scholarly journals Membrane permeabilization is mediated by distinct epitopes in mouse and human orthologs of the necroptosis effector, MLKL

2021 ◽  
Author(s):  
Ashish Sethi ◽  
Christopher R Horne ◽  
Cheree Fitzgibbon ◽  
Karyn L Wilde ◽  
Katherine A Davies ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Cell Death ◽  
Inflammatory Diseases ◽  
Upstream Regulator ◽  
Cell Death Pathway ◽  
Lytic Function ◽  
Human Orthologs ◽  
Binding Epitope ◽  
Kinase Phosphorylation ◽  
Terminal Effector

Necroptosis is a lytic programmed cell death pathway with origins in innate immunity that is frequently dysregulated in inflammatory diseases. The terminal effector of the pathway, MLKL, is licensed to kill following phosphorylation of its pseudokinase domain by the upstream regulator, RIPK3 kinase. Phosphorylation provokes the unleashing of MLKL's N-terminal four-helix bundle (4HB or HeLo) domain, which binds and permeabilizes the plasma membrane to cause cell death. The precise mechanism by which the 4HB domain permeabilizes membranes, and how the mechanism differs between species, remains unclear. Here, we identify the membrane binding epitope of mouse MLKL using NMR spectroscopy. Using liposome permeabilization and cell death assays, we validate K69 in the α3 helix, W108 in the α4 helix, and R137/Q138 in the first brace helix as crucial residues for necroptotic signaling. This epitope differs from the phospholipid binding site reported for human MLKL, which comprises basic residues primarily located in the α1 and α2 helices. In further contrast to human and plant MLKL orthologs, in which the α3-α4 loop forms a helix, this loop is unstructured in mouse MLKL in solution. Together, these findings illustrate the versatility of the 4HB domain fold, whose lytic function can be mediated by distinct epitopes in different orthologs.

scholarly journals Human RIPK3 maintains MLKL in an inactive conformation prior to cell death by necroptosis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yanxiang Meng ◽  
Katherine A. Davies ◽  
Cheree Fitzgibbon ◽  
Samuel N. Young ◽  
Sarah E. Garnish ◽  
...  
Keyword(s):  
Cell Death ◽  
Host Defense ◽  
Inflammatory Diseases ◽  
Kinase Domain ◽  
Human Cells ◽  
Complex Assembly ◽  
Inactive Conformation ◽  
Upstream Regulator ◽  
Widespread Interest ◽  
Terminal Effector

AbstractThe ancestral origins of the lytic cell death mode, necroptosis, lie in host defense. However, the dysregulation of necroptosis in inflammatory diseases has led to widespread interest in targeting the pathway therapeutically. This mode of cell death is executed by the terminal effector, the MLKL pseudokinase, which is licensed to kill following phosphorylation by its upstream regulator, RIPK3 kinase. The precise molecular details underlying MLKL activation are still emerging and, intriguingly, appear to mechanistically-diverge between species. Here, we report the structure of the human RIPK3 kinase domain alone and in complex with the MLKL pseudokinase. These structures reveal how human RIPK3 structurally differs from its mouse counterpart, and how human RIPK3 maintains MLKL in an inactive conformation prior to induction of necroptosis. Residues within the RIPK3:MLKL C-lobe interface are crucial to complex assembly and necroptotic signaling in human cells, thereby rationalizing the strict species specificity governing RIPK3 activation of MLKL.

scholarly journals The necroptotic cell death pathway operates in megakaryocytes, but not in platelet synthesis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Diane Moujalled ◽  
Pradnya Gangatirkar ◽  
Maria Kauppi ◽  
Jason Corbin ◽  
Marion Lebois ◽  
...  
Keyword(s):  
Cell Death ◽  
Inflammatory Cell ◽  
Kinase Domain ◽  
Cell Swelling ◽  
Platelet Production ◽  
Upstream Regulator ◽  
Cell Death Pathway ◽  
Cell Death Program ◽  
Signalling Cascade ◽  
Terminal Effector

AbstractNecroptosis is a pro-inflammatory cell death program executed by the terminal effector, mixed lineage kinase domain-like (MLKL). Previous studies suggested a role for the necroptotic machinery in platelets, where loss of MLKL or its upstream regulator, RIPK3 kinase, impacted thrombosis and haemostasis. However, it remains unknown whether necroptosis operates within megakaryocytes, the progenitors of platelets, and whether necroptotic cell death might contribute to or diminish platelet production. Here, we demonstrate that megakaryocytes possess a functional necroptosis signalling cascade. Necroptosis activation leads to phosphorylation of MLKL, loss of viability and cell swelling. Analyses at steady state and post antibody-mediated thrombocytopenia revealed that platelet production was normal in the absence of MLKL, however, platelet activation and haemostasis were impaired with prolonged tail re-bleeding times. We conclude that MLKL plays a role in regulating platelet function and haemostasis and that necroptosis signalling in megakaryocytes is dispensable for platelet production.

NETosis: how vital is it?

Blood ◽  
2013 ◽  
Vol 122 (16) ◽  
pp. 2784-2794 ◽  
Author(s):  
Bryan G. Yipp ◽  
Paul Kubes
Keyword(s):  
Innate Immunity ◽  
Cell Death ◽  
Critical Role ◽  
Neutrophil Extracellular Traps ◽  
Live Cell ◽  
Extracellular Traps ◽  
Cell Death Pathway ◽  
A Cell ◽  
Net Release

Abstract In this review, we examine the evidence that neutrophil extracellular traps (NETs) play a critical role in innate immunity. We summarize how NETs are formed in response to various stimuli and provide evidence that NETosis is not universally a cell death pathway. Here we describe at least 2 different mechanisms by which NETs are formed, including a suicide lytic NETosis and a live cell or vital NETosis. We also evaluate the evidence for NETs in catching and killing pathogens. Finally, we examine how infections are related to the development of autoimmune and vasculitic diseases through unintended but detrimental bystander damage resulting from NET release.

scholarly journals Identification of MLKL membrane translocation as a checkpoint in necroptotic cell death using Monobodies

2020 ◽  
Vol 117 (15) ◽  
pp. 8468-8475 ◽  
Author(s):  
Emma J. Petrie ◽  
Richard W. Birkinshaw ◽  
Akiko Koide ◽  
Eric Denbaum ◽  
Joanne M. Hildebrand ◽  
...  
Keyword(s):  
Cell Death ◽  
Binding Sites ◽  
Inflammatory Diseases ◽  
Adaptor Proteins ◽  
Kinase Domain ◽  
Membrane Translocation ◽  
Mixed Lineage Kinase ◽  
X Ray ◽  
Cell Death Pathway ◽  
Pathway Activation

The necroptosis cell death pathway has been implicated in host defense and in the pathology of inflammatory diseases. While phosphorylation of the necroptotic effector pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) by the upstream protein kinase RIPK3 is a hallmark of pathway activation, the precise checkpoints in necroptosis signaling are still unclear. Here we have developed monobodies, synthetic binding proteins, that bind the N-terminal four-helix bundle (4HB) “killer” domain and neighboring first brace helix of human MLKL with nanomolar affinity. When expressed as genetically encoded reagents in cells, these monobodies potently block necroptotic cell death. However, they did not prevent MLKL recruitment to the “necrosome” and phosphorylation by RIPK3, nor the assembly of MLKL into oligomers, but did block MLKL translocation to membranes where activated MLKL normally disrupts membranes to kill cells. An X-ray crystal structure revealed a monobody-binding site centered on the α4 helix of the MLKL 4HB domain, which mutational analyses showed was crucial for reconstitution of necroptosis signaling. These data implicate the α4 helix of its 4HB domain as a crucial site for recruitment of adaptor proteins that mediate membrane translocation, distinct from known phospholipid binding sites.

scholarly journals Statins as anti-pyroptotic agents

2021 ◽  
Vol 17 (5) ◽  
pp. 1414-1417
Author(s):  
Masoomeh Khalifeh ◽  
Peter Penson ◽  
Maciej Banach ◽  
Amirhossein Sahebkar
Keyword(s):  
Innate Immunity ◽  
Cell Death ◽  
Tissue Damage ◽  
Inflammatory Responses ◽  
Inflammatory Diseases ◽  
Potential Value ◽  
Inflammatory Caspases ◽  
Pleiotropic Actions

IntroductionPyroptosis is a regulated form of cell death, which is often a consequence of the activation of inflammatory caspases.Material and methodsAppropriate inflammatory responses and the induction of pyroptosis enhance the clearance of pathogens and increase innate immunity.ResultsHowever, excessive pyroptosis contributes to a hyperinflammatory response and aggravates tissue damage, thereby causing inflammatory diseases. There have been recent reports on the modulation of pyroptosis by statins, which may explain part of the pleiotropic actions of these drugs in inflammatory diseases and cancer.ConclusionsHerein, the extant evidence for the potential value of statins in targeting pyroptosis in various diseases is reviewed.

A Cell Death Pathway Induced by Antibody-Mediated Cross-Linking of CD45 on Lymphocytes

2003 ◽  
Vol 23 (5-6) ◽  
pp. 421-440 ◽  
Author(s):  
Ann-Muriel Steff ◽  
Marylene Fortin ◽  
Fabianne Philippoussis ◽  
Sylvie Lesage ◽  
Chantal Arguin ◽  
...  
Keyword(s):  
Cell Death ◽  
Cross Linking ◽  
Cell Death Pathway ◽  
A Cell

Dynamical analysis of the programmed cell death pathway

2007 ◽  
Author(s):  
Luciano Carotenuto ◽  
Vincenza Pace ◽  
Dina Bellizzi ◽  
Giovanna De Benedictis
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Dynamical Analysis ◽  
Cell Death Pathway

Humoral factors of innate immunity in the saliva of patients with infectious inflammatory respiratory diseases

2018 ◽  
pp. 13-17
Author(s):  
Oleg Melnikov ◽  
Diana Zabolotnaya ◽  
Alexander Bredun ◽  
Bogdan Bil ◽  
Oksana Rylska ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Chronic Rhinosinusitis ◽  
Inflammatory Diseases ◽  
Activity Level ◽  
Chronic Tonsillitis ◽  
Control Group ◽  
Quantitative Composition ◽  
Upper Airways ◽  
Humoral Factors ◽  
Immune Modulators

Introduction: In recently ears factors of innate immunity both cellular and humoral have been paid considerable attention as they are a protective barrier of a fast response and that is why they are largely concentrated at the intersection of the digestive tract and airways. The data concerning the activity level of factors of innate immunity in the upper airways affected by nonspecific inflammatory processes is insufficient and sparse and therefore the purpose of this research was to study the content of humoral factors of innate immunity in the oropharyngeal secretion (ORS) of patients with chronic infectious inflammatory diseases of the upper airways in remission. Materials and Methods: There was an examination of 16 patients with chronic rhinosinusitis (CRS) of bacterial genesis (15-40 years old), 12 patients with rhinopharyngitis of post-viral genesis (8-16 years old), 12 people with scleroma from 30 tо 52 years of age (atrophic form), 10 patients with chronic tonsillitis in remission (from 10 to 33 years of age) and 11 patients of a control group (practically healthy donors from 12 tо 40 years of age). The content of MIP-1b, defensin-1β, lactoferrin, lysozyme, α-interferon was studied in the nonstimulated OPS. Statistics were carried out using Mann-Whitney U-test. Results: The greatest number of deviations in the decrease in the content of the examined nonspecific protective factors was found in cases of scleroma, chronic tonsillitis and chronic rhinosinusitis (p<0,05). The lack of protective humoral factors of innate immunity can be evidence of local immunodeficiency even in remission, which is a pathophysiological component of the maintenance of chronic inflammation. Conclusion: The decrease int he quantitative composition of factors of innate immunity in the oropharyngeal secretion of patients with chronic infectious inflammatory diseases of the airways is an objective ground not only for a replacement therapy, but also for the use of immune response modifiers from photo-immune modulators to “genuine immune modulators” controlling the state of the factors of both innate immunity and immunoglobulins, primarily of secretory type, the level and functionality of various groups of immunocompetent and accessory cells.

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