scholarly journals The immunoproteasome, the 20S proteasome and the PA28αβ proteasome regulator are oxidative-stress-adaptive proteolytic complexes

2010 ◽  
Vol 432 (3) ◽  
pp. 585-595 ◽  
Author(s):  
Andrew M. Pickering ◽  
Alison L. Koop ◽  
Cheryl Y. Teoh ◽  
Gennady Ermak ◽  
Tilman Grune ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Dna Replication ◽  
De Novo ◽  
Physical Activation ◽  
20S Proteasome ◽  
Oxidized Proteins ◽  
Pre Treatment ◽  
Sirna Knockdown ◽  
Rna Knockdown ◽  
Interfering Rna

Oxidized cytoplasmic and nuclear proteins are normally degraded by the proteasome, but accumulate with age and disease. We demonstrate the importance of various forms of the proteasome during transient (reversible) adaptation (hormesis), to oxidative stress in murine embryonic fibroblasts. Adaptation was achieved by ‘pre-treatment’ with very low concentrations of H2O2, and tested by measuring inducible resistance to a subsequent much higher ‘challenge’ dose of H2O2. Following an initial direct physical activation of pre-existing proteasomes, the 20S proteasome, immunoproteasome and PA28αβ regulator all exhibited substantially increased de novo synthesis during adaptation over 24 h. Cellular capacity to degrade oxidatively damaged proteins increased with 20S proteasome, immunoproteasome and PA28αβ synthesis, and was mostly blocked by the 20S proteasome, immunoproteasome and PA28 siRNA (short interfering RNA) knockdown treatments. Additionally, PA28αβ-knockout mutants achieved only half of the H2O2-induced adaptive increase in proteolytic capacity of wild-type controls. Direct comparison of purified 20S proteasome and immunoproteasome demonstrated that the immunoproteasome can selectively degrade oxidized proteins. Cell proliferation and DNA replication both decreased, and oxidized proteins accumulated, during high H2O2 challenge, but prior H2O2 adaptation was protective. Importantly, siRNA knockdown of the 20S proteasome, immunoproteasome or PA28αβ regulator blocked 50–100% of these adaptive increases in cell division and DNA replication, and immunoproteasome knockdown largely abolished protection against protein oxidation.

scholarly journals Activation of Chaperone-mediated Autophagy during Oxidative Stress

2004 ◽  
Vol 15 (11) ◽  
pp. 4829-4840 ◽  
Author(s):  
Roberta Kiffin ◽  
Christopher Christian ◽  
Erwin Knecht ◽  
Ana Maria Cuervo
Keyword(s):  
Oxidative Stress ◽  
De Novo ◽  
Cell Types ◽  
Substrate Uptake ◽  
De Novo Synthesis ◽  
Efficient Removal ◽  
Oxidized Proteins ◽  
Lysosomal Membrane Protein ◽  
Almost All ◽  
Chaperone Mediated Autophagy

Oxidatively damaged proteins accumulate with age in almost all cell types and tissues. The activity of chaperone-mediated autophagy (CMA), a selective pathway for the degradation of cytosolic proteins in lysosomes, decreases with age. We have analyzed the possible participation of CMA in the removal of oxidized proteins in rat liver and cultured mouse fibroblasts. Added to the fact that CMA substrates, when oxidized, are more efficiently internalized into lysosomes, we have found a constitutive activation of CMA during oxidative stress. Oxidation-induced activation of CMA correlates with higher levels of several components of the lysosomal translocation complex, but in particular of the lumenal chaperone, required for substrate uptake, and of the lysosomal membrane protein (lamp) type 2a, previously identified as a receptor for this pathway. In contrast with the well characterized mechanism of CMA activation during nutritional stress, which does not require de novo synthesis of the receptor, oxidation-induced activation of CMA is attained through transcriptional up-regulation of lamp2a. We conclude that CMA is activated during oxidative stress and that the higher activity of this pathway under these conditions, along with the higher susceptibility of the oxidized proteins to be taken up by lysosomes, both contribute to the efficient removal of oxidized proteins.

scholarly journals The Pep4p vacuolar proteinase contributes to the turnover of oxidized proteins but PEP4 overexpression is not sufficient to increase chronological lifespan in Saccharomyces cerevisiae

Microbiology ◽  
2006 ◽  
Vol 152 (12) ◽  
pp. 3595-3605 ◽  
Author(s):  
Marta Marques ◽  
Dominik Mojzita ◽  
Maria A. Amorim ◽  
Teresa Almeida ◽  
Stefan Hohmann ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Protein Turnover ◽  
Yeast Cells ◽  
Global Changes ◽  
20S Proteasome ◽  
Oxidized Proteins ◽  
Genes Encoding ◽  
Induced Genes

Turnover of damaged molecules is considered to play a key role in housekeeping of cells exposed to oxidative stress, and during the progress of ageing. In this work, global changes in the transcriptome were analysed during recovery of yeast cells after H2O2 stress. Regarding induced genes, those associated with protein fate were the most significantly over-represented. In addition to genes encoding subunits of the 20S proteasome, genes related to vacuolar proteolysis (PEP4 and LAP4), protein sorting into the vacuole, and vacuolar fusion were found to be induced. The upregulation of PEP4 gene expression was associated with an increase in Pep4p activity. The induction of genes related to proteolysis was correlated with an increased protein turnover after H2O2-induced oxidation. Furthermore, protein degradation and the removal of oxidized proteins decreased in Pep4p-deficient cells. Pep4p activity also increased during chronological ageing, and cells lacking Pep4p displayed a shortened lifespan associated with higher levels of carbonylated proteins. PEP4 overexpression prevented the accumulation of oxidized proteins, but did not increase lifespan. These results indicate that Pep4p is important for protein turnover after oxidative damage; however, increased removal of oxidized proteins is not sufficient to enhance lifespan.

scholarly journals Vesicle-Associated Membrane Protein 4 and Syntaxin 6 Interactions at the Chlamydial Inclusion

2013 ◽  
Vol 81 (9) ◽  
pp. 3326-3337 ◽  
Author(s):  
Emily J. Kabeiseman ◽  
Kyle Cichos ◽  
Ted Hackstadt ◽  
Andrea Lucas ◽  
Elizabeth R. Moore
Keyword(s):  
Membrane Protein ◽  
De Novo ◽  
Snare Proteins ◽  
Dependent Manner ◽  
Binding Partner ◽  
And Function ◽  
Sirna Knockdown ◽  
Interfering Rna ◽  
Chlamydial Inclusion

ABSTRACTThe predominant players in membrane fusion events are thesolubleN-ethylmaleimide-sensitive factorattachment proteinreceptor (SNARE) family of proteins. We hypothesize that SNARE proteins mediate fusion events at the chlamydial inclusion and are important for chlamydial lipid acquisition. We have previously demonstrated thattrans-Golgi SNARE syntaxin 6 localizes to the chlamydial inclusion. To investigate the role of syntaxin 6 at the chlamydial inclusion, we examined the localization and function of anothertrans-Golgi SNARE and syntaxin 6-binding partner, vesicle-associated membrane protein 4 (VAMP4), at the chlamydial inclusion. In this study, we demonstrate that syntaxin 6 and VAMP4 colocalize to the chlamydial inclusion and interact at the chlamydial inclusion. Furthermore, in the absence of VAMP4, syntaxin 6 is not retained at the chlamydial inclusion. Small interfering RNA (siRNA) knockdown of VAMP4 inhibited chlamydial sphingomyelin acquisition, correlating with a log decrease in infectious progeny. VAMP4 retention at the inclusion was shown to be dependent onde novochlamydial protein synthesis, but unlike syntaxin 6, VAMP4 recruitment is observed in a species-dependent manner. Notably, VAMP4 knockdown inhibits sphingomyelin trafficking only to inclusions in which it localizes. These data support the hypothesis that VAMP proteins play a central role in mediating eukaryotic vesicular interactions at the chlamydial inclusion and, thus, support chlamydial lipid acquisition and chlamydial development.

scholarly journals Genome-wide identification of endothelial cell–enriched genes in the mouse embryo

Blood ◽  
2012 ◽  
Vol 120 (4) ◽  
pp. 914-923 ◽  
Author(s):  
Haruka Takase ◽  
Ken Matsumoto ◽  
Rie Yamadera ◽  
Yoshiaki Kubota ◽  
Ayaka Otsu ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
De Novo ◽  
Vascular Development ◽  
Tube Formation ◽  
Vascular Plexus ◽  
Genome Wide ◽  
A Genome ◽  
Human Orthologs ◽  
Rna Knockdown ◽  
Interfering Rna

Abstract The early blood vessels of the embryo and yolk sac in mammals develop by aggregation of de novo–forming angioblasts into a primitive vascular plexus, which then undergoes a complex remodeling process. Angiogenesis is also important for disease progression in the adult. However, the precise molecular mechanism of vascular development remains unclear. It is therefore of great interest to determine which genes are specifically expressed in developing endothelial cells (ECs). Here, we used Flk1-deficient mouse embryos, which lack ECs, to perform a genome-wide survey for genes related to vascular development. We identified 184 genes that are highly enriched in developing ECs. The human orthologs of most of these genes were also expressed in HUVECs, and small interfering RNA knockdown experiments on 22 human orthologs showed that 6 of these genes play a role in tube formation by HUVECs. In addition, we created Arhgef15 knockout and RhoJ knockout mice by a gene-targeting method and found that Arhgef15 and RhoJ were important for neonatal retinal vascularization. Thus, the genes identified in our survey show high expression in ECs; further analysis of these genes should facilitate our understanding of the molecular mechanisms of vascular development in the mouse.

scholarly journals Hemeoxygenase-1 Mediates an Adaptive Response to Spermidine-Induced Cell Death in Human Endothelial Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Hana Yang ◽  
Seung Eun Lee ◽  
Gun-Dong Kim ◽  
Hye Rim Park ◽  
Yong Seek Park
Keyword(s):  
Oxidative Stress ◽  
Endothelial Cells ◽  
Amine Oxidase ◽  
Umbilical Vein ◽  
Human Endothelial Cells ◽  
Polymerase Chain ◽  
Living Organisms ◽  
Umbilical Vein Endothelial Cells ◽  
Rna Knockdown ◽  
Interfering Rna

Spermidine (SPD) is a ubiquitous polycation that is commonly distributed in living organisms. Intracellular levels of SPD are tightly regulated, and SPD controls cell proliferation and death. However, SPD undergoes oxidation in the presence of serum, producing aldehydes, hydrogen peroxide, and ammonia, which exert cytotoxic effect on cells. Hemeoxygenase-1 (HO-1) is thought to have a protective effect against oxidative stress. Upregulation of HO-1 in endothelial cells is considered to be beneficial in the cardiovascular disease. In the present study, we demonstrate that the ubiquitous polyamine, SPD, induces HO-1 in human umbilical vein endothelial cells (HUVECs). SPD-induced HO-1 expression was examined by Western blot and reverse transcription-polymerase chain reaction (RT-PCR). Involvement of reactive oxygen species, serum amine oxidase, PI3K/Akt signaling pathway, and transcription factor Nrf2 in the induction of HO-1 by SPD was also investigated. Furthermore, small interfering RNA knockdown of Nrf2 or HO-1 and treatment with the specific HO-1 inhibitor ZnPP exhibited a noteworthy increase of death of SPD-stimulated HUVECs. In conclusion, these results suggest that SPD induces PI3K/Akt-Nrf2-mediated HO-1 expression in human endothelial cells, which may have a role in cytoprotection of the cells against oxidative stress-induced death.

PrimPol (Primase/Polymerase), replicating enzyme – A mini review

2020 ◽  
Vol 2 (4) ◽  
pp. 89-92
Author(s):  
Muhammad Amir ◽  
Sabeera Afzal ◽  
Alia Ishaq
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Genetic Information ◽  
Nuclear Dna ◽  
De Novo ◽  
Dna Polymerases ◽  
Test Site ◽  
Mitochondrial Dna Replication ◽  
Dna Template ◽  
Preliminary Phase

Polymerases were revealed first in 1970s. Most important to the modest perception the enzyme responsible for nuclear DNA replication that was pol , for DNA repair pol and for mitochondrial DNA replication pol  DNA construction and renovation done by DNA polymerases, so directing both the constancy and discrepancy of genetic information. Replication of genome initiate with DNA template-dependent fusion of small primers of RNA. This preliminary phase in replication of DNA demarcated as de novo primer synthesis which is catalyzed by specified polymerases known as primases. Sixteen diverse DNA-synthesizing enzymes about human perspective are devoted to replication, reparation, mutilation lenience, and inconsistency of nuclear DNA. But in dissimilarity, merely one DNA polymerase has been called in mitochondria. It has been suggest that PrimPol is extremely acting the roles by re-priming DNA replication in mitochondria to permit an effective and appropriate way replication to be accomplished. Investigations from a numeral of test site have significantly amplified our appreciative of the role, recruitment and regulation of the enzyme during DNA replication. Though, we are simply just start to increase in value the versatile roles that play PrimPol in eukaryote.

scholarly journals GCN2 drives macrophage and MDSC function and immunosuppression in the tumor microenvironment

2019 ◽  
Vol 4 (42) ◽  
pp. eaax8189 ◽  
Author(s):  
Marie Jo Halaby ◽  
Kebria Hezaveh ◽  
Sara Lamorte ◽  
M. Teresa Ciudad ◽  
Andreas Kloetgen ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Macrophage Polarization ◽  
Suppressor Cells ◽  
Interferon Γ ◽  
Mass Cytometry ◽  
General Control ◽  
Immune Attack ◽  
Environmental Sensor ◽  
Rna Knockdown ◽  
Interfering Rna

General control nonderepressible 2 (GCN2) is an environmental sensor controlling transcription and translation in response to nutrient availability. Although GCN2 is a putative therapeutic target for immuno-oncology, its role in shaping the immune response to tumors is poorly understood. Here, we used mass cytometry, transcriptomics, and transcription factor–binding analysis to determine the functional impact of GCN2 on the myeloid phenotype and immune responses in melanoma. We found that myeloid-lineage deletion of GCN2 drives a shift in the phenotype of tumor-associated macrophages and myeloid-derived suppressor cells (MDSCs) that promotes antitumor immunity. Time-of-flight mass cytometry (CyTOF) and single-cell RNA sequencing showed that this was due to changes in the immune microenvironment with increased proinflammatory activation of macrophages and MDSCs and interferon-γ expression in intratumoral CD8+ T cells. Mechanistically, GCN2 altered myeloid function by promoting increased translation of the transcription factor CREB-2/ATF4, which was required for maturation and polarization of macrophages and MDSCs in both mice and humans, whereas targeting Atf4 by small interfering RNA knockdown reduced tumor growth. Last, analysis of patients with cutaneous melanoma showed that GCN2-dependent transcriptional signatures correlated with macrophage polarization, T cell infiltrates, and overall survival. Thus, these data reveal a previously unknown dependence of tumors on myeloid GCN2 signals for protection from immune attack.

scholarly journals Hydralazine protects the heart against acute ischaemia/reperfusion injury by inhibiting Drp1-mediated mitochondrial fission

2021 ◽  
Author(s):  
Siavash Beikoghli Kalkhoran ◽  
Janos Kriston-Vizi ◽  
Sauri Hernandez-Resendiz ◽  
Gustavo E Crespo-Avilan ◽  
Ayeshah A Rosdah ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Infarct Size ◽  
Myocardial Infarct ◽  
Mitochondrial Fission ◽  
Vehicle Control ◽  
Myocardial Infarct Size ◽  
Ischaemia Reperfusion Injury ◽  
Ischaemia Reperfusion ◽  
Pre Treatment ◽  
Apoptotic Effects

Abstract Aims Genetic and pharmacological inhibition of mitochondrial fission induced by acute myocardial ischaemia/reperfusion injury (IRI) has been shown to reduce myocardial infarct size. The clinically used anti-hypertensive and heart failure medication, hydralazine, is known to have anti-oxidant and anti-apoptotic effects. Here, we investigated whether hydralazine confers acute cardioprotection by inhibiting Drp1-mediated mitochondrial fission. Methods and results Pre-treatment with hydralazine was shown to inhibit both mitochondrial fission and mitochondrial membrane depolarisation induced by oxidative stress in HeLa cells. In mouse embryonic fibroblasts (MEFs), pre-treatment with hydralazine attenuated mitochondrial fission and cell death induced by oxidative stress, but this effect was absent in MEFs deficient in the mitochondrial fission protein, Drp1. Molecular docking and surface plasmon resonance studies demonstrated binding of hydralazine to the GTPase domain of the mitochondrial fission protein, Drp1 (KD 8.6±1.0 µM), and inhibition of Drp1 GTPase activity in a dose-dependent manner. In isolated adult murine cardiomyocytes subjected to simulated IRI, hydralazine inhibited mitochondrial fission, preserved mitochondrial fusion events, and reduced cardiomyocyte death (hydralazine 24.7±2.5% vs. control 34.1±1.5%, P=0.0012). In ex vivo perfused murine hearts subjected to acute IRI, pre-treatment with hydralazine reduced myocardial infarct size (as % left ventricle: hydralazine 29.6±6.5% vs. vehicle control 54.1±4.9%, P=0.0083), and in the murine heart subjected to in vivo IRI, the administration of hydralazine at reperfusion, decreased myocardial infarct size (as % area-at-risk: hydralazine 28.9±3.0% vs. vehicle control 58.2±3.8%, P<0.001). Conclusion We show that, in addition to its antioxidant and anti-apoptotic effects, hydralazine, confers acute cardioprotection by inhibiting IRI-induced mitochondrial fission, raising the possibility of repurposing hydralazine as a novel cardioprotective therapy for improving post-infarction outcomes.

scholarly journals Mechanism of miR-218-5p in autophagy, apoptosis and oxidative stress in rheumatoid arthritis synovial fibroblasts is mediated by KLF9 and JAK/STAT3 pathways

2021 ◽  
pp. jim-2020-001437
Author(s):  
Ming Chen ◽  
Minghui Li ◽  
Na Zhang ◽  
Wenwen Sun ◽  
Hui Wang ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Oxidative Stress ◽  
Signaling Pathway ◽  
Synovial Tissue ◽  
Synovial Fibroblasts ◽  
Reverse Transcription Pcr ◽  
Stat3 Signaling ◽  
Stat3 Signaling Pathway ◽  
Interfering Rna ◽  
And Oxidative Stress

This study was aimed to investigate the effects of miR-218-5p on the proliferation, apoptosis, autophagy, and oxidative stress of rheumatoid arthritis synovial fibroblasts (RASFs), and the related mechanisms. Quantitative reverse transcription–PCR showed that the expression of miR-218-5p in rheumatoid arthritis synovial tissue was significantly higher than that in healthy synovial tissue. Compared with healthy synovial fibroblasts, miR-218-5p expression was obviously upregulated in RASFs, while KLF9 protein expression was markedly downregulated. Mechanistically, miR-218-5p could directly bind to the 3′ untranslated region of KLF9 to inhibit the expression of KLF9. Additionally, transfection of miR-218-5p small interfering RNA (siRNA) inhibited the proliferation but promoted apoptosis and autophagy of RASFs. Simultaneously, miR-218-5p silencing reduced reactive oxygen species and malondialdehyde levels and increased superoxide dismutase and glutathione peroxidase activity to improve oxidative stress in RASFs. More importantly, the introduction of KLF9 siRNA reversed the effects of miR-218-5p siRNA transfection on RASF proliferation, apoptosis, autophagy, and oxidative stress. What is more, silencing miR-218-5p inhibited the activation of JAK2/STAT3 signaling pathway by targeting KLF9. Collectively, knockdown of miR-218-5p could regulate the proliferation, apoptosis, autophagy and oxidative stress of RASFs by increasing the expression of KLF9 and inhibiting the activation of the JAK2/STAT3 signaling pathway, which may provide a potential target for the mechanism research of RA.

scholarly journals Amino Acid Transporter LAT1 (SLC7A5) Mediates MeHg-Induced Oxidative Stress Defense in the Human Placental Cell Line HTR-8/SVneo

2021 ◽  
Vol 22 (4) ◽  
pp. 1707
Author(s):  
Sebastian Granitzer ◽  
Raimund Widhalm ◽  
Martin Forsthuber ◽  
Isabella Ellinger ◽  
Gernot Desoye ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Amino Acid ◽  
De Novo ◽  
Structural Similarity ◽  
Amino Acid Transporter ◽  
Cell Number ◽  
Mehg Exposure ◽  
Acid Transporter ◽  
And Oxidative Stress

The placental barrier can protect the fetus from contact with harmful substances. The potent neurotoxin methylmercury (MeHg), however, is very efficiently transported across the placenta. Our previous data suggested that L-type amino acid transporter (LAT)1 is involved in placental MeHg uptake, accepting MeHg-L-cysteine conjugates as substrate due to structural similarity to methionine. The aim of the present study was to investigate the antioxidant defense of placental cells to MeHg exposure and the role of LAT1 in this response. When trophoblast-derived HTR-8/SVneo cells were LAT1 depleted by siRNA-mediated knockdown, they accumulated less MeHg. However, they were more susceptible to MeHg-induced toxicity. This was evidenced in decreased cell viability at a usually noncytotoxic concentration of 0.03 µM MeHg (~6 µg/L). Treatment with ≥0.3 µM MeHg increased cytotoxicity, apoptosis rate, and oxidative stress of HTR-8/SVneo cells. These effects were enhanced under LAT1 knockdown. Reduced cell number was seen when MeHg-exposed cells were cultured in medium low in cysteine, a constituent of the tripeptide glutathione (GSH). Because LAT1-deficient HTR-8/SVneo cells have lower GSH levels than control cells (independent of MeHg treatment), we conclude that LAT1 is essential for de novo synthesis of GSH, required to counteract oxidative stress. Genetic predisposition to decreased LAT1 function combined with MeHg exposure could increase the risk of placental damage.

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