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Hepatitis B virus (HBV) is the prototype member of the family Hepadnaviridae and replicates via episomal copies of a covalently closed circular DNA (cccDNA) genome of approximately 3.2 kb. The chromatinization of this small viral genome, with overlapping open reading frames and regulatory elements, suggests an important role for epigenetic pathways to regulate HBV transcription. However, the host pathways that regulate HBV transcription and the temporal nature of promoter usage in infected cells are not well understood, in part due to the compact genome structure and overlapping open reading frames. To address this we developed a simple and cost-effective PCR assay to quantify the major viral RNAs and validated this technique using current state-of-art de novo HBV infection model systems. Our PCR method is three orders of magnitude more sensitive than Northern blot and requires relatively small amounts of starting material, making this an attractive tool for assessing HBV transcription.
Male sex chromosomal complement exacerbates the pathogenicity of Th17 cells in a chronic model of central nervous system autoimmunity.
Sex differences in multiple sclerosis (MS) incidence and severity have long been recognized. However, the underlying cellular and molecular mechanisms for why male sex is associated with more aggressive disease remain poorly defined. Using a T cell adoptive transfer model of chronic experimental autoimmune encephalomyelitis (EAE), we find that male Th17 cells induce disease of increased severity relative to female Th17 cells, irrespective of whether transferred to male or female recipients. Throughout the disease course, a greater frequency of male Th17 cells produce IFNγ, a hallmark of pathogenic Th17 responses. Intriguingly, XY chromosomal complement increases the pathogenicity of male Th17 cells. An X-linked immune regulator, Jarid1c, is downregulated in pathogenic male murine Th17 cells, and functional experiments reveal that it represses the severity of Th17-mediated EAE. Furthermore, Jarid1c expression is downregulated in CD4<sup>+</sup> T cells from MS-affected individuals. Our data indicate that male sex chromosomal complement critically regulates Th17 cell pathogenicity.
RHO to the DOCK for GDP disembarking: structural insights into the DOCK GTPase nucleotide exchange factors.
The human dedicator of cytokinesis (DOCK) family consists of 11 structurally conserved proteins that serve as atypical RHO guanine nucleotide exchange factors (RHO GEFs). These regulatory proteins act as mediators in numerous cellular cascades that promote cytoskeletal remodelling, playing roles in various crucial processes such as differentiation, migration, polarisation and axon growth in neurons. At the molecular level, DOCK DHR2 domains facilitate nucleotide dissociation from small GTPases, a process which is otherwise too slow for rapid spatiotemporal control of cellular signalling. Here, we provide an overview of the biological and structural characteristics for the various DOCK proteins and describe how they differ from other RHO GEFs and between DOCK sub-families. The expression of the family varies depending on cell or tissue type, and they are consequently implicated in a broad range of disease phenotypes, particularly in the brain. A growing body of available structural information reveals the mechanism by which the catalytic DHR2 domain elicits nucleotide dissociation and also indicates strategies for the discovery and design of high-affinity small molecule inhibitors. Such compounds could serve as chemical probes to interrogate the cellular function and provide starting points for drug discovery of this important class of enzymes.
Ubiquitin is a versatile posttranslational modification, which is covalently attached to protein targets either as a single moiety or as a ubiquitin chain. In contrast to K48 and K63-linked chains, which have been extensively studied, the regulation and function of most atypical ubiquitin chains are only starting to emerge. The deubiquitinase TRABID/ZRANB1 is tuned for the recognition and cleavage of K29 and K33-linked chains. Yet, substrates of TRABID and the cellular functions of these atypical ubiquitin signals remain unclear. We determined the interactome of two TRABID constructs rendered catalytic dead either through a point mutation in the catalytic cysteine residue or through removal of the OTU catalytic domain. We identified 50 proteins trapped by both constructs and which therefore represent candidate substrates of TRABID. The E3 ubiquitin ligase HECTD1 was then validated as a substrate of TRABID and used UbiCREST and Ub-AQUA proteomics to show that HECTD1 preferentially assembles K29- and K48-linked ubiquitin chains. Further in vitro autoubiquitination assays using ubiquitin mutants established that while HECTD1 can assemble short homotypic K29 and K48-linked chains, it requires branching at K29/K48 in order to achieve its full ubiquitin ligase activity. We next used transient knockdown and genetic knockout of TRABID in mammalian cells in order to determine the functional relationship between TRABID and HECTD1. This revealed that upon TRABID depletion, HECTD1 is readily degraded. Thus, this study identifies HECTD1 as a mammalian E3 ligase that assembles branched K29/K48 chains and also establishes TRABID-HECTD1 as a DUB/E3 pair regulating K29 linkages.
The origins, prevalence and nature of dairying have been long debated by archaeologists. Within the last decade, new advances in high-resolution mass spectrometry have allowed for the direct detection of milk proteins from archaeological remains, including ceramic residues, dental calculus, and preserved dairy products. Proteins recovered from archaeological remains are susceptible to post-excavation and laboratory contamination, a particular concern for ancient dairying studies as milk proteins such as beta-lactoglobulin (BLG) and caseins are potential laboratory contaminants. Here, we examine how site-specific rates of deamidation (i.e., deamidation occurring in specific positions in the protein chain) can be used to elucidate patterns of peptide degradation, and authenticate ancient milk proteins. First, we characterize site-specific deamidation patterns in modern milk products and experimental samples, confirming that deamidation occurs primarily at low half-time sites. We then compare this to previously published palaeoproteomic data from six studies reporting ancient milk peptides. We confirm that site-specific deamidation rates, on average, are more advanced in BLG recovered from ancient dental calculus and pottery residues. Nevertheless, deamidation rates displayed a high degree of variability, making it challenging to authenticate samples with relatively few milk peptides. We demonstrate that site-specific deamidation is a useful tool for identifying modern contamination but highlight the need for multiple lines of evidence to authenticate ancient protein data.
Network Analysis of the CSF Proteome Characterizes Convergent Pathways of Cellular Dysfunction in ALS.
<h4>Background</h4>Amyotrophic lateral sclerosis is a clinical syndrome with complex biological determinants, but which in most cases is characterized by TDP-43 pathology. The identification in CSF of a protein signature of TDP-43 network dysfunction would have the potential to inform the identification of new biomarkers and therapeutic targets.<h4>Methods</h4>We compared CSF proteomic data from patients with ALS (<i>n</i> = 41), Parkinson's disease (<i>n</i> = 19) and healthy control participants (<i>n</i> = 20). Weighted correlation network analysis was used to identify modules within the CSF protein network and combined with gene ontology enrichment analysis to functionally annotate module proteins. Analysis of module eigenproteins and differential correlation analysis of the CSF protein network was used to compare ALS and Parkinson's disease protein co-correlation with healthy controls. In order to monitor temporal changes in the CSF proteome, we performed longitudinal analysis of the CSF proteome in a subset of ALS patients.<h4>Results</h4>Weighted correlation network analysis identified 10 modules, including those enriched for terms involved in gene expression including nucleic acid binding, RNA metabolism and translation; humoral immune system function, including complement pathways; membrane proteins, axonal outgrowth and adherence; and glutamatergic synapses. Immune system module eigenproteins were increased in ALS, whilst axonal module eigenproteins were decreased in ALS. The 19 altered protein correlations in ALS were enriched for gene expression (OR 3.05, <i>p</i> = 0.017) and membrane protein modules (OR 17.48, <i>p</i> = 0.011), including intramodular hub proteins previously identified as TDP-43 interactors. Proteins decreasing over longitudinal analysis ALS were enriched in glutamatergic synapse and axonal outgrowth modules. Protein correlation network disruptions in Parkinson's disease showed no module enrichment.<h4>Conclusions</h4>Alterations in the co-correlation network in CSF samples identified a set of pathways known to be associated with TDP-43 dysfunction in the pathogenesis of ALS, with important implications for therapeutic targeting and biomarker development.
ABPP-HT - High-Throughput Activity-Based Profiling of Deubiquitylating Enzyme Inhibitors in a Cellular Context.
The potency and selectivity of a small molecule inhibitor are key parameters to assess during the early stages of drug discovery. In particular, it is very informative for characterizing compounds in a relevant cellular context in order to reveal potential off-target effects and drug efficacy. Activity-based probes are valuable tools for that purpose, however, obtaining cellular target engagement data in a high-throughput format has been particularly challenging. Here, we describe a new methodology named ABPP-HT (high-throughput-compatible activity-based protein profiling), implementing a semi-automated proteomic sample preparation workflow that increases the throughput capabilities of the classical ABPP workflow approximately ten times while preserving its enzyme profiling characteristics. Using a panel of deubiquitylating enzyme (DUB) inhibitors, we demonstrate the feasibility of ABPP-HT to provide compound selectivity profiles of endogenous DUBs in a cellular context at a fraction of time as compared to previous methodologies.
Subtraction-free and bisulfite-free specific sequencing of 5-methylcytosine and its oxidized derivatives at base resolution
<jats:title>Abstract</jats:title><jats:p>Although various methods have been developed for sequencing cytosine modifications, it is still challenging for specific and quantitative sequencing of individual modification at base-resolution. For example, to obtain both true 5-methylcytosine (5mC) and true 5-hydroxymethylcytosine (5hmC) information, the two major epigenetic modifications, it usually requires subtraction of two methods, which increases noise and requires high sequencing depth. Recently, we developed TET-assisted pyridine borane sequencing (TAPS) for bisulfite-free direct sequencing of 5mC and 5hmC. Here we demonstrate that two sister methods, TAPSβ and chemical-assisted pyridine borane sequencing (CAPS), can be effectively used for subtraction-free and specific whole-genome sequencing of 5mC and 5hmC, respectively. We also demonstrate pyridine borane sequencing (PS) for whole-genome profiling of 5-formylcytosine and 5-carboxylcytosine, the further oxidized derivatives of 5mC and 5hmC. This work completes the set of versatile borane reduction chemistry-based methods as a comprehensive toolkit for direct and quantitative sequencing of all four cytosine epigenetic modifications.</jats:p>
Germline and somatic genetic variants in the p53 pathway interact to affect cancer risk, progression, and drug response.
Insights into oncogenesis derived from cancer susceptibility loci (single nucleotide polymorphisms, SNP) hold the potential to facilitate better cancer management and treatment through precision oncology. However, therapeutic insights have thus far been limited by our current lack of understanding regarding both interactions of these loci with somatic cancer driver mutations and their influence on tumorigenesis. For example, while both germline and somatic genetic variation to the p53 tumor suppressor pathway are known to promote tumorigenesis, little is known about the extent to which such variants cooperate to alter pathway activity. Here we hypothesize that cancer risk-associated germline variants interact with somatic TP53 mutational status to modify cancer risk, progression, and response to therapy. Focusing on a cancer risk SNP (rs78378222) with a well-documented ability to directly influence p53 activity as well as integration of germline datasets relating to cancer susceptibility with tumor data capturing somatically-acquired genetic variation provided supportive evidence for this hypothesis. Integration of germline and somatic genetic data enabled identification of a novel entry point for therapeutic manipulation of p53 activities. A cluster of cancer risk SNPs resulted in increased expression of pro-survival p53 target gene KITLG and attenuation of p53-mediated responses to genotoxic therapies, which were reversed by pharmacological inhibition of the pro-survival c-KIT signal. Together, our results offer evidence of how cancer susceptibility SNPs can interact with cancer driver genes to affect cancer progression and identify novel combinatorial therapies.
Terminating the SARS-CoV-2 pandemic relies upon pan-global vaccination. Current vaccines elicit neutralizing antibody responses to the virus spike derived from early isolates. However, new strains have emerged with multiple mutations, including P.1 from Brazil, B.1.351 from South Africa, and B.1.1.7 from the UK (12, 10, and 9 changes in the spike, respectively). All have mutations in the ACE2 binding site, with P.1 and B.1.351 having a virtually identical triplet (E484K, K417N/T, and N501Y), which we show confer similar increased affinity for ACE2. We show that, surprisingly, P.1 is significantly less resistant to naturally acquired or vaccine-induced antibody responses than B.1.351, suggesting that changes outside the receptor-binding domain (RBD) impact neutralization. Monoclonal antibody (mAb) 222 neutralizes all three variants despite interacting with two of the ACE2-binding site mutations. We explain this through structural analysis and use the 222 light chain to largely restore neutralization potency to a major class of public antibodies.
T cell assays differentiate clinical and subclinical SARS-CoV-2 infections from cross-reactive antiviral responses.
Identification of protective T cell responses against SARS-CoV-2 requires distinguishing people infected with SARS-CoV-2 from those with cross-reactive immunity to other coronaviruses. Here we show a range of T cell assays that differentially capture immune function to characterise SARS-CoV-2 responses. Strong ex vivo ELISpot and proliferation responses to multiple antigens (including M, NP and ORF3) are found in 168 PCR-confirmed SARS-CoV-2 infected volunteers, but are rare in 119 uninfected volunteers. Highly exposed seronegative healthcare workers with recent COVID-19-compatible illness show T cell response patterns characteristic of infection. By contrast, >90% of convalescent or unexposed people show proliferation and cellular lactate responses to spike subunits S1/S2, indicating pre-existing cross-reactive T cell populations. The detection of T cell responses to SARS-CoV-2 is therefore critically dependent on assay and antigen selection. Memory responses to specific non-spike proteins provide a method to distinguish recent infection from pre-existing immunity in exposed populations.
Inhaled budesonide for COVID-19 in people at higher risk of adverse outcomes in the community: interim analyses from the PRINCIPLE trial
<jats:p>BACKGROUND Inhaled budesonide has shown efficacy for treating COVID-19 in the community but has not yet been tested in effectiveness trials. METHODS We performed a multicenter, open-label, multi-arm, adaptive platform randomized controlled trial involving people aged ≥65 years, or ≥50 years with comorbidities, and unwell ≤14 days with suspected COVID-19 in the community (PRINCIPLE). Participants were randomized to usual care, usual care plus inhaled budesonide (800μg twice daily for 14 days), or usual care plus other interventions. The co-primary endpoints are time to first self-reported recovery, and hospitalization/death related to COVID-19, both measured over 28 days from randomisation and analysed using Bayesian models. RESULTS The trial opened on April 2, 2020. Randomization to inhaled budesonide began on November 27, 2020 and was stopped on March 31, 2021 based on an interim analysis using data from March 4, 2021. Here, we report updated interim analysis data from March 25, 2021, at which point the trial had randomized 4663 participants with suspected COVID-19. Of these, 2617 (56.1%) tested SARS-CoV-2 positive and contributed data to this interim budesonide primary analysis; 751 budesonide, 1028 usual care and 643 to other interventions. Time to first self-reported recovery was shorter in the budesonide group compared to usual care (hazard ratio 1.208 [95% BCI 1.076 - 1.356], probability of superiority 0.999, estimated benefit [95% BCI] of 3.011 [1.134 - 5.41] days). Among those in the interim budesonide primary analysis who had the opportunity to contribute data for 28 days follow up, there were 59/692 (8.5%) COVID-19 related hospitalizations/deaths in the budesonide group vs 100/968 (10.3%) in the usual care group (estimated percentage benefit, 2.1% [95% BCI -0.7% - 4.8%], probability of superiority 0.928). CONCLUSIONS In this updated interim analysis, inhaled budesonide reduced time to recovery by a median of 3 days in people with COVID-19 with risk factors for adverse outcomes. Once 28 day follow up is complete for all participants randomized to budesonide, final analyses of time to recovery and hospitalization/death will be published. (Funded by the National Institute of Health Research/ United Kingdom Research Innovation [MC_PC_19079]; PRINCIPLE ISRCTN number, <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="isrctn" xlink:href="86534580">ISRCTN86534580</jats:ext-link>.)</jats:p>
<p>Chemical probes are of great use for investigating target safety, viability, and translation. Alongside this, they act as useful structural templates to inspire drug discovery.</p>