ADHD, results are consistent with heightened activities in the amygdala and non-dopaminergic neurotransmitter nuclei preventing reinforcement learning.

neuroscience↗

bioRxiv2018

Ring nucleases deactivate Type III CRISPR ribonucleases by degrading cyclic oligoadenylate

The CRISPR-Cas system provides adaptive immunity against mobile genetic elements in prokaryotes, utilising small CRISPR RNAs which direct effector complexes to degrade invading entities. Type III effector complexes were recently demonstrated to synthesise a novel second messenger, cyclic oligoadenylate (cOA), on binding target RNA. cOA in turn binds to and activates a range of downstream effector proteins including ribonucleases (Csm6/Csx1) and transcription factors via a CARF (CRISPR associated Rossman Fold) domain, inducing an antiviral state in the cell that is important for immunity. The mechanism of the \"off-switch\" that resets the system is not understood. Here, we report the identification of the nuclease that degrades these cOA ring molecules. The \"Ring nuclease\" is itself a CARF family protein with a metal independent mechanism, which cleaves cOA4 rings to generate linear di-adenylate species and switches off the antiviral state. The identification of Ring nucleases adds an important insight to the CRISPR-Cas system.

biochemistry↗

bioRxiv2017

Variation in context dependent foraging behavior across pollinators

Pollinator foraging behavior has direct consequences for plant reproduction and has been implicated in driving floral trait evolution. Exploring the degree to which pollinators exhibit flexibility in foraging behavior will add to a mechanistic understanding of how pollinators can impact selection on plant traits. Although plants have evolved suites of floral traits to attract pollinators, flower color is a particularly important aspect of the floral display. Some pollinators show strong innate color preference, but many pollinators display flexibility in preference due to learning associations between rewards and color, or due to variable perception of color in different environments or plant communities. This study examines the flexibility in flower color preference of two groups of native butterfly pollinators under natural field conditions. Our study reveals that pipevine swallowtails and skippers, the predominate pollinators of the two native Texas Phlox species, display distinct patterns of color preferences across different contexts. Pipevine swallowtails exhibit highly flexible color preferences and likely utilize other floral traits to make foraging decisions. In contrast, skippers have consistent color preferences and likely use flower color as a primary cue for foraging. As a result of this variation in color preference flexibility, the two pollinator groups impose concordant selection on flower color in some contexts but discordant selection in other contexts. This variability could have profound implications for how flower traits respond to pollinator-mediated selection. Our findings suggest that studying dynamics of behavior in natural field conditions is important for understanding plant-pollinator interactions.

animal behavior and cognition↗