Monitoring Changes in the Oxidizing Milieu in the Endoplasmic Reticulum of Mammalian Cells Using HyPerER.

The production of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress are tightly linked. The generation of ROS can be both the cause and a consequence of ER stress pathways, and an increasing number of human diseases are characterized by tissue atrophy in response to ER stress and oxidative injury. For the assessment of modulators of ER luminal ROS generation and for mechanistic studies, methods to monitor changes in ER reduction-oxidation (redox) states in a time-resolved and organelle-specific manner are needed. This has been greatly facilitated by the development of genetically encoded fluorescent probes, which can be targeted to different subcellular locations by specific amino acid extensions. One of these probes is the yellow fluorescent protein-based redox biosensor, HyPer. Here, we provide a protocol for the time-resolved monitoring of the oxidizing milieu in the ER of adherent mammalian cells using the ratiometric sensor, HyPerER, which is specifically targeted to the ER lumen.

Ca2+ mobilization-dependent reduction of the endoplasmic reticulum lumen is due to influx of cytosolic glutathione.

BACKGROUND: The lumen of the endoplasmic reticulum (ER) acts as a cellular Ca2+ store and a site for oxidative protein folding, which is controlled by the reduced glutathione (GSH) and glutathione-disulfide (GSSG) redox pair. Although depletion of luminal Ca2+ from the ER provokes a rapid and reversible shift towards a more reducing poise in the ER, the underlying molecular basis remains unclear. RESULTS: We found that Ca2+ mobilization-dependent ER luminal reduction was sensitive to inhibition of GSH synthesis or dilution of cytosolic GSH by selective permeabilization of the plasma membrane. A glutathione-centered mechanism was further indicated by increased ER luminal glutathione levels in response to Ca2+ efflux. Inducible reduction of the ER lumen by GSH flux was independent of the Ca2+-binding chaperone calreticulin, which has previously been implicated in this process. However, opening the translocon channel by puromycin or addition of cyclosporine A mimicked the GSH-related effect of Ca2+ mobilization. While the action of puromycin was ascribable to Ca2+ leakage from the ER, the mechanism of cyclosporine A-induced GSH flux was independent of calcineurin and cyclophilins A and B and remained unclear. CONCLUSIONS: Our data strongly suggest that ER influx of cytosolic GSH, rather than inhibition of local oxidoreductases, is responsible for the reductive shift upon Ca2+ mobilization. We postulate the existence of a Ca2+- and cyclosporine A-sensitive GSH transporter in the ER membrane. These findings have important implications for ER redox homeostasis under normal physiology and ER stress.

Glucose Transport and Transporters in the Endomembranes.

Glucose is a basic nutrient in most of the creatures; its transport through biological membranes is an absolute requirement of life. This role is fulfilled by glucose transporters, mediating the transport of glucose by facilitated diffusion or by secondary active transport. GLUT (glucose transporter) or SLC2A (Solute carrier 2A) families represent the main glucose transporters in mammalian cells, originally described as plasma membrane transporters. Glucose transport through intracellular membranes has not been elucidated yet; however, glucose is formed in the lumen of various organelles. The glucose-6-phosphatase system catalyzing the last common step of gluconeogenesis and glycogenolysis generates glucose within the lumen of the endoplasmic reticulum. Posttranslational processing of the oligosaccharide moiety of glycoproteins also results in intraluminal glucose formation in the endoplasmic reticulum (ER) and Golgi. Autophagic degradation of polysaccharides, glycoproteins, and glycolipids leads to glucose accumulation in lysosomes. Despite the obvious necessity, the mechanism of glucose transport and the molecular nature of mediating proteins in the endomembranes have been hardly elucidated for the last few years. However, recent studies revealed the intracellular localization and functional features of some glucose transporters; the aim of the present paper was to summarize the collected knowledge.

A Systems Biological View of Life-and-Death Decision with Respect to Endoplasmic Reticulum Stress-The Role of PERK Pathway.

Accumulation of misfolded/unfolded proteins in the endoplasmic reticulum (ER) leads to the activation of three branches (Protein kinase (RNA)-like endoplasmic reticulum kinase [PERK], Inositol requiring protein 1 [IRE-1] and Activating trascription factor 6 [ATF6], respectively) of unfolded protein response (UPR). The primary role of UPR is to try to drive back the system to the former or a new homeostatic state by self-eating dependent autophagy, while excessive level of ER stress results in apoptotic cell death. Our study focuses on the role of PERK- and IRE-1-induced arms of UPR in life-or-death decision. Here we confirm that silencing of PERK extends autophagy-dependent survival, whereas the IRE-1-controlled apoptosis inducer is downregulated during ER stress. We also claim that the proper order of surviving and self-killing mechanisms is controlled by a positive feedback loop between PERK and IRE-1 branches. This regulatory network makes possible a smooth, continuous activation of autophagy with respect to ER stress, while the induction of apoptosis is irreversible and switch-like. Using our knowledge of molecular biological techniques and systems biological tools we give a qualitative description about the dynamical behavior of PERK- and IRE-1-controlled life-or-death decision. Our model claims that the two arms of UPR accomplish an altered upregulation of autophagy and apoptosis inducers during ER stress. Since ER stress is tightly connected to aging and age-related degenerative disorders, studying the signaling pathways of UPR and their role in maintaining ER proteostasis have medical importance.

On the role of 4-hydroxynonenal in health and disease.

Polyunsaturated fatty acids are susceptible to peroxidation and they yield various degradation products, including the main α,ß-unsaturated hydroxyalkenal, 4-hydroxy-2,3-trans-nonenal (HNE) in oxidative stress. Due to its high reactivity, HNE interacts with various macromolecules of the cell, and this general toxicity clearly contributes to a wide variety of pathological conditions. In addition, growing evidence suggests a more specific function of HNE in electrophilic signaling as a second messenger of oxidative/electrophilic stress. It can induce antioxidant defense mechanisms to restrain its own production and to enhance the cellular protection against oxidative stress. Moreover, HNE-mediated signaling can largely influence the fate of the cell through modulating major cellular processes, such as autophagy, proliferation and apoptosis. This review focuses on the molecular mechanisms underlying the signaling and regulatory functions of HNE. The role of HNE in the pathophysiology of cancer, cardiovascular and neurodegenerative diseases is also discussed.

Physiological functions of presenilins; beyond γ-secretase.

Presenilin (PS) was identified in screens for mutations causing the early onset forms of familial Alzheimer's disease (FAD) in 1995. As catalytic units of the γ-secretase complex, presenilins participate in the processing of amyloid beta protein (Aß), the main component of deposits in brain of patients with AD. The more than 90 substrates of γ-secretase isolated so far demonstrate its contribution to wide range of cellular processes and signaling events. However, recent findings have revealed numerous γ-secretase-independent presenilin functions, including involvement in calcium homeostasis, endoplasmic reticulum (ER) stress and autophagy. This mini-review attempts to summarize the multiple physiological and pathological functions of presenilin.

Dissection of structural and functional requirements that underlie the interaction of ERdj3 protein with substrates in the endoplasmic reticulum.

ERdj3, a mammalian endoplasmic reticulum (ER) Hsp40/DnaJ family member, binds unfolded proteins, transfers them to BiP, and concomitantly stimulates BiP ATPase activity. However, the requirements for ERdj3 binding to and release from substrates in cells are not well understood. We found that ERdj3 homodimers that cannot stimulate the ATPase activity of BiP (QPD mutants) bound to unfolded ER proteins under steady state conditions in much greater amounts than wild-type ERdj3. This was due to reduced release from these substrates as opposed to enhanced binding, although in both cases dimerization was strictly required for substrate binding. Conversely, heterodimers consisting of one wild-type and one mutant ERdj3 subunit bound substrates at levels comparable with wild-type ERdj3 homodimers, demonstrating that release requires only one protomer to be functional in stimulating BiP ATPase activity. Co-expressing wild-type ERdj3 and a QPD mutant, which each exclusively formed homodimers, revealed that the release rate of wild-type ERdj3 varied according to the relative half-lives of substrates, suggesting that ERdj3 release is an important step in degradation of unfolded client proteins in the ER. Furthermore, pulse-chase experiments revealed that the binding of QPD mutant homodimers remained constant as opposed to increasing, suggesting that ERdj3 does not normally undergo reiterative binding cycles with substrates.

Natural mutations lead to enhanced proteasomal degradation of human Ncb5or, a novel flavoheme reductase.

NADH cytochrome b5 oxidoreductase (Ncb5or) protects ß-cells against oxidative stress and lipotoxicity. The predominant phenotype of lean Ncb5or-null mouse is insulin-dependent diabetes due to ß-cell death. This suggests the putative role of NCB5OR polymorphism in human diabetes. Therefore, we aimed to investigate the effect of natural missense mutations on the expression of human NCB5OR. Protein and mRNA levels of five non-synonymous coding variants were analyzed in transfected HEK293 and HepG2 cells. Although the mRNA levels were only slightly affected by the mutations, the amount of Ncb5or protein was largely reduced upon two Glu to Gly replacements in the third exon (p.E87G, p.E93G). These two mutations remarkably and synergistically shortened the half-life of Ncb5or and their effect could be attenuated by proteasome inhibitors. Our results strongly indicate that p.E87G, p.E93G mutations lead to enhanced proteasomal degradation due to manifest conformational alterations in the b5 domain. These data provide first evidence for natural mutations in NCB5OR gene resulting in decreased protein levels and hence having potential implications in human pathology.

Life and death of a BiP substrate.

BiP is the mammalian endoplasmic reticulum (ER) Hsp70 orthologue that plays a major role in all functions of this organelle including the seemingly opposing functions of aiding the maturation of unfolded nascent proteins and identifying and targeting chronically unfolded proteins for degradation. The recent identification of mammalian BiP co-factors combined with delineation of the ER degradation machinery and data suggesting that the ER is subdivided into unique regions helps explain how these different functions can occur in the same organelle and raises some unresolved issues.

Metyrapone prevents cortisone-induced preadipocyte differentiation by depleting luminal NADPH of the endoplasmic reticulum.

Preadipocyte differentiation is greatly affected by prereceptorial glucocorticoid activation catalyzed by 11beta-hydroxysteroid dehydrogenase type 1 in the lumen of the endoplasmic reticulum. The role of the local NADPH pool in this process was investigated using metyrapone as an NADPH-depleting agent. Metyrapone administered at low micromolar concentrations caused the prompt oxidation of the endogenous NADPH, inhibited the reduction of cortisone and enhanced the oxidation of cortisol in native rat liver microsomal vesicles. However, in permeabilized microsomes, it only slightly decreased both NADPH-dependent cortisone reduction and NADP(+)-dependent cortisol oxidation. Accordingly, metyrapone administration caused a switch in 11beta-hydroxysteroid dehydrogenase activity from reductase to dehydrogenase in both 3T3-L1-derived and human stem cell-derived differentiated adipocytes. Metyrapone greatly attenuated the induction of 11beta-hydroxysteroid dehydrogenase type 1 and the accumulation of lipid droplets during preadipocyte differentiation when 3T3-L1 cells were stimulated with cortisone, while it was much less effective in case of cortisol or dexamethasone. In conclusion, the positive feedback of glucocorticoid activation during preadipocyte differentiation is interrupted by metyrapone, which depletes NADPH in the endoplasmic reticulum. The results also indicate that the reduced state of luminal pyridine nucleotides in the endoplasmic reticulum is important in the process of adipogenesis.

The translocon and the non-specific transport of small molecules in the endoplasmic reticulum (Review).

Transmembrane fluxes are major determinants of several enzyme activities localized in the luminal compartment of the endoplasmic reticulum (ER). Although a large number of metabolites were shown to be transported across the ER membrane, only a few transporters have been identified so far. It can be assumed that the basal permeability of ER membrane vesicles (microsomes) to a variety of small molecules is due to the presence of a low-selectivity channel or pore rather than many strictly specific transporters. The translocon complex is a possible candidate for this role because it transitionally forms an open channel in the ER membrane and an increasing amount of evidence shows the permeation of small compounds through this channel. It seems plausible that the translocon pore is not only responsible for inward and outward peptide translocation but also contributes to basal Ca(2+) leakage from the ER and ensures the substrate supply for certain luminal ER enzymes.

Transport and transporters in the endoplasmic reticulum.

Enzyme activities localized in the luminal compartment of the endoplasmic reticulum are integrated into the cellular metabolism by transmembrane fluxes of their substrates, products and/or cofactors. Most compounds involved are bulky, polar or even charged; hence, they cannot be expected to diffuse through lipid bilayers. Accordingly, transport processes investigated so far have been found protein-mediated. The selective and often rate-limiting transport processes greatly influence the activity, kinetic features and substrate specificity of the corresponding luminal enzymes. Therefore, the phenomenological characterization of endoplasmic reticulum transport contributes largely to the understanding of the metabolic functions of this organelle. Attempts to identify the transporter proteins have only been successful in a few cases, but recent development in molecular biology promises a better progress in this field.

Translocon pores in the endoplasmic reticulum are permeable to small anions.

Contribution of translocon peptide channels to the permeation of low molecular mass anions was investigated in rat liver microsomes. Puromycin, which purges translocon pores of nascent polypeptides, creating additional empty pores, raised the microsomal uptake of radiolabeled UDP-glucuronic acid, while it did not increase the uptake of glucose-6-phosphate or glutathione. The role of translocon pores in the transport of small anions was also investigated by measuring the effect of puromycin on the activity of microsomal enzymes with intraluminal active sites. The mannose-6-phosphatase activity of glucose-6-phosphatase and the activity of UDP-glucuronosyltransferase were elevated upon addition of puromycin, but glucose-6-phosphatase and beta-glucuronidase activities were not changed. The increase in enzyme activities was due to a better access of the substrates to the luminal compartment rather than to activation of the enzymes. Antibody against Sec61 translocon component decreased the activity of UDP-glucuronosyltransferase and antagonized the effect of puromycin. Similarly, the addition of the puromycin antagonist anisomycin or treatments of microsomes, resulting in the release of attached ribosomes, prevented the puromycin-dependent increase in the activity. Mannose-6-phosphatase and UDP-glucuronosyltransferase activities of smooth microsomal vesicles showed higher basal latencies that were not affected by puromycin. In conclusion, translationally inactive, ribosome-bound translocons allow small anions to cross the endoplasmic reticulum membrane. This pathway can contribute to the nonspecific substrate supply of enzymes with intraluminal active centers.

Uncoupled redox systems in the lumen of the endoplasmic reticulum. Pyridine nucleotides stay reduced in an oxidative environment.

The redox state of the intraluminal pyridine nucleotide pool was investigated in rat liver microsomal vesicles. The vesicles showed cortisone reductase activity in the absence of added reductants, which was dependent on the integrity of the membrane. The intraluminal pyridine nucleotide pool could be oxidized by the addition of cortisone or metyrapone but not of glutathione. On the other hand, intraluminal pyridine nucleotides were slightly reduced by cortisol or glucose 6-phosphate, although glutathione was completely ineffective. Redox state of microsomal protein thiols/disulfides was not altered either by manipulations affecting the redox state of pyridine nucleotides or by the addition of NAD(P)+ or NAD(P)H. The uncoupling of the thiol/disulfide and NAD(P)+/NAD(P)H redox couples was not because of their subcompartmentation, because enzymes responsible for the intraluminal oxidoreduction of pyridine nucleotides were distributed equally in smooth and rough microsomal subfractions. Instead, the phenomenon can be explained by the negligible representation of glutathione reductase in the endoplasmic reticulum lumen. The results demonstrated the separate existence of two redox systems in the endoplasmic reticulum lumen, which explains the contemporary functioning of oxidative folding and of powerful reductive reactions.