scholarly journals Proteasomal degradation restricts the nuclear lifespan of AID

2008 ◽  
Vol 205 (6) ◽  
pp. 1357-1368 ◽  
Author(s):  
Said Aoufouchi ◽  
Ahmad Faili ◽  
Carole Zober ◽  
Orietta D'Orlando ◽  
Sandra Weller ◽  
...  
Keyword(s):  
Cell Line ◽  
Cytidine Deaminase ◽  
Proteasome Inhibitors ◽  
Proteasomal Degradation ◽  
Protein Accumulation ◽  
Restricted Form ◽  
Lysine Residues ◽  
Immunoglobulin Repertoire ◽  
The Stability ◽  
Specific Degradation

Activation-induced cytidine deaminase (AID) initiates all postrearrangement processes that diversify the immunoglobulin repertoire by specific deamination of cytidines at the immunoglobulin (Ig) locus. As uncontrolled expression of AID is potentially mutagenic, different types of regulation, particularly nucleocytoplasmic shuttling, restrict the likelihood of AID–deoxyribonucleic acid encounters. We studied additional mechanisms of regulation affecting the stability of the AID protein. No modulation of protein accumulation according to the cell cycle was observed in a Burkitt's lymphoma cell line. In contrast, the half-life of AID was markedly reduced in the nucleus, and this destabilization was accompanied by a polyubiquitination that was revealed in the presence of proteasome inhibitors. The same compartment-specific degradation was observed in activated mouse B cells, and also in a non–B cell line. No specific lysine residues could be linked to this degradation, so it remains unclear whether polyubiquitination proceeds through several alternatives sites or through the protein N terminus. The nuclear-restricted form of AID displayed enhanced mutagenicity at both Ig and non-Ig loci, most notably at TP53, suggesting that modulation of nuclear AID content through proteasomal degradation may represent another level of control of AID activity.

scholarly journals Modulation of IGF2 Expression in the Murine Thymus and Thymic Epithelial Cells Following Coxsackievirus-B4 Infection

2021 ◽  
Vol 9 (2) ◽  
pp. 402
Author(s):  
Hélène Michaux ◽  
Aymen Halouani ◽  
Charlotte Trussart ◽  
Chantal Renard ◽  
Hela Jaïdane ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cell Line ◽  
Thymic Epithelial Cells ◽  
Altered Expression ◽  
Coxsackievirus B4 ◽  
Gene Coding ◽  
Self Tolerance ◽  
Stat3 Phosphorylation ◽  
The Stability

Coxsackievirus B4 (CV-B4) can infect human and murine thymic epithelial cells (TECs). In a murine TEC cell line, CV-B4 can downregulate the transcription of the insulin-like growth factor 2 (Igf2) gene coding for the self-peptide of the insulin family. In this study, we show that CV-B4 infections of a murine TEC cell line decreased Igf2 P3 promoter activity by targeting a region near the transcription start site; however, the stability of Igf2 transcripts remained unchanged, indicating a regulation of Igf2 transcription. Furthermore, CV-B4 infections decreased STAT3 phosphorylation in vitro. We also showed that mice infected with CV-B4 had an altered expression of Igf2 isoforms as detected in TECs, followed by a decrease in the pro-IGF2 precursor in the thymus. Our study sheds new light on the intrathymic regulation of Igf2 transcription during CV-B4 infections and supports the hypothesis that a viral infection can disrupt central self-tolerance to insulin by decreasing Igf2 transcription in the thymic epithelium.

scholarly journals A phosphoinositide-binding cluster in cavin1 acts as a molecular sensor for cavin1 degradation

2015 ◽  
Vol 26 (20) ◽  
pp. 3561-3569 ◽  
Author(s):  
Vikas A. Tillu ◽  
Oleksiy Kovtun ◽  
Kerrie-Ann McMahon ◽  
Brett M. Collins ◽  
Robert G. Parton
Keyword(s):  
Proteasomal Degradation ◽  
Resting Cells ◽  
Coat Proteins ◽  
Cellular Processes ◽  
Molecular Sensor ◽  
Lysine Residues ◽  
Low Levels ◽  
Membrane Stretching ◽  
Signaling Modules ◽  
Site Release

Caveolae are abundant surface organelles implicated in a range of cellular processes. Two classes of proteins work together to generate caveolae: integral membrane proteins termed caveolins and cytoplasmic coat proteins called cavins. Caveolae respond to membrane stress by releasing cavins into the cytosol. A crucial aspect of this model is tight regulation of cytosolic pools of cavin under resting conditions. We now show that a recently identified region of cavin1 that can bind phosphoinositide (PI) lipids is also a major site of ubiquitylation. Ubiquitylation of lysines within this site leads to rapid proteasomal degradation. In cells that lack caveolins and caveolae, cavin1 is cytosolic and rapidly degraded as compared with cells in which cavin1 is associated with caveolae. Membrane stretching causes caveolar disassembly, release of cavin complexes into the cytosol, and increased proteasomal degradation of wild-type cavin1 but not mutant cavin1 lacking the major ubiquitylation site. Release of cavin1 from caveolae thus leads to exposure of key lysine residues in the PI-binding region, acting as a trigger for cavin1 ubiquitylation and down-regulation. This mutually exclusive PI-binding/ubiquitylation mechanism may help maintain low levels of cytosolic cavin1 in resting cells, a prerequisite for cavins acting as signaling modules following release from caveolae.

scholarly journals Impaired RIPK1 ubiquitination sensitizes mice to TNF toxicity and inflammatory cell death

2020 ◽  
Author(s):  
Matthias Kist ◽  
László G. Kőműves ◽  
Tatiana Goncharov ◽  
Debra L. Dugger ◽  
Charles Yu ◽  
...  
Keyword(s):  
Cell Death ◽  
Inflammatory Responses ◽  
Mapk Signaling ◽  
Embryonic Lethality ◽  
Mapk Activation ◽  
Interacting Protein ◽  
Signaling Complex ◽  
Ubiquitination Site ◽  
Lysine Residues ◽  
The Stability

Abstract Receptor-interacting protein 1 (RIP1; RIPK1) is a key regulator of multiple signaling pathways that mediate inflammatory responses and cell death. TNF-TNFR1 triggered signaling complex formation, subsequent NF-κB and MAPK activation and induction of cell death involve RIPK1 ubiquitination at several lysine residues including Lys376 and Lys115. Here we show that mutating the ubiquitination site K376 of RIPK1 (K376R) in mice activates cell death resulting in embryonic lethality. In contrast to Ripk1K376R/K376R mice, Ripk1K115R/K115R mice reached adulthood and showed slightly higher responsiveness to TNF-induced death. Cell death observed in Ripk1K376R/K376R embryos relied on RIPK1 kinase activity as administration of RIPK1 inhibitor GNE684 to pregnant heterozygous mice effectively blocked cell death and prolonged survival. Embryonic lethality of Ripk1K376R/K376R mice was prevented by the loss of TNFR1, or by simultaneous deletion of caspase-8 and RIPK3. Interestingly, elimination of the wild-type allele from adult Ripk1K376R/cko mice was tolerated. However, adult Ripk1K376R/cko mice were exquisitely sensitive to TNF-induced hypothermia and associated lethality. Absence of the K376 ubiquitination site diminished K11-linked, K63-linked, and linear ubiquitination of RIPK1, and promoted the assembly of death-inducing cellular complexes, suggesting that multiple ubiquitin linkages contribute to the stability of the RIPK1 signaling complex that stimulates NF-κB and MAPK activation. In contrast, mutating K115 did not affect RIPK1 ubiquitination or TNF stimulated NF-κB and MAPK signaling. Overall, our data indicate that selective impairment of RIPK1 ubiquitination can lower the threshold for RIPK1 activation by TNF resulting in cell death and embryonic lethality.

scholarly journals SILVER NANOPARTICLES FROM MEDICINALLY IMPORTANT EUPHORBIA CYATHOPHORA EXTRACT: BIOSYNTHESIS, CHARACTERIZATION, AND ANTICANCER ACTIVITY.

2018 ◽  
Vol 11 (2) ◽  
pp. 154
Author(s):  
Robert Lotha ◽  
Aravind Sivasubramanian ◽  
Meenakshi Sundaram Muthuraman
Keyword(s):  
Electron Microscopy ◽  
Silver Nanoparticles ◽  
Cell Line ◽  
Aqueous Extract ◽  
Visible Spectroscopy ◽  
X Ray ◽  
Transmission Electron ◽  
The Stability ◽  
Ht 29 ◽  
Scanning Electron

Objective: The present study was aimed at the biosynthesis of silver nanoparticles (AgNPs) using aqueous extract of Euphorbia cyathophora leavesand testing their anticancer potential using HT-29 cell line model.Methods: Green synthesis of silver nanoparticles was obtained with the aqueous extract of E. cyathophora. The synthesized nanoparticles wereconfirmed initially by ultraviolet-visible spectroscopy. Further, scanning electron microscopy, transmission electron microscopy, and X-Ray diffractionstudies also ensured the presence of silver nanoparticles. Zeta potential studies revealed the stability of the silver nanoparticles.Results: Antioxidant and anticancer studies of the nanoparticles against HT-29 cell line exhibited remarkable results.Conclusion: This ensures that the synthesized nanoparticles play an important role in medicinal biology.

Dual regulation of transcription factor Nrf2 by Keap1 and by the combined actions of β-TrCP and GSK-3

2015 ◽  
Vol 43 (4) ◽  
pp. 611-620 ◽  
Author(s):  
John D. Hayes ◽  
Sudhir Chowdhry ◽  
Albena T. Dinkova-Kostova ◽  
Calum Sutherland
Keyword(s):  
Transcription Factor ◽  
Growth Factors ◽  
Ubiquitin Ligase ◽  
Molecular Mechanisms ◽  
Glycogen Synthase ◽  
Proteasomal Degradation ◽  
Regulation Of Transcription ◽  
Related Factor ◽  
Nrf2 Target Gene ◽  
The Stability

Nuclear factor-erythroid 2 p45 (NF-E2 p45)-related factor 2 (Nrf2) is a master regulator of redox homoeostasis that allows cells to adapt to oxidative stress and also promotes cell proliferation. In this review, we describe the molecular mechanisms by which oxidants/electrophilic agents and growth factors increase Nrf2 activity. In the former case, oxidants/electrophiles increase the stability of Nrf2 by antagonizing the ability of Kelch-like ECH-associated protein 1 (Keap1) to target the transcription factor for proteasomal degradation via the cullin-3 (Cul3)–RING ubiquitin ligase CRLKeap1. In the latter case, we speculate that growth factors increase the stability of Nrf2 by stimulating phosphoinositide 3-kinase (PI3K)−protein kinase B (PKB)/Akt signalling, which in turn results in inhibitory phosphorylation of glycogen synthase kinase-3 (GSK-3) and in doing so prevents the formation of a DSGIS motif-containing phosphodegron in Nrf2 that is recognized by the β-transducin repeat-containing protein (β-TrCP) Cul1-based E3 ubiquitin ligase complex SCFβ-TrCP. We present data showing that in the absence of Keap1, the electrophile tert-butyl hydroquinone (tBHQ) can stimulate Nrf2 activity and induce the Nrf2-target gene NAD(P)H:quinone oxidoreductase-1 (NQO1), whilst simultaneously causing inhibitory phosphorylation of GSK-3β at Ser9. Together, these observations suggest that tBHQ can suppress the ability of SCFβ-TrCP to target Nrf2 for proteasomal degradation by increasing PI3K−PKB/Akt signalling. We also propose a scheme that explains how other protein kinases that inhibit GSK-3 could stimulate induction of Nrf2-target genes by preventing formation of the DSGIS motif-containing phosphodegron in Nrf2.

scholarly journals The Structural Interface between HIV-1 Vif and Human APOBEC3H

2016 ◽  
Vol 91 (5) ◽  
Author(s):  
Marcel Ooms ◽  
Michael Letko ◽  
Viviana Simon
Keyword(s):  
Binding Site ◽  
Drug Targets ◽  
Cytidine Deaminase ◽  
Interaction Model ◽  
Proteasomal Degradation ◽  
Large Set ◽  
Structure Model ◽  
Contact Points ◽  
Vif Protein ◽  
Hiv 1

ABSTRACT Human APOBEC3H (A3H) is a cytidine deaminase that inhibits HIV-1 replication. To evade this restriction, the HIV-1 Vif protein binds A3H and mediates its proteasomal degradation. To date, little information on the Vif-A3H interface has been available. To decipher how both proteins interact, we first mapped the Vif-binding site on A3H by functionally testing a large set of A3H mutants in single-cycle infectivity and replication assays. Our data show that the two A3H α-helixes α3 and α4 represent the Vif-binding site of A3H. We next used viral adaptation and a set of Vif mutants to identify novel, reciprocal Vif variants that rescued viral infectivity in the presence of two Vif-resistant A3H mutants. These A3H-Vif interaction points were used to generate the first A3H-Vif structure model, which revealed that the A3H helixes α3 and α4 interact with the Vif β-sheet (β2-β5). This model is in good agreement with previously reported Vif and A3H amino acids important for interaction. Based on the predicted A3H-Vif interface, we tested additional points of contact, which validated our model. Moreover, these experiments showed that the A3H and A3G binding sites on HIV-1 Vif are largely distinct, with both host proteins interacting with Vif β-strand 2. Taken together, this virus-host interface model explains previously reported data and will help to identify novel drug targets to combat HIV-1 infection. IMPORTANCE HIV-1 needs to overcome several intracellular restriction factors in order to replicate efficiently. The human APOBEC3 locus encodes seven proteins, of which A3D, A3F, A3G, and A3H restrict HIV-1. HIV encodes the Vif protein, which binds to the APOBEC3 proteins and leads to their proteasomal degradation. No HIV-1 Vif-APOBEC3 costructure exists to date despite extensive research. We and others previously generated HIV-1 Vif costructure models with A3G and A3F by mapping specific contact points between both proteins. Here, we applied a similar approach to HIV-1 Vif and A3H and successfully generated a Vif-A3H interaction model. Importantly, we find that the HIV-1 Vif-A3H interface is distinct from the Vif-A3G and Vif-A3F interfaces, with a small Vif region being important for recognition of both A3G and A3H. Our Vif-A3H structure model informs on how both proteins interact and could guide toward approaches to block the Vif-A3H interface to target HIV replication.

scholarly journals Image-Based Screening for the Identification of Novel Proteasome Inhibitors

2007 ◽  
Vol 12 (2) ◽  
pp. 203-210 ◽  
Author(s):  
Linda Rickardson ◽  
Malin Wickström ◽  
Rolf Larsson ◽  
Henrik Lövborg
Keyword(s):  
Cell Line ◽  
Fluorescent Protein ◽  
Proteasome Inhibitors ◽  
Myeloma Cell ◽  
Cancer Drug ◽  
Screening Assay ◽  
Hek 293 ◽  
Compound Libraries ◽  
Human Embryo Kidney ◽  
Green Fluorescent

The proteasome is a new, interesting target in cancer drug therapy, and the proteasome inhibitor bortezomib has shown an effect in myeloma patients. It is of interest to efficiently discover and evaluate new proteasome inhibitors. The authors describe the development of an image-based screening assay for the identification of compounds with proteasome-inhibiting activity. The stably transfected human embryo kidney cell line HEK 293 ZsGreen Proteasome Sensor Cell Line expressing the ZsProSensor-1 fusion protein was used for screening and evaluation of proteasome inhibitors. Inhibition of the proteasome leads to accumulation of the green fluorescent protein ZsGreen, which is measured in the ArrayScan® High Content Screening system, in which cell morphology is studied simultaneously. When screening the LOPAC1280 substance library, several compounds with effect on the proteasome were found; among the hits were disulfiram and ammonium pyrrolidinedithiocarbamate (PDTC). Cytotoxic analysis of disulfiram and PDTC showed that the compounds induced cytotoxicity in the myeloma cell line RPMI 8226. The average Z' value for the assay was 0.66. The results indicate that the assay rapidly identifies new proteasome-inhibiting substances, and it will be further used as a tool for image-based screening of other chemically diverse compound libraries.

scholarly journals Incomplete proteasomal degradation of green fluorescent proteins in the context of tandem fluorescent protein timers

2015 ◽  
Author(s):  
Anton Khmelinskii ◽  
Matthias Meurer ◽  
Chi-Ting Ho ◽  
Birgit Besenbeck ◽  
Julia Fueller ◽  
...  
Keyword(s):  
Protein Turnover ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Proteasomal Degradation ◽  
Time Range ◽  
Green Fluorescent Proteins ◽  
Red Fluorescent Proteins ◽  
Dependent Change ◽  
The Stability ◽  
Green Fluorescent

Tandem fluorescent protein timers (tFTs) report on protein age through time-dependent change in color, which can be exploited to study protein turnover and trafficking. Each tFT, composed of two fluorescent proteins (FPs) that differ in maturation kinetics, is suited to follow protein dynamics within a specific time range determined by the maturation rates of both FPs. So far tFTs were constructed by combining different slower-maturing red fluorescent proteins (redFPs) with the same faster-maturing superfolder green fluorescent protein (sfGFP). Towards a comprehensive characterization of tFTs, we compare here tFTs composed of different faster-maturing greenFPs, while keeping the slower-maturing redFP constant (mCherry). Our results indicate that the greenFP maturation kinetics influences the time range of a tFT. Moreover, we observe that commonly used greenFPs can partially withstand proteasomal degradation due to the stability of the FP fold, which results in accumulation of tFT fragments in the cell. Depending on the order of FPs in the timer, incomplete proteasomal degradation either shifts the time range of the tFT towards slower time scales or precludes its use for measurements of protein turnover. We identify greenFPs that are efficiently degraded by the proteasome and provide simple guidelines for design of new tFTs.

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