scholarly journals The RHIM within the M45 protein from murine cytomegalovirus forms heteromeric amyloid fibrils with RIPK1 and RIPK3

2018 ◽  
Author(s):  
Chi L. L. Pham ◽  
Merryn Strange ◽  
Ailis O’ Carroll ◽  
Nirukshan Shanmugam ◽  
Emma Sierecki ◽  
...  
Keyword(s):  
Cell Death ◽  
Biological Activity ◽  
Self Assembly ◽  
Amyloid Fibrils ◽  
Human Cells ◽  
Terminal Region ◽  
Murine Cytomegalovirus ◽  
Homotypic Interaction ◽  
Amyloid Assembly

AbstractThe M45 protein from murine cytomegalovirus protects infected murine cells from death by necroptosis and can protect human cells from necroptosis induced by TNFR activation, when heterologously expressed. We show that the N-terminal 90 residues of the M45 protein, which contain a RIP Homotypic Interaction Motif (RHIM), are sufficient to confer protection against TNFR-induced necroptosis. This N-terminal region of M45 drives rapid self-assembly into homo-oligomeric amyloid fibrils and interacts with the RHIMs of human RIPK1 and RIPK3 kinases to form heteromeric amyloid fibrils in vitro. An intact RHIM core tetrad is required for the inhibition of cell death by M45 and we show that mutation of those key tetrad residues abolishes homo- and hetero-amyloid assembly by M45 in vitro, suggesting that the amyloidogenic nature of the M45 RHIM underlies its biological activity. Our results indicate that M45 mimics the interactions made by RIPK1 with RIPK3 in forming heteromeric amyloid structures.

scholarly journals TAT-RHIM: a more complex issue than expected

2022 ◽  
Author(s):  
Benedikt Kolbrink ◽  
Theresa Riebeling ◽  
Nikolas K. Teiwes ◽  
Claudia Steinem ◽  
Hubert Kalbacher ◽  
...  
Keyword(s):  
Cell Death ◽  
Self Assembly ◽  
Amyloid Fibrils ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Murine Cytomegalovirus ◽  
Target Cells ◽  
Regulated Cell Death ◽  
Regulated Necrosis ◽  
Wide Range

Murine cytomegalovirus protein M45 contains a RIP homotypic interaction motif (RHIM) that is sufficient to confer protection of infected cells against necroptotic cell death. Mechanistically, the N-terminal region of M45 drives rapid self-assembly into homo-oligomeric amyloid fibrils, and interacts with the endogenous RHIM domains of receptor-interacting protein kinases (RIPK) 1, RIPK3, Z-DNA binding protein 1, and TIR domain-containing adaptor-inducing interferon-β. Remarkably, all four mammalian proteins harbouring such a RHIM domain are key components of inflammatory signalling and regulated cell death processes. Immunogenic cell death by regulated necrosis causes extensive tissue damage in a wide range of diseases, including ischemia reperfusion injury, myocardial infarction, sepsis, stroke and organ transplantation. To harness the cell death suppression properties of M45 protein in a therapeutically usable manner, we developed a synthetic peptide encompassing only the RHIM domain of M45. To trigger delivery of RHIM into target cells, we fused the transactivator protein transduction domain of human immunodeficiency virus 1 to the N-terminus of the peptide. The fused peptide could efficiently penetrate eukaryotic cells, but unexpectedly it killed all tested cancer cell lines and primary cells irrespective of species without further stimulus through a necrosis-like cell death. Typical inhibitors of different forms of regulated cell death cannot impede this process, which appears to involve a direct disruption of biomembranes. Nevertheless, our finding has potential clinical relevance; reliable induction of a necrotic form of cell death distinct from all known forms of regulated cell death may offer a novel therapeutic approach to combat resistant tumour cells.

scholarly journals Individual Expression of Hepatitis A Virus 3C Protease Induces Ferroptosis in Human Cells In Vitro

2021 ◽  
Vol 22 (15) ◽  
pp. 7906
Author(s):  
Alexey A. Komissarov ◽  
Maria A. Karaseva ◽  
Marina P. Roschina ◽  
Andrey V. Shubin ◽  
Nataliya A. Lunina ◽  
...  
Keyword(s):  
Cell Death ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Proteolytic Enzymes ◽  
Morphological Changes ◽  
Human Cells ◽  
Mitochondrial Potential ◽  
3C Protease ◽  
Wide Range

Regulated cell death (RCD) is a fundamental process common to nearly all living beings and essential for the development and tissue homeostasis in animals and humans. A wide range of molecules can induce RCD, including a number of viral proteolytic enzymes. To date, numerous data indicate that picornaviral 3C proteases can induce RCD. In most reported cases, these proteases induce classical caspase-dependent apoptosis. In contrast, the human hepatitis A virus 3C protease (3Cpro) has recently been shown to cause caspase-independent cell death accompanied by previously undescribed features. Here, we expressed 3Cpro in HEK293, HeLa, and A549 human cell lines to characterize 3Cpro-induced cell death morphologically and biochemically using flow cytometry and fluorescence microscopy. We found that dead cells demonstrated necrosis-like morphological changes including permeabilization of the plasma membrane, loss of mitochondrial potential, as well as mitochondria and nuclei swelling. Additionally, we showed that 3Cpro-induced cell death was efficiently blocked by ferroptosis inhibitors and was accompanied by intense lipid peroxidation. Taken together, these results indicate that 3Cpro induces ferroptosis upon its individual expression in human cells. This is the first demonstration that a proteolytic enzyme can induce ferroptosis, the recently discovered and actively studied type of RCD.

scholarly journals Observation of an α-synuclein liquid droplet state and its maturation into Lewy body-like assemblies

2021 ◽  
Author(s):  
Maarten C Hardenberg ◽  
Tessa Sinnige ◽  
Sam Casford ◽  
Samuel Dada ◽  
Chetan Poudel ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Self Assembly ◽  
Lewy Body ◽  
Amyloid Fibrils ◽  
Liquid Droplet ◽  
Large Body ◽  
Lewy Bodies ◽  
Cellular Components

Abstract Misfolded α-synuclein is a major component of Lewy bodies, which are a hallmark of Parkinson’s disease. A large body of evidence shows that α-synuclein can aggregate into amyloid fibrils, but the relationship between α-synuclein self-assembly and Lewy body formation remains unclear. Here we show, both in vitro and in a Caenorhabditis elegans model of Parkinson’s disease, that α-synuclein undergoes liquid‒liquid phase separation by forming a liquid droplet state, which converts into an amyloid-rich hydrogel with Lewy-body-like properties. This maturation process towards the amyloid state is delayed in the presence of model synaptic vesicles in vitro. Taken together, these results suggest that the formation of Lewy bodies may be linked to the arrested maturation of α-synuclein condensates in the presence of lipids and other cellular components.

scholarly journals Modulation of Amyloidogenic Protein Self-Assembly Using Tethered Small Molecules

2020 ◽  
Author(s):  
Emma Cawood ◽  
Nicolas Guthertz ◽  
Jessica Ebo ◽  
Theodoros Karamanos ◽  
Sheena E. Radford FRS ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Structural Characterization ◽  
Self Assembly ◽  
Molecular Mechanisms ◽  
Amyloid Fibrils ◽  
Fibril Formation ◽  
X Ray Crystallography ◽  
Starting Point ◽  
Amyloid Assembly

<p></p><p>Protein-protein interactions (PPIs) are involved in many of life’s essential biological functions yet are also an underlying cause of several human diseases, including amyloidosis. The modulation of PPIs presents opportunities to gain mechanistic insights into amyloid assembly, particularly through the use of methods which can trap specific intermediates for detailed study. Such information can also provide a starting point for drug discovery. Here, we demonstrate that covalently tethered small molecule fragments can be used to stabilize specific oligomers during amyloid fibril formation, facilitating the structural characterization of these assembly intermediates. We exemplify the power of covalent tethering using the naturally occurring truncated variant (ΔN6) of the human protein β2-microglobulin (β2m), which assembles into amyloid fibrils associated with dialysis-related amyloidosis. Using this approach, we have trapped tetramers formed by ΔN6 under conditions which would normally lead to fibril formation and found that the degree of tetramer stabilization depends on the site of the covalent tether and the nature of the protein-fragment interaction. The covalent protein-ligand linkage enabled structural characterization of these trapped oligomeric species using X-ray crystallography and NMR, providing insight into why tetramer stabilization inhibits amyloid assembly. Our findings highlight the power of “post-translational chemical modification" as a tool to study biological molecular mechanisms. </p><br><p></p>

scholarly journals Kinetic Transition in Amyloid Assembly as a Screening Assay for Oligomer-Selective Dyes

Biomolecules ◽  
2019 ◽  
Vol 9 (10) ◽  
pp. 539 ◽  
Author(s):  
Jeremy Barton ◽  
D. Sebastian Arias ◽  
Chamani Niyangoda ◽  
Gustavo Borjas ◽  
Nathan Le ◽  
...  
Keyword(s):  
Crystal Violet ◽  
Amyloid Fibrils ◽  
Amyloid Β ◽  
Fibril Formation ◽  
Thioflavin T ◽  
Screening Assay ◽  
Kinetic Transition ◽  
Amyloid Assembly

Assembly of amyloid fibrils and small globular oligomers is associated with a significant number of human disorders that include Alzheimer’s disease, senile systemic amyloidosis, and type II diabetes. Recent findings implicate small amyloid oligomers as the dominant aggregate species mediating the toxic effects in these disorders. However, validation of this hypothesis has been hampered by the dearth of experimental techniques to detect, quantify, and discriminate oligomeric intermediates from late-stage fibrils, in vitro and in vivo. We have shown that the onset of significant oligomer formation is associated with a transition in thioflavin T kinetics from sigmoidal to biphasic kinetics. Here we showed that this transition can be exploited for screening fluorophores for preferential responses to oligomer over fibril formation. This assay identified crystal violet as a strongly selective oligomer-indicator dye for lysozyme. Simultaneous recordings of amyloid kinetics with thioflavin T and crystal violet enabled us to separate the combined signals into their underlying oligomeric and fibrillar components. We provided further evidence that this screening assay could be extended to amyloid-β peptides under physiological conditions. Identification of oligomer-selective dyes not only holds the promise of biomedical applications but provides new approaches for unraveling the mechanisms underlying oligomer versus fibril formation in amyloid assembly.

scholarly journals Heterotypic Aβ interactions facilitate amyloid assembly and modify amyloid structure

2021 ◽  
Author(s):  
Katerina Konstantoulea ◽  
Patricia Guerreiro ◽  
Meine Ramakers ◽  
Nikolaos Louros ◽  
Liam Aubrey ◽  
...  
Keyword(s):  
Self Assembly ◽  
Brain Regions ◽  
Amyloid Deposition ◽  
Proteomics Data ◽  
Aβ Amyloid ◽  
Heterotypic Interactions ◽  
Human Proteins ◽  
Fibril Morphology ◽  
Amyloid Assembly

AbstractIt is still unclear why pathological amyloid deposition initiates in specific brain regions, nor why specific cells or tissues are more susceptible than others. Amyloid deposition is determined by the self-assembly of short protein segments called aggregation-prone regions (APRs) that favour cross-β structure. Here we investigated whether Aβ amyloid assembly can be modified by heterotypic interactions between Aβ APRs and short homologous segments in otherwise unrelated human proteins. We identified heterotypic interactions that accelerate Aβ assembly, modify fibril morphology and affect its pattern of deposition in vitro. Moreover, we found that co-expression of these proteins in an Aβ reporter cell line promotes Aβ amyloid aggregation. Importantly, reanalysis of proteomics data of Aβ plaques from AD patients revealed an enrichment in proteins that share homologous sequences to the Aβ APRs, suggesting heterotypic amyloid interactions may occur in patients. Strikingly, we did not find such a bias in plaques from overexpression models in mouse. Based on these data, we propose that heterotypic APR interactions may play a hitherto unrealised role in amyloid-deposition diseases.

scholarly journals Diatom-Derived Polyunsaturated Aldehydes Activate Similar Cell Death Genes in Two Different Systems: Sea Urchin Embryos and Human Cells

2020 ◽  
Vol 21 (15) ◽  
pp. 5201 ◽  
Author(s):  
Christian Galasso ◽  
Susanna Celentano ◽  
Maria Costantini ◽  
Salvatore D’Aniello ◽  
Adrianna Ianora ◽  
...  
Keyword(s):  
Cell Death ◽  
Sea Urchin ◽  
Marine Organism ◽  
Sea Urchins ◽  
Paracentrotus Lividus ◽  
Human Cell Line ◽  
Human Cells ◽  
Similar Cell ◽  
Polyunsaturated Aldehydes

Programmed cell death, such as apoptosis and autophagy, are key processes that are activated early on during development, leading to remodelling in embryos and homeostasis in adult organisms. Genomic conservation of death factors has been largely investigated in the animal and plant kingdoms. In this study, we analysed, for the first time, the expression profile of 11 genes involved in apoptosis (extrinsic and intrinsic pathways) and autophagy in sea urchin Paracentrotus lividus embryos exposed to antiproliferative polyunsaturated aldehydes (PUAs), and we compared these results with those obtained on the human cell line A549 treated with the same molecules. We found that sea urchins and human cells activated, at the gene level, a similar cell death response to these compounds. Despite the evolutionary distance between sea urchins and humans, we observed that the activation of apoptotic and autophagic genes in response to cytotoxic compounds is a conserved process. These results give first insight on death mechanisms of P. lividus death mechanisms, also providing additional information for the use of this marine organism as a useful in vitro model for the study of cell death signalling pathways activated in response to chemical compounds.

scholarly journals Mechanism of Cellular Formation and In Vivo Seeding Effects of Hexameric β-Amyloid Assemblies

2021 ◽  
Author(s):  
Céline Vrancx ◽  
Devkee M. Vadukul ◽  
Nuria Suelves ◽  
Sabrina Contino ◽  
Ludovic D’Auria ◽  
...  
Keyword(s):  
Cell Lines ◽  
Self Assembly ◽  
Amyloid Fibrils ◽  
Senile Plaques ◽  
Amyloid Peptide ◽  
Cellular Models ◽  
Aβ Aggregation ◽  
Β Amyloid

AbstractThe β-amyloid peptide (Aβ) is found as amyloid fibrils in senile plaques, a typical hallmark of Alzheimer’s disease (AD). However, intermediate soluble oligomers of Aβ are now recognized as initiators of the pathogenic cascade leading to AD. Studies using recombinant Aβ have shown that hexameric Aβ in particular acts as a critical nucleus for Aβ self-assembly. We recently isolated hexameric Aβ assemblies from a cellular model, and demonstrated their ability to enhance Aβ aggregation in vitro. Here, we report the presence of similar hexameric-like Aβ assemblies across several cellular models, including neuronal-like cell lines. In order to better understand how they are produced in a cellular context, we investigated the role of presenilin-1 (PS1) and presenilin-2 (PS2) in their formation. PS1 and PS2 are the catalytic subunits of the γ-secretase complex that generates Aβ. Using CRISPR-Cas9 to knockdown each of the two presenilins in neuronal-like cell lines, we observed a direct link between the PS2-dependent processing pathway and the release of hexameric-like Aβ assemblies in extracellular vesicles. Further, we assessed the contribution of hexameric Aβ to the development of amyloid pathology. We report the early presence of hexameric-like Aβ assemblies in both transgenic mice brains exhibiting human Aβ pathology and in the cerebrospinal fluid of AD patients, suggesting hexameric Aβ as a potential early AD biomarker. Finally, cell-derived hexameric Aβ was found to seed other human Aβ forms, resulting in the aggravation of amyloid deposition in vivo and neuronal toxicity in vitro.

scholarly journals Individual expression of hepatitis A virus 3C protease induces ferroptosis in human cells in vitro

2021 ◽  
Author(s):  
Alexey A. Komissarov ◽  
Maria A. Karaseva ◽  
Marina P. Roschina ◽  
Andrey V. Shubin ◽  
Nataliya A. Lunina ◽  
...  
Keyword(s):  
Cell Death ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Proteolytic Enzymes ◽  
Human Cells ◽  
3C Protease ◽  
Regulated Cell Death ◽  
Wide Range ◽  
Numerous Data

Regulated cell death (RCD) is a fundamental process common to nearly all living beings and essential for the development and tissue homeostasis in animals and humans. A wide range of molecules can induce RCD including a number of viral proteolytic enzymes. To date, numerous data indicate that picornaviral 3C proteases can induce RCD. In most reported cases, these proteases induce classical caspase-dependent apoptosis. In contrast, the human hepatitis A virus 3C protease (3Cpro) has recently been shown to cause caspase-independent cell death accompanied by previously undescribed features. Here, we describe the 3Cpro effect on human cells in vitro and demonstrate that the enzyme induces ferroptosis which is mediated by iron ions and cause oxidative stress in the cells. This is the first demonstration that proteolytic enzyme can induce ferroptosis, the recently discovered and actively studied type of RCD.

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