Zhenbo Cao

Crystal Structure of Reduced and of Oxidized Peroxiredoxin IV Enzyme Reveals a Stable Oxidized Decamer and a Non-disulfide-bonded Intermediate in the Catalytic Cycle.

Background: Peroxiredoxin IV metabolizes endoplasmic reticulum-derived hydrogen peroxide. Results: Peroxiredoxin IV structures reveal an unusually stable decamer and a sulfenylated intermediate in the enzymatic cycle. Conclusion: The enzymatic cycle of peroxiredoxin IV involves destabilization of the active site followed by formation of a stable disulfide-bonded decamer. Significance: Elucidating peroxiredoxin structures is required to understand how they function during oxidative stress. Peroxiredoxin IV (PrxIV) is an endoplasmic reticulum-localized enzyme that metabolizes the hydrogen peroxide produced by endoplasmic reticulum oxidase 1 (Ero1). It has been shown to play a role in de novo disulfide formation, oxidizing members of the protein disulfide isomerase family of enzymes, and is a member of the typical 2-Cys peroxiredoxin family. We have determined the crystal structure of both reduced and disulfide-bonded, as well as a resolving cysteine mutant of human PrxIV. We show that PrxIV has a similar structure to other typical 2-Cys peroxiredoxins and undergoes a conformational change from a fully folded to a locally unfolded form following the formation of a disulfide between the peroxidatic and resolving cysteine residues. Unlike other mammalian typical 2-Cys peroxiredoxins, we show that human PrxIV forms a stable decameric structure even in its disulfide-bonded state. In addition, the structure of a resolving cysteine mutant reveals an intermediate in the reaction cycle that adopts the locally unfolded conformation. Interestingly the peroxidatic cysteine in the crystal structure is sulfenylated rather than sulfinylated or sulfonylated. In addition, the peroxidatic cysteine in the resolving cysteine mutant is resistant to hyper-oxidation following incubation with high concentrations of hydrogen peroxide. These results highlight some unique properties of PrxIV and suggest that the equilibrium between the fully folded and locally unfolded forms favors the locally unfolded conformation upon sulfenylation of the peroxidatic cysteine residue.

Lack of an efficient endoplasmic reticulum-localized recycling system protects peroxiredoxin IV from hyperoxidation.

Background: Peroxiredoxin IV metabolizes endoplasmic reticulum-derived hydrogen peroxide. Results: In contrast to other peroxiredoxins, peroxiredoxin IV is insensitive to hyperoxidation within its cellular environment. Conclusion: There is a lack of a robust peroxiredoxin IV recycling system within the endoplasmic reticulum. Significance: Studying the reduction of peroxiredoxin IV highlights the different mechanisms for maintaining redox status within different cellular compartments. Typical 2-Cys peroxiredoxins are required to remove hydrogen peroxide from several different cellular compartments. Their activity can be regulated by hyperoxidation and consequent inactivation of the active-site peroxidatic cysteine. Here we developed a simple assay to quantify the hyperoxidation of peroxiredoxins. Hyperoxidation of peroxiredoxins can only occur efficiently in the presence of a recycling system, usually involving thioredoxin and thioredoxin reductase. We demonstrate that there is a marked difference in the sensitivity of the endoplasmic reticulum-localized peroxiredoxin to hyperoxidation compared with either the cytosolic or mitochondrial enzymes. Each enzyme is equally sensitive to hyperoxidation in the presence of a robust recycling system. Our results demonstrate that peroxiredoxin IV recycling in the endoplasmic reticulum is much less efficient than in the cytosol or mitochondria, leading to the protection of peroxiredoxin IV from hyperoxidation.

Cytosolic thioredoxin reductase 1 is required for correct disulfide formation in the ER

Folding of proteins entering the secretory pathway in mammalian cells frequently requires the insertion of disulfide bonds. Disulfide insertion can result in covalent linkages found in the native structure as well as those that are not, so‐called non‐native disulfides. The pathways for disulfide formation are well characterized, but our understanding of how non‐native disulfides are reduced so that the correct or native disulfides can form is poor. Here, we use a novel assay to demonstrate that the reduction in non‐native disulfides requires NADPH as the ultimate electron donor, and a robust cytosolic thioredoxin system, driven by thioredoxin reductase 1 (TrxR1 or TXNRD1). Inhibition of this reductive pathway prevents the correct folding and secretion of proteins that are known to form non‐native disulfides during their folding. Hence, we have shown for the first time that mammalian cells have a pathway for transferring reducing equivalents from the cytosol to the ER, which is required to ensure correct disulfide formation in proteins entering the secretory pathway.

The membrane topology of vitamin K epoxide reductase is conserved between human isoforms and the bacterial enzyme

The membrane topology of vitamin K epoxide reductase (VKOR) is controversial with data supporting both a three transmembrane and a four transmembrane model. The positioning of the transmembrane domains and the loops between these domains is critical if we are to understand the mechanism of vitamin K oxidation and its recycling by members of the thioredoxin family of proteins and the mechanism of action of warfarin, an inhibitor of VKOR. Here we show that both mammalian VKOR isoforms adopt the same topology, with the large loop between transmembrane one and two facing the lumen of the endoplasmic reticulum (ER). We used a redox sensitive green fluorescent protein (GFP) fused to the N- or C-terminus to show that these regions face the cytosol, and introduction of glycosylation sites along with mixed disulfide formation with thioredoxin-like transmembrane protein (TMX) to demonstrate ER localization of the major loop. The topology is identical with the bacterial homologue from Synechococcussp., for which the structure and mechanism of recycling has been characterized. Our results provide a resolution to the membrane topology controversy and support previous results suggesting a role for members of the ER protein disulfide isomerase (PDI) family in recycling VKOR.

Improved Catenated Structures of Bovine Peroxiredoxin III F190L Reveal Details of Ring-Ring Interactions and a Novel Conformational State.

Mitochondrial 2-cys peroxiredoxin III (PrxIII) is a key player in antioxidant defence reducing locally-generated H2O2 to H2O. A Phe to Leu (F190L) mutation in the C-terminal α-helix of PrxIII, mimicking that found in some bacteria and parasites, increases its resistance to hyperoxidation but has no obvious influence on peroxidase activity. Here we report on the oxidized and reduced crystal structures of bovine PrxIII F190L at 2.4 Å and 2.2 Å, respectively. Both structures exist as two-ring catenanes with their dodecameric rings inclined at 55o to each other, similar to that previously reported for PrxIII C168S. The new higher-resolution structures reveal details of the complex network of H-bonds stabilising the inter-toroid contacts. In addition, Arg123, the key conserved residue, that normally interacts with the catalytic cys (Cp, cys 47) is found in a distinct conformation extending away from the Cp while the characteristic Arg-Glu-Arg network, underpinning the active-site geometry also displays a distinctive arrangement, not observed previously. This novel active-site organisation may provide new insights into the dynamics of the large-scale conformational changes occurring between oxidized and reduced states.

Regulating the level of intracellular hydrogen peroxide: the role of peroxiredoxin IV

Hydrogen peroxide (H2O2) can act as a signalling molecule affecting the cell cycle as well as contributing towards the oxidative stress response. The primary target of this molecule is oxidation-sensitive cysteine residues in proteins such as protein tyrosine phosphatases. The cell has robust mechanisms to remove H2O2 that need to be regulated for H2O2 to react with and modify protein thiols. In particular, the family of peroxiredoxins are capable of the rapid removal of even trace amounts of this molecule. It has been suggested that the inactivation of peroxiredoxins by hyperoxidation may allow H2O2 levels to increase in cells and thereby modify critical thiol groups in proteins. We have been studying how the H2O2 produced during disulfide formation in the ER (endoplasmic reticulum) is metabolized and have shown that ER-resident peroxiredoxin IV not only can remove H2O2, but also contributes to de novo disulfide formation. In the present article, we review recent data on the structure and function of this enzyme as well as its sensitivity to hyperoxidation.

The Peroxiredoxin Family: An Unfolding Story

Peroxiredoxins (Prxs) are a large and conserved family of peroxidases that are considered to be the primary cellular guardians against oxidative stress in all living organisms. Prxs share a thioredoxin fold and contain a highly-reactive peroxidatic cysteine in a specialised active-site environment that is able to reduce their peroxide substrates. The minimal functional unit for Prxs are either monomers or dimers, but many dimers assemble into decameric rings. Ring structures can further form a variety of high molecular weight complexes. Many eukaryotic Prxs contain a conserved GGLG and C-terminal YF motif that confer sensitivity to elevated levels of peroxide, leading to hyperoxidation and inactivation. Inactive forms of Prxs can be re-reduced by the enzyme sulfiredoxin, in an ATP-dependent reaction. Cycles of hyperoxidation and reactivation are considered to play an integral role in a variety of H2O2-mediated cell signalling pathways in both stress and non-stress conditions. Prxs are also considered to exhibit chaperone-like properties when cells are under oxidative or thermal stress. The roles of various types of covalent modifications, e.g. acetylation and phosphorylation are also discussed. The ability of Prxs to assemble into ordered arrays such as nanotubes is currently being exploited in nanotechnology.

Detecting peroxiredoxin hyperoxidation by one-dimensional isoelectric focusing

The activity of typical 2-cys peroxiredoxin (Prxs) can be regulated by hyperoxidation with a consequent loss of redox activity. Here we developed a simple assay to monitor the level of hyperoxidation of different typical 2-cys prxs simultaneously. This assay only requires standard equipment and can compare different samples on the same gel. It requires much less time than conventional 2D gels and gives more information than Western blotting with an antibody specific for hyperoxidized peroxiredoxin. This method could also be used to monitor protein modification with a charge difference such as phosphorylation.

Vascular Nox (NADPH Oxidase) Compartmentalization, Protein Hyperoxidation, and Endoplasmic Reticulum Stress Response in Hypertension

Vascular Nox (NADPH oxidase)-derived reactive oxygen species and endoplasmic reticulum (ER) stress have been implicated in hypertension. However, relationships between these processes are unclear. We hypothesized that Nox isoforms localize in a subcellular compartment-specific manner, contributing to oxidative and ER stress, which influence the oxidative proteome and vascular function in hypertension. Nox compartmentalization (cell fractionation), O2− (lucigenin), H2O2 (amplex red), reversible protein oxidation (sulfenylation), irreversible protein oxidation (protein tyrosine phosphatase, peroxiredoxin oxidation), and ER stress (PERK [protein kinase RNA-like endoplasmic reticulum kinase], IRE1&agr; [inositol-requiring enzyme 1], and phosphorylation/oxidation) were studied in spontaneously hypertensive rat (SHR) vascular smooth muscle cells (VSMCs). VSMC proliferation was measured by fluorescence-activated cell sorting, and vascular reactivity assessed in stroke-prone SHR arteries by myography. Noxs were downregulated by short interfering RNA and pharmacologically. In SHR, Noxs were localized in specific subcellular regions: Nox1 in plasma membrane and Nox4 in ER. In SHR, oxidative stress was associated with increased protein sulfenylation and hyperoxidation of protein tyrosine phosphatases and peroxiredoxins. Inhibition of Nox1 (NoxA1ds), Nox1/4 (GKT137831), and ER stress (4-phenylbutyric acid/tauroursodeoxycholic acid) normalized SHR vascular reactive oxygen species generation. GKT137831 reduced IRE1&agr; sulfenylation and XBP1 (X-box binding protein 1) splicing in SHR. Increased VSMC proliferation in SHR was normalized by GKT137831, 4-phenylbutyric acid, and STF083010 (IRE1–XBP1 disruptor). Hypercontractility in the stroke-prone SHR was attenuated by 4-phenylbutyric acid. We demonstrate that protein hyperoxidation in hypertension is associated with oxidative and ER stress through upregulation of plasmalemmal-Nox1 and ER-Nox4. The IRE1–XBP1 pathway of the ER stress response is regulated by Nox4/reactive oxygen species and plays a role in the hyperproliferative VSMC phenotype in SHR. Our study highlights the importance of Nox subcellular compartmentalization and interplay between cytoplasmic reactive oxygen species and ER stress response, which contribute to the VSMC oxidative proteome and vascular dysfunction in hypertension.

Methionine Sulfoxide Reductase B3 Requires Resolving Cysteine Residues for full Activity and can act as a Stereospecific Methionine Oxidase.

The oxidation of methionine residues in proteins occurs during oxidative stress and can lead to an alteration in protein function. The enzyme methionine sulfoxide reductase (Msr) reverses this modification. Here, we characterise the mammalian enzyme Msr B3. There are two splice variants of this enzyme that differ only in their N-terminal signal sequence, which directs the protein to either the endoplasmic reticulum (ER) or mitochondria. We demonstrate here that the enzyme can complement a bacterial strain, which is dependent on methionine sulfoxide reduction for growth, that the purified recombinant protein is enzymatically active showing stereospecificity towards R-methionine sulfoxide, and identify the active site and two resolving cysteine residues. The enzyme is efficiently recycled by thioredoxin only in the presence of both resolving cysteine residues. These results show that for this isoform of Msrs, the reduction cycle most likely proceeds through a three-step process. This involves an initial sulfenylation of the active site thiol followed by the formation of an intrachain disulfide with a resolving thiol group and completed by the reduction of this disulfide by a thioredoxin-like protein to regenerate the active site thiol. Interestingly, the enzyme can also act as an oxidase catalysing the stereospecific formation of R-methionine sulfoxide. This result has important implications for the role of this enzyme in the reversible modification of ER and mitochondrial proteins.