Publications by authors named "Dene Littler"

Enzyme promiscuity is the ability of an enzyme to catalyze an unexpected side reaction in addition to its main reaction. Here, we describe a biocatalytic process to produce non-hydrolyzable NAD+ analogs based on the ADP-ribosyltransferase (ART) activity of pertussis toxin PtxS1 subunit. First, in identical manner to normal catalysis, PtxS1 activates NAD+ to form the reactive oxocarbenium cation.

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Long COVID occurs in a small but important minority of patients following COVID-19, reducing quality of life and contributing to healthcare burden. Although research into underlying mechanisms is evolving, immunity is understudied. SARS-CoV-2-specific T cell responses are of key importance for viral clearance and COVID-19 recovery.

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Article Synopsis
  • Most COVID-19 vaccines focus on the Spike protein, but mutations in the virus highlight the need for broader vaccine development.
  • The study uses mass spectrometry to identify immunopeptides from seven stable SARS-CoV-2 proteins, mapping their interactions with various Human Leukocyte Antigens (HLA) worldwide.
  • Out of 248 unique peptides found, over half are novel, and testing shows T cell responses to several peptides, which could help create improved COVID vaccines targeting multiple virus proteins.
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T cells in jawed vertebrates comprise two lineages, αβ T cells and γδ T cells, defined by the antigen receptors they express-that is, αβ and γδ T cell receptors (TCRs), respectively. The two lineages have different immunological roles, requiring that γδ TCRs recognize more structurally diverse ligands. Nevertheless, the receptors use shared CD3 subunits to initiate signalling.

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Respiratory infections cause significant morbidity and mortality, yet it is unclear why some individuals succumb to severe disease. In patients hospitalized with avian A(H7N9) influenza, we investigated early drivers underpinning fatal disease. Transcriptomics strongly linked oleoyl-acyl-carrier-protein (ACP) hydrolase (OLAH), an enzyme mediating fatty acid production, with fatal A(H7N9) early after hospital admission, persisting until death.

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Article Synopsis
  • Influenza B viruses (IBVs) significantly impact health, but the immune response, especially involving CD8 T-cells, is not well understood; only 18 T-cell epitopes have been identified so far.
  • This study identifies 9 new highly conserved CD8 T-cell epitopes linked to certain HLAs, which could enhance the development of more effective cross-reactive T-cell vaccines against IBVs.
  • Additionally, the research reveals that while the frequency of IBV-specific CD8 T-cells decreases with age, they retain a memory phenotype and exhibit unique T-cell receptor repertoires based on specific epitopes.
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The search for effective antiviral agents against SARS-CoV-2 remains a critical global endeavor. In this study, we focused on the viral nucleocapsid protein Nsp9, which is a key player in viral RNA replication and an attractive drug target. Employing a two-pronged approach, an in-house natural product library was screened using native mass spectrometry to identify compounds capable of binding to Nsp9.

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Article Synopsis
  • - The parasite Plasmodium falciparum, responsible for severe malaria, invades red blood cells by exporting hundreds of proteins that modify the host cell to enhance parasite growth and evade the immune system.
  • - These exported proteins contain a specific motif (PEXEL) that signals their processing and export, involving a proteolytic cleavage step in the parasite’s endoplasmic reticulum, which assists in the release of proteins into the host cell's vacuole.
  • - The study reveals that the PEXEL's sequence and a 'spacer' region between the PEXEL and functional protein regions are crucial for the protein's recognition and efficient transport by the PTEX complex into the red blood cells.
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Psoriasis is a chronic skin disease characterized by hyperproliferative epidermal lesions infiltrated by autoreactive T cells. Individuals expressing the human leukocyte antigen (HLA) C∗06:02 allele are at highest risk for developing psoriasis. An autoreactive T cell clone (termed Vα3S1/Vβ13S1) isolated from psoriatic plaques is selective for HLA-C∗06:02, presenting a peptide derived from the melanocyte-specific autoantigen ADAMTSL5 (VRSRRCLRL).

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Hyphenated mass spectrometry has been used to identify ligands binding to proteins. It involves mixing protein and compounds, separation of protein-ligand complexes from unbound compounds, dissociation of the protein-ligand complex, separation to remove protein, and injection of the supernatant into a mass spectrometer to observe the ligand. Here we report collision-induced affinity selection mass spectrometry (CIAS-MS), which allows separation and dissociation inside the instrument.

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Nsp9 is a conserved accessory component of the coronaviral replication and transcription complex. It is the predominant substrate of nsp12's nucleotidylation activity while also serving to recruit proteins required for viral 5'-capping. Anti-nsp9 specific nanobodies have been isolated previously.

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Bordetella pertussis is the causative agent of whooping cough, a highly contagious respiratory disease. Pertussis toxin (PT), a major virulence factor secreted by B. pertussis, is an AB5-type protein complex topologically related to cholera toxin.

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Heterotrimeric G proteins are the main signalling effectors for G protein-coupled receptors. Understanding the distinct functions of different G proteins is key to understanding how their signalling modulates physiological responses. Pertussis toxin, a bacterial AB toxin, inhibits Gα G proteins and has proven useful for interrogating inhibitory G protein signalling.

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SARS-CoV-2 (COVID-19) has infected over 219 million people and caused the death of over 4.55 million worldwide. In a previous screen of a natural product library against purified SARS-CoV-2 Nsp9 using a native mass spectrometry-based approach, we identified an kaurane natural product, oridonin (), with micromolar affinities.

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Unlike conventional αβ T cells, γδ T cells typically recognize nonpeptide ligands independently of major histocompatibility complex (MHC) restriction. Accordingly, the γδ T cell receptor (TCR) can potentially recognize a wide array of ligands; however, few ligands have been described to date. While there is a growing appreciation of the molecular bases underpinning variable (V)δ1 and Vδ2 γδ TCR-mediated ligand recognition, the mode of Vδ3 TCR ligand engagement is unknown.

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The Nsp9 replicase is a conserved coronaviral protein that acts as an essential accessory component of the multi-subunit viral replication/transcription complex. Nsp9 is the predominant substrate for the essential nucleotidylation activity of Nsp12. Compounds specifically interfering with this viral activity would facilitate its study.

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The coronaviral nonstructural protein 9 (Nsp9) is essential for viral replication; it is the primary substrate of Nsp12's pseudokinase domain within the viral replication transcription complex, an association that also recruits other components during different stages of RNA reproduction. In the unmodified state, Nsp9 forms an obligate homodimer via an essential GxxxG protein-interaction motif, but its ssRNA-binding mechanism remains unknown. Using structural biological techniques, here we show that a base-mimicking compound identified from a small molecule fragment screen engages Nsp9 via a tetrameric Pi-Pi stacking interaction that induces the formation of a parallel trimer-of-dimers.

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The race to identify a successful treatment for COVID19 will be defined by fundamental research into the replication cycle of the SARS-CoV-2 virus. This has identified five distinct stages from which numerous vaccination and clinical trials have emerged alongside an innumerable number of drug discovery studies currently in development for disease intervention. Informing every step of the viral replication cycle has been an unprecedented 'call-to-arms' by the global structural biology community.

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Many of the SARS-CoV-2 proteins have related counterparts across the Severe Acute Respiratory Syndrome (SARS-CoV) family. One such protein is non-structural protein 9 (Nsp9), which is thought to mediate viral replication, overall virulence, and viral genomic RNA reproduction. We sought to better characterize the SARS-CoV-2 Nsp9 and subsequently solved its X-ray crystal structure, in an apo form and, unexpectedly, in a peptide-bound form with a sequence originating from a rhinoviral 3C protease sequence (LEVL).

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Article Synopsis
  • Bacteria have developed specialized systems to absorb essential nutrients like iron, particularly those that are pathogens such as Pectobacterium which target ferredoxin produced by plants.
  • The ferredoxin uptake system (Fus) in these bacteria is a gene cluster that helps transport and process ferredoxin inside the bacterial cell.
  • Research shows that proteins similar to Fus, specifically YddB and PqqL from E. coli, share structural and functional traits with the components of the Fus system, suggesting that such protease-associated import systems are common among Gram-negative bacteria.
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Iron is essential for life. Accessing iron from the environment can be a limiting factor that determines success in a given environmental niche. For bacteria, access of chelated iron from the environment is often mediated by TonB-dependent transporters (TBDTs), which are β-barrel proteins that form sophisticated channels in the outer membrane.

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Pertussis-like toxins are secreted by several bacterial pathogens during infection. They belong to the AB virulence factors, which bind to glycans on host cell membranes for internalization. Host cell recognition and internalization are mediated by toxin B subunits sharing a unique pentameric ring-like assembly.

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Fuculose-1-phosphate aldolase (FucA) catalyses the reversible cleavage of L-fuculose 1-phosphate to dihydroxyacetone phosphate (DHAP) and L-lactaldehyde. This enzyme from mesophiles and thermophiles has been extensively studied; however, there is no report on this enzyme from a psychrophile. In this study, the gene encoding FucA from Glaciozyma antarctica PI12 (GaFucA) was cloned and the enzyme was overexpressed in Escherichia coli, purified and crystallized.

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The ubiquitous second messenger cAMP mediates signal transduction processes in the malarial parasite that regulate host erythrocyte invasion and the proliferation of merozoites. In Plasmodium falciparum, the central receptor for cAMP is the single regulatory subunit (R) of protein kinase A (PKA). To aid the development of compounds that can selectively dysregulate parasite PKA signaling, we solved the structure of the PKA regulatory subunit in complex with cAMP and a related analogue that displays antimalarial activity, (S)-2-Cl-cAMPS.

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Central to malaria pathogenesis is the invasion of human red blood cells by Plasmodium falciparum parasites. Following each cycle of intracellular development and replication, parasites activate a cellular program to egress from their current host cell and invade a new one. The orchestration of this process critically relies upon numerous organised phospho-signaling cascades, which are mediated by a number of central kinases.

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