A truncated form of HpARI stabilises IL-33, amplifying responses to the cytokine

1AbstractThe murine intestinal nematode Heligmosomoides polygyrus releases the H. polygyrus Alarmin Release Inhibitor (HpARI) - a protein which binds to IL-33 and to DNA, effectively tethering the cytokine in the nucleus of necrotic cells. Previous work showed that a non-natural truncation consisting of the first 2 domains of HpARI (HpARI_CCP1/2) retains binding to both DNA and IL-33, and inhibited IL-33 release in vivo. Here, we show that the affinity of HpARI_CCP1/2 for IL-33 is significantly lower than that of the full-length protein, and that HpARI_CCP1/2 lacks the ability to prevent interaction of IL-33 with its receptor. When HpARI_CCP1/2 was applied in vivo it potently amplified IL-33-dependent immune responses to Alternaria alternata allergen, Nippostrongylus brasiliensis infection and recombinant IL-33 injection. Mechanistically, we found that HpARI_CCP1/2 is able to bind to and stabilise IL-33, preventing its degradation and maintaining the cytokine in its active form. This study highlights the importance of IL-33 inactivation, the potential for IL-33 stabilisation in vivo, and describes a new tool for IL-33 research.

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A helminth-derived suppressor of ST2 blocks allergic responses

eLife ◽

10.7554/elife.54017 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 4

Author(s):

Francesco Vacca ◽

Caroline Chauché ◽

Abhishek Jamwal ◽

Elizabeth C Hinchy ◽

Graham Heieis ◽

...

Keyword(s):

Immune Responses ◽

Heligmosomoides Polygyrus ◽

Human Pbmc ◽

Complement Control Protein ◽

High Affinity ◽

Intestinal Nematode ◽

Control Protein

The IL-33-ST2 pathway is an important initiator of type 2 immune responses. We previously characterised the HpARI protein secreted by the model intestinal nematode Heligmosomoides polygyrus, which binds and blocks IL-33. Here, we identify H. polygyrus Binds Alarmin Receptor and Inhibits (HpBARI) and HpBARI_Hom2, both of which consist of complement control protein (CCP) domains, similarly to the immunomodulatory HpARI and Hp-TGM proteins. HpBARI binds murine ST2, inhibiting cell surface detection of ST2, preventing IL-33-ST2 interactions, and inhibiting IL-33 responses in vitro and in an in vivo mouse model of asthma. In H. polygyrus infection, ST2 detection is abrogated in the peritoneal cavity and lung, consistent with systemic effects of HpBARI. HpBARI_Hom2 also binds human ST2 with high affinity, and effectively blocks human PBMC responses to IL-33. Thus, we show that H. polygyrus blocks the IL-33 pathway via both HpARI which blocks the cytokine, and also HpBARI which blocks the receptor.

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Nutritional control of gastrointestinal parasitism in lactating rats

Proceedings of the British Society of Animal Science ◽

10.1017/s1752756200009224 ◽

2005 ◽

Vol 2005 ◽

pp. 11-11 ◽

Cited By ~ 1

Author(s):

J.G.M. Houdijk ◽

N.S. Jessop ◽

D.P. Knox ◽

I. Kyriazakis

Keyword(s):

Immune Responses ◽

Indirect Evidence ◽

Host Protein ◽

Nippostrongylus Brasiliensis ◽

Host Immune Responses ◽

Protein Nutrition ◽

Intestinal Nematode ◽

Nematode Parasites ◽

Periparturient Period ◽

Protein Supply

Small ruminant studies have shown that a reduction in protein scarcity, through either an increase in protein supply or reduction in protein demand, results in reduced nematode egg excretion and worm burdens during the periparturient period (Houdijk and Athanasiadou, 2003). Whilst this reduced degree of parasitism indirectly suggests that such nutritional effects are mediated through changes in host immune responses, there is only limited direct evidence for this. A rodent model may be used for directly assessing immune responses that underlie nutritional control of nematode parasites. There is indirect evidence that lactating rats undergo a breakdown of immunity to the intestinal nematode Nippostrongylus brasiliensis (Houdijk et al., 2003). Provided that this breakdown is sensitive to protein nutrition, this model may be used for elucidating interactions between nutrition and immunity to parasites. Therefore, we assessed whether breakdown of immunity to N. brasiliensis in the lactating rat is sensitive to host protein nutrition.

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Novel WD-Repeat Protein Mip1p Facilitates Function of the Meiotic Regulator Mei2p in Fission Yeast

Molecular and Cellular Biology ◽

10.1128/mcb.20.4.1234-1242.2000 ◽

2000 ◽

Vol 20 (4) ◽

pp. 1234-1242 ◽

Cited By ~ 33

Author(s):

Satoko Shinozaki-Yabana ◽

Yoshinori Watanabe ◽

Masayuki Yamamoto

Keyword(s):

Fission Yeast ◽

Sexual Development ◽

Rna Binding ◽

Genomic Library ◽

Active Form ◽

Functional Expression ◽

Truncated Form ◽

Wd Repeat ◽

Two Hybrid

ABSTRACT In fission yeast, the onset of meiosis is triggered by activation of the RNA-binding protein Mei2p. We screened for a high-copy-number suppressor of the ectopic meiosis induced by expression of an active form of Mei2p. Consequently we isolated a truncated form of a novel gene, named mip1, from a fission yeast genomic library. Themip1 gene encoded a protein of 1,313 amino acids which carried a WD-repeat motif in the C-terminal region and was apparently conserved among eukaryotes. Mip1p was cytoplasmic, and two-hybrid and immunoprecipitation analyses demonstrated that Mip1p was bound to Mei2p in vivo. Genetic evidence indicated that wild-type Mip1p was required for the function of Mei2p to induce meiosis and that the truncated form of it (Mip1-15p) dominantly interfered with Mei2p. Mip1p appeared to be involved also in conjugation, associating with Ste11p, which is a key transcription factor for sexual development. Furthermore, Mip1p was essential for cell growth, to which neither Mei2p nor Ste11p is relevant. These results suggest that Mip1p assists functional expression of a number of proteins required for proliferation and sexual development in fission yeast.

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IL-21 receptor signaling is integral to the development of Th2 effector responses in vivo

Blood ◽

10.1182/blood-2006-05-021600 ◽

2006 ◽

Vol 109 (5) ◽

pp. 2023-2031 ◽

Cited By ~ 110

Author(s):

Anja Fröhlich ◽

Benjamin J. Marsland ◽

Ivo Sonderegger ◽

Michael Kurrer ◽

Martin R. Hodge ◽

...

Keyword(s):

Leishmania Major ◽

Allergic Airway Inflammation ◽

Nippostrongylus Brasiliensis ◽

Autoimmune Myocarditis ◽

Heligmosomoides Polygyrus ◽

Peripheral Tissues ◽

Th2 Cell ◽

Th2 Responses

Abstract Interleukin 21 (IL-21) is a member of the common γ-chain family of cytokines, which influence a broad spectrum of immunologic responses. A number of studies have examined the function of IL-21, but its specific role in Th1/Th2-cell differentiation and related effector responses remains to be clarified. Thus, we generated IL-21R–deficient mice and have investigated the role of IL-21R signaling using a series of in vivo experimentally induced disease models. We first addressed the role of IL-21R signaling in Th2 immune responses by examining allergic airway inflammation, and Nippostrongylus brasiliensis and Heligmosomoides polygyrus antihelminth responses. In each of these systems, IL-21R signaling played a clear role in the development of Th2 responses. Comparatively, IL-21R signaling was not required for the containment of Leishmania major infection or the development of experimental autoimmune myocarditis, indicative of competent Th1 and Th17 responses, respectively. Adoptive transfer of T cells and analysis of IL-21R+/+/IL-21R−/− chimera mice revealed that IL-21R–signaling was central to Th2-cell survival or migration to peripheral tissues. Overall, our data show IL-21 plays a crucial role in supporting polarized Th2 responses in vivo, while appearing superfluous for Th1 and Th17 responses.

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Phosphatase PP2C6 negatively regulates phosphorylation status of Pti1b kinase, a regulator of flagellin-triggered immunity in tomato

10.1101/613612 ◽

2019 ◽

Author(s):

Fabian Giska ◽

Gregory B. Martin

Keyword(s):

Immune Responses ◽

Plant Immunity ◽

Active Form ◽

Control Plant ◽

Negative Regulator ◽

Response To Treatment ◽

Positive Regulators ◽

Molecular Patterns

AbstractPlant immune responses, including the production of reactive oxygen species (ROS), are triggered when pattern recognition receptors (PRR) become activated upon detection of microbe-associated molecular patterns (MAMPs). Receptor-like cytoplasmic kinases are key components of PRR-dependent signaling pathways. In tomato two such kinases, Pti1a and Pti1b, are important positive regulators of the plant immune response. However, it is unknown how these kinases control plant immunity at the molecular level, and how their activity is regulated. To investigate these issues, we used mass spectrometry to search for interactors of Pti1b in Nicotiana benthamiana leaves and identified a protein phosphatase, PP2C6. An in vitro pull-down assay and in vivo split luciferase complementation assay verified this interaction. Pti1b was found to autophosphorylate on threonine-233 and this phosphorylation was abolished in the presence of PP2C6. An arginine-to-cysteine substitution at position 240 in the Arabidopsis MARIS kinase was previously reported to convert it into a constitutive-active form. The analogous substitution in Pti1b made it resistant to PP2C6 phosphatase activity, although it still interacted with PP2C6. Treatment of N. benthamiana leaves with the MAMP flg22 induced threonine phosphorylation of Pti1b. Expression of PP2C6, but not a phosphatase-inactive variant of this protein, in N. benthamiana leaves greatly reduced ROS production in response to treatment with MAMPs flg22 or csp22. The results indicate that PP2C6 acts as a negative regulator by dephosphorylating the Pti1b kinase, thereby interfering with its ability to activate plant immune responses.

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Spatial and temporal regulation of cytokine expression in Type 2 immune responses

10.26686/wgtn.17013620.v1 ◽

2021 ◽

Author(s):

◽

Ryan Kyle

Keyword(s):

T Cells ◽

Lymph Node ◽

Immune Responses ◽

Cd4 T Cells ◽

Helminth Infection ◽

Allergic Inflammation ◽

Nippostrongylus Brasiliensis ◽

Reporter Mice

<p>Type 2 immune responses are generated to provide protection against parasitic helminth infections, however these responses also cause the pathologies associated with allergic inflammation. Studies of the cell types and signalling pathways that mediate Type 2 immune responses have been previously undertaken with the goals of efficient development of vaccines against helminths, and identification of pathways that can be inhibited to decrease the damage caused by allergic inflammation. The cytokines interleukin-4 (IL-4) and interleukin-13 (IL-13) mediate many of the downstream effector functions of the Type 2 immune response. To study the mechanisms that control expression of these two cytokines I have used a novel dual cytokine IL-4 and IL-13 transgenic reporter mouse. Utilising this tool along with other IL-4 reporter mice I have discovered that the amount of T cell receptor (TCR) signalling modulates the allelic expression of IL-4 by CD4⁺ T cells. The transgenic IL-4 reporter mouse has for the first time allowed independent measurement of the effects of IL-4 deficiency on the expression of IL-4 in vivo. Using this system I have found that IL- 4 is not required for the in vivo generation or expansion of IL-4 producing CD4⁺ T cells. Th2 differentiated CD4⁺ T cells also expresses IL-13, however the dual reporter mice have demonstrated that IL-13 is expressed consistently later than IL-4 in vitro, and IL-13 requires constant, or multiple exposures to TCR stimulus for expression to be induced. IL-13 expression is absent from lymph node CD4⁺ T cells during exposure to allergens or helminth infection. Sequestration of CD4⁺ T cells in the lymph node does not impact the number of IL-13 expressing CD4⁺ T cells in the lung during a helminth infection, indicating that adaptive immune cell derived IL-13 may be entirely produced by lung resident cells not requiring transit through the lymph node. I have characterised a population of innate lymphoid cells (ILCs) within the skin and found that the proportion of these cells that constitutively express IL-13 decreases with age. These cells did not drastically change in numbers or IL-13 responses in a range of inflammatory conditions including a model of atopic dermatitis. Basophils were found to respond to the atopic dermatitis model by migrating specifically to the treated skin site and draining lymph node, and producing IL-4 in a thymic stromal lymphopoietin dependant manner. Treatment with exogenous cytokines induced IL-13 expression from group 2 ILCs (ILC2s) in the lung and these cells promoted protective immune responses against Nippostrongylus brasiliensis infection. The immune response generated during a primary infection by Nippostrongylus brasiliensis provides protection from re-infection. Long-term protection is dependent on CD4⁺ T cells but when sufficiently stimulated by cytokine, ILC2s can rescue the protection lost by the depletion of CD4⁺ T cells. This thesis has shown that CD4⁺ T cells and populations of innate immune cells differentially regulate the expression of the closely related Type 2 cytokines IL-4 and IL- 13. These discoveries will help direct future research aiming to boost the effectiveness of anti-helminth vaccines, or decrease the pathology caused by allergic diseases by targeting specific cytokine expression.</p>

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Selective disruption of E-cadherin function in early Xenopus embryos by a dominant negative mutant

Development ◽

10.1242/dev.120.4.901 ◽

1994 ◽

Vol 120 (4) ◽

pp. 901-909 ◽

Cited By ~ 10

Author(s):

E. Levine ◽

C.H. Lee ◽

C. Kintner ◽

B.M. Gumbiner

Keyword(s):

Early Embryo ◽

Dominant Negative ◽

Full Length ◽

Dominant Negative Mutant ◽

Wild Type ◽

Truncated Form ◽

Adhesive Properties ◽

Negative Mutant ◽

E Cadherin

E-cadherin function was disrupted in vivo in developing Xenopus laevis embryos through the expression of a mutant E-cadherin protein lacking its cytoplasmic tail. This truncated form of E-cadherin was designed to act as a dominant negative mutant by competing with the extracellular interactions of wild-type endogenous E-cadherin. Expression of truncated E-cadherin in the early embryo causes lesions to develop in the ectoderm during gastrulation. In contrast, expression of a similarly truncated N-cadherin protein failed to cause the lesions. The ectodermal defect caused by the truncated E-cadherin is rescued by overexpression of wild-type E-cadherin, by co-injection of full-length E-cadherin RNA along with the RNA for the truncated form. Overexpression of full-length C-cadherin, however, is unable to compensate for the disruption of E-cadherin function and can actually cause similar ectodermal lesions when injected alone, suggesting that there is a specific requirement for E-cadherin. Therefore, E-cadherin seems to be specifically required for maintaining the integrity of the ectoderm during epiboly in the gastrulating Xenopus embryo. Differential cadherin expression reflects, therefore, the requirement for distinct adhesive properties during different morphogenetic cell behaviors.

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PP2C phosphatase Pic1 negatively regulates the phosphorylation status of Pti1b kinase, a regulator of flagellin-triggered immunity in tomato

Biochemical Journal ◽

10.1042/bcj20190299 ◽

2019 ◽

Vol 476 (11) ◽

pp. 1621-1635 ◽

Cited By ~ 3

Author(s):

Fabian Giska ◽

Gregory B. Martin

Keyword(s):

Immune Responses ◽

Plant Immunity ◽

Active Form ◽

Control Plant ◽

Negative Regulator ◽

Response To Treatment ◽

Positive Regulators ◽

Molecular Patterns

Abstract Plant immune responses, including the production of reactive oxygen species (ROS), are triggered when pattern recognition receptors (PRRs) become activated upon detection of microbe-associated molecular patterns (MAMPs). Receptor-like cytoplasmic kinases are key components of PRR-dependent signaling pathways. In tomato, two such kinases, Pti1a and Pti1b, are important positive regulators of the plant immune response. However, it is unknown how these kinases control plant immunity at the molecular level and how their activity is regulated. To investigate these issues, we used mass spectrometry to search for interactors of Pti1b in Nicotiana benthamiana leaves and identified a PP2C protein phosphatase, referred to as Pic1. An in vitro pull-down assay and in vivo split-luciferase complementation assay verified this interaction. Pti1b was found to autophosphorylate on threonine-233, and this phosphorylation was abolished in the presence of Pic1. An arginine-to-cysteine substitution at position 240 in the Arabidopsis MARIS kinase was previously reported to convert it into a constitutive-active form. The analogous substitution in Pti1b made it resistant to Pic1 phosphatase activity, although it still interacted with Pic1. Treatment of N. benthamiana leaves with the MAMP flg22 induced threonine phosphorylation of Pti1b. The expression of Pic1, but not a phosphatase-inactive variant of this protein, in N. benthamiana leaves greatly reduced ROS production in response to treatment with MAMPs flg22 or csp22. The results indicate that Pic1 acts as a negative regulator by dephosphorylating the Pti1b kinase, thereby interfering with its ability to activate plant immune responses.

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Allosteric activation of preformed EGF receptor dimers by a single ligand binding event

10.21203/rs.3.rs-182557/v2 ◽

2021 ◽

Author(s):

Endang R. Purba ◽

Ei-ichiro Saita ◽

Reetesh R. Akhouri ◽

Lars-Göran Öfverstedt ◽

Gunnar Wilken ◽

...

Keyword(s):

Ligand Binding ◽

Single Molecule ◽

Molecular Mechanisms ◽

Egf Receptor ◽

Active Form ◽

Growth Factor Receptor ◽

Full Length ◽

Receptor Dimer

Abstract Aberrant activation of the epidermal growth factor receptor (EGFR) by mutations has been implicated in a variety of human cancers. Elucidation of the structure of the full-length receptor is essential to understand the molecular mechanisms underlying its activation. Unlike previously anticipated, here, we report that purified full-length EGFR adopts a homodimeric form in vitro before and after ligand binding. Cryo-electron tomography analysis of the purified receptor also showed that the extracellular domains of the receptor dimer, which are conformationally flexible before activation, are stabilized by ligand binding. This conformational flexibility stabilization most likely accompanies rotation of the entire extracellular domain and the transmembrane a-helix, resulting in dissociation of the intracellular kinase dimer and, thus, rearranging it into an active form. Consistently, mutations of amino acid residues at the interface of the inactive, symmetric kinase dimer spontaneously activate the receptor in vivo. Optical single-molecule observation also demonstrated that binding of only one ligand activates the receptor dimer on the cell surface. Based on these results, we propose an allosteric model for the activation of EGFR dimers by ligand binding. Our results demonstrate how oncogenic mutations spontaneously activate the receptor and shed light on the development of novel cancer therapies.

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Biochemical and Genetic Interactions betweenDrosophila Caspases and the Proapoptotic Genesrpr, hid, and grim

Molecular and Cellular Biology ◽

10.1128/mcb.20.8.2907-2914.2000 ◽

2000 ◽

Vol 20 (8) ◽

pp. 2907-2914 ◽

Cited By ~ 71

Author(s):

Zhiwei Song ◽

Bo Guan ◽

Andreas Bergman ◽

Donald W. Nicholson ◽

Nancy A. Thornberry ◽

...

Keyword(s):

Caspase 3 ◽

Cultured Cells ◽

Ectopic Expression ◽

Full Length ◽

Truncated Form ◽

Effector Caspase ◽

Death Effector ◽

Human Caspase

ABSTRACT In Drosophila melanogaster, the induction of apoptosis requires three closely linked genes, reaper(rpr), head involution defective(hid), and grim. The products of these genes induce apoptosis by activating a caspase pathway. Two very similarDrosophila caspases, DCP-1 and drICE, have been previously identified. We now show that DCP-1 has a substrate specificity that is remarkably similar to those of human caspase 3 and Caenorhabditis elegans CED-3, suggesting that DCP-1 is a death effector caspase. drICE and DCP-1 have similar yet different enzymatic specificities. Although expression of either in cultured cells induces apoptosis, neither protein was able to induce DNA fragmentation inDrosophila SL2 cells. Ectopic expression of a truncated form of dcp-1 (ΔN-dcp-1) in the developingDrosophila retina under an eye-specific promoter resulted in a small and rough eye phenotype, whereas expression of the full-length dcp-1 (fl-dcp-1) had little effect. On the other hand, expression of either full-length drICE(fl-drICE) or truncated drICE(ΔN-drICE) in the retina showed no obvious eye phenotype. Although active DCP-1 protein cleaves full-length DCP-1 and full-length drICE in vitro, GMR-ΔN-dcp-1 did not enhance the eye phenotype of GMR-fl-dcp-1 or GMR-fl-drICEflies. Significantly, GMR-rpr and GMR-grim, but not GMR-hid, dramatically enhanced the eye phenotype of GMR-fl-dcp-1 flies. These results indicate that Reaper and Grim, but not HID, can activate DCP-1 in vivo.

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