An Atlas of Peroxiredoxins Created Using an Active Site Profile-Based Approach to Functionally Relevant Clustering of Proteins.

Peroxiredoxins (Prxs or Prdxs) are a large protein superfamily of antioxidant enzymes that rapidly detoxify damaging peroxides and/or affect signal transduction and, thus, have roles in proliferation, differentiation, and apoptosis. Prx superfamily members are widespread across phylogeny and multiple methods have been developed to classify them. Here we present an updated atlas of the Prx superfamily identified using a novel method called MISST (Multi-level Iterative Sequence Searching Technique). MISST is an iterative search process developed to be both agglomerative, to add sequences containing similar functional site features, and divisive, to split groups when functional site features suggest distinct functionally-relevant clusters. Superfamily members need not be identified initially-MISST begins with a minimal representative set of known structures and searches GenBank iteratively. Further, the method's novelty lies in the manner in which isofunctional groups are selected; rather than use a single or shifting threshold to identify clusters, the groups are deemed isofunctional when they pass a self-identification criterion, such that the group identifies itself and nothing else in a search of GenBank. The method was preliminarily validated on the Prxs, as the Prxs presented challenges of both agglomeration and division. For example, previous sequence analysis clustered the Prx functional families Prx1 and Prx6 into one group. Subsequent expert analysis clearly identified Prx6 as a distinct functionally relevant group. The MISST process distinguishes these two closely related, though functionally distinct, families. Through MISST search iterations, over 38,000 Prx sequences were identified, which the method divided into six isofunctional clusters, consistent with previous expert analysis. The results represent the most complete computational functional analysis of proteins comprising the Prx superfamily. The feasibility of this novel method is demonstrated by the Prx superfamily results, laying the foundation for potential functionally relevant clustering of the universe of protein sequences.

A peroxiredoxin-based proteinaceous scaffold for the growth and differentiation of neuronal cells and tumour stem cells in the absence of prodifferentiation agents.

The use of nanoscale materials in the design of scaffolds for CNS tissue is increasing, due to their ability to promote cell adhesion, to mimic an extracellular matrix microenvironment and to interact with neuronal membranes. In this framework, one of the major challenges when using undifferentiated neural cells is how to control the differentiation process. Here we report the characterization of a scaffold based on the self-assembled nanotubes of a mutant of the protein peroxiredoxin (from Schistosoma mansoni or Bos taurus), which allows the growth and differentiation of a model neuronal cell line (SHSY5Y). The results obtained demonstrate that SHSY5Y cells grow without any sign of toxicity and develop a neuronal phenotype, as shown by the expression of neuronal differentiation markers, without the use of any differentiation supplement, even in the presence of serum. The prodifferentiation effect is demonstrated to be dependent on the formation of the protein nanotube, since a wild-type (WT) form of the peroxiredoxin from Schistosoma mansoni does not induce any differentiation. The protein scaffold was also able to induce the spread of glioblastoma cancer stem cells growing in neurospheres and allowing the acquisition of a neuron-like morphology, as well as of immature rat cortical neurons. This protein used here as coating agent may be suggested for the development of scaffolds for tissue regeneration or anti-tumour devices. Copyright © 2016 John Wiley & Sons, Ltd.

Site-specific mutagenesis of yeast 2-Cys peroxiredoxin improves heat or oxidative stress tolerance by enhancing its chaperone or peroxidase function.

Yeast peroxiredoxin II (yPrxII) is an antioxidant enzyme that plays a protective role against the damage caused by reactive oxygen species (ROS) in Saccharomyces cerevisiae. This enzyme consists of 196 amino acids containing 2-Cys Prx with highly conserved two active cysteine residues at positions 48 and 171. The yPrxII has dual enzymatic functions as a peroxidase and molecular chaperone. To understand the effect of additional cysteine residues on dual functions of yPrxII, S79C-yPrxII and S109C-yPrxII, the substitution of Ser with Cys residue at 79 and 109 positions, respectively, was generated. S109C-yPrxII and S79C-yPrxII showed 3.7- and 2.7-fold higher chaperone and peroxidase activity, respectively, than the wild type (WT). The improvement in enzyme activity was found to be closely associated with structural changes in proteins. S109C-yPrxII had increased ß-sheet in its secondary structure and formed high-molecular-weight (HMW) as well as low-molecular-weight (LMW) complexes, but S79C-yPrxII formed only LMW complexes. HMW complexes predominantly exhibited a chaperone function, and LMW complexes showed a peroxidase function. In addition, transgenic yeast cells over-expressing Cys-substituted yPrxII showed greater tolerance against heat and oxidative stress compared to WT-yPrxII.

Optimized enzymatic dual functions of PaPrx protein by proton irradiation.

We investigated the effects of proton irradiation on the function and structure of the Pseudomonas aeruginosa peroxiredoxin (PaPrx). Polyacrylamide gel demonstrated that PaPrx proteins exposed to proton irradiation at several doses exhibited simultaneous formation of high molecular weight (HMW) complexes and fragmentation. Size-exclusion chromatography (SEC) analysis revealed that the number of fragments and very low molecular weight (LMW) structures increased as the proton irradiation dose increased. The peroxidase activity of irradiated PaPrx was preserved, and its chaperone activity was significantly increased by increasing the proton irradiation dose. The chaperone activity increased about 3-4 fold after 2.5 kGy proton irradiation, compared with that of non-irradiated PaPrx, and increased to almost the maximum activity after 10 kGy proton irradiation. We previously obtained functional switching in PaPrx proteins, by using gamma rays and electron beams as radiation sources, and found that the proteins exhibited increased chaperone activity but decreased peroxidase activity. Interestingly, in this study we newly found that proton irradiation could enhance both peroxidase and chaperone activities. Therefore, we can suggest proton irradiation as a novel protocol for conserved 2-Cys protein engineering.

Switching between the alternative structures and functions of a 2-Cys peroxiredoxin, by site-directed mutagenesis.

Members of the typical 2-Cys peroxiredoxin (Prx) subfamily represent an intriguing example of protein moonlighting behavior since this enzyme shifts function: indeed, upon chemical stimuli, such as oxidative stress, Prx undergoes a switch from peroxidase to molecular chaperone, associated to a change in quaternary structure from dimers/decamers to higher-molecular-weight (HMW) species. In order to detail the structural mechanism of this switch at molecular level, we have designed and expressed mutants of peroxiredoxin I from Schistosoma mansoni (SmPrxI) with constitutive HMW assembly and molecular chaperone activity. By a combination of X-ray crystallography, transmission electron microscopy and functional experiments, we defined the structural events responsible for the moonlighting behavior of 2-Cys Prx and we demonstrated that acidification is coupled to local structural variations localized at the active site and a change in oligomerization to HMW forms, similar to those induced by oxidative stress. Moreover, we suggest that the binding site of the unfolded polypeptide is at least in part contributed by the hydrophobic surface exposed by the unfolding of the active site. We also find an inverse correlation between the extent of ring stacking and molecular chaperone activity that is explained assuming that the binding occurs at the extremities of the nanotube, and the longer the nanotube is, the lesser the ratio binding sites/molecular mass is.

Synergistic cooperation of PDI family members in peroxiredoxin 4-driven oxidative protein folding.

The mammalian endoplasmic reticulum (ER) harbors disulfide bond-generating enzymes, including Ero1α and peroxiredoxin 4 (Prx4), and nearly 20 members of the protein disulfide isomerase family (PDIs), which together constitute a suitable environment for oxidative protein folding. Here, we clarified the Prx4 preferential recognition of two PDI family proteins, P5 and ERp46, and the mode of interaction between Prx4 and P5 thioredoxin domain. Detailed analyses of oxidative folding catalyzed by the reconstituted Prx4-PDIs pathways demonstrated that, while P5 and ERp46 are dedicated to rapid, but promiscuous, disulfide introduction, PDI is an efficient proofreader of non-native disulfides. Remarkably, the Prx4-dependent formation of native disulfide bonds was accelerated when PDI was combined with ERp46 or P5, suggesting that PDIs work synergistically to increase the rate and fidelity of oxidative protein folding. Thus, the mammalian ER seems to contain highly systematized oxidative networks for the efficient production of large quantities of secretory proteins.

Functional switching of a novel prokaryotic 2-Cys peroxiredoxin (PpPrx) under oxidative stress.

Many proteins have been isolated from eukaryotes as redox-sensitive proteins, but whether these proteins are present in prokaryotes is not clear. Redox-sensitive proteins contain disulfide bonds, and their enzymatic activity is modulated by redox in vivo. In the present study, we used thiol affinity purification and mass spectrometry to isolate and identify 19 disulfide-bond-containing proteins in Pseudomonas putida exposed to potential oxidative damages. Among these proteins, we found that a typical 2-Cys Prx-like protein (designated PpPrx) displays diversity in structure and apparent molecular weight (MW) and can act as both a peroxidase and a molecular chaperone. We also identified a regulatory factor involved in this structural and functional switching. Exposure of pseudomonads to hydrogen peroxide (H(2)O(2)) caused the protein structures of PpPrx to convert from high MW complexes to low MW forms, triggering a chaperone-to-peroxidase functional switch. This structural switching was primarily guided by the thioredoxin system. Thus, the peroxidase efficiency of PpPrx is clearly associated with its ability to form distinct protein structures in response to stress.

The malarial parasite Plasmodium falciparum imports the human protein peroxiredoxin 2 for peroxide detoxification.

Coevolution of the malarial parasite and its human host has resulted in a complex network of interactions contributing to the homeodynamics of the host-parasite unit. As a rapidly growing and multiplying organism, Plasmodium falciparum depends on an adequate antioxidant defense system that is efficient despite the absence of genuine catalase and glutathione peroxidase. Using different experimental approaches, we demonstrate that P. falciparum imports the human redox-active protein peroxiredoxin 2 (hPrx-2, hTPx1) into its cytosol. As shown by confocal microscopy and immunogold electron microscopy, hPrx-2 is also present in the Maurer's clefts, organelles that are described as being involved in parasite protein export. Enzyme kinetic analyses prove that hPrx-2 accepts Plasmodium cytosolic thioredoxin 1 as a reducing substrate. hPrx-2 accounts for roughly 50% of thioredoxin peroxidase activity in parasite extracts, thus indicating a functional role of hPrx-2 as an enzymatic scavenger of peroxides in the parasite. Under chloroquine treatment, a drug promoting oxidative stress, the abundance of hPrx-2 in the parasite increases significantly. P. falciparum has adapted to adopt the hPrx-2, thereby using the host protein for its own purposes.

Reconstitution of the mitochondrial PrxIII antioxidant defence pathway: general properties and factors affecting PrxIII activity and oligomeric state.

The mitochondrial 2-Cys peroxiredoxin PrxIII serves as a thioredoxin-dependent peroxidase operating in tandem with its cognate partners, an organelle-specific thioredoxin (Trx2) and NADP-linked thioredoxin reductase (TRR2). This PrxIII pathway is emerging as a primary regulator of intracellular H(2)O(2) levels with dual roles in antioxidant defence and H(2)O(2)-mediated signalling. Here we describe the reconstitution of the mammalian PrxIII pathway in vitro from its purified recombinant components and investigate some of its overall properties. Employing the site-directed PrxIII mutants C47S, C66S and C168S, the putative N and C-terminal catalytic cysteine residues are shown to be essential for function whereas the C66S mutant retains full activity. The pathway attains maximal capacity at low H(2)O(2) concentrations (<10 microM) and is progressively inhibited in the range 0.1 mM to 1.0 mM peroxide. Damage to PrxIII caused by over-oxidation is confirmed by the appearance of abnormal oxidised species of PrxIII on SDS-PAGE at elevated H(2)O(2) levels. The presence of an N-terminal His-tag on PrxIII markedly enhances dodecamer stability, particularly apparent in its oxidised state. Its removal promotes oxidised PrxIII dissociation into dimers and leads to a 3.0-3.5-fold stimulation in peroxidase activity. The unusual concatenated crystal structure of PrxIII consisting of two-interlocked dodecameric rings is also evident in dilute solution employing transmission electron microscopy; however, it represents only 3-5% of the population with most molecules present as single toroids. Moreover, concatenated PrxIII C168S reverts to single toroids on crystal dissolution indicating that these higher-order structures are produced dynamically during the crystallisation process.