Hepatocyte proteomes reveal the role of protein disulfide isomerase 4 in alpha 1-antitrypsin deficiency.

BACKGROUND & AIMS: A single point mutation in the Z-variant of alpha 1-antitrypsin (Z-AAT) alone can lead to both a protein folding and trafficking defect, preventing its exit from the endoplasmic reticulum (ER), and the formation of aggregates that are retained as inclusions within the ER of hepatocytes. These defects result in a systemic AAT deficiency (AATD) that causes lung disease, whereas the ER-retained aggregates can induce severe liver injury in patients with ZZ-AATD. Unfortunately, therapeutic approaches are still limited and liver transplantation represents the only curative treatment option. To overcome this limitation, a better understanding of the molecular basis of ER aggregate formation could provide new strategies for therapeutic intervention. METHODS: Our functional and omics approaches here based on human hepatocytes from patients with ZZ-AATD have enabled the identification and characterisation of the role of the protein disulfide isomerase (PDI) A4/ERP72 in features of AATD-mediated liver disease. RESULTS: We report that 4 members of the PDI family (PDIA4, PDIA3, P4HB, and TXNDC5) are specifically upregulated in ZZ-AATD liver samples from adult patients. Furthermore, we show that only PDIA4 knockdown or alteration of its activity by cysteamine treatment can promote Z-AAT secretion and lead to a marked decrease in Z aggregates. Finally, detailed analysis of the Z-AAT interactome shows that PDIA4 silencing provides a more conducive environment for folding of the Z mutant, accompanied by reduction of Z-AAT-mediated oxidative stress, a feature of AATD-mediated liver disease. CONCLUSIONS: PDIA4 is involved in AATD-mediated liver disease and thus represents a therapeutic target for inhibition by drugs such as cysteamine. PDI inhibition therefore represents a potential therapeutic approach for treatment of AATD. LAY SUMMARY: Protein disulfide isomerase (PDI) family members, and particularly PDIA4, are upregulated and involved in alpha 1-antitrypsin deficiency (AATD)-mediated liver disease in adults. PDI inhibition upon cysteamine treatment leads to improvements in features of AATD and hence represents a therapeutic approach for treatment of AATD-mediated liver disease.

Emerging Evidence Highlighting the Importance of Redox Dysregulation in the Pathogenesis of Amyotrophic Lateral Sclerosis (ALS).

The cellular redox state, or balance between cellular oxidation and reduction reactions, serves as a vital antioxidant defence system that is linked to all important cellular activities. Redox regulation is therefore a fundamental cellular process for aerobic organisms. Whilst oxidative stress is well described in neurodegenerative disorders including amyotrophic lateral sclerosis (ALS), other aspects of redox dysfunction and their contributions to pathophysiology are only just emerging. ALS is a fatal neurodegenerative disease affecting motor neurons, with few useful treatments. Hence there is an urgent need to develop more effective therapeutics in the future. Here, we discuss the increasing evidence for redox dysregulation as an important and primary contributor to ALS pathogenesis, which is associated with multiple disease mechanisms. Understanding the connection between redox homeostasis, proteins that mediate redox regulation, and disease pathophysiology in ALS, may facilitate a better understanding of disease mechanisms, and lead to the design of better therapeutic strategies.

Experimental Nonalcoholic Steatohepatitis and Liver Fibrosis Are Ameliorated by Pharmacologic Activation of Nrf2 (NF-E2 p45-Related Factor 2).

BACKGROUND & AIMS: Nonalcoholic steatohepatitis (NASH) is associated with oxidative stress. We surmised that pharmacologic activation of NF-E2 p45-related factor 2 (Nrf2) using the acetylenic tricyclic bis(cyano enone) TBE-31 would suppress NASH because Nrf2 is a transcriptional master regulator of intracellular redox homeostasis. METHODS: Nrf2+/+ and Nrf2-/- C57BL/6 mice were fed a high-fat plus fructose (HFFr) or regular chow diet for 16 weeks or 30 weeks, and then treated for the final 6 weeks, while still being fed the same HFFr or regular chow diets, with either TBE-31 or dimethyl sulfoxide vehicle control. Measures of whole-body glucose homeostasis, histologic assessment of liver, and biochemical and molecular measurements of steatosis, endoplasmic reticulum (ER) stress, inflammation, apoptosis, fibrosis, and oxidative stress were performed in livers from these animals. RESULTS: TBE-31 treatment reversed insulin resistance in HFFr-fed wild-type mice, but not in HFFr-fed Nrf2-null mice. TBE-31 treatment of HFFr-fed wild-type mice substantially decreased liver steatosis and expression of lipid synthesis genes, while increasing hepatic expression of fatty acid oxidation and lipoprotein assembly genes. Also, TBE-31 treatment decreased ER stress, expression of inflammation genes, and markers of apoptosis, fibrosis, and oxidative stress in the livers of HFFr-fed wild-type mice. By comparison, TBE-31 did not decrease steatosis, ER stress, lipogenesis, inflammation, fibrosis, or oxidative stress in livers of HFFr-fed Nrf2-null mice. CONCLUSIONS: Pharmacologic activation of Nrf2 in mice that had already been rendered obese and insulin resistant reversed insulin resistance, suppressed hepatic steatosis, and mitigated against NASH and liver fibrosis, effects that we principally attribute to inhibition of ER, inflammatory, and oxidative stress.

Differential activity of rice protein disulfide isomerase family members for disulfide bond formation and reduction.

Protein disulfide isomerases (PDIs), a family of thiol-disulfide oxidoreductases that are ubiquitous in all eukaryotes, are the principal catalysts for disulfide bond formation. Here, we investigated three rice (Oryza sativa) PDI family members (PDIL1;1, PDIL1;4, and PDIL2;3) and found that PDIL1;1 exhibited the highest catalytic activity for both disulfide bond formation and disulfide bond reduction. The activity of PDIL1;1-catalyzed disulfide bond reduction, in which two redox-active sites were involved, was enhanced by increasing the glutathione concentration. These results suggest that PDIL1;1 plays primary roles in both disulfide bond formation and disulfide bond reduction, which allow for redox control of protein quality and packaging.

Molecular chaperones and proteostasis regulation during redox imbalance.

Free radicals originate from both exogenous environmental sources and as by-products of the respiratory chain and cellular oxygen metabolism. Sustained accumulation of free radicals, beyond a physiological level, induces oxidative stress that is harmful for the cellular homeodynamics as it promotes the oxidative damage and stochastic modification of all cellular biomolecules including proteins. In relation to proteome stability and maintenance, the increased concentration of oxidants disrupts the functionality of cellular protein machines resulting eventually in proteotoxic stress and the deregulation of the proteostasis (homeostasis of the proteome) network (PN). PN curates the proteome in the various cellular compartments and the extracellular milieu by modulating protein synthesis and protein machines assembly, protein recycling and stress responses, as well as refolding or degradation of damaged proteins. Molecular chaperones are key players of the PN since they facilitate folding of nascent polypeptides, as well as holding, folding, and/or degradation of unfolded, misfolded, or non-native proteins. Therefore, the expression and the activity of the molecular chaperones are tightly regulated at both the transcriptional and post-translational level at organismal states of increased oxidative and, consequently, proteotoxic stress, including ageing and various age-related diseases (e.g. degenerative diseases and cancer). In the current review we present a synopsis of the various classes of intra- and extracellular chaperones, the effects of oxidants on cellular homeodynamics and diseases and the redox regulation of chaperones.

Protein disulfide isomerase may facilitate the efflux of nitrite derived S-nitrosothiols from red blood cells.

Protein disulfide isomerase (PDI) is an abundant protein primarily found in the endoplasmic reticulum and also secreted into the blood by a variety of vascular cells. The evidence obtained here, suggests that PDI could directly participate in the efflux of NO(+) from red blood cells (RBC). PDI was detected both in RBC membranes and in the cytosol. PDI was S-nitrosylated when RBCs were exposed to nitrite under ∼50% oxygen saturation but not under ∼100% oxygen saturation. Furthermore, it was observed that hemoglobin (Hb) could promote PDI S-nitrosylation in the presence of ∼600 nM nitrite. In addition, three lines of evidence were obtained for PDI-Hb interactions: (1) Hb co-immunoprecipitated with PDI; (2) Hb quenched the intrinsic PDI fluorescence in a saturable manner; and (3) Hb-Fe(II)-NO absorption spectrum decreased in a [PDI]-dependent manner. Finally, PDI was detected on the surface RBC under ∼100% oxygen saturation and released as soluble under ∼50% oxygen saturation. The soluble PDI detected under ∼50% oxygen saturation was S-nitrosylated. Based on these data it is proposed that PDI is taken up by RBC and forms a complex with Hb. Hb-Fe(II)-NO that is formed from nitrite reduction under ∼50% O2, then transfers NO(+) to either Hb-Cys ß93 or directly to PDI resulting in S-nitroso-PDI which transverses the RBC membrane and attaches to the RBC surface. When RBCs enter tissues the S-nitroso-PDI is released from the RBC-surface into the blood where its NO(+) is transferred into the endothelium thereby inducing vasodilation, suggesting local oxygen-dependent dynamic interplays between nitrite, NO and S-nitrosylation.

Intracellular glutathione pools are heterogeneously concentrated.

Glutathione is present in millimolar concentrations in the cell, but its relative distribution among cellular compartments remains elusive. We have chosen the endoplasmic reticulum (ER) as an example organelle to study compartment-specific glutathione levels. Using a glutaredoxin sensor (sCGrx1pER), which rapidly and specifically equilibrates with the reduced glutathione (GSH)-glutathione disulfide (GSSG) redox couple with known equilibrium constant, we showed that the [GSH]:[GSSG] ratio in the ER of intact HeLa cells is less than 7:1. Taking into consideration the previously determined value for [GSH](2):[GSSG] in the ER of 83 mM, this translates into a total glutathione concentration in the ER ([GStot]=[GSH]+2[GSSG]) of greater than 15 mM. Since the integrated, intracellular [GStot] was measured as ~7 mM, we conclude the existence of a [GStot] gradient across the ER membrane. A possible homeostatic mechanism by which cytosol-derived glutathione is trapped in the ER is discussed. We propose a high [GStot] as a distinguishing feature of the ER environment compared to the extracellular space.

Oxidized lipids activate autophagy in a JNK-dependent manner by stimulating the endoplasmic reticulum stress response.

Excessive production of unsaturated aldehydes from oxidized lipoproteins and membrane lipids is a characteristic feature of cardiovascular disease. Our previous studies show that unsaturated lipid peroxidation-derived aldehydes such as 4-hydroxy-trans-2-nonenal (HNE) promote autophagy in rat aortic smooth muscle cells (RASMC). In this study, we examined the mechanism by which HNE induces autophagy. Exposure of RASMC to HNE led to the modification of several proteins, most of which were identified by mass spectrometry and confocal microscopy to be localized to the endoplasmic reticulum (ER). HNE stimulated the phosphorylation of PKR-like ER kinase and eukaryotic initiation factor 2α and increased heme oxygenase-1 (HO-1) abundance. HNE treatment also increased LC3-II formation and the phosphorylation of JNK and p38. Pharmacological inhibition of JNK, but not p38, prevented HNE-induced HO-1 expression and LC3-II formation. Inhibition of JNK increased cell death in HNE-treated cells. Pretreatment with the chemical chaperone phenylbutryic acid prevented LC3-II formation as well as JNK phosphorylation and HO-1 induction. Taken together, these data suggest that autophagic responses triggered by unsaturated aldehydes could be attributed, in part, to ER stress, which stimulates autophagy by a JNK-dependent mechanism and promotes cell survival during oxidative stress.