Reduction of the endoplasmic reticulum accompanies the oxidative damage of diabetes mellitus

The endoplasmic reticulum (ER), similary to other subcompartments of the eukaryotic cell possesses a relatively oxidizing environment. The special milieu of ER lumen is important for many ER-specific processes (redox protein folding, glycoprotein synthesis, quality control of secreted proteins, antigen presentation, etc.). Despite of the vital importance of redox regulation in the ER, we have a surprisingly fragmented knowledge about the mechanisms responsible for the ER redox balance. Moreover, new observations on disulfide bridge synthesis and on glutathione functions urge us to revise our recent theories based on many indirect and in vitro results. We have also very little information about the effects of different pathological conditions on the thiol metabolism and redox folding in the ER. Examining the role of molecular chaperones in the cellular pathology of diabetes mellitus we found that the ER redox environment shifted to a more reducing state, which was followed by changes of the thiol metabolism and structural-functional changes of the protein machinery involved in the redox folding process in diabetes. The possible consequences of these unexpected changes are also discussed.     

Molecular chaperones,stress proteins and redox homeostasis

Protection against oxidative stress is highly interrelated with the function of the most ancient cellular defense system, the network of molecular chaperones, heat shock, or stress-proteins. These ubiquitous, conserved proteins help other proteins and macromolecules to fold or re-fold and reach their final, native conformation. Redox regulation of protein folding becomes especially important during the preparation of extracellular proteins to the outside oxidative milieu, which should take place in a gradual and step-by-step controlled manner in the endoplasmic reticulum or in the periplasm. Several chaperones, such as members of the Hsp33 family in yeast and the plethora of small heat shock proteins as well as one of the major chaperones, Hsp70 are able to act against cytoplasmic oxidative damage. Abrupt changes of cellular redox status lead to chaperone induction. The function of several chaperones is tightly regulated by the surrounding redox conditions. Moreover, our recent data suggest that chaperones may act as a central switchboard for the transmission of redox changes in the life of the cell.     

FAD oxidizes the ERO1-PDI electron transfer chain: the role of membrane integrity

The molecular steps of the electron transfer in the endoplasmic reticulum from the secreted proteins during their oxidation are relatively unknown. We present here that flavine adenine dinucleotide (FAD) is a powerful oxidizer of the oxidoreductase system, Ero1 and PDI, besides the proteins of rat liver microsomes and HepG2 hepatoma cells. Inhibition of FAD transport hindered the action of FAD. Microsomal membrane integrity was mandatory for all FAD-related oxidation steps downstream of Ero1. The PDI inhibitor bacitracin could inhibit FAD-mediated oxidation of microsomal proteins and PDI, but did not hinder the FAD-driven oxidation of Ero1. Our data demonstrated that Ero1 can utilize FAD as an electron acceptor and that FAD-driven protein oxidation goes through the Ero1-PDI pathway and requires the integrity of the endoplasmic reticulum membrane. Our findings prompt further studies to elucidate the membrane-dependent steps of PDI oxidation and the role of FAD in redox folding.     

Protein-disulfide isomerase- and protein thiol-dependent dehydroascorbate reduction and ascorbate accumulation in the lumen of the endoplasmic reticulum

The transport and intraluminal reduction of dehydroascorbate was investigated in microsomal vesicles from various tissues. The highest rates of transport and intraluminal isotope accumulation (using radiolabeled compound and a rapid filtration technique) were found in hepatic microsomes. These microsomes contain the highest amount of protein-disulfide isomerase, which is known to have a dehydroascorbate reductase activity. The steady-state level of intraluminal isotope accumulation was more than 2-fold higher in hepatic microsomes prepared from spontaneously diabetic BioBreeding/Worcester rats and was very low in fetal hepatic microsomes although the initial rate of transport was not changed. In these microsomes, the amount of protein-disulfide isomerase was similar, but the availability of protein thiols was different and correlated with dehydroascorbate uptake. The increased isotope accumulation was accompanied by a higher rate of dehydroascorbate reduction and increased protein thiol oxidation in microsomes from diabetic animals. The results suggest that both the activity of protein-disulfide isomerase and the availability of protein thiols as reducing equivalents can play a crucial role in the accumulation of ascorbate in the lumen of the endoplasmic reticulum. These findings also support the fact that dehydroascorbate can act as an oxidant in the protein-disulfide isomerase-catalyzed protein disulfide formation.     

Reactive cysteines of the 90-kDa heat shock protein, Hsp90

The 90-kDa heat shock protein (Hsp90) is the most abundant molecular chaperone of the eukaryotic cytoplasm. Its cysteine groups participate in the interactions of Hsp90 with the heme-regulated eIF-2alpha kinase and molybdate, a stabilizer of Hsp90-protein complexes. In our present studies we investigated the reactivity of the sulfhydryl groups of Hsp90. Our data indicate that Hsp90 as well as two Hsp90 peptides containing Cys-521 and Cys-589/590 are able to reduce cytochrome c. The effect of Hsp90 can be blocked by sulfhydryl reagents including arsenite and cadmium, which indicates the involvement of the vicinal cysteines Cys589/590 in the reduction of cytochrome c. Hsp90 neither reduces the disulfide bonds of insulin nor possesses a NADPH:quinone oxidoreductase activity. Oxidizing conditions impair the chaperone activity of Hsp90 toward citrate synthase. The high and specific reactivity of Hsp90 cysteine groups toward cytochrome c may indicate a role of this chaperone in modulation of the redox status of the cytosol in resting and apoptotic cells.     

Roles Played by the Na+/Ca2+ Exchanger and Hypothermia in the Prevention of Ischemia-Induced Carrier-Mediated Efflux of Catecholamines into the Extracellular Space: Implications for Stroke Therapy

The release of [³H]dopamine ([³H]DA) and [³H]noradrenaline ([³H]NA) in acutely perfused rat striatal and cortical slice preparations was measured at 37 °C and 17 °C under ischemic conditions. The ischemia was simulated by the removal of oxygen and glucose from the Krebs solution. At 37 °C, resting release rates in response to ischemia were increased; in contrast, at 17 °C, resting release rates were significantly reduced, or resting release was completely prevented. The removal of extracellular Ca²⁺ further increased the release rates of [³H]DA and [³H]NA induced by ischemic conditions. This finding indicated that the Na⁺/Ca²⁺ exchanger (NCX), working in reverse in the absence of extracellular Ca²⁺, fails to trigger the influx of Ca²⁺ in exchange for Na⁺ and fails to counteract ischemia by further increasing the intracellular Na⁺ concentration ([Na⁺]_i). KB-R7943, an inhibitor of NCX, significantly reduced the cytoplasmic resting release rate of catecholamines under ischemic conditions and under conditions where Ca²⁺ was removed. Hypothermia inhibited the excessive release of [³H]DA in response to ischemia, even in the absence of Ca²⁺. These findings further indicate that the NCX plays an important role in maintaining a high [Na⁺]_i, a condition that may lead to the reversal of monoamine transporter functions; this effect consequently leads to the excessive cytoplasmic tonic release of monoamines and the reversal of the NCX. Using HPLC combined with scintillation spectrometry, hypothermia, which enhances the stimulation-evoked release of DA, was found to inhibit the efflux of toxic DA metabolites, such as 3,4-dihydroxyphenylacetaldehyde (DOPAL). In slices prepared from human cortical brain tissue removed during elective neurosurgery, the uptake and release values for [³H]NA did not differ from those measured at 37 °C in slices that were previously maintained under hypoxic conditions at 8 °C for 20 h. This result indicates that hypothermia preserves the functions of the transport and release mechanisms, even under hypoxic conditions. Oxidative stress (H₂O₂), a mediator of ischemic brain injury enhanced the striatal resting release of [³H]DA and its toxic metabolites (DOPAL, quinone). The study supports our earlier findings that during ischemia transmitters are released from the cytoplasm. In addition, the major findings of this study that hypothermia of brain slice preparations prevents the extracellular calcium concentration ([Ca²⁺]_o)-independent non-vesicular transmitter release induced by ischemic insults, inhibiting Na⁺/Cl^?-dependent membrane transport of monoamines and their toxic metabolites into the extracellular space, where they can exert toxic effects.

     

FAD transport and FAD-dependent protein thiol oxidation in rat liver microsomes

The transport of FAD and its effect on disulfide bond formation was investigated in rat liver microsomal vesicles. By measuring the intravesicular FAD-accessible space, we observed that FAD permeates across the microsomal membrane and accumulates in the lumen. Rapid filtration experiments also demonstrated the uptake and efflux of the compound, which could be inhibited by atractyloside and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. FAD entering the lumen promoted the oxidation of protein thiols and increased the intraluminal oxidation of glucose-6-phosphate. These findings support the notion that, similar to yeast, free FAD may have a decisive role in the mechanism of oxidative protein folding in the endoplasmic reticulum lumen of mammalian cells.     

Characterization of the 90 kDa heat shock protein (HSP90)-associated ATP/GTPase

The 90 kDa heat shock protein (HSP90) is an ATP-binding molecular chaperone with an associated ATPase activity having nucleoplasmin and HSP70-binding homology domains and containing Ca-binding EF-hands and a nuclear localization signal. Here we characterize the HSP90-associated ATPase and show that it is (i) a P-type ATPase inhibited by molybdate and vanadate, (ii) able to hydrolyze methylfluorescein phosphate with a 5–6-fold higher affinity, (iii) a 3-times better GTPase than ATPase in the presence of calcium and (iv) HSP27 and F-actin, but not HSP10 can “convert” the HSP90-associated ATPase activity to HSP90 autokinase activity. The HSP90-associated ATP/GTPase may participate in the regulation of complex formation of HSP90 with other proteins, such as F-actin, tubulin and heat shock proteins.     

Integration of neuronally predifferentiated human dental pulp stem cells into rat brain in vivo

Pluripotency and their neural crest origin make dental pulp stem cells (DPSCs) an attractive donor source for neuronal cell replacement. Despite recent encouraging results in this field, little is known about the integration of transplanted DPSC derived neuronal pecursors into the central nervous system. To address this issue, neuronally predifferentiated DPSCs, labeled with a vital cell dye Vybrant DiD were introduced into postnatal rat brain. DPSCs were transplanted into the cerebrospinal fluid of 3-day-old male Wistar rats. Cortical lesion was induced by touching a cold (−60 °C) metal stamp to the calvaria over the forelimb motor cortex. Four weeks later cell localization was detected by fluorescent microscopy and neuronal cell markers were studied by immunohistochemistry. To investigate electrophysiological properties of engrafted, fluorescently labeled DPSCs, 300 μm-thick horizontal brain slices were prepared and the presence of voltage-dependent sodium and potassium channels were recorded by patch clamping.Predifferentiated donor DPSCs injected into the cerebrospinal fluid of newborn rats migrated as single cells into a variety of brain regions. Most of the cells were localized in the normal neural progenitor zones of the brain, the subventricular zone (SVZ), subgranular zone (SGZ) and subcallosal zone (SCZ). Immunohistochemical analysis revealed that transplanted DPSCs expressed the early neuronal marker N-tubulin, the neuronal specific intermediate filament protein NF-M, the postmitotic neuronal marker NeuN, and glial GFAP. Moreover, the cells displayed TTX sensitive voltage dependent (VD) sodium currents (I_Na) and TEA sensitive delayed rectifier potassium currents (K_DR). Four weeks after injury, fluorescently labeled cells were detected in the lesioned cortex. Neurospecific marker expression was increased in DPSCs found in the area of the cortical lesions compared to that in fluorescent cells of uninjured brain. TTX sensitive VD sodium currents and TEA sensitive K_DR significantly increased in labeled cells of the cortically injured area. In conclusion, our data demonstrate that engrafted DPSC-derived cells integrate into the host brain and show neuronal properties not only by expressing neuron-specific markers but also by exhibiting voltage dependent sodium and potassium channels. This proof of concept study reveals that predifferentiated hDPSCs may serve as useful sources of neuro- and gliogenesis in vivo, especially when the brain is injured.     

Diabetic changes in the redox status of the microsomal protein folding machinery

Changes in assisted protein folding are largely unexplored in diabetes. In the present studies, we have identified a reductive shift in the redox status of rat liver microsomes after 4 weeks of streptozotocin-induced diabetes. This change was reflected by a significant increase in the total- and protein-sulfhydryl content, as well as in the free sulfhydryl groups of the major protein disulfide isomerases (PDIs), the 58 kDa PDI and the 57 kDa ERp57 but not other chaperones. A parallel decrease of the protein-disulfide oxidoreductase activity was detected in the microsomal fraction of diabetic livers. The oxidant of PDI, Ero1-Lalpha showed a more oxidized status in diabetic rats. Our results reveal major changes in the redox status of the endoplasmic reticulum and its redox chaperones in diabetic rats, which may contribute to the defective protein secretion of the diabetic liver.