Biology subjects

Bird, P. I.

Publications and source records attributed to Bird, P. I..

4 recordsLinked to original sources

bioRxiv2021

An inconvenient association between granzyme A and Nicotinamide Nucleotide Transhydrogenase

Granzyme A (GzmA) is a serine protease secreted by cytotoxic lymphocytes, with GzmA-/- mouse studies informing our understanding of GzmAs physiological function. We show herein that GzmA-/- mice have a mixed C57BL/6J and C57BL/6N background and retain the full length Nicotinamide Nucleotide Transhydrogenase (Nnt) gene, whereas Nnt is truncated in C57BL/6J mice. Chikungunya viral arthritis was substantially ameliorated in GzmA-/- mice; however, the presence of Nnt, rather than loss of GzmA, was responsible for this phenotype by constraining lymphocyte infiltration. A new CRISPR active site mutant C57BL/6J GzmAS211A mouse provided the first insights into GzmAs bioactivity free of background issues, with circulating proteolytically active GzmA promoting immune-stimulating and pro-inflammatory signatures. Remarkably, k-mer mining of the Sequence Read Archive illustrated that {approx}27% of Run Accessions and {approx}38% of Bioprojects listing C57BL/6J as the mouse strain, had Nnt sequencing reads inconsistent with a C57BL/6J background. The Nnt issue has clearly complicated our understanding of GzmA and may similarly have influenced studies across a broad range of fields.

immunology↗

bioRxiv2020

Granzyme A inhibition reduces inflammation and increases survival during abdominal sepsis.

Sepsis is a serious syndrome characterised by a dysregulated systemic inflammatory response. Here we have analysed the role and the therapeutic potential of Granzyme A (GzmA) in the pathogenesis of peritoneal sepsis using the Cecal Ligation and Puncture (CLP) polymicrobial sepsis model and samples from humans undergoing abdominal sepsis. Elevated GzmA was observed in serum from patients with abdominal sepsis suggesting that GzmA plays an important role in this pathology. In the CLP model GzmA deficient mice, or WT mice treated with an extracellular GzmA inhibitor, showed increased survival, which correlated with a reduction in proinflammatory markers in both serum and peritoneal lavage fluid. GzmA deficiency did not influence bacterial load in blood and spleen indicating that GzmA has no role in bacterial control. Mechanistically, we found that extracellular active GzmA acts as a proinflammatory mediator in macrophages by inducing the TLR4-dependent expression of IL-6 and TNF. Our findings implicate GzmA as a key regulator of the inflammatory response during abdominal sepsis, and suggest that it could be targeted for treatment of this severe pathology.

immunology↗

bioRxiv2019

Detection of active Granzyme A in NK92 cells with fluorescent activity-based probe

Cytotoxic T-lymphocytes (CTLs) and natural killer cells (NKs) kill compromised cells to defend against tumor and viral infections. Both effector cell types use multiple strategies to induce target cell death including Fas/CD95 activation; and the release of perforin and a group of lymphocyte granule serine proteases called granzymes. Granzymes have relatively broad and overlapping substrate specificities and may hydrolyze a wide range of peptidic epitopes; it is therefore challenging to identify their natural and synthetic substrates and to distinguish their localization and functions. Here, we present a specific and potent substrate, an inhibitor, and an activity-based probe of Granzyme A (GrA) that can be used to follow functional GrA in cells.

biochemistry↗

bioRxiv2019

The structure of Macrophage Expressed Gene-1, a phagolysosome immune effector that is activated upon acidification.

Macrophage Expressed Gene-1 (MPEG-1; also termed Perforin-2) is an endosomal / phagolysosomal perforin-like protein that is conserved across the metazoan kingdom and that functions within the phagolysosome to damage engulfed microbes. Like the Membrane Attack Complex and perforin, MPEG-1 has been postulated to form pores in target membranes, however, its mode of action remains to be established. We used single particle cryo-Electron Microscopy to determine the 2.4 [A] structure of a hexadecameric assembly of MPEG-1 that displays the expected features of a soluble pre-pore complex. We further discovered that the MPEG-1 pre-pore-like assemblies can be induced to perforate membranes through mild acidification, such as would occur within maturing phagolysosomes. We next solved the 3.6 [A] cryo-EM structure of MPEG-1 in complex with liposomes. Remarkably these data revealed that a C-terminal Multi-vesicular body of 12 kDa (MVB12)-associated {beta}-prism (MABP) domain interacts with target membranes in a mode that positions the pore forming machinery of MPEG-1 to point away from the bound membrane. This unexpected mechanism of membrane interaction raises the intriguing possibility that MPEG-1 may be able to remain bound to the phagolysosome membrane while simultaneously forming pores in engulfed bacterial targets.

biochemistry↗