Publications by authors named "Phillip I Bird"

95 Publications

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

Theranostics 2021 30;11(8):3781-3795. Epub 2021 Jan 30.

Instituto de Carboquímica ICB-CSIC, 50018, Zaragoza, Spain.

Peritonitis is one of the most common causes of sepsis, a serious syndrome characterized by a dysregulated systemic inflammatory response. Recent evidence suggests that Granzyme A (GzmA), a serine protease mainly expressed by NK and T cells, could act as a proinflammatory mediator and could play an important role in the pathogenesis of sepsis. This work aims to analyze the role and the therapeutic potential of GzmA in the pathogenesis of peritoneal sepsis. The level of extracellular GzmA as well as GzmA activity were analyzed in serum from healthy volunteers and patients with confirmed peritonitis and were correlated with the Sequential Organ Failure Assessment (SOFA) score. Peritonitis was induced in C57Bl/6 (WT) and GzmA mice by cecal ligation and puncture (CLP). Mice were treated intraperitoneally with antibiotics alone or in combination serpinb6b, a specific GzmA inhibitor, for 5 days. Mouse survival was monitored during 14 days, levels of some proinflammatory cytokines were measured in serum and bacterial load and diversity was analyzed in blood and spleen at different times. Clinically, 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 and GzmA did not affect bacterial replication in macrophages indicating that GzmA has no role in bacterial control. Analysis of GzmA in lymphoid cells following CLP showed that it was mainly expressed by NK cells. Mechanistically, we found that extracellular active GzmA acts as a proinflammatory mediator in macrophages by inducing the TLR4-dependent expression of IL-6 and TNFα. Our findings implicate GzmA as a key regulator of the inflammatory response during abdominal sepsis and provide solid evidences about its therapeutic potential for the treatment of this severe pathology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7150/thno.49288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7914344PMC
January 2021

Increased susceptibility to acoustic trauma in a mouse model of non-syndromic sensorineural deafness, DFNB91.

Eur J Neurosci 2021 Mar 7;53(5):1638-1651. Epub 2020 Nov 7.

Department of Biochemistry & Molecular Biology, Monash University, Clayton, Vic., Australia.

Inactivating mutations of SERPINB6 in humans result in progressive hearing loss starting in early adulthood (DFNB91). We have previously shown that C57BL/6J mice lacking the orthologous gene, Serpinb6a, exhibit progressive hearing loss, which is associated with progressive loss of distinct cell types in the organ of Corti beginning with outer hair cells (OHCs). However, deafness in these animals occurs much earlier than expected, possibly because C57BL/6J mice also carry an age-related hearing loss mutation in the cadherin 23 gene (Cdh23 ) that causes late onset hearing loss. The CBA/CaH strain of mice does not carry Cdh23 and may represent a better model of the human DFNB91 patients. Here, we show that transfer of the mutant Serpinb6a allele onto the Cdh23 normal CBA/CaH background markedly delays onset of hearing loss, more closely phenocopying DFNB91, without altering the pattern of cellular loss. Young, pre-symptomatic mice of this genotype exposed to acoustic trauma exhibit permanent hearing loss, compared to controls, associated with the disappearance of OHCs. We conclude that Serpinb6 helps to maintain hearing by protecting hair cells from stress.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/ejn.15011DOI Listing
March 2021

Extracellular Granzyme A Promotes Colorectal Cancer Development by Enhancing Gut Inflammation.

Cell Rep 2020 07;32(1):107847

Fundación Instituto de Investigación Sanitaria Aragón (IIS Aragón), Biomedical Research Centre of Aragon (CIBA), 50009 Zaragoza, Spain; Aragon I+D Foundation (ARAID), Zaragoza, Spain; Nanoscience Institute of Aragon (INA), University of Zaragoza, 50018 Zaragoza, Spain; Department Biochemistry and Molecular and Cell Biology and Department Microbiology, Preventive Medicine and Public Health, University of Zaragoza, 50009 Zaragoza, Spain; CIBER-BBN, Madrid, Spain. Electronic address:

If not properly regulated, the inflammatory immune response can promote carcinogenesis, as evident in colorectal cancer (CRC). Aiming to gain mechanistic insight into the link between inflammation and CRC, we perform transcriptomics analysis of human CRC, identifying a strong correlation between expression of the serine protease granzyme A (GzmA) and inflammation. In a dextran sodium sulfate and azoxymethane (DSS/AOM) mouse model, deficiency and pharmacological inhibition of extracellular GzmA both attenuate gut inflammation and prevent CRC development, including the initial steps of cell transformation and epithelial-to-mesenchymal transition. Mechanistically, extracellular GzmA induces NF-κB-dependent IL-6 production in macrophages, which in turn promotes STAT3 activation in cultured CRC cells. Accordingly, colon tissues from DSS/AOM-treated, GzmA-deficient animals present reduced levels of pSTAT3. By identifying GzmA as a proinflammatory protease that promotes CRC development, these findings provide information on mechanisms that link immune cell infiltration to cancer progression and present GzmA as a therapeutic target for CRC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2020.107847DOI Listing
July 2020

Noninvasive optical detection of granzyme B from natural killer cells with enzyme-activated fluorogenic probes.

J Biol Chem 2020 07 21;295(28):9567-9582. Epub 2020 May 21.

Wroclaw University of Science and Technology, Department of Chemical Biology and Bioimaging, Wroclaw, Poland

Natural killer (NK) cells are key innate immunity effectors that combat viral infections and control several cancer types. For their immune function, human NK cells rely largely on five different cytotoxic proteases, called granzymes (A/B/H/K/M). Granzyme B (GrB) initiates at least three distinct cell death pathways, but key aspects of its function remain unexplored because selective probes that detect its activity are currently lacking. In this study, we used a set of unnatural amino acids to fully map the substrate preferences of GrB, demonstrating previously unknown GrB substrate preferences. We then used these preferences to design substrate-based inhibitors and a GrB-activatable activity-based fluorogenic probe. We show that our GrB probes do not significantly react with caspases, making them ideal for in-depth analyses of GrB localization and function in cells. Using our quenched fluorescence substrate, we observed GrB within the cytotoxic granules of human YT cells. When used as cytotoxic effectors, YT cells loaded with GrB attacked MDA-MB-231 target cells, and active GrB influenced its target cell-killing efficiency. In summary, we have developed a set of molecular tools for investigating GrB function in NK cells and demonstrate noninvasive visual detection of GrB with an enzyme-activated fluorescent substrate.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA120.013204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7363135PMC
July 2020

Detection of Active Granzyme A in NK92 Cells with Fluorescent Activity-Based Probe.

J Med Chem 2020 03 16;63(6):3359-3369. Epub 2020 Mar 16.

Wrocław University of Science and Technology, Department of Chemical Biology and Bioimaging, Wyb. Wyspiańskiego 29, 50-370 Wroclaw, Poland.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.9b02042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7590976PMC
March 2020

Granzyme A in Chikungunya and Other Arboviral Infections.

Front Immunol 2019 14;10:3083. Epub 2020 Jan 14.

QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia.

Granzyme A (GzmA) is secreted by cytotoxic lymphocytes and has traditionally been viewed as a mediator of cell death. However, a growing body of data suggests the physiological role of GzmA is promotion of inflammation. Here, we show that GzmA is significantly elevated in the sera of chikungunya virus (CHIKV) patients and that GzmA levels correlated with viral loads and disease scores in these patients. Serum GzmA levels were also elevated in CHIKV mouse models, with NK cells the likely source. Infection of mice deficient in type I interferon responses with CHIKV, Zika virus, or dengue virus resulted in high levels of circulating GzmA. We also show that subcutaneous injection of enzymically active recombinant mouse GzmA was able to mediate inflammation, both locally at the injection site as well as at a distant site. Protease activated receptors (PARs) may represent targets for GzmA, and we show that treatment with PAR antagonist ameliorated GzmA- and CHIKV-mediated inflammation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2019.03083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6971054PMC
November 2020

SerpinB1 controls encephalitogenic T helper cells in neuroinflammation.

Proc Natl Acad Sci U S A 2019 10 23;116(41):20635-20643. Epub 2019 Sep 23.

The Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA 02115;

SerpinB1, a protease inhibitor and neutrophil survival factor, was recently linked with IL-17-expressing T cells. Here, we show that () is dramatically induced in a subset of effector CD4 cells in experimental autoimmune encephalomyelitis (EAE). Despite normal T cell priming, mice are resistant to EAE with a paucity of T helper (T) cells that produce two or more of the cytokines, IFNγ, GM-CSF, and IL-17. These multiple cytokine-producing CD4 cells proliferate extremely rapidly; highly express the cytolytic granule proteins perforin-A, granzyme C (GzmC), and GzmA and surface receptors IL-23R, IL-7Rα, and IL-1R1; and can be identified by the surface marker CXCR6. In mice, CXCR6 T cells are generated but fail to expand due to enhanced granule protease-mediated mitochondrial damage leading to suicidal cell death. Finally, anti-CXCR6 antibody treatment, like deletion, dramatically reverts EAE, strongly indicating that the CXCR6 T cells are the drivers of encephalitis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1905762116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6789640PMC
October 2019

The cryo-EM structure of the acid activatable pore-forming immune effector Macrophage-expressed gene 1.

Nat Commun 2019 09 19;10(1):4288. Epub 2019 Sep 19.

ARC Centre of Excellence in Advanced Molecular Imaging, Monash University, Melbourne, VIC, 3800, Australia.

Macrophage-expressed gene 1 (MPEG1/Perforin-2) is a perforin-like protein that functions within the phagolysosome to damage engulfed microbes. MPEG1 is thought to form pores in target membranes, however, its mode of action remains unknown. We use cryo-Electron Microscopy (cryo-EM) to determine the 2.4 Å structure of a hexadecameric assembly of MPEG1 that displays the expected features of a soluble prepore complex. We further discover that MPEG1 prepore-like assemblies can be induced to perforate membranes through acidification, such as would occur within maturing phagolysosomes. We next solve the 3.6 Å cryo-EM structure of MPEG1 in complex with liposomes. These data reveal that a multi-vesicular body of 12 kDa (MVB12)-associated β-prism (MABP) domain binds membranes such that the pore-forming machinery of MPEG1 is oriented away from the bound membrane. This unexpected mechanism of membrane interaction suggests that MPEG1 remains bound to the phagolysosome membrane while simultaneously forming pores in engulfed bacterial targets.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-12279-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6753088PMC
September 2019

A transgenic zebrafish model of hepatocyte function in human Z α1-antitrypsin deficiency.

Biol Chem 2019 11;400(12):1603-1616

Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Melbourne 3800, Victoria, Australia.

In human α1-antitrypsin deficiency, homozygous carriers of the Z (E324K) mutation in the gene SERPINA1 have insufficient circulating α1-antitrypsin and are predisposed to emphysema. Misfolding and accumulation of the mutant protein in hepatocytes also causes endoplasmic reticulum stress and underpins long-term liver damage. Here, we describe transgenic zebrafish (Danio rerio) expressing the wildtype or the Z mutant form of human α1-antitrypsin in hepatocytes. As observed in afflicted humans, and in rodent models, about 80% less α1-antitrypsin is evident in the circulation of zebrafish expressing the Z mutant. Although these zebrafish also show signs of liver stress, they do not accumulate α1-antitrypsin in hepatocytes. This new zebrafish model will provide useful insights into understanding and treatment of α1-antitrypsin deficiency.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1515/hsz-2018-0391DOI Listing
November 2019

Granzyme K Expressed by Classically Activated Macrophages Contributes to Inflammation and Impaired Remodeling.

J Invest Dermatol 2019 04 3;139(4):930-939. Epub 2018 Nov 3.

International Collaboration On Repair Discoveries (ICORD) Centre, Vancouver Coastal Health Research Institute, University of British Columbia, Vancouver, British Columbia, Canada; Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada; British Columbia Professional Firefighters' Burn and Wound Healing Group, Vancouver, British Columbia, Canada. Electronic address:

Granzyme K (GzmK), traditionally described as a pro-apoptotic, granule-secreted serine protease, has been proposed to promote inflammation. Found at low levels in the plasma of healthy individuals, GzmK is markedly elevated in response to sepsis and infection. In this study we investigated the role of GzmK in inflammation and remodeling in response to thermal injury. In human burn tissue, GzmK was elevated compared with normal skin, with expression predominantly found in macrophages. GzmK was expressed and secreted by cultured human classically activated macrophages. To assess the role of GzmK in response to skin wounding, wild-type or GzmK mice were subjected to grade 2 thermal injury. GzmK mice exhibited improved wound closure, matrix organization, and tensile strength compared with wild-type mice. Reduced proinflammatory IL-6, ICAM-1, VCAM-1, and MCP-1 expressions were observed at 3 days after injury. Additionally, GzmK induced IL-6 expression in keratinocytes and skin fibroblasts that was dependent on PAR-1 activation. Re-epithelialization showed the greatest degree of improvement of all healing parameters, suggesting that keratinocytes are sensitive to GzmK-mediated proteolysis. In support, keratinocytes, but not skin fibroblasts, exposed to GzmK showed impaired wound healing in vitro. In summary, GzmK influences wound healing by augmenting inflammation and impeding epithelialization.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jid.2018.09.031DOI Listing
April 2019

An Essential Role of Maspin in Embryogenesis and Tumor Suppression-Letter.

Cancer Res 2017 09 6;77(18):5207. Epub 2017 Sep 6.

Department of Biochemistry and Molecular Biology, Biomedical Discovery Institute, Monash University, Victoria, Australia.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-17-0178DOI Listing
September 2017

Granzyme A Deficiency Breaks Immune Tolerance and Promotes Autoimmune Diabetes Through a Type I Interferon-Dependent Pathway.

Diabetes 2017 12 21;66(12):3041-3050. Epub 2017 Jul 21.

St. Vincent's Institute, Fitzroy, Victoria, Australia

Granzyme A is a protease implicated in the degradation of intracellular DNA. Nucleotide complexes are known triggers of systemic autoimmunity, but a role in organ-specific autoimmune disease has not been demonstrated. To investigate whether such a mechanism could be an endogenous trigger for autoimmunity, we examined the impact of granzyme A deficiency in the NOD mouse model of autoimmune diabetes. Granzyme A deficiency resulted in an increased incidence in diabetes associated with accumulation of ssDNA in immune cells and induction of an interferon response in pancreatic islets. Central tolerance to proinsulin in transgenic NOD mice was broken on a granzyme A-deficient background. We have identified a novel endogenous trigger for autoimmune diabetes and an in vivo role for granzyme A in maintaining immune tolerance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2337/db17-0517DOI Listing
December 2017

A pro-survival role for the intracellular granzyme B inhibitor Serpinb9 in natural killer cells during poxvirus infection.

Immunol Cell Biol 2017 11 15;95(10):884-894. Epub 2017 Aug 15.

Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia.

Intracellular serpins are proposed to inactivate proteases released from lysosome-related organelles into the host cell interior, preventing cell death. Serpinb9 opposes the immune cytotoxic protease, granzyme B, and in a number of settings protects cells against granzyme B-mediated cell death. Using a knockout mouse line engineered to express green fluorescent protein under the serpbinb9 promoter, we demonstrate that serpinb9 is vital for host survival during Ectromelia virus infection by maintaining both mature natural killer NK) cells, and activated CD8 T cells. Serpinb9 expression parallels granzyme B expression within both populations during infection. Maturing serpinb9-null NK cells exhibit higher levels of granzyme B-mediated apoptosis during infection; hence there are fewer mature NK cells, and these cells also have lower cytotoxic potential. Thus the serpinb9-granzyme B axis is important for homeostasis of both major cytotoxic effector cell populations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/icb.2017.59DOI Listing
November 2017

Neurodevelopmental MACPFs: The vertebrate astrotactins and BRINPs.

Semin Cell Dev Biol 2017 12 12;72:171-181. Epub 2017 May 12.

Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, 3800, Australia.

Astrotactins (ASTNs) and Bone morphogenetic protein/retinoic acid inducible neural-specific proteins (BRINPs) are two groups of Membrane Attack Complex/Perforin (MACPF) superfamily proteins that show overlapping expression in the developing and mature vertebrate nervous system. ASTN(1-2) and BRINP(1-3) genes are found at conserved loci in humans that have been implicated in neurodevelopmental disorders (NDDs). Here we review the tissue distribution and cellular localization of these proteins, and discuss recent studies that provide insight into their structure and interactions. We highlight the genetic relationships and co-expression of Brinps and Astns; and review recent knock-out mouse phenotypes that indicate a possible overlap in protein function between ASTNs and BRINPs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.semcdb.2017.05.005DOI Listing
December 2017

Granzyme K-deficient mice show no evidence of impaired antiviral immunity.

Immunol Cell Biol 2017 09 21;95(8):676-683. Epub 2017 Apr 21.

Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia.

The biological role of granzyme K, a serine protease of cytotoxic T lymphocytes (CTL), is controversial. It has been reported to induce perforin-mediated cell death in vitro, but is also reported to be non-cytotoxic and to operate in inflammatory processes. To elucidate the biological role of this protease, we have deleted the granzyme K gene in mice (mutant allele: Gzmk; MGI:5636646). Gzmk mice are healthy, anatomically normal, fecund and show normal hematopoietic development. Gzmk mice readily recover from lymphocytic choriomeningitis virus and mouse pox Ectromelia virus infection. Ex vivo, virus-specific granzyme K-deficient CTL are indistinguishable from those of wild-type mice in apoptosis induction of target cells. These data suggest that granzyme K does not play an essential role in viral immunity or cytotoxicity. Our granzyme K knockout line completes the collection of mouse models for the human granzymes, and will further our understanding of their biological roles and relationships.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/icb.2017.35DOI Listing
September 2017

RNA-Seq analysis of chikungunya virus infection and identification of granzyme A as a major promoter of arthritic inflammation.

PLoS Pathog 2017 02 16;13(2):e1006155. Epub 2017 Feb 16.

QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia.

Chikungunya virus (CHIKV) is an arthritogenic alphavirus causing epidemics of acute and chronic arthritic disease. Herein we describe a comprehensive RNA-Seq analysis of feet and lymph nodes at peak viraemia (day 2 post infection), acute arthritis (day 7) and chronic disease (day 30) in the CHIKV adult wild-type mouse model. Genes previously shown to be up-regulated in CHIKV patients were also up-regulated in the mouse model. CHIKV sequence information was also obtained with up to ≈8% of the reads mapping to the viral genome; however, no adaptive viral genome changes were apparent. Although day 2, 7 and 30 represent distinct stages of infection and disease, there was a pronounced overlap in up-regulated host genes and pathways. Type I interferon response genes (IRGs) represented up to ≈50% of up-regulated genes, even after loss of type I interferon induction on days 7 and 30. Bioinformatic analyses suggested a number of interferon response factors were primarily responsible for maintaining type I IRG induction. A group of genes prominent in the RNA-Seq analysis and hitherto unexplored in viral arthropathies were granzymes A, B and K. Granzyme A-/- and to a lesser extent granzyme K-/-, but not granzyme B-/-, mice showed a pronounced reduction in foot swelling and arthritis, with analysis of granzyme A-/- mice showing no reductions in viral loads but reduced NK and T cell infiltrates post CHIKV infection. Treatment with Serpinb6b, a granzyme A inhibitor, also reduced arthritic inflammation in wild-type mice. In non-human primates circulating granzyme A levels were elevated after CHIKV infection, with the increase correlating with viral load. Elevated granzyme A levels were also seen in a small cohort of human CHIKV patients. Taken together these results suggest granzyme A is an important driver of arthritic inflammation and a potential target for therapy.

Trial Registration: ClinicalTrials.gov NCT00281294.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1006155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5312928PMC
February 2017

Epigenetic control of mitochondrial cell death through PACS1-mediated regulation of BAX/BAK oligomerization.

Cell Death Differ 2017 06 6;24(6):961-970. Epub 2017 Jan 6.

Cancer Immunology Program, Peter MacCallum Cancer Centre, East Melbourne 3002, Australia.

PCAF and ADA3 associate within the same macromolecular complexes to control the transcription of many genes, including some that regulate apoptosis. Here we show that PCAF and ADA3 regulate the expression of PACS1, whose protein product is a key component of the machinery that sorts proteins among the trans-Golgi network and the endosomal compartment. We describe a novel role for PACS1 as a regulator of the intrinsic pathway of apoptosis and mitochondrial outer membrane permeabilization. Cells with decreased PACS1 expression were refractory to cell death mediated by a variety of stimuli that operate through the mitochondrial pathway, including human granzyme B, staurosporine, ultraviolet radiation and etoposide, but remained sensitive to TRAIL receptor ligation. The mitochondria of protected cells failed to release cytochrome c as a result of perturbed oligomerization of BAX and BAK. We conclude that PCAF and ADA3 transcriptionally regulate PACS1 and that PACS1 is a key regulator of BAX/BAK oligomerization and the intrinsic (mitochondrial) pathway to apoptosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/cdd.2016.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5442465PMC
June 2017

Mice Lacking or , or Both, Exhibit Behaviors Consistent with Neurodevelopmental Disorders.

Front Behav Neurosci 2016 25;10:196. Epub 2016 Oct 25.

Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University Clayton, VIC, Australia.

, and Astrotactins () , are members of the Membrane Attack Complex/Perforin (MACPF) superfamily that are predominantly expressed in the mammalian brain during development. Genetic variation at the human and loci has been implicated in neurodevelopmental disorders. We, and others, have previously shown that mice exhibit behavior reminiscent of autism spectrum disorder (ASD) and attention deficit hyperactivity disorder (ADHD). We created mice and mice via the Cre-mediated LoxP system to investigate the effect of gene deletion on anatomy and behavior. Additionally, double knock-out mice were generated by interbreeding and mice. Genomic validation was carried out for each knock-out line, followed by histological, weight and behavioral examination. triple knock-out mice were also generated by crossing double knock-out mice with previously generated mice, and examined by weight and histological analysis. and mice differ in their behavior: mice are hyperactive, whereas mice exhibit marked changes in anxiety-response on the elevated plus maze. mice also show evidence of altered sociability. Both and mice have normal short-term memory, olfactory responses, pre-pulse inhibition, and motor learning. The double knock-out mice show behaviors of and mice, without evidence of new or exacerbated phenotypes. is important in moderation of anxiety, with potential relevance to anxiety disorders. dysfunction resulting in hyperactivity may be relevant to the association of ADHD with chromosome locus 1q25.2. and genes do not compensate in the mammalian brain and likely have distinct molecular or cell-type specific functions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fnbeh.2016.00196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5079073PMC
October 2016

Characterization of Lgr5+ progenitor cell transcriptomes in the apical and basal turns of the mouse cochlea.

Oncotarget 2016 Jul;7(27):41123-41141

State Key Laboratory of Bioelectronics, Institute of Life Sciences, Southeast University, Nanjing, China.

Lgr5+ supporting cells (SCs) are enriched hair cell (HC) progenitors in the cochlea, and several studies have shown a difference in the proliferation and HC regeneration ability of SCs between the apical and basal turns. However, the detailed differences between the transcriptomes of the apical and basal Lgr5+ SCs have not yet been investigated. We found that when isolated by FACS, Lgr5+ cells from the apex generated significantly more HCs and had significantly higher proliferation and mitotic HC regeneration ability compared to those from the base. Next, we used microarray analysis to determine the transcriptome expression profiles of Lgr5+ progenitors from the apex and the base. We first analyzed the genes that were enriched and differentially expressed in Lgr5+ progenitors from the apex and the base. Then we analyzed the cell cycle genes and the transcription factors that might regulate the proliferation and differentiation of Lgr5+ progenitors. Lastly, to further analyze the role of differentially expressed genes and to gain an overall view of the gene network in cochlear HC regeneration, we created a protein-protein interaction network. Our datasets suggest the possible genes that might regulate the proliferation and HC regeneration ability of Lgr5+ progenitors, and these genes might provide new therapeutic targets for HC regeneration in the future.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.18632/oncotarget.8636DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5173047PMC
July 2016

Brinp1(-/-) mice exhibit autism-like behaviour, altered memory, hyperactivity and increased parvalbumin-positive cortical interneuron density.

Mol Autism 2016 31;7:22. Epub 2016 Mar 31.

Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC 3800 Australia.

Background: BMP/RA-inducible neural-specific protein 1 (Brinp1) is highly conserved in vertebrates, and continuously expressed in the neocortex, hippocampus, olfactory bulb and cerebellum from mid-embryonic development through to adulthood.

Methods: Brinp1 knock-out (Brinp1(-/-)) mice were generated by Cre-recombinase-mediated removal of the third exon of Brinp1. Knock-out mice were characterised by behavioural phenotyping, immunohistochemistry and expression analysis of the developing and adult brain.

Results: Absence of Brinp1 during development results in a behavioural phenotype resembling autism spectrum disorder (ASD), in which knock-out mice show reduced sociability and changes in vocalisation capacity. In addition, Brinp1(-/-) mice exhibit hyper-locomotor activity, have impaired short-term memory, and exhibit poor reproductive success. Brinp1(-/-) mice show increased density of parvalbumin-expressing interneurons in the adult mouse brain. Brinp1(-/-) mice do not show signs of altered neural precursor proliferation or increased apoptosis during late embryonic brain development. The expression of the related neuronal migration genes Astn1 and Astn2 is increased in the brains of Brinp1(-/-) mice, suggesting that they may ameliorate the effects of Brinp1 loss.

Conclusions: Brinp1 plays an important role in normal brain development and function by influencing neuronal distribution within the cortex. The increased cortical PV-positive interneuron density and altered behaviour of Brinp1(-/-) mice resemble features of a subset of human neurological disorders; namely autism spectrum disorder (ASD) and the hyperactivity aspect of attention deficit hyperactivity disorder (ADHD).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13229-016-0079-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4818446PMC
December 2016

A Novel Serpin Regulatory Mechanism: SerpinB9 IS REVERSIBLY INHIBITED BY VICINAL DISULFIDE BOND FORMATION IN THE REACTIVE CENTER LOOP.

J Biol Chem 2016 Feb 15;291(7):3626-38. Epub 2015 Dec 15.

From the Department of Biochemistry & Molecular Biology, Biomedicine Discovery Institute, Monash University Clayton, Clayton, Victoria 3800 Australia and

The intracellular protease inhibitor Sb9 (SerpinB9) is a regulator of the cytotoxic lymphocyte protease GzmB (granzyme B). Although GzmB is primarily involved in the destruction of compromised cells, recent evidence suggests that it is also involved in lysosome-mediated death of the cytotoxic lymphocyte itself. Sb9 protects the cell from GzmB released from lysosomes into the cytosol. Here we show that reactive oxygen species (ROS) generated within cytotoxic lymphocytes by receptor stimulation are required for lyososomal permeabilization and release of GzmB into the cytosol. Importantly, ROS also inactivate Sb9 by oxidizing a highly conserved cysteine pair (P1-P1' in rodents and P1'-P2' in other mammals) in the reactive center loop to form a vicinal disulfide bond. Replacement of the P4-P3' reactive center loop residues of the prototype serpin, SERPINA1, with the P4-P5' residues of Sb9 containing the cysteine pair is sufficient to convert SERPINA1 into a ROS-sensitive GzmB inhibitor. Conversion of the cysteine pair to serines in either human or mouse Sb9 results in a functional serpin that inhibits GzmB and resists ROS inactivation. We conclude that ROS sensitivity of Sb9 allows the threshold for GzmB-mediated suicide to be lowered, as part of a conserved post-translational homeostatic mechanism regulating lymphocyte numbers or activity. It follows, for example, that antioxidants may improve NK cell viability in adoptive immunotherapy applications by stabilizing Sb9.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M115.699298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4751400PMC
February 2016

Analysis of Perforin Assembly by Quartz Crystal Microbalance Reveals a Role for Cholesterol and Calcium-independent Membrane Binding.

J Biol Chem 2015 Dec 5;290(52):31101-12. Epub 2015 Nov 5.

From the Department of Biochemistry and Molecular Biology,

Perforin is an essential component in the cytotoxic lymphocyte-mediated cell death pathway. The traditional view holds that perforin monomers assemble into pores in the target cell membrane via a calcium-dependent process and facilitate translocation of cytotoxic proteases into the cytoplasm to induce apoptosis. Although many studies have examined the structure and role of perforin, the mechanics of pore assembly and granzyme delivery remain unclear. Here we have employed quartz crystal microbalance with dissipation monitoring (QCM-D) to investigate binding and assembly of perforin on lipid membranes, and show that perforin monomers bind to the membrane in a cooperative manner. We also found that cholesterol influences perforin binding and activity on intact cells and model membranes. Finally, contrary to current thinking, perforin efficiently binds membranes in the absence of calcium. When calcium is added to perforin already on the membrane, the QCM-D response changes significantly, indicating that perforin becomes membranolytic only after calcium binding.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M115.683078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4692234PMC
December 2015

Bone morphogenetic protein/retinoic acid inducible neural-specific protein (brinp) expression during Danio rerio development.

Gene Expr Patterns 2015 May-Jul;18(1-2):37-43. Epub 2015 May 14.

Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria, Australia. Electronic address:

Prototype Membrane Attack Complex/Perforin (MACPF) superfamily proteins such as complement and perforin play crucial roles in immune defense where they drive lytic pore formation. However, it is evident that other MACPF family members are important in the central nervous system. For example, three bone morphogenetic protein/retinoic acid inducible neural-specific proteins (Brinp1, Brinp2 and Brinp3) are present in developing and mature mammalian neurons, but their molecular function is unknown. In this study we have identified and cloned full-length orthologues of all three human brinps from Danio rerio (zebrafish). Zebrafish and human brinps show very high sequence conservation, and the chromosomal loci are syntenic. We also identified two additional brinp3 paralogues at a separate locus in the zebrafish genome. The spatiotemporal expression of all five zebrafish brinps was determined by RT-PCR and whole mount RNA in situ hybridisation. Each brinp is expressed broadly in the developing nervous system at early stages (24 hours post fertilisation), but localises to specific structures in older embryos (48-72 hpf), as has been reported in mice. The conserved structures and spatiotemporal expression patterns of brinps reported in this study suggest that zebrafish will be useful for generating loss of function phenotypes to assist in determining the molecular role of these proteins.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.gep.2015.05.002DOI Listing
March 2016

Zebrafish models for nemaline myopathy reveal a spectrum of nemaline bodies contributing to reduced muscle function.

Acta Neuropathol 2015 Sep 1;130(3):389-406. Epub 2015 May 1.

School of Biological Sciences, Monash University, Melbourne, VIC, Australia.

Nemaline myopathy is characterized by muscle weakness and the presence of rod-like (nemaline) bodies. The genetic etiology of nemaline myopathy is becoming increasingly understood with mutations in ten genes now known to cause the disease. Despite this, the mechanism by which skeletal muscle weakness occurs remains elusive, with previous studies showing no correlation between the frequency of nemaline bodies and disease severity. To investigate the formation of nemaline bodies and their role in pathogenesis, we generated overexpression and loss-of-function zebrafish models for skeletal muscle α-actin (ACTA1) and nebulin (NEB). We identify three distinct types of nemaline bodies and visualize their formation in vivo, demonstrating these nemaline bodies not only exhibit different subcellular origins, but also have distinct pathological consequences within the skeletal muscle. One subtype is highly dynamic and upon breakdown leads to the accumulation of cytoplasmic actin contributing to muscle weakness. Examination of a Neb-deficient model suggests this mechanism may be common in nemaline myopathy. Another subtype results from a reduction of actin and forms a more stable cytoplasmic body. In contrast, the final type originates at the Z-disk and is associated with myofibrillar disorganization. Analysis of zebrafish and muscle biopsies from ACTA1 nemaline myopathy patients demonstrates that nemaline bodies also possess a different protein signature. In addition, we show that the ACTA1(D286G) mutation causes impaired actin incorporation and localization in the sarcomere. Together these data provide a novel examination of nemaline body origins and dynamics in vivo and identifies pathological changes that correlate with muscle weakness.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00401-015-1430-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4541704PMC
September 2015

Granzyme B promotes cytotoxic lymphocyte transmigration via basement membrane remodeling.

Immunity 2014 Dec 28;41(6):960-72. Epub 2014 Nov 28.

Department of Biochemistry and Molecular Biology, Monash University, Melbourne, VIC 3800, Australia. Electronic address:

Granzyme B (GzmB) is a protease with a well-characterized intracellular role in targeted destruction of compromised cells by cytotoxic lymphocytes. However, GzmB also cleaves extracellular matrix components, suggesting that it influences the interplay between cytotoxic lymphocytes and their environment. Here, we show that GzmB-null effector T cells and natural killer (NK) cells exhibited a cell-autonomous homing deficit in mouse models of inflammation and Ectromelia virus infection. Intravital imaging of effector T cells in inflamed cremaster muscle venules revealed that GzmB-null cells adhered normally to the vessel wall and could extend lamellipodia through it but did not cross it efficiently. In vitro migration assays showed that active GzmB was released from migrating cytotoxic lymphocytes and enabled chemokine-driven movement through basement membranes. Finally, proteomic analysis demonstrated that GzmB cleaved basement membrane constituents. Our results highlight an important role for GzmB in expediting cytotoxic lymphocyte diapedesis via basement membrane remodeling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.immuni.2014.11.012DOI Listing
December 2014

A natural genetic variant of granzyme B confers lethality to a common viral infection.

PLoS Pathog 2014 Dec 11;10(12):e1004526. Epub 2014 Dec 11.

Immunology and Virology Program, Centre for Ophthalmology and Visual Science, The University of Western Australia, Crawley, Western Australia, Australia; Centre for Experimental Immunology, Lions Eye Institute, Nedlands, Western Australia, Australia.

Many immune response genes are highly polymorphic, consistent with the selective pressure imposed by pathogens over evolutionary time, and the need to balance infection control with the risk of auto-immunity. Epidemiological and genomic studies have identified many genetic variants that confer susceptibility or resistance to pathogenic micro-organisms. While extensive polymorphism has been reported for the granzyme B (GzmB) gene, its relevance to pathogen immunity is unexplored. Here, we describe the biochemical and cytotoxic functions of a common allele of GzmB (GzmBW) common in wild mouse. While retaining 'Asp-ase' activity, GzmBW has substrate preferences that differ considerably from GzmBP, which is common to all inbred strains. In vitro, GzmBW preferentially cleaves recombinant Bid, whereas GzmBP activates pro-caspases directly. Recombinant GzmBW and GzmBP induced equivalent apoptosis of uninfected targets cells when delivered with perforin in vitro. Nonetheless, mice homozygous for GzmBW were unable to control murine cytomegalovirus (MCMV) infection, and succumbed as a result of excessive liver damage. Although similar numbers of anti-viral CD8 T cells were generated in both mouse strains, GzmBW-expressing CD8 T cells isolated from infected mice were unable to kill MCMV-infected targets in vitro. Our results suggest that known virally-encoded inhibitors of the intrinsic (mitochondrial) apoptotic pathway account for the increased susceptibility of GzmBW mice to MCMV. We conclude that different natural variants of GzmB have a profound impact on the immune response to a common and authentic viral pathogen.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1004526DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4263754PMC
December 2014

Assembly of streptolysin O pores assessed by quartz crystal microbalance and atomic force microscopy provides evidence for the formation of anchored but incomplete oligomers.

Biochim Biophys Acta 2015 Jan;1848(1 Pt A):115-26

Streptolysin O (SLO) is a bacterial pore forming protein that is part of the cholesterol dependent cytolysin (CDC) family. We have used quartz crystal microbalance with dissipation monitoring (QCM-D) to examine SLO membrane binding and pore formation. In this system, SLO binds tightly to cholesterol-containing membranes, and assembles into partial and complete pores confirmed by atomic force microscopy. SLO binds to the lipid bilayer at a single rate consistent with the Langmuir isotherm model of adsorption. Changes in dissipation illustrate that SLO alters the viscoelastic properties of the bilayer during pore formation, but there is no loss of material from the bilayer as reported for small membrane-penetrating peptides. SLO mutants were used to further dissect the assembly and insertion processes by QCM-D. This shows the signature of SLO in QCM-D changes when pore formation is inhibited, and that bound and inserted SLO forms can be distinguished. Furthermore a pre-pore locked SLO mutant binds reversibly to lipid, suggesting that the partially complete wtSLO forms observed by AFM are anchored to the membrane.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbamem.2014.10.012DOI Listing
January 2015

Blessing or curse? Proteomics in granzyme research.

Proteomics Clin Appl 2014 Jun;8(5-6):351-81

Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.

Granzymes (gzms) are a group of serine proteases that play an important role in innate and adaptive immunity, blood coagulation, apoptosis, and inflammation, but are also connected to atherosclerosis, diabetes, cardiovascular and inflammatory lung diseases, cancer, and sepsis. Humans have five gzms (gzms A, B, H, K, and M), which differ in their substrate specificity. It is widely accepted that they are delivered from cytotoxic lymphocytes via perforin into the cytoplasm of target cells where they initiate cell death, modulate cytokine signaling, or inactivate pathogen proteins. However, more recent evidence indicates gzms also act extracellularly in noncytotoxic processes. Proteomic approaches are directed at mapping gzm cleavage specificity, identifying substrates, and unraveling the (patho-) physiological role of these proteases. These studies have refined our understanding of gzm species specificity, and collectively uncovered an enormous number of new substrates. However, with the exception of a very few human gzmB substrates supported by independent data (Bid, DNA-PK, PARP, ICAD, and procaspase 7), it is presently unclear which are physiologically relevant. This review aims to summarize and analyze the different proteomic approaches used and discuss both their convincing and controversial outcomes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/prca.201300096DOI Listing
June 2014

The perforin pore facilitates the delivery of cationic cargos.

J Biol Chem 2014 Mar 20;289(13):9172-81. Epub 2014 Feb 20.

From the Department of Biochemistry and Molecular Biology.

Cytotoxic lymphocytes eliminate virally infected or neoplastic cells through the action of cytotoxic proteases (granzymes). The pore-forming protein perforin is essential for delivery of granzymes into the cytoplasm of target cells; however the mechanism of this delivery is incompletely understood. Perforin contains a membrane attack complex/perforin (MACPF) domain and oligomerizes to form an aqueous pore in the plasma membrane; therefore the simplest (and best supported) model suggests that granzymes passively diffuse through the perforin pore into the cytoplasm of the target cell. Here we demonstrate that perforin preferentially delivers cationic molecules while anionic and neutral cargoes are delivered inefficiently. Furthermore, another distantly related pore-forming MACPF protein, pleurotolysin (from the oyster mushroom), also favors the delivery of cationic molecules, and efficiently delivers human granzyme B. We propose that this facilitated diffusion is due to conserved features of oligomerized MACPF proteins, which may include an anionic lumen.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M113.544890DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3979413PMC
March 2014

Identification of Serpinb6b as a species-specific mouse granzyme A inhibitor suggests functional divergence between human and mouse granzyme A.

J Biol Chem 2014 Mar 6;289(13):9408-17. Epub 2014 Feb 6.

From the Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.

The granzyme family serine proteases are key effector molecules expressed by cytotoxic lymphocytes. The physiological role of granzyme (Gzm) A is controversial, with significant debate over its ability to induce death in target cells. Here, we investigate the natural inhibitors of GzmA. We employed substrate phage display and positional proteomics to compare substrate specificities of mouse (m) and human (h) GzmA at the peptide and proteome-wide levels and we used the resulting substrate specificity profiles to search for potential inhibitors from the intracellular serpin family. We identified Serpinb6b as a potent inhibitor of mGzmA. Serpinb6b interacts with mGzmA, but not hGzmA, with an association constant of 1.9 ± 0.8 × 10(5) M(-1) s(-1) and a stoichiometry of inhibition of 1.8. Mouse GzmA is over five times more cytotoxic than hGzmA when delivered into P815 target cells with streptolysin O, whereas transfection of target cells with a Serpinb6b cDNA increases the EC50 value of mGzmA 13-fold, without affecting hGzmA cytotoxicity. Unexpectedly, we also found that Serpinb6b employs an exosite to specifically inhibit dimeric but not monomeric mGzmA. The identification of an intracellular inhibitor specific for mGzmA only indicates that a lineage-specific increase in GzmA cytotoxic potential has driven cognate inhibitor evolution.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M113.525808DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3979379PMC
March 2014