scholarly journals The proteasome cap RPT5/Rpt5p subunit prevents aggregation of unfolded ricin A chain

2013 ◽  
Vol 453 (3) ◽  
pp. 435-445 ◽  
Author(s):  
Paola Pietroni ◽  
Nishi Vasisht ◽  
Jonathan P. Cook ◽  
David M. Roberts ◽  
J. Michael Lord ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Proteasome Inhibitors ◽  
Retrograde Transport ◽  
26S Proteasome ◽  
Atpase Subunit ◽  
Aaa Atpase ◽  
A Chain ◽  
Ricin A

The plant cytotoxin ricin enters mammalian cells by receptor-mediated endocytosis, undergoing retrograde transport to the ER (endoplasmic reticulum) where its catalytic A chain (RTA) is reductively separated from the holotoxin to enter the cytosol and inactivate ribosomes. The currently accepted model is that the bulk of ER-dislocated RTA is degraded by proteasomes. We show in the present study that the proteasome has a more complex role in ricin intoxication than previously recognized, that the previously reported increase in sensitivity of mammalian cells to ricin in the presence of proteasome inhibitors simply reflects toxicity of the inhibitors themselves, and that RTA is a very poor substrate for proteasomal degradation. Denatured RTA and casein compete for a binding site on the regulatory particle of the 26S proteasome, but their fates differ. Casein is degraded, but the mammalian 26S proteasome AAA (ATPase associated with various cellular activities)-ATPase subunit RPT5 acts as a chaperone that prevents aggregation of denatured RTA and stimulates recovery of catalytic RTA activity in vitro. Furthermore, in vivo, the ATPase activity of Rpt5p is required for maximal toxicity of RTA dislocated from the Saccharomyces cerevisiae ER. The results of the present study implicate RPT5/Rpt5p in the triage of substrates in which either activation (folding) or inactivation (degradation) pathways may be initiated.

scholarly journals Altered phosphorylation of the proteasome subunit Rpt6 has minimal impact on synaptic plasticity and learning

2019 ◽  
Author(s):  
Samantha L. Scudder ◽  
Frankie R. Gonzales ◽  
Kristin K. Howell ◽  
Ivar S. Stein ◽  
Lara E. Dozier ◽  
...  
Keyword(s):  
Synaptic Plasticity ◽  
Long Term Potentiation ◽  
26S Proteasome ◽  
Compensatory Mechanisms ◽  
Atpase Subunit ◽  
Minimal Impact ◽  
Proteasome Subunit ◽  
Aaa Atpase

ABSTRACTDynamic control of protein degradation via the ubiquitin proteasome system is thought to play a crucial role in neuronal function and synaptic plasticity. The proteasome subunit Rpt6, an AAA ATPase subunit of the 19S regulatory particle, has emerged as an important site for regulation of 26S proteasome function in neurons. Phosphorylation of Rpt6 on serine 120 (S120) can stimulate the catalytic rate of substrate degradation by the 26S proteasome and this site is targeted by the plasticity-related kinase calcium/calmodulin-dependent kinase II (CaMKII), making it an attractive candidate for regulation of proteasome function in neurons. Several in vitro studies have shown that altered Rpt6 S120 phosphorylation can affect the structure and function of synapses. To evaluate the importance of Rpt6 S120 phosphorylation in vivo, we created two mouse models which feature mutations at S120 that block or mimic phosphorylation at this site. We find that peptidase and ATPase activities are upregulated in the phospho-mimetic mutant and downregulated in the phospho-dead mutant (S120 mutated to aspartic acid (S120D) or alanine (S120A), respectively). Surprisingly, these mutations had no effect on basal synaptic transmission, long-term potentiation, and dendritic spine dynamics and density in the hippocampus. Furthermore, these mutants displayed no deficits in cued and contextual fear memory. Thus, in a mouse model that blocks or mimics phosphorylation at this site, either compensatory mechanisms negate these effects, or small variations in proteasome activity are not enough to induce significant changes in synaptic structure, plasticity, or behavior.

Immunotoxins up to the present day

1989 ◽  
Vol 9 (2) ◽  
pp. 139-156 ◽  
Author(s):  
Francis A. Drobniewski
Keyword(s):  
Clinical Studies ◽  
Polyclonal Antibodies ◽  
Cell Binding ◽  
Ricin A Chain ◽  
Future Role ◽  
A Chain ◽  
Ricin A

Immunotoxins consist of monoclonal or polyclonal antibodies conjugated to bacterial or plant toxins. The toxins used are typically of the A-B type in which a toxic A chain is coupled to a B chain responsible for cell binding and facilitation of A chain entry into the cytosol. Two broad strategies have been followed: coupling intact toxins, or A chains alone, to antibodies. This review examines current progress in in vitro and in vivo research, including recent clinical studies, concentrating principally on ricin or ricin A chain conjugates. The future role of conjugates using membrane-acting toxins, immunolysins, is also discussed.

scholarly journals Cytotoxicity of a cell-reactive immunotoxin containing ricin A chain is potentiated by an anti-immunotoxin containing ricin B chain.

1984 ◽  
Vol 160 (1) ◽  
pp. 341-346 ◽  
Author(s):  
E S Vitetta ◽  
R J Fulton ◽  
J W Uhr
Keyword(s):  
Cell Surface ◽  
Cell Line ◽  
Ricin A Chain ◽  
Daudi Cell ◽  
A Cell ◽  
A Chain ◽  
Ricin A

In vitro killing of the human Daudi cell line by either univalent [F(ab')] or divalent (IgG) forms of rabbit anti-human Ig (RAHIg) coupled to ricin A chain can be specifically potentiated by a "piggyback" treatment with ricin B chain coupled to goat anti-rabbit Ig (GARIg). When cells are treated with univalent immunotoxin (IT) [F(ab') RAHIg-A] and then cultured, IT can be detected on the cell surface for at least 5 h, since GARIg-B can still enhance killing at this time. These results provide a strategy for in vivo use of A chain- and B chain-containing IT.

scholarly journals CRISPR/Cas9-mediated base-editing enables a chain reaction through sequential repair of sgRNA scaffold mutations

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tsuyoshi Fukushima ◽  
Yosuke Tanaka ◽  
Keito Adachi ◽  
Nanami Masuyama ◽  
Akiho Tsuchiya ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Regulatory Networks ◽  
Chain Reaction ◽  
Reaction Systems ◽  
Chain Reactions ◽  
Base Editing ◽  
A Chain ◽  
Loxp Sequence

AbstractCell behavior is controlled by complex gene regulatory networks. Although studies have uncovered diverse roles of individual genes, it has been challenging to record or control sequential genetic events in living cells. In this study, we designed two cellular chain reaction systems that enable sequential sgRNA activation in mammalian cells using a nickase Cas9 tethering of a cytosine nucleotide deaminase (nCas9-CDA). In these systems, thymidine (T)-to-cytosine (C) substitutions in the scaffold region of the sgRNA or the TATA box-containing loxP sequence (TATAloxP) are corrected by the nCas9-CDA, leading to activation of the next sgRNA. These reactions can occur multiple times, resulting in cellular chain reactions. As a proof of concept, we established a chain reaction by repairing sgRNA scaffold mutations in 293 T cells. Importantly, the results obtained in yeast or in vitro did not match those obtained in mammalian cells, suggesting that in vivo chain reactions need to be optimized in appropriate cellular contexts. Our system may lay the foundation for building cellular chain reaction systems that have a broad utility in the future biomedical research.

Cyclosporin A and cyclosporin SDZ PSC 833 enhance anti-CD5 ricin A- chain immunotoxins in human leukemic T cells

Blood ◽  
1994 ◽  
Vol 83 (2) ◽  
pp. 482-489 ◽  
Author(s):  
JP Jaffrezou ◽  
BI Sikic ◽  
G Laurent
Keyword(s):  
Cyclosporin A ◽  
Small Population ◽  
High Dose ◽  
Ricin A Chain ◽  
Psc 833 ◽  
A Chain ◽  
Ricin A ◽  
Sdz Psc 833

Abstract Recent studies have shown that cyclosporin A (CsA) may affect ricin A- chain immunotoxin (RTA-IT) therapy. In this study, we evaluated the ability of CsA and its nonimmunosuppressive analog, SDZ PSC 833, to enhance anti-CD5 T101 RTA-ITs in vitro. Both 4 mumol/L CsA and 4 mumol/L SDZ PSC 833 significantly and specifically enhanced the cytotoxic activity of T101 RTA-IT on the human lymphoblastic T-cell line, CEM III (101-fold and 105-fold, respectively). Furthermore, these Cs also enhanced the cytotoxicity of the more potent T101 F(ab')2 RTA- IT (ninefold and eightfold, respectively). The effect of human plasma, originating from four patients enrolled in a phase I high-dose CsA regimen, was examined on T101 RTA-IT cytotoxicity on CEM III cells. In each case, with plasma CsA levels between 3,090 and 4,860 ng/mL (2.5 to 4 mumol/L), a significant increase in T101 RTA-IT-mediated cytotoxicity was observed ranging from 31% to 60%. Neither CsA nor SDZ PSC 833 affected the rate of RTA-IT binding, internalization, intracellular trafficking, or degradation. Analysis of internalized T101 RTA-IT molecules showed that these were essentially intact, which suggests that these enhancers may act only on a small population of RTA-ITs that escapes present investigational techniques. In conclusion, because the concentrations used are clinically achievable, Cs appear to be promising agents for in vivo enhancement of RTA-ITs.

scholarly journals Cyclosporin A and cyclosporin SDZ PSC 833 enhance anti-CD5 ricin A- chain immunotoxins in human leukemic T cells

Blood ◽  
1994 ◽  
Vol 83 (2) ◽  
pp. 482-489
Author(s):  
JP Jaffrezou ◽  
BI Sikic ◽  
G Laurent
Keyword(s):  
Cyclosporin A ◽  
Small Population ◽  
High Dose ◽  
Ricin A Chain ◽  
Psc 833 ◽  
A Chain ◽  
Ricin A ◽  
Sdz Psc 833

Recent studies have shown that cyclosporin A (CsA) may affect ricin A- chain immunotoxin (RTA-IT) therapy. In this study, we evaluated the ability of CsA and its nonimmunosuppressive analog, SDZ PSC 833, to enhance anti-CD5 T101 RTA-ITs in vitro. Both 4 mumol/L CsA and 4 mumol/L SDZ PSC 833 significantly and specifically enhanced the cytotoxic activity of T101 RTA-IT on the human lymphoblastic T-cell line, CEM III (101-fold and 105-fold, respectively). Furthermore, these Cs also enhanced the cytotoxicity of the more potent T101 F(ab')2 RTA- IT (ninefold and eightfold, respectively). The effect of human plasma, originating from four patients enrolled in a phase I high-dose CsA regimen, was examined on T101 RTA-IT cytotoxicity on CEM III cells. In each case, with plasma CsA levels between 3,090 and 4,860 ng/mL (2.5 to 4 mumol/L), a significant increase in T101 RTA-IT-mediated cytotoxicity was observed ranging from 31% to 60%. Neither CsA nor SDZ PSC 833 affected the rate of RTA-IT binding, internalization, intracellular trafficking, or degradation. Analysis of internalized T101 RTA-IT molecules showed that these were essentially intact, which suggests that these enhancers may act only on a small population of RTA-ITs that escapes present investigational techniques. In conclusion, because the concentrations used are clinically achievable, Cs appear to be promising agents for in vivo enhancement of RTA-ITs.

scholarly journals Leucine 232 and hydrophobic residues at the ribosomal P stalk binding site are critical for biological activity of ricin

2019 ◽  
Vol 39 (10) ◽  
Author(s):  
Yijun Zhou ◽  
Xiao-Ping Li ◽  
Jennifer N. Kahn ◽  
John E. McLaughlin ◽  
Nilgun E. Tumer
Keyword(s):  
Biological Activity ◽  
Mammalian Cells ◽  
Electrostatic Interactions ◽  
Active Site Residues ◽  
Hydrophobic Pocket ◽  
Hydrophobic Residues ◽  
A Chain ◽  
Ribosomal P

Abstract Ricin interacts with the ribosomal P stalk to cleave a conserved adenine from the α-sarcin/ricin loop (SRL) of the rRNA. Ricin toxin A chain (RTA) uses Arg235 as the most critical arginine for binding to the P stalk through electrostatic interactions to facilitate depurination. Structural analysis showed that a P2 peptide binds to a hydrophobic pocket on RTA and the last two residues form hydrogen bonds with Arg235. The importance of hydrophobic residues relative to Arg235 in the interaction with the P stalk in vivo and on the toxicity of RTA is not known. Here, we mutated residues in the hydrophobic pocket to analyze their contribution to toxicity and depurination activity in yeast and in mammalian cells. We found that Leu232, Tyr183 and Phe240 contribute cumulatively to toxicity, with Leu232 being the most significant. A quadruple mutant, Y183A/L232A/R235A/F240A, which combined mutations in critical hydrophobic residues with R235A completely abolished the activity of RTA, indicating that Arg235 and hydrophobic residues are required for full biological activity. Y183A and F240A mutants had reduced activity on RNA, but higher activity on ribosomes compared with R235A in vitro, suggesting that they could partially regain activity upon interaction with ribosomes. These results expand the region of interaction between RTA and the P stalk critical for cellular activity to include the hydrophobic pocket and provide the first evidence that interaction of P stalk with the hydrophobic pocket promotes a conformational rearrangement of RTA to correctly position the active site residues for catalytic attack on the SRL.

scholarly journals Dissociation of Coatomer from Membranes Is Required for Brefeldin A–induced Transfer of Golgi Enzymes to the Endoplasmic Reticulum

1997 ◽  
Vol 137 (2) ◽  
pp. 319-333 ◽  
Author(s):  
Jochen Scheel ◽  
Rainer Pepperkok ◽  
Martin Lowe ◽  
Gareth Griffiths ◽  
Thomas E. Kreis
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Transport ◽  
Golgi Complex ◽  
Mammalian Cells ◽  
Brefeldin A ◽  
Retrograde Transport ◽  
Marker Proteins ◽  
Kdel Receptor

Addition of brefeldin A (BFA) to mammalian cells rapidly results in the removal of coatomer from membranes and subsequent delivery of Golgi enzymes to the endoplasmic reticulum (ER). Microinjected anti-EAGE (intact IgG or Fab-fragments), antibodies against the “EAGE”-peptide of β-COP, inhibit BFA-induced redistribution of β-COP in vivo and block transfer of resident proteins of the Golgi complex to the ER; tubulo-vesicular clusters accumulate and Golgi membrane proteins concentrate in cytoplasmic patches containing β-COP. These patches are devoid of marker proteins of the ER, the intermediate compartment (IC), and do not contain KDEL receptor. Interestingly, relocation of KDEL receptor to the IC, where it colocalizes with ERGIC53 and ts-O45-G, is not inhibited under these conditions. While no stacked Golgi cisternae remain in these injected cells, reassembly of stacks of Golgi cisternae following BFA wash-out is inhibited to only ∼50%. Mono- or divalent anti-EAGE stabilize binding of coatomer to membranes in vitro, at least as efficiently as GTPγS. Taken together these results suggest that enhanced binding of coatomer to membranes completely inhibits the BFA-induced retrograde transport of Golgi resident proteins to the ER, probably by inhibiting fusion of Golgi with ER membranes, but does not interfere with the disassembly of the stacked Golgi cisternae and recycling of KDEL receptor to the IC. These results confirm our previous results suggesting that COPI is involved in anterograde membrane transport from the ER/IC to the Golgi complex (Pepperkok et al., 1993), and corroborate that COPI regulates retrograde membrane transport between the Golgi complex and ER in mammalian cells.

scholarly journals CRISPR/Cas9-mediated Base-editing Enables a Chain Reaction Through Sequential Repair of sgRNA Scaffold Mutations

2021 ◽  
Author(s):  
Tsuyoshi Fukushima ◽  
Yosuke Tanaka ◽  
Keito Adachi ◽  
Nanami Masuyama ◽  
Shuhei Asada ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Regulatory Networks ◽  
Chain Reaction ◽  
Cellular Behavior ◽  
Reaction Systems ◽  
Chain Reactions ◽  
Base Editing ◽  
A Chain

Abstract Cellular behavior is governed by the complex gene regulatory networks. Although studies have revealed diverse roles of individual genes, it has been a challenge to record or control the sequential genetic events in living cells. In this study, we designed two cellular chain reaction systems that enable sequential sgRNA expression in mammalian cells using a nickase Cas9 tethering of a cytosine nucleotide deaminase (nCas9-CDA). In these systems, the thymidine (T)-to-cytosine (C) substitutions in the scaffold region of sgRNA or TATA box containing loxP sequence (TATAloxP) are corrected by the nCas9-CDA, which leads to expression of next sgRNA. These reactions can proceed several times, thus generating cellular chain reactions. As a proof of the concept, we established a chain reaction through the repair of sgRNA scaffold mutations in 293T cells. Importantly, the results obtained in yeast or in vitro were not consistent with those in mammalian cells, suggesting that the in vivo chain reactions need to be optimized in appropriate cellular contexts. Our system may lay the foundation for building cellular chain reaction systems that have a broad utility in the future biomedical research.

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