scholarly journals Neuroserpin Inclusion Bodies in a FENIB Yeast Model

2021 ◽  
Vol 9 (7) ◽  
pp. 1498
Author(s):  
Valentina Vapore ◽  
Corrado Mazzaglia ◽  
Diego Sibilia ◽  
Mara Del Vecchio ◽  
Gernot Fruhmann ◽  
...  
Keyword(s):  
Cell Death ◽  
Mammalian Cells ◽  
Inclusion Bodies ◽  
Point Mutations ◽  
Monogenic Disease ◽  
Lag Phase ◽  
Cell Toxicity ◽  
Wild Type ◽  
Familial Encephalopathy ◽  
Human Wild Type

FENIB (familial encephalopathy with neuroserpin inclusion bodies) is a human monogenic disease caused by point mutations in the SERPINI1 gene, characterized by the intracellular deposition of polymers of neuroserpin (NS), which leads to proteotoxicity and cell death. Despite the different cell and animal models developed thus far, the exact mechanism of cell toxicity elicited by NS polymers remains unclear. Here, we report that human wild-type NS and the polymerogenic variant G392E NS form protein aggregates mainly localized within the endoplasmic reticulum (ER) when expressed in the yeast S. cerevisiae. The expression of NS in yeast delayed the exit from the lag phase, suggesting that NS inclusions cause cellular stress. The cells also showed a higher resistance following mild oxidative stress treatments when compared to control cells. Furthermore, the expression of NS in a pro-apoptotic mutant strain-induced cell death during aging. Overall, these data recapitulate phenotypes observed in mammalian cells, thereby validating S. cerevisiae as a model for FENIB.

scholarly journals G392E neuroserpin causing the dementia FENIB is secreted from cells but is not synaptotoxic

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Thies Ingwersen ◽  
Christian Linnenberg ◽  
Emanuela D’Acunto ◽  
Shabnam Temori ◽  
Irene Paolucci ◽  
...  
Keyword(s):  
Cell Death ◽  
Hippocampal Neurons ◽  
Inclusion Bodies ◽  
Cell Model ◽  
Point Mutations ◽  
Mutant Form ◽  
Hek 293 ◽  
Extracellular Milieu ◽  
Familial Encephalopathy ◽  
And Oxidative Stress

AbstractFamilial encephalopathy with neuroserpin inclusion bodies (FENIB) is a progressive neurodegenerative disease caused by point mutations in the gene for neuroserpin, a serine protease inhibitor of the nervous system. Different mutations are known that are responsible for mutant neuroserpin polymerization and accumulation as inclusion bodies in many cortical and subcortical neurons, thereby leading to cell death, dementia and epilepsy. Many efforts have been undertaken to elucidate the molecular pathways responsible for neuronal death. Most investigations have concentrated on analysis of intracellular mechanisms such as endoplasmic reticulum (ER) stress, ER-associated protein degradation (ERAD) and oxidative stress. We have generated a HEK-293 cell model of FENIB by overexpressing G392E-mutant neuroserpin and in this study we examine trafficking and toxicity of this polymerogenic variant. We observed that a small fraction of mutant neuroserpin is secreted via the ER-to-Golgi pathway, and that this release can be pharmacologically regulated. Overexpression of the mutant form of neuroserpin did not stimulate cell death in the HEK-293 cell model. Finally, when treating primary hippocampal neurons with G392E neuroserpin polymers, we did not detect cytotoxicity or synaptotoxicity. Altogether, we report here that a polymerogenic mutant form of neuroserpin is secreted from cells but is not toxic in the extracellular milieu.

scholarly journals Conserved Motifs within Ebola and Marburg Virus VP40 Proteins Are Important for Stability, Localization, and Subsequent Budding of Virus-Like Particles

2009 ◽  
Vol 84 (5) ◽  
pp. 2294-2303 ◽  
Author(s):  
Yuliang Liu ◽  
Luis Cocka ◽  
Atsushi Okumura ◽  
Yong-An Zhang ◽  
J. Oriol Sunyer ◽  
...  
Keyword(s):  
Self Assembly ◽  
Mammalian Cells ◽  
Ebola Virus ◽  
Intracellular Localization ◽  
Gel Filtration ◽  
Point Mutations ◽  
Marburg Virus ◽  
Structure Stability ◽  
Wild Type ◽  
Virus Like Particles

ABSTRACT The filovirus VP40 protein is capable of budding from mammalian cells in the form of virus-like particles (VLPs) that are morphologically indistinguishable from infectious virions. Ebola virus VP40 (eVP40) contains well-characterized overlapping L domains, which play a key role in mediating efficient virus egress. L domains represent only one component required for efficient budding and, therefore, there is a need to identify and characterize additional domains important for VP40 function. We demonstrate here that the 96LPLGVA101 sequence of eVP40 and the corresponding 84LPLGIM89 sequence of Marburg virus VP40 (mVP40) are critical for efficient release of VP40 VLPs. Indeed, deletion of these motifs essentially abolished the ability of eVP40 and mVP40 to bud as VLPs. To address the mechanism by which the 96LPLGVA101 motif of eVP40 contributes to egress, a series of point mutations were introduced into this motif. These mutants were then compared to the eVP40 wild type in a VLP budding assay to assess budding competency. Confocal microscopy and gel filtration analyses were performed to assess their pattern of intracellular localization and ability to oligomerize, respectively. Our results show that mutations disrupting the 96LPLGVA101 motif resulted in both altered patterns of intracellular localization and self-assembly compared to wild-type controls. Interestingly, coexpression of either Ebola virus GP-WT or mVP40-WT with eVP40-ΔLPLGVA failed to rescue the budding defective eVP40-ΔLPLGVA mutant into VLPs; however, coexpression of eVP40-WT with mVP40-ΔLPLGIM successfully rescued budding of mVP40-ΔLPLGIM into VLPs at mVP40-WT levels. In sum, our findings implicate the LPLGVA and LPLGIM motifs of eVP40 and mVP40, respectively, as being important for VP40 structure/stability and budding.

scholarly journals Proteolytic processing of Semliki Forest virus-specific non-structural polyprotein by nsP2 protease

2001 ◽  
Vol 82 (4) ◽  
pp. 765-773 ◽  
Author(s):  
Andres Merits ◽  
Lidia Vasiljeva ◽  
Tero Ahola ◽  
Leevi Kääriäinen ◽  
Petri Auvinen
Keyword(s):  
Mammalian Cells ◽  
Active Sites ◽  
Insect Cells ◽  
Semliki Forest Virus ◽  
Point Mutations ◽  
Proteolytic Processing ◽  
Wild Type ◽  
In Trans ◽  
Polyprotein Precursor

The RNA replicase proteins of Semliki Forest virus (SFV) are translated as a P1234 polyprotein precursor that contains two putative autoproteases. Point mutations introduced into the predicted active sites of both proteases nsP2 (P2) and nsP4 (P4), separately or in combination, completely abolished virus replication in mammalian cells. The effects of these mutations on polyprotein processing were studied by in vitro translation and by expression of wild-type polyproteins P1234, P123, P23, P34 and their mutated counterparts in insect cells using recombinant baculoviruses. A mutation in the catalytic site of the P2 protease, C478A, (P2CA) completely abolished the processing of P12CA34, P12CA3 and P2CA3. Co-expression of P23 and P12CA34 in insect cells resulted in in trans cleavages at the P2/3 and P3/4 sites. Co-expression of P23 and P34 resulted in cleavage at the P3/4 site. In contrast, a construct with a mutation in the active site of the putative P4 protease, D6A, (P1234DA) was processed like the wild-type protein. P34 or its truncated forms were not processed when expressed alone. In insect cells, P4 was rapidly destroyed unless an inhibitor of proteosomal degradation was used. It is concluded that P2 is the only protease needed for the processing of SFV polyprotein P1234. Analysis of the cleavage products revealed that P23 or P2 could not cleave the P1/2 site in trans.

scholarly journals Molecular Nature of Spontaneous Modifications in gacS Which Cause Colony Phase Variation in Pseudomonas sp. Strain PCL1171

2005 ◽  
Vol 187 (2) ◽  
pp. 593-600 ◽  
Author(s):  
Daan van den Broek ◽  
Thomas F. C. Chin-A-Woeng ◽  
Guido V. Bloemberg ◽  
Ben J. J. Lugtenberg
Keyword(s):  
Phase I ◽  
Phase Ii ◽  
High Frequency ◽  
Point Mutations ◽  
Phase Variation ◽  
Lag Phase ◽  
Spontaneous Mutations ◽  
Wild Type ◽  
Type Population ◽  
Molecular Nature

ABSTRACT Pseudomonas sp. strain PCL1171 displays colony phase variation between opaque phase I and translucent phase II colonies, thereby regulating the production of secondary metabolites and exoenzymes. Complementation and sequence analysis of 26 phase II mutants and of 13 wild-type phase II sectors growing out of phase I colonies showed that in all these cases the phase II phenotype is caused by spontaneous mutations in gacA or/and gacS. Mutation of gac reduced both the length of the lag phase and the generation time. Isolation and sequencing of the gacS genes from the phase II bacteria revealed one insertion as well as several random point mutations, deletions, and DNA rearrangements. Most phase II colonies reverted with a high frequency, resulting in wild-type gacA and gacS genes and a phase I phenotype. Some phase II bacteria retained the phase II phenotype but changed genotypically as a result of (re)introduction of mutations in either gacA or gacS. The reversion of gacA or gacS to the wild type was not affected by mutation of recA and recB. We conclude that in Pseudomonas sp. strain PCL1171, mutations in gacA and gacS are the basis for phase variation from phase I to phase II colonies and that, since these mutations are efficiently removed, mutations in gac result in dynamic switches between the “wild-type” population and the subpopulations harboring spontaneous mutations in gacA and or gacS, thereby enabling both populations to be maintained.

scholarly journals Embelin as Lead Compound for New Neuroserpin Polymerization Inhibitors

Life ◽  
2020 ◽  
Vol 10 (7) ◽  
pp. 111
Author(s):  
Cristina Visentin ◽  
Loana Musso ◽  
Luca Broggini ◽  
Francesca Bonato ◽  
Rosaria Russo ◽  
...  
Keyword(s):  
Inclusion Bodies ◽  
Point Mutations ◽  
Lead Compound ◽  
Specific Point ◽  
Familial Encephalopathy ◽  
The Central Nervous System ◽  
The One ◽  
Interaction Surface ◽  
Better Than

Familial encephalopathy with neuroserpin inclusion bodies (FENIB) is a severe and lethal neurodegenerative disease. Upon specific point mutations in the SERPINI1gene-coding for the human protein neuroserpin (NS) the resulting pathologic NS variants polymerize and accumulate within the endoplasmic reticulum of neurons in the central nervous system. To date, embelin (EMB) is the only known inhibitor of NS polymerization in vitro. This molecule is capable of preventing NS polymerization and dissolving preformed polymers. Here, we show that lowering EMB concentration results in increasing size of NS oligomers in vitro. Moreover, we observe that in cells expressing NS, the polymerization of G392E NS is reduced, but this effect is mediated by an increased proteasomal degradation rather than polymerization impairment. For these reasons we designed a systematic chemical evolution of the EMB scaffold aimed to improve its anti-polymerization properties. The effect of EMB analogs against NS polymerization was assessed in vitro. None of the EMB analogs displayed an anti-polymerization activity better than the one reported for EMB, indicating that the EMB–NS interaction surface is very specific and highly optimized. Thus, our results indicate that EMB is, to date, still the best candidate for developing a treatment against NS polymerization.

scholarly journals Calcium signalling in mammalian cell lines expressing wild type and mutant human α1-Antitrypsin

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Nancy T. Malintan ◽  
Steven D. Buckingham ◽  
David A. Lomas ◽  
David B. Sattelle
Keyword(s):  
Cell Lines ◽  
Inclusion Bodies ◽  
Autosomal Dominant ◽  
Calcium Signalling ◽  
Wild Type ◽  
Dominant Form ◽  
Mammalian Cell Lines ◽  
Autosomal Dominant Form ◽  
Familial Encephalopathy ◽  
Sense And Respond

AbstractA possible role for calcium signalling in the autosomal dominant form of dementia, familial encephalopathy with neuroserpin inclusion bodies (FENIB), has been proposed, which may point towards a mechanism by which cells could sense and respond to the accumulation of mutant serpin polymers in the endoplasmic reticulum (ER). We therefore explored possible defects in Ca2+-signalling, which may contribute to the pathology associated with another serpinopathy, α1-antitrypsin (AAT) deficiency. Using CHO K1 cell lines stably expressing a wild type human AAT (MAAT) and a disease-causing polymer-forming variant (ZAAT) and the truncated variant (NHK AAT), we measured basal intracellular free Ca2+, its responses to thapsigargin (TG), an ER Ca2+-ATPase blocker, and store-operated Ca2+-entry (SOCE). Our fura2 based Ca2+ measurements detected no differences between these 3 parameters in cell lines expressing MAAT and cell lines expressing ZAAT and NHK AAT mutants. Thus, in our cell-based models of α1-antitrypsin (AAT) deficiency, unlike the case for FENIB, we were unable to detect defects in calcium signalling.

The P-MAPA immunomodulator partially prevents apoptosis induced by Zika virus infection in THP-1 cells

2020 ◽  
Vol 21 ◽  
Author(s):  
Morganna C. Lima ◽  
Elisa A. N. Azevedo ◽  
Clarice N. L. de Morais ◽  
Larissa I. O. de Sousa ◽  
Bruno M. Carvalho ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Death ◽  
Virus Infection ◽  
Virus Replication ◽  
Zika Virus ◽  
Mammalian Cells ◽  
Caspase 3 ◽  
Neurological Complications ◽  
Zika Virus Infection

Background: Zika virus is an emerging arbovirus of global importance. ZIKV infection is associated with a range of neurological complications such as the Congenital Zika Syndrome and Guillain Barré Syndrome. Despite the magnitude of recent outbreaks, there is no specific therapy to prevent or to alleviate disease pathology. Objective: To investigate the role of P-MAPA immunomodulator in Zika-infected THP-1 cells. Methods: THP-1 cells were subjected at Zika virus infection (Multiplicity of Infection = 0.5) followed by treatment with P-MAPA for until 96 hours post-infection. After that, the cell death was analyzed by annexin+/ PI+ and caspase 3/ 7+ staining by flow cytometry. In addition, the virus replication and cell proliferation were accessed by RT-qPCR and Ki67 staining, respectively. Results: We demonstrate that P-MAPA in vitro treatment significantly reduces Zika virus-induced cell death and caspase-3/7 activation on THP-1 infected cells, albeit it has no role in virus replication and cell proliferation. Conclusions: Our study reveals that P-MAPA seems to be a satisfactory alternative to inhibits the effects of Zika virus infection in mammalian cells.

scholarly journals Salicylic Acid Accumulation Controlled by LSD1 Is Essential in Triggering Cell Death in Response to Abiotic Stress

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 962
Author(s):  
Maciej Jerzy Bernacki ◽  
Anna Rusaczonek ◽  
Weronika Czarnocka ◽  
Stanisław Karpiński
Keyword(s):  
Cell Death ◽  
Salicylic Acid ◽  
Abiotic Stress ◽  
Wild Type ◽  
Pr Genes ◽  
Genes Expression ◽  
Salicylic Acid Accumulation ◽  
Cell Death Phenotype ◽  
Insight Into

Salicylic acid (SA) is well known hormonal molecule involved in cell death regulation. In response to a broad range of environmental factors (e.g., high light, UV, pathogens attack), plants accumulate SA, which participates in cell death induction and spread in some foliar cells. LESION SIMULATING DISEASE 1 (LSD1) is one of the best-known cell death regulators in Arabidopsis thaliana. The lsd1 mutant, lacking functional LSD1 protein, accumulates SA and is conditionally susceptible to many biotic and abiotic stresses. In order to get more insight into the role of LSD1-dependent regulation of SA accumulation during cell death, we crossed the lsd1 with the sid2 mutant, caring mutation in ISOCHORISMATE SYNTHASE 1(ICS1) gene and having deregulated SA synthesis, and with plants expressing the bacterial nahG gene and thus decomposing SA to catechol. In response to UV A+B irradiation, the lsd1 mutant exhibited clear cell death phenotype, which was reversed in lsd1/sid2 and lsd1/NahG plants. The expression of PR-genes and the H2O2 content in UV-treated lsd1 were significantly higher when compared with the wild type. In contrast, lsd1/sid2 and lsd1/NahG plants demonstrated comparability with the wild-type level of PR-genes expression and H2O2. Our results demonstrate that SA accumulation is crucial for triggering cell death in lsd1, while the reduction of excessive SA accumulation may lead to a greater tolerance toward abiotic stress.

scholarly journals Uncoupling Salicylic Acid-Dependent Cell Death and Defense-Related Responses From Disease Resistance in the Arabidopsis Mutant acd5

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 341-350
Author(s):  
Jean T Greenberg ◽  
F Paul Silverman ◽  
Hua Liang
Keyword(s):  
Cell Death ◽  
Salicylic Acid ◽  
Disease Resistance ◽  
Pseudomonas Syringae ◽  
Higher Plants ◽  
Ethylene Signaling ◽  
Symptom Development ◽  
Wild Type ◽  
Dependent Cell ◽  
Arabidopsis Mutant

Abstract Salicylic acid (SA) is required for resistance to many diseases in higher plants. SA-dependent cell death and defense-related responses have been correlated with disease resistance. The accelerated cell death 5 mutant of Arabidopsis provides additional genetic evidence that SA regulates cell death and defense-related responses. However, in acd5, these events are uncoupled from disease resistance. acd5 plants are more susceptible to Pseudomonas syringae early in development and show spontaneous SA accumulation, cell death, and defense-related markers later in development. In acd5 plants, cell death and defense-related responses are SA dependent but they do not confer disease resistance. Double mutants with acd5 and nonexpressor of PR1, in which SA signaling is partially blocked, show greatly attenuated cell death, indicating a role for NPR1 in controlling cell death. The hormone ethylene potentiates the effects of SA and is important for disease symptom development in Arabidopsis. Double mutants of acd5 and ethylene insensitive 2, in which ethylene signaling is blocked, show decreased cell death, supporting a role for ethylene in cell death control. We propose that acd5 plants mimic P. syringae-infected wild-type plants and that both SA and ethylene are normally involved in regulating cell death during some susceptible pathogen infections.

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