scholarly journals Apoptotic stress induces Bax-dependent, caspase-independent redistribution of LINC complex nesprins

2020 ◽  
Vol 6 (1) ◽  
Author(s):  
Liora Lindenboim ◽  
Dan Grozki ◽  
Ayelet R. Amsalem-Zafran ◽  
Aida Peña-Blanco ◽  
Gregg G. Gundersen ◽  
...  
Keyword(s):  
Membrane Integrity ◽  
Mitochondrial Outer Membrane ◽  
Linc Complex ◽  
Mitochondrial Targeting ◽  
Double Knockout ◽  
Binding Partner ◽  
Mouse Embryo Fibroblasts ◽  
Bh3 Domain ◽  
The Family ◽  
Close Proximity

Abstract The canonical function of Bcl-2 family proteins is to regulate mitochondrial membrane integrity. In response to apoptotic signals the multi-domain pro-apoptotic proteins Bax and Bak are activated and perforate the mitochondrial outer membrane by a mechanism which is inhibited by their interaction with pro-survival members of the family. However, other studies have shown that Bax and Bak may have additional, non-canonical functions, which include stress-induced nuclear envelope rupture and discharge of nuclear proteins into the cytosol. We show here that the apoptotic stimuli cisplatin and staurosporine induce a Bax/Bak-dependent degradation and subcellular redistribution of nesprin-1 and nesprin-2 but not nesprin-3, of the linker of nucleoskeleton and cytoskeleton (LINC) complex. The degradation and redistribution were caspase-independent and did not occur in Bax/Bak double knockout (DKO) mouse embryo fibroblasts (MEFs). Re-expression of Bax in Bax/Bak DKO MEFs restored stress-induced redistribution of nesprin-2 by a mechanism which requires Bax membrane localization and integrity of the α helices 5/6, and the Bcl-2 homology 3 (BH3) domain. We found that nesprin-2 interacts with Bax in close proximity to perinuclear mitochondria in mouse and human cells. This interaction requires the mitochondrial targeting and N-terminal region but not the BH3 domain of Bax. Our results identify nesprin-2 as a Bax binding partner and also a new function of Bax in impairing the integrity of the LINC complex.

scholarly journals Involvement of Noxa in Cellular Apoptotic Responses to Interferon, Double-stranded RNA, and Virus Infection

2005 ◽  
Vol 280 (16) ◽  
pp. 15561-15568 ◽  
Author(s):  
Yaping Sun ◽  
Douglas W. Leaman
Keyword(s):  
Mitochondrial Protein ◽  
Membrane Integrity ◽  
Ectopic Expression ◽  
Human Tumor ◽  
Mitochondrial Outer Membrane ◽  
Dependent Manner ◽  
Mitochondrial Targeting ◽  
Double Stranded Rna ◽  
Bh3 Domain ◽  
Apoptosis Protein

Double-stranded RNA (dsRNA) accumulates in virally infected cells, leading to induction of genes encoding proteins involved in signaling, apoptosis, protein synthesis/processing, and cell metabolism. Noxa is a BH3-containing mitochondrial protein that contributes to apoptosis by disrupting mitochondrial outer membrane integrity. Here we demonstrate potent induction of Noxa expression by exposure of cells to dsRNA, interferon (IFN), and virus. Noxa induction was confirmed by using reverse transcriptase-PCR and immunoblot analyses in multiple human tumor cell lines. Importantly, Noxa regulation by IFN and dsRNA was independent of p53, thereby identifying a novel mechanism of Noxa induction. Ectopic expression of Noxa in HT1080 fibrosarcoma cells enhanced cellular sensitivity to viral or dsRNA/actinomycin D-induced apoptosis, typified by enhanced cytochromecrelease from the mitochondrial to the cytosolic fraction and increased cleavage of caspases 3 and 9. Point and deletion mutations of Noxa confirmed that both the BH3 domain and the mitochondrial-targeting domain were necessary for enhanced cellular apoptotic responses to dsRNA, IFN, or virus. Treatment of cells with dsRNA or virus, but not etoposide, induced interaction between Noxa and Bax that required an intact Noxa BH3 domain. Interestingly, the Noxa mitochondrial-targeting domain deletion mutant interacted with Bax in a dsRNA-dependent manner and redirected Bax away from the mitochondria, thus acting as a dominant-negative protein. Together, these data suggest that Noxa is an important component of the innate immune response of cells to viral infection, leading to enhanced cellular apoptosis that may play a role in limiting viral dissemination.

scholarly journals Tom20 Mediates Localization of mRNAs to Mitochondria in a Translation-Dependent Manner

2009 ◽  
Vol 30 (1) ◽  
pp. 284-294 ◽  
Author(s):  
Erez Eliyahu ◽  
Lilach Pnueli ◽  
Daniel Melamed ◽  
Tanja Scherrer ◽  
André P. Gerber ◽  
...  
Keyword(s):  
Large Scale ◽  
Dna Microarrays ◽  
Protein Transport ◽  
Yeast Cells ◽  
Mitochondrial Proteins ◽  
Mitochondrial Outer Membrane ◽  
Dependent Manner ◽  
Mitochondrial Targeting ◽  
Double Knockout ◽  
Mitochondrial Fractions

ABSTRACT mRNAs encoding mitochondrial proteins are enriched in the vicinity of mitochondria, presumably to facilitate protein transport. A possible mechanism for enrichment may involve interaction of the translocase of the mitochondrial outer membrane (TOM) complex with the precursor protein while it is translated, thereby leading to association of polysomal mRNAs with mitochondria. To test this hypothesis, we isolated mitochondrial fractions from yeast cells lacking the major import receptor, Tom20, and compared their mRNA repertoire to that of wild-type cells by DNA microarrays. Most mRNAs encoding mitochondrial proteins were less associated with mitochondria, yet the extent of decrease varied among genes. Analysis of several mRNAs revealed that optimal association of Tom20 target mRNAs requires both translating ribosomes and features within the encoded mitochondrial targeting signal. Recently, Puf3p was implicated in the association of mRNAs with mitochondria through interaction with untranslated regions. We therefore constructed a tom20Δ puf3Δ double-knockout strain, which demonstrated growth defects under conditions where fully functional mitochondria are required. Mislocalization effects for few tested mRNAs appeared stronger in the double knockout than in the tom20Δ strain. Taken together, our data reveal a large-scale mRNA association mode that involves interaction of Tom20p with the translated mitochondrial targeting sequence and may be assisted by Puf3p.

scholarly journals Structural Mechanism for Regulation of Bcl-2 protein Noxa by phosphorylation

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Christine B. Karim ◽  
L. Michel Espinoza-Fonseca ◽  
Zachary M. James ◽  
Eric A. Hanse ◽  
Jeffrey S. Gaynes ◽  
...  
Keyword(s):  
Md Simulations ◽  
Salt Bridges ◽  
Binding Assays ◽  
Peptide Backbone ◽  
Structural Mechanism ◽  
Binding Partner ◽  
Flexible Loop ◽  
Bh3 Domain ◽  
N Terminus ◽  
Β Sheets

Abstract We showed previously that phosphorylation of Noxa, a 54-residue Bcl-2 protein, at serine 13 (Ser13) inhibited its ability to promote apoptosis through interactions with canonical binding partner, Mcl-1. Using EPR spectroscopy, molecular dynamics (MD) simulations and binding assays, we offer evidence that a structural alteration caused by phosphorylation partially masks Noxa’s BH3 domain, inhibiting the Noxa-Mcl-1 interaction. EPR of unphosphorylated Noxa, with spin-labeled amino acid TOAC incorporated within the BH3 domain, revealed equilibrium between ordered and dynamically disordered states. Mcl-1 further restricted the ordered component for non-phosphorylated Noxa, but left the pSer13 Noxa profile unchanged. Microsecond MD simulations indicated that the BH3 domain of unphosphorylated Noxa is housed within a flexible loop connecting two antiparallel β-sheets, flanked by disordered N- and C-termini and Ser13 phosphorylation creates a network of salt-bridges that facilitate the interaction between the N-terminus and the BH3 domain. EPR showed that a spin label inserted near the N-terminus was weakly immobilized in unphosphorylated Noxa, consistent with a solvent-exposed helix/loop, but strongly constrained in pSer13 Noxa, indicating a more ordered peptide backbone, as predicted by MD simulations. Together these studies reveal a novel mechanism by which phosphorylation of a distal serine inhibits a pro-apoptotic BH3 domain and promotes cell survival.

The Modern Life of the East African Native

Africa ◽  
1932 ◽  
Vol 5 (1) ◽  
pp. 50-60 ◽  
Author(s):  
H. M. T. Kayamba
Keyword(s):  
Family Life ◽  
The Other ◽  
East African ◽  
Modern Life ◽  
Full Control ◽  
The Family ◽  
Close Proximity ◽  
Heavy Work ◽  
The Village

The family life of an African is primarily based on polygamy and patriarchy. Each family has its own village and the head of the family is elder of the village. As soon as a youth gets married and has children he thinks of establishing his own village in order to obtain sufficient land for cultivation for himself and his children. This is the start of a native village. He calls the village by some name which comes to his fancy. Probably after a few months few people join him at the newly established village; thus the village grows and the founder is called the elder of the village. The next thought of the African after he has acquired a little wealth is to increase the number of his wives to the number that his wealth can provide him. Very often he keeps them in different huts and at different villages which he calls Mtaa, meaning a quarter. He spends days and nights proportionately at each hut, usually three nights at each hut if they are in close proximity or seven days if they are far. He calls it Kugawa ngono, which means the distribution of conjugal rights. Each wife has her own farm which she cultivates with her children, her husband doing the heavy work. The husband has his own farm over which he has authority. Each wife harvests and keeps her own food in her granary in the hut. She has full control over her own food. She feeds and clothes her children from the proceeds of the sale of her crop; and feeds her husband with the same food when he stays with her. The crop from the husband's farm is stored in the senior wife's hut. The senior wife is the first wife in marriage. She keeps all the money of the husband and distributes food to the other wives from the husband's store when the other wives have run short of food.

scholarly journals The Toxoplasma gondii protein ROP2 mediates host organelle association with the parasitophorous vacuole membrane

2001 ◽  
Vol 154 (1) ◽  
pp. 95-108 ◽  
Author(s):  
Anthony P. Sinai ◽  
Keith A. Joiner
Keyword(s):  
Toxoplasma Gondii ◽  
Fluorescent Protein ◽  
Parasitophorous Vacuole ◽  
Host Cells ◽  
In Vitro Assays ◽  
Mitochondrial Outer Membrane ◽  
Mitochondrial Targeting ◽  
Parasitophorous Vacuole Membrane ◽  
Parasite Protein ◽  
Vacuole Membrane

Toxoplasma gondii replicates within a specialized vacuole surrounded by the parasitophorous vacuole membrane (PVM). The PVM forms intimate interactions with host mitochondria and endoplasmic reticulum (ER) in a process termed PVM–organelle association. In this study we identify a likely mediator of this process, the parasite protein ROP2. ROP2, which is localized to the PVM, is secreted from anterior organelles termed rhoptries during parasite invasion into host cells. The NH2-terminal domain of ROP2 (ROP2hc) within the PVM is exposed to the host cell cytosol, and has characteristics of a mitochondrial targeting signal. In in vitro assays, ROP2hc is partially translocated into the mitochondrial outer membrane and behaves like an integral membrane protein. Although ROP2hc does not translocate across the ER membrane, it does exhibit carbonate-resistant binding to this organelle. In vivo, ROP2hc expressed as a soluble fragment in the cytosol of uninfected cells associates with both mitochondria and ER. The 30–amino acid (aa) NH2-terminal sequence of ROP2hc, when fused to green fluorescent protein (GFP), is sufficient for mitochondrial targeting. Deletion of the 30-aa NH2-terminal signal from ROP2hc results in robust localization of the truncated protein to the ER. These results demonstrate a new mechanism for tight association of different membrane-bound organelles within the cell cytoplasm.

scholarly journals Contamination of Sandpits with Soil-Transmitted Helminths Eggs in an Urban Environment

2019 ◽  
Vol 63 (1) ◽  
pp. 60-63
Author(s):  
J. Bystrianska ◽  
I. Papajová ◽  
J. Šoltys ◽  
N. Sasáková
Keyword(s):  
Public Health ◽  
Urban Environment ◽  
Toxocara Canis ◽  
The Public ◽  
Soil Transmitted Helminths ◽  
Helminth Eggs ◽  
Faecal Samples ◽  
The Family ◽  
Close Proximity ◽  
Toxascaris Leonina

Abstract The aim of this study was to monitor the occurrence of the propagative stages of intestinal endoparasites in dog excrements collected within the close proximity of sandpits in an urban environment (Košice, Slovakia) and to determine the level of sandpits contamination with soil-transmitted helminths (STHs). A total of 201 dog faecal samples were examined for the presence of helminth eggs with 10.95 % of the samples being positive. In faeces the most prevalent eggs were those of Toxocara canis (7.46 %). The contamination of sand with STH eggs in 84 sandpits was also investigated. Toxocara spp. eggs were found in 21.43 % of the sandpits. The eggs from the family Ancylostomatidae and Toxascaris leonina were also present. Taenia type eggs and Trichuris sp. eggs occurred less frequently. In some samples, not only monoinfection but also co-infection with eggs of 2‒3 helminth species were detected. In conclusion, the environmental contamination of sandpits with STHs eggs might pose a significant threat to the public health.

scholarly journals Paradoxical implication of BAX/BAK in the persistence of tetraploid cells

2021 ◽  
Vol 12 (11) ◽  
Author(s):  
Jiayin Deng ◽  
Lucía G. Gutiérrez ◽  
Gautier Stoll ◽  
Omar Motiño ◽  
Isabelle Martins ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Kinase Inhibitors ◽  
Mitochondrial Outer Membrane ◽  
Double Knockout ◽  
Microtubule Inhibitor ◽  
Bcl2 Family ◽  
Mitochondrial Outer Membrane Permeabilization ◽  
Rate Limiting Step ◽  
Low Incidence ◽  
Senescence Program

AbstractPro-apoptotic multi-domain proteins of the BCL2 family such as BAX and BAK are well known for their important role in the induction of mitochondrial outer membrane permeabilization (MOMP), which is the rate-limiting step of the intrinsic pathway of apoptosis. Human or mouse cells lacking both BAX and BAK (due to a double knockout, DKO) are notoriously resistant to MOMP and cell death induction. Here we report the surprising finding that BAX/BAK DKO cells proliferate less than control cells expressing both BAX and BAK (or either BAX or BAK) when they are driven into tetraploidy by transient exposure to the microtubule inhibitor nocodazole. Mechanistically, in contrast to their BAX/BAK-sufficient controls, tetraploid DKO cells activate a senescent program, as indicated by the overexpression of several cyclin-dependent kinase inhibitors and the activation of β-galactosidase. Moreover, DKO cells manifest alterations in ionomycin-mobilizable endoplasmic reticulum (ER) Ca2+ stores and store-operated Ca2+ entry that are affected by tetraploidization. DKO cells manifested reduced expression of endogenous sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (Serca2a) and transfection-enforced reintroduction of Serca2a, or reintroduction of an ER-targeted variant of BAK into DKO cells reestablished the same pattern of Ca2+ fluxes as observed in BAX/BAK-sufficient control cells. Serca2a reexpression and ER-targeted BAK also abolished the tetraploidy-induced senescence of DKO cells, placing ER Ca2+ fluxes downstream of the regulation of senescence by BAX/BAK. In conclusion, it appears that BAX/BAK prevent the induction of a tetraploidization-associated senescence program. Speculatively, this may contribute to the low incidence of cancers in BAX/BAK DKO mice and explain why human cancers rarely lose the expression of both BAX and BAK.

scholarly journals Mitochondrial residence of the apoptosis inducer BAX is more important than BAX oligomerization in promoting membrane permeabilization

2020 ◽  
Vol 295 (6) ◽  
pp. 1623-1636 ◽  
Author(s):  
Tomomi Kuwana ◽  
Louise E. King ◽  
Katia Cosentino ◽  
Julian Suess ◽  
Ana J. Garcia-Saez ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Membrane Permeabilization ◽  
Size Exclusion ◽  
Mitochondrial Outer Membrane ◽  
Intermembrane Space ◽  
Double Knockout ◽  
Apoptosis Pathway ◽  
Bax Protein ◽  
Intrinsic Apoptosis Pathway

Permeabilization of the mitochondrial outer membrane is a key step in the intrinsic apoptosis pathway, triggered by the release of mitochondrial intermembrane space proteins into the cytoplasm. The BCL-2–associated X apoptosis regulator (BAX) protein critically contributes to this process by forming pores in the mitochondrial outer membrane. However, the relative roles of the mitochondrial residence of BAX and its oligomerization in promoting membrane permeabilization are unclear. To this end, using both cell-free and cellular experimental systems, including membrane permeabilization, size-exclusion chromatography-based oligomer, and retrotranslocation assays, along with confocal microscopy analysis, here we studied two BAX C-terminal variants, T182I and G179P. Neither variant formed large oligomers when activated in liposomes. Nevertheless, the G179P variant could permeabilize liposome membranes, suggesting that large BAX oligomers are not essential for the permeabilization. However, when G179P was transduced into BAX/BCL2 agonist killer (BAK) double-knockout mouse embryonic fibroblasts, its location was solely cytoplasmic, and it then failed to mediate cell death. In contrast, T182I was inefficient in both liposome insertion and permeabilization. Yet, when transduced into cells, BAXT182I resided predominantly on mitochondria, because of its slow retrotranslocation and mediated apoptosis as efficiently as WT BAX. We conclude that BAX's mitochondrial residence in vivo, regulated by both targeting and retrotranslocation, is more significant for its pro-apoptotic activity than its ability to insert and to form higher-order oligomers in model membranes. We propose that this finding should be taken into account when developing drugs that modulate BAX activity.

scholarly journals Mdm2 Directs the Ubiquitination of β-Arrestin-sequestered cAMP Phosphodiesterase-4D5

2009 ◽  
Vol 284 (24) ◽  
pp. 16170-16182 ◽  
Author(s):  
Xiang Li ◽  
George S. Baillie ◽  
Miles D. Houslay
Keyword(s):  
Terminal Region ◽  
Scaffolding Protein ◽  
Double Knockout ◽  
Camp Phosphodiesterase ◽  
Small Interference Rna ◽  
Mouse Embryo Fibroblasts ◽  
C Terminus ◽  
Rna Knockdown ◽  
Degrading System

β-Arrestin plays a key role in regulating β2-adrenoreceptor signaling by interdicting activation of adenylyl cyclase and selectively sequestering cAMP phosphodiesterase-4D5 (PDE4D5) for delivery of an active cAMP degrading system to the site of cAMP synthesis. Here we show that the β-agonist, isoprenaline, triggers the rapid and transient ubiquitination of PDE4D5 in primary cardiomyocytes, mouse embryo fibroblasts, and HEK293B2 cells constitutively expressing β2-adrenoceptors. Reconstitution analyses in β-arrestin1/2 double knockout cells plus small interference RNA knockdown studies indicate that a β-arrestin-scaffolded pool of the E3-ubiquitin ligase, Mdm2, mediates PDE4D5 ubiquitination. Critical for this is the ubiquitin-interacting motif located in the extreme C terminus of PDE4D5, which is specific to the PDE4D sub-family. In vitro SUMOylation of a PDE4D5 spot-immobilized peptide array, followed by a mutagenesis strategy, showed that PDE4D5 ubiquitination occurs at Lys-48, Lys-53, and Lys-78, which are located within its isoform-specific N-terminal region, as well as at Lys-140 located within its regulatory UCR1 module. We suggest that mono-ubiquitination at Lys-140 primes PDE4D5 for a subsequent cascade of polyubiquitination occurring within its isoform-specific N-terminal region at Lys-48, Lys-53, and Lys-78. PDE4D5 interacts with a non-ubiquitinated β-arrestin sub-population that is likely to be protected from Mdm2-mediated ubiquitination due to steric hindrance caused by sequestered PDE4D5. Ubiquitination of PDE4D5 elicits an increase in the fraction of PDE4D5 sequestered by β-arrestin in cells, thereby contributing to the fidelity of PDE4D5-β-arrestin interaction, as well as decreasing the fraction of PDE4D5 sequestered by the scaffolding protein, RACK1.

scholarly journals A Fanci knockout mouse model reveals common and distinct functions for FANCI and FANCD2

2019 ◽  
Vol 47 (14) ◽  
pp. 7532-7547 ◽  
Author(s):  
Emilie L Dubois ◽  
Laure Guitton-Sert ◽  
Mariline Béliveau ◽  
Kalindi Parmar ◽  
Jalila Chagraoui ◽  
...  
Keyword(s):  
Meiotic Recombination ◽  
Loop Formation ◽  
Mouse Development ◽  
Double Knockout ◽  
Knockout Mouse Model ◽  
Binding Partner ◽  
Meiotic Chromosomes ◽  
Cellular Sensitivity ◽  
D Loop ◽  
Different Levels

AbstractFanconi Anemia (FA) clinical phenotypes are heterogenous and rely on a mutation in one of the 22 FANC genes (FANCA-W) involved in a common interstrand DNA crosslink-repair pathway. A critical step in the activation of FA pathway is the monoubiquitination of FANCD2 and its binding partner FANCI. To better address the clinical phenotype associated with FANCI and the epistatic relationship with FANCD2, we created the first conditional inactivation model for FANCI in mouse. Fanci −/− mice displayed typical FA features such as delayed development in utero, microphtalmia, cellular sensitivity to mitomycin C, occasional limb abnormalities and hematological deficiencies. Interestingly, the deletion of Fanci leads to a strong meiotic phenotype and severe hypogonadism. FANCI was localized in spermatocytes and spermatids and in the nucleus of oocytes. Both FANCI and FANCD2 proteins co-localized with RPA along meiotic chromosomes, albeit at different levels. Consistent with a role in meiotic recombination, FANCI interacted with RAD51 and stimulated D-loop formation, unlike FANCD2. The double knockout Fanci−/− Fancd2−/− also showed epistatic relationship for hematological defects while being not epistatic with respect to generating viable mice in crosses of double heterozygotes. Collectively, this study highlights common and distinct functions of FANCI and FANCD2 during mouse development, meiotic recombination and hematopoiesis.

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