N-Ethylmaleimide-Modified Hsp70 Inhibits Protein Folding

Hermawan, Aynih; Chirico, William J.

doi:10.1006/abbi.1999.1354

Cited by 15 publications

(18 citation statements)

References 44 publications

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“…For example, in vitro alkylation of the three cysteines in the cytosolic yeast Hsp70 Ssa1 triggers a loss of ATPase activity and augments the capacity of Ssa1 to prevent aggregation of denatured luciferase (Liu et al, 1996; Hermawan and Chirico, 1999). Cys15 in Ssa1 is equivalent to the conserved BiP cysteine, and changes in chaperone activity for alkylated Ssa1 could occur through a mechanism similar to what we describe for BiP.…”

Section: Discussionmentioning

confidence: 99%

Redox signaling via the molecular chaperone BiP protects cells against endoplasmic reticulum-derived oxidative stress

Wang

Pareja

Kaiser

et al. 2014

eLife

101

156

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Oxidative protein folding in the endoplasmic reticulum (ER) has emerged as a potentially significant source of cellular reactive oxygen species (ROS). Recent studies suggest that levels of ROS generated as a byproduct of oxidative folding rival those produced by mitochondrial respiration. Mechanisms that protect cells against oxidant accumulation within the ER have begun to be elucidated yet many questions still remain regarding how cells prevent oxidant-induced damage from ER folding events. Here we report a new role for a central well-characterized player in ER homeostasis as a direct sensor of ER redox imbalance. Specifically we show that a conserved cysteine in the lumenal chaperone BiP is susceptible to oxidation by peroxide, and we demonstrate that oxidation of this conserved cysteine disrupts BiP's ATPase cycle. We propose that alteration of BiP activity upon oxidation helps cells cope with disruption to oxidative folding within the ER during oxidative stress.DOI: http://dx.doi.org/10.7554/eLife.03496.001

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Section: Discussionmentioning

confidence: 99%

Redox signaling via the molecular chaperone BiP protects cells against endoplasmic reticulum-derived oxidative stress

Wang

Pareja

Kaiser

et al. 2014

eLife

101

156

View full text Add to dashboard Cite

show abstract

“…, 1996). NEM-Ssa1 is incompetent for protein folding and competitively inhibits folding by unmodified Ssa1 (Hermawan and Chirico, 1999). These results provide biochemical support for our findings that substitution of C264 and C303 with aspartic acid caused apparent inactivation of Ssa1 as judged by complementation, resulting in constitutive Hsf1 derepression.…”

Section: Discussionmentioning

confidence: 99%

The yeast Hsp70 Ssa1 is a sensor for activation of the heat shock response by thiol-reactive compounds

Wang

Gibney

West

et al. 2012

MBoC

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“…Second, if cysteines are not important for the UL11-binding site, then why does NEM block the interaction? The most likely explanation is that the addition of hydrophobic NEM moieties distorts the UL11-binding site, similar to its affect on the conformation of other proteins, such as Hsp70 chaperone Ssa1p from Saccharomyces cerevisiae (10,27,41). However, NEM does not globally disrupt the conformation of UL16, because the modified protein is still capable of binding UL21 (26) and gE (74).…”

Section: Discussionmentioning

confidence: 99%

Regulated Interaction of Tegument Proteins UL16 and UL11 from Herpes Simplex Virus

Chadha

Han

Starkey

et al. 2012

J Virol

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It is well known that proteins in the tegument (located between the viral capsid and envelope proteins) play critical roles in the assembly and budding of herpesviruses. Tegument proteins UL16 and UL11 of herpes simplex virus (HSV) are conserved among all the Herpesviridae. Although these proteins directly interact in vitro, UL16 was found to colocalize poorly with UL11 in cotransfected cells. To explain this discrepancy, we hypothesized that UL16 is initially made in an inactive form and is artificially transformed to the binding-competent state when cells are disrupted. Consistent with a regulated interaction, UL16 was able to fully colocalize with UL11 when a large C-terminal segment of UL16 was removed, creating mutant UL16 (1-155). Moreover, membrane flotation assays revealed a massive movement of this mutant to the top of sucrose gradients in the presence of UL11, whereas both the full-length UL16 and the C-terminal fragment (residues 156 to 373) remained at the bottom. Further evidence for the presence of a C-terminal regulatory domain was provided by single-amino-acid substitutions at conserved cysteines (C269S, C271S, and C357S), which enabled the efficient interaction of full-length UL16 with UL11. Lastly, the binding site for UL11 was further mapped to residues 81 to 155, and to our surprise, the 5 Cys residues within UL16(1-155) are not required, even though the modification of free cysteines in UL16 with N-ethylmaleimide does in fact prevent binding. Collectively, these results reveal a regulatory function within the C-terminal region of UL16 that controls an N-terminal UL11-binding activity.

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N-Ethylmaleimide-Modified Hsp70 Inhibits Protein Folding

Cited by 15 publications

References 44 publications

Redox signaling via the molecular chaperone BiP protects cells against endoplasmic reticulum-derived oxidative stress

Redox signaling via the molecular chaperone BiP protects cells against endoplasmic reticulum-derived oxidative stress

The yeast Hsp70 Ssa1 is a sensor for activation of the heat shock response by thiol-reactive compounds

Regulated Interaction of Tegument Proteins UL16 and UL11 from Herpes Simplex Virus

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