Keratin 8 is a scaffolding and regulatory protein of ERAD complexes.

Early recognition and enhanced degradation of misfolded proteins by the endoplasmic reticulum (ER) quality control and ER-associated degradation (ERAD) cause defective protein secretion and membrane targeting, as exemplified for Z-alpha-1-antitrypsin (Z-A1AT), responsible for alpha-1-antitrypsin deficiency (A1ATD) and F508del-CFTR (cystic fibrosis transmembrane conductance regulator) responsible for cystic fibrosis (CF). Prompted by our previous observation that decreasing Keratin 8 (K8) expression increased trafficking of F508del-CFTR to the plasma membrane, we investigated whether K8 impacts trafficking of soluble misfolded Z-A1AT protein. The subsequent goal of this study was to elucidate the mechanism underlying the K8-dependent regulation of protein trafficking, focusing on the ERAD pathway. The results show that diminishing K8 concentration in HeLa cells enhances secretion of both Z-A1AT and wild-type (WT) A1AT with a 13-fold and fourfold increase, respectively. K8 down-regulation triggers ER failure and cellular apoptosis when ER stress is jointly elicited by conditional expression of the µs heavy chains, as previously shown for Hrd1 knock-out. Simultaneous K8 silencing and Hrd1 knock-out did not show any synergistic effect, consistent with K8 acting in the Hrd1-governed ERAD step. Fractionation and co-immunoprecipitation experiments reveal that K8 is recruited to ERAD complexes containing Derlin2, Sel1 and Hrd1 proteins upon expression of Z/WT-A1AT and F508del-CFTR. Treatment of the cells with c407, a small molecule inhibiting K8 interaction, decreases K8 and Derlin2 recruitment to high-order ERAD complexes. This was associated with increased Z-A1AT secretion in both HeLa and Z-homozygous A1ATD patients' respiratory cells. Overall, we provide evidence that K8 acts as an ERAD modulator. It may play a scaffolding protein role for early-stage ERAD complexes, regulating Hrd1-governed retrotranslocation initiation/ubiquitination processes. Targeting K8-containing ERAD complexes is an attractive strategy for the pharmacotherapy of A1ATD.

Ouabain and chloroquine trigger senolysis of BRAF-V600E-induced senescent cells by targeting autophagy.

The expression of BRAF-V600E triggers oncogene-induced senescence in normal cells and is implicated in the development of several cancers including melanoma. Here, we report that cardioglycosides such as ouabain are potent senolytics in BRAF senescence. Sensitization by ATP1A1 knockdown and protection by supplemental potassium showed that senolysis by ouabain was mediated by the Na,K-ATPase pump. Both ion transport inhibition and signal transduction result from cardioglycosides binding to Na,K-ATPase. An inhibitor of the pump that does not trigger signaling was not senolytic despite blocking ion transport, demonstrating that signal transduction is required for senolysis. Ouabain triggered the activation of Src, p38, Akt, and Erk in BRAF-senescent cells, and signaling inhibitors prevented cell death. The expression of BRAF-V600E increased ER stress and autophagy in BRAF-senescent cells and sensitized the cell to senolysis by ouabain. Ouabain inhibited autophagy flux, which was restored by signaling inhibitors. Consequently, we identified autophagy inhibitor chloroquine as a novel senolytic in BRAF senescence based on the mode of action of cardioglycosides. Our work underlies the interest of characterizing the mechanisms of senolytics to discover novel compounds and identifies the endoplasmic reticulum stress-autophagy tandem as a new vulnerability in BRAF senescence that can be exploited for the development of further senolytic strategies.

Physiologically relevant reconstitution of iron-sulfur cluster biosynthesis uncovers persulfide-processing functions of ferredoxin-2 and frataxin.

Iron-sulfur (Fe-S) clusters are essential protein cofactors whose biosynthetic defects lead to severe diseases among which is Friedreich's ataxia caused by impaired expression of frataxin (FXN). Fe-S clusters are biosynthesized on the scaffold protein ISCU, with cysteine desulfurase NFS1 providing sulfur as persulfide and ferredoxin FDX2 supplying electrons, in a process stimulated by FXN but not clearly understood. Here, we report the breakdown of this process, made possible by removing a zinc ion in ISCU that hinders iron insertion and promotes non-physiological Fe-S cluster synthesis from free sulfide in vitro. By binding zinc-free ISCU, iron drives persulfide uptake from NFS1 and allows persulfide reduction into sulfide by FDX2, thereby coordinating sulfide production with its availability to generate Fe-S clusters. FXN stimulates the whole process by accelerating persulfide transfer. We propose that this reconstitution recapitulates physiological conditions which provides a model for Fe-S cluster biosynthesis, clarifies the roles of FDX2 and FXN and may help develop Friedreich's ataxia therapies.

Endoplasmic Reticulum Transport of Glutathione by Sec61 Is Regulated by Ero1 and Bip.

In the endoplasmic reticulum (ER), Ero1 catalyzes disulfide bond formation and promotes glutathione (GSH) oxidation to GSSG. Since GSSG cannot be reduced in the ER, maintenance of the ER glutathione redox state and levels likely depends on ER glutathione import and GSSG export. We used quantitative GSH and GSSG biosensors to monitor glutathione import into the ER of yeast cells. We found that glutathione enters the ER by facilitated diffusion through the Sec61 protein-conducting channel, while oxidized Bip (Kar2) inhibits transport. Increased ER glutathione import triggers H2O2-dependent Bip oxidation through Ero1 reductive activation, which inhibits glutathione import in a negative regulatory loop. During ER stress, transport is activated by UPR-dependent Ero1 induction, and cytosolic glutathione levels increase. Thus, the ER redox poise is tuned by reciprocal control of glutathione import and Ero1 activation. The ER protein-conducting channel is permeable to small molecules, provided the driving force of a concentration gradient.

Reexamining the Function of Glutathione in Oxidative Protein Folding and Secretion.

SIGNIFICANCE: Disturbance of glutathione (GSH) metabolism is a hallmark of numerous diseases, yet GSH functions are poorly understood. One key to this question is to consider its functional compartmentation. GSH is present in the endoplasmic reticulum (ER), where it competes with substrates for oxidation by the oxidative folding machinery, composed in eukaryotes of the thiol oxidase Ero1 and proteins from the disulfide isomerase family (protein disulfide isomerase). Yet, whether GSH is required for proper ER oxidative protein folding is a highly debated question. Recent Advances: Oxidative protein folding has been thoroughly dissected over the past decades, and its actors and their mode of action elucidated. Genetically encoded GSH probes have recently provided an access to subcellular redox metabolism, including the ER. CRITICAL ISSUES: Of the few often-contradictory models of the role of GSH in the ER, the most popular suggest it serves as reducing power. Yet, as a reductant, GSH also activates Ero1, which questions how GSH can nevertheless support protein reduction. Hence, whether GSH operates in the ER as a reductant, an oxidant, or just as a "blank" compound mirroring ER/periplasm redox activity is a highly debated question, which is further stimulated by the puzzling occurrence of GSH in the Escherichia coli periplasmic "secretory" compartment, aside from the Dsb thiol-reducing and oxidase pathways. FUTURE DIRECTIONS: Addressing the mechanisms controlling GSH traffic in and out of the ER/periplasm and its recycling will help address GSH function in secretion. In addition, as thioredoxin reductase was recently implicated in ER oxidative protein folding, the relative contribution of each of these two reducing pathways should now be addressed. Antioxid. Redox Signal. 27, 1178-1199.

A scaffold protein that chaperones a cysteine-sulfenic acid in H2O2 signaling.

In Saccharomyces cerevisiae, Yap1 regulates an H2O2-inducible transcriptional response that controls cellular H2O2 homeostasis. H2O2 activates Yap1 by oxidation through the intermediary of the thiol peroxidase Orp1. Upon reacting with H2O2, Orp1 catalytic cysteine oxidizes to a sulfenic acid, which then engages into either an intermolecular disulfide with Yap1, leading to Yap1 activation, or an intramolecular disulfide that commits the enzyme into its peroxidatic cycle. How the first of these two competing reactions, which is kinetically unfavorable, occurs was previously unknown. We show that the Yap1-binding protein Ybp1 brings together Orp1 and Yap1 into a ternary complex that selectively activates condensation of the Orp1 sulfenylated cysteine with one of the six Yap1 cysteines while inhibiting Orp1 intramolecular disulfide formation. We propose that Ybp1 operates as a scaffold protein and as a sulfenic acid chaperone to provide specificity in the transfer of oxidizing equivalents by a reactive sulfenic acid species.

The antioxidant machinery of the endoplasmic reticulum: Protection and signaling.

Cellular metabolism is inherently linked to the production of oxidizing by-products, including reactive oxygen species (ROS) hydrogen peroxide (H2O2). When present in excess, H2O2 can damage cellular biomolecules, but when produced in coordinated fashion, it typically serves as a mobile signaling messenger. It is therefore not surprising that cell health critically relies on both low-molecular-weight and enzymatic antioxidant components, which protect from ROS-mediated damage and shape the propagation and duration of ROS signals. This review focuses on H2O2-antioxidant cross talk in the endoplasmic reticulum (ER), which is intimately linked to the process of oxidative protein folding. ER-resident or ER-regulated sources of H2O2 and other ROS, which are subgrouped into constitutive and stimulated sources, are discussed and set into context with the diverse antioxidant mechanisms in the organelle. These include two types of peroxide-reducing enzymes, a high concentration of glutathione derived from the cytosol, and feedback-regulated thiol-disulfide switches, which negatively control the major ER oxidase ER oxidoreductin-1. Finally, new evidence highlighting emerging principles of H2O2-based cues at the ER will likely set a basis for establishing ER redox processes as a major line of future signaling research. A fundamental problem that remains to be solved is the specific, quantitative, time resolved, and targeted detection of H2O2 in the ER and in specialized ER subdomains.

RNF185 is a novel E3 ligase of endoplasmic reticulum-associated degradation (ERAD) that targets cystic fibrosis transmembrane conductance regulator (CFTR).

In the endoplasmic reticulum (ER), misfolded or improperly assembled proteins are exported to the cytoplasm and degraded by the ubiquitin-proteasome pathway through a process called ER-associated degradation (ERAD). ER-associated E3 ligases, which coordinate substrate recognition, export, and proteasome targeting, are key components of ERAD. Cystic fibrosis transmembrane conductance regulator (CFTR) is one ERAD substrate targeted to co-translational degradation by the E3 ligase RNF5/RMA1. RNF185 is a RING domain-containing polypeptide homologous to RNF5. We show that RNF185 controls the stability of CFTR and of the CFTRΔF508 mutant in a RING- and proteasome-dependent manner but does not control that of other classical ERAD model substrates. Reciprocally, its silencing stabilizes CFTR proteins. Turnover analyses indicate that, as RNF5, RNF185 targets CFTR to co-translational degradation. Importantly, however, simultaneous depletion of RNF5 and RNF185 profoundly blocks CFTRΔF508 degradation not only during translation but also after synthesis is complete. Our data thus identify RNF185 and RNF5 as a novel E3 ligase module that is central to the control of CFTR degradation.

Functions and cellular compartmentation of the thioredoxin and glutathione pathways in yeast.

SIGNIFICANCE: The thioredoxin (TRX) and glutathione (GSH) pathways are universally conserved thiol-reductase systems that drive an array of cellular functions involving reversible disulfide formation. Here we consider these pathways in Saccharomyces cerevisiae, focusing on their cell compartment-specific functions, as well as the mechanisms that explain extreme differences of redox states between compartments. RECENT ADVANCES: Recent work leads to a model in which the yeast TRX and GSH pathways are not redundant, in contrast to Escherichia coli. The cytosol possesses full sets of both pathways, of which the TRX pathway is dominant, while the GSH pathway acts as back up of the former. The mitochondrial matrix also possesses entire sets of both pathways, in which the GSH pathway has major role in redox control. In both compartments, GSH has also nonredox functions in iron metabolism, essential for viability. The endoplasmic reticulum (ER) and mitochondrial intermembrane space (IMS) are sites of intense thiol oxidation, but except GSH lack thiol-reductase pathways. CRITICAL ISSUES: What are the thiol-redox links between compartments? Mitochondria are totally independent, and insulated from the other compartments. The cytosol is also totally independent, but also provides reducing power to the ER and IMS, possibly by ways of reduced and oxidized GSH entering and exiting these compartments. FUTURE DIRECTIONS: Identifying the mechanisms regulating fluxes of GSH and oxidized glutathione between cytosol and ER, IMS, and possibly also peroxisomes, vacuole is needed to establish the proposed model of eukaryotic thiol-redox homeostasis, which should facilitate exploration of this system in mammals and plants.

Glutathione revisited: a vital function in iron metabolism and ancillary role in thiol-redox control.

Glutathione contributes to thiol-redox control and to extra-mitochondrial iron-sulphur cluster (ISC) maturation. To determine the physiological importance of these functions and sort out those that account for the GSH requirement for viability, we performed a comprehensive analysis of yeast cells depleted of or containing toxic levels of GSH. Both conditions triggered an intense iron starvation-like response and impaired the activity of extra-mitochondrial ISC enzymes but did not impact thiol-redox maintenance, except for high glutathione levels that altered oxidative protein folding in the endoplasmic reticulum. While iron partially rescued the ISC maturation and growth defects of GSH-depleted cells, genetic experiments indicated that unlike thioredoxin, glutathione could not support by itself the thiol-redox duties of the cell. We propose that glutathione is essential by its requirement in ISC assembly, but only serves as a thioredoxin backup in cytosolic thiol-redox maintenance. Glutathione-high physiological levels are thus meant to insulate its cytosolic function in iron metabolism from variations of its concentration during redox stresses, a model challenging the traditional view of it as prime actor in thiol-redox control.