Different interaction modes for protein-disulfide isomerase (PDI) as an efficient regulator and a specific substrate of endoplasmic reticulum oxidoreductin-1α (Ero1α).

Protein-disulfide isomerase (PDI) and sulfhydryl oxidase endoplasmic reticulum oxidoreductin-1α (Ero1α) constitute the pivotal pathway for oxidative protein folding in the mammalian endoplasmic reticulum (ER). Ero1α oxidizes PDI to introduce disulfides into substrates, and PDI can feedback-regulate Ero1α activity. Here, we show the regulatory disulfide of Ero1α responds to the redox fluctuation in ER very sensitively, relying on the availability of redox active PDI. The regulation of Ero1α is rapidly facilitated by either a or a' catalytic domain of PDI, independent of the substrate binding domain. On the other hand, activated Ero1α specifically binds to PDI via hydrophobic interactions and preferentially catalyzes the oxidation of domain a'. This asymmetry ensures PDI to function simultaneously as an oxidoreductase and an isomerase. In addition, several PDI family members are also characterized to be potent regulators of Ero1α. The novel modes for PDI as a competent regulator and a specific substrate of Ero1α govern efficient and faithful oxidative protein folding and maintain the ER redox homeostasis.

Regulation of urea cycle by reversible high-stoichiometry lysine succinylation.

The post-translational modification lysine succinylation is implicated in the regulation of various metabolic pathways. However, its biological relevance remains uncertain due to methodological difficulties in determining high-impact succinylation sites. Here, using stable isotope labelling and data-independent acquisition mass spectrometry, we quantified lysine succinylation stoichiometries in mouse livers. Despite the low overall stoichiometry of lysine succinylation, several high-stoichiometry sites were identified, especially upon deletion of the desuccinylase SIRT5. In particular, multiple high-stoichiometry lysine sites identified in argininosuccinate synthase (ASS1), a key enzyme in the urea cycle, are regulated by SIRT5. Mutation of the high-stoichiometry lysine in ASS1 to succinyl-mimetic glutamic acid significantly decreased its enzymatic activity. Metabolomics profiling confirms that SIRT5 deficiency decreases urea cycle activity in liver. Importantly, SIRT5 deficiency compromises ammonia tolerance, which can be reversed by the overexpression of wild-type, but not succinyl-mimetic, ASS1. Therefore, lysine succinylation is functionally important in ammonia metabolism.

Expression of selenocysteine-containing glutathione S-transferase in eukaryote.

Glutathione peroxidase (GPX) is a crucial antioxidant selenocysteine (Sec) containing enzyme which plays a significant role in protecting cells against oxidative damage by catalyzing the reduction of hydroperoxides with glutathione (GSH). Several methods have been used to generate GPX mimics, however, only a few of these methods involved genetic engineering and none of them have achieved specific site-directed incorporation of Sec without other modifications, which has hampered further structure-function studies. Here, we report for the first time the conversion of human glutathione transferase Zeta (hGSTZ1-1) into seleno-hGSTZ1-1 by means of genetic engineering in eukaryotes. Fluorescence microscopy images of the expression of Seleno-GST-green fluorescent protein chimaera indicated that we successfully achieved the read-through of the UGA codon to specifically incorporate Sec. Therefore, we achieved the conversion of human glutathione transferase Zeta (hGSTZ1-1) into a seleno-GST (seleno-hGSTZ1-1) by means of genetic engineering in eukaryotes. These results show that recombinant selenoproteins with incorporation of specific selenocysteine residues may be heterologously produced in eukaryotes by using a Sec insertion sequence in the 3' untranslated region (3'-UTR) of the mRNA, and the recombinant selenoproteins is single catalytically active residue and well-characterized structure. In this case a novel GPX activity of 2050±225 U/µmol was introduced into hGSTZ1-1 by substitution of serine 15 by Sec 15. This result will lay a foundation for preparing much smaller GPX mimics with higher activity.

Effects of sugars, fatty acids and amino acids on cytosolic and mitochondrial hydrogen peroxide release from liver cells.

The rates of formation of superoxide and hydrogen peroxide at different electron-donating sites in isolated mitochondria are critically dependent on the substrates that are added, through their effects on the reduction level of each site and the components of the protonmotive force. However, in intact cells the acute effects of added substrates on different sites of cytosolic and mitochondrial hydrogen peroxide production are unclear. Here we tested the effects of substrate addition on cytosolic and mitochondrial hydrogen peroxide release from intact AML12 liver cells. In 30-min starved cells replete with endogenous substrates, addition of glucose, fructose, palmitate, alanine, leucine or glutamine had no effect on the rate or origin of cellular hydrogen peroxide release. However, following 150-min starvation of the cells to deplete endogenous glycogen (and other substrates), cellular hydrogen peroxide production, particularly from NADPH oxidases (NOXs), was decreased, GSH/GSSH ratio increased, and antioxidant gene expression was unchanged. Addition of glucose or glutamine (but not the other substrates) increased hydrogen peroxide release. There were similar relative increases from each of the three major sites of production: mitochondrial sites IQ and IIIQo, and cytosolic NOXs. Glucose supplementation also restored ATP production and mitochondrial NAD reduction level, suggesting that the increased rates of hydrogen peroxide release from the mitochondrial sites were driven by increases in the protonmotive force and the degree of reduction of the electron transport chain. Long-term (24 h) glucose or glutamine deprivation also diminished hydrogen peroxide release rate, ATP production rate and (for glucose deprivation) NAD reduction level. We conclude that the rates of superoxide and hydrogen peroxide production from mitochondrial sites in liver cells are insensitive to extra added substrates when endogenous substrates are not depleted, but these rates are decreased when endogenous substrates are lowered by 150 min of starvation, and can be enhanced by restoring glucose or glutamine supply through improvements in mitochondrial energetic state.

Production of superoxide and hydrogen peroxide in the mitochondrial matrix is dominated by site IQ of complex I in diverse cell lines.

Understanding how mitochondria contribute to cellular oxidative stress and drive signaling and disease is critical, but quantitative assessment is difficult. Our previous studies of cultured C2C12 cells used inhibitors of specific sites of superoxide and hydrogen peroxide production to show that mitochondria generate about half of the hydrogen peroxide released by the cells, and site IQ of respiratory complex I produces up to two thirds of the superoxide and hydrogen peroxide generated in the mitochondrial matrix. Here, we used the same approach to measure the engagement of these sites in seven diverse cell lines to determine whether this pattern is specific to C2C12 cells, or more general. These diverse cell lines covered primary, immortalized, and cancerous cells, from seven tissues (liver, cervix, lung, skin, neuron, heart, bone) of three species (human, rat, mouse). The rate of appearance of hydrogen peroxide in the extracellular medium spanned a 30-fold range from HeLa cancer cells (3 pmol/min/mg protein) to AML12 liver cells (84 pmol/min/mg protein). The mean contribution of identified mitochondrial sites to this extracellular hydrogen peroxide signal was 30 ± 7% SD; the mean contribution of NADPH oxidases was 60 ± 14%. The relative contributions of different sites in the mitochondrial electron transport chain were broadly similar in all seven cell types (and similar to published results for C2C12 cells). 70 ± 4% of identified superoxide/hydrogen peroxide generation in the mitochondrial matrix was from site IQ; 30 ± 4% was from site IIIQo. We conclude that although absolute rates vary considerably, the relative contributions of different sources of hydrogen peroxide production are similar in nine diverse cell types under unstressed conditions in vitro. Identified mitochondrial sites account for one third of total cellular hydrogen peroxide production (half each from sites IQ and IIIQo); in the mitochondrial matrix the majority (two thirds) of superoxide/hydrogen peroxide is from site IQ.

Metformin alleviates human cellular aging by upregulating the endoplasmic reticulum glutathione peroxidase 7.

Metformin, an FDA-approved antidiabetic drug, has been shown to elongate lifespan in animal models. Nevertheless, the effects of metformin on human cells remain unclear. Here, we show that low-dose metformin treatment extends the lifespan of human diploid fibroblasts and mesenchymal stem cells. We report that a low dose of metformin upregulates the endoplasmic reticulum-localized glutathione peroxidase 7 (GPx7). GP×7 expression levels are decreased in senescent human cells, and GPx7 depletion results in premature cellular senescence. We also indicate that metformin increases the nuclear accumulation of nuclear factor erythroid 2-related factor 2 (Nrf2), which binds to the antioxidant response elements in the GPX7 gene promoter to induce its expression. Moreover, the metformin-Nrf2-GPx7 pathway delays aging in worms. Our study provides mechanistic insights into the beneficial effects of metformin on human cellular aging and highlights the importance of the Nrf2-GPx7 pathway in pro-longevity signaling.

Structural insights into the redox-regulated dynamic conformations of human protein disulfide isomerase.

AIM: Human protein disulfide isomerase (hPDI) is a key enzyme and a redox-regulated chaperone responsible for oxidative protein folding in the endoplasmic reticulum. This work aims to reveal the molecular mechanism underlying the redox-regulated functions of hPDI by determining the crystal structures of hPDI in different redox states. RESULTS: The structures of hPDI (abb'xa') in both the reduced and oxidized states showed that the four thioredoxin domains of a, b, b', and a' are arranged as a horseshoe shape with two CGHC active sites, respectively, in domains a and a' facing each other at the two ends. In reduced hPDI, domains a, b, and b' line up in the same plane, whereas domain a' twists ∼45° out. The two active sites are 27.6 Å apart. In oxidized hPDI, the four domains are differently organized to stay in the same plane, and the distance between the active sites increases to 40.3 Å. In contrast to the closed conformation of reduced hPDI, oxidized hPDI exists in an open state with more exposed hydrophobic areas and a larger cleft with potential for substrate binding. INNOVATION: This is the first report of the high-resolution structures of hPDI containing all four domains in both the reduced and the oxidized states. It reveals the redox-regulated structural dynamic properties of the protein. CONCLUSION: The redox-regulated open/closed conformational switch of hPDI endows the protein with versatile target-binding capacities for its enzymatic and chaperone functions.