Entamoeba histolytica: biochemical characterization of a protein disulfide isomerase

Protein disulfide isomerase (PDI) enzymes are eukaryotic oxidoreductases that catalyze oxidation, reduction and isomerization of disulfide bonds in polypeptide substrates. Here, we report the biochemical characterization of a PDI enzyme from the protozoan parasite Entamoeba histolytica (EhPDI). Our results show that EhPDI behaves mainly as an oxidase/isomerase and can be inhibited by bacitracin, a known PDI inhibitor; moreover, it exhibits chaperone-like activity. Albeit its physiological role in the life style of the parasite (including virulence and survival) remains to be studied, EhPDI could represent a potential drug target for anti-amebic therapy.     

Activity,stability and folding analysis of the chitinase from Entamoeba histolytica

Human amebiasis, caused by the parasitic protozoan Entamoeba histolytica, remains as a significant public health issue in developing countries. The life cycle of the parasite compromises two main stages, trophozoite and cyst, linked by two major events: encystation and excystation. Interestingly, the cyst stage has a chitin wall that helps the parasite to withstand harsh environmental conditions. Since the amebic chitinase, EhCHT1, has been recognized as a key player in both encystation and excystation, it is plausible to consider that specific inhibition could arrest the life cycle of the parasite and, thus, stop the infection. However, to selectively target EhCHT1 it is important to recognize its unique biochemical features to have the ability to control its cellular function. Hence, to gain further insights into the structure–function relationship, we conducted an experimental approach to examine the effects of pH, temperature, and denaturant concentration on the enzymatic activity and protein stability. Additionally, dependence on in vivo oxidative folding was further studied using a bacterial model. Our results attest the potential of EhCHT1 as a target for the design and development of new or improved anti-amebic therapeutics. Likewise, the potential of the oxidoreductase EhPDI, involved in oxidative folding of amebic proteins, was also confirmed.     

Gene sequencing, modelling and immunolocalization of the protein disulfide isomerase from Plasmodium chabaudi

Malaria remains one of the major human parasitic diseases, particularly in subtropical regions. Most of the fatal cases are caused by Plasmodium falciparum. The rodent parasite Plasmodium chabaudi has been the model of choice in research due to its similarities to human malaria, including developmental cycle, preferential invasion of mature erythrocytes, synchrony of asexual development, antigenic variation, gene sinteny as well as similar resistance mechanisms. Protein disulfide isomerase (PDI) is an essential catalyst of the endoplasmic reticulum in different biological systems with folding and chaperone activities. Most of the proteins exported by parasites have to pass through the endoplasmic reticulum before reaching their final destination and their correct folding is critical for parasite survival. PDI constitutes a potential target for the development of alternative therapy strategies based on the inhibition of folding and chaperoning of exported proteins. We here describe the sequencing of the gene coding for the PDI from P. chabaudi and analyse the relationship to its counterpart enzymes, particularly with the PDI from other Plasmodium species. The model constructed, based on the recent model deduced from the crystallographic structure 2B5E, was compared with the previous theoretical model for the whole PDI molecule constructed by threading. A recombinant PDI from P. chabaudi was also produced and used as an antigen for monoclonal antibody production for application in PDI immunolocalization.     

Gaussia princeps luciferase: a bioluminescent substrate for oxidative protein folding

Gaussia princeps luciferase (GLuc) generates an intense burst of blue light when exposed to coelenterazine in the absence of ATP. Here we show that this 5‐disulfide containing enzyme can be used as a facile and convenient substrate for studies of oxidative protein folding. Reduced GLuc (rGLuc), with 10 free cysteine residues, is completely inactive as a luciferase but >60% bioluminescence activity, compared to controls, can be recovered using a range of oxidizing regimens in the absence of the exogenous shuffling activity of protein disulfide isomerase (PDI). The sulfhydryl oxidase QSOX1 can be assayed using rGLuc in a simple bioluminescence plate reader format. Similarly, low concentrations of rGLuc can be oxidized by millimolar levels of dehydroascorbate, hydrogen peroxide or much lower concentrations of sodium tetrathionate. The oxidative refolding of rGLuc in the presence of a range of glutathione redox buffers is only marginally accelerated by micromolar levels of PDI. This modest rate enhancement probably results from a relatively simple disulfide connectivity in native GLuc; reflecting two homologous domains each carrying two disulfide bonds with a single interdomain disulfide. When GLuc is reoxidized under denaturing conditions the resulting scrambled protein (sGLuc) can be used in a sensitive bioluminescence assay for reduced PDI in the absence of added exogenous thiols. Finally, the general facility by which rGLuc can recover bioluminescent activity in vitro provides a sensitive method for the assessment of inhibitors of oxidative protein folding.     

The contributions of protein disulfide isomerase and its homologues to oxidative protein folding in the yeast endoplasmic reticulum

In vitro, protein disulfide isomerase (Pdi1p) introduces disulfides into proteins (oxidase activity) and provides quality control by catalyzing the rearrangement of incorrect disulfides (isomerase activity). Protein disulfide isomerase (PDI) is an essential protein in Saccharomyces cerevisiae, but the contributions of the catalytic activities of PDI to oxidative protein folding in the endoplasmic reticulum (ER) are unclear. Using variants of Pdi1p with impaired oxidase or isomerase activity, we show that isomerase-deficient mutants of PDI support wild-type growth even in a strain in which all of the PDI homologues of the yeast ER have been deleted. Although the oxidase activity of PDI is sufficient for wild-type growth, pulse-chase experiments monitoring the maturation of carboxypeptidase Y reveal that oxidative folding is greatly compromised in mutants that are defective in isomerase activity. Pdi1p and one or more of its ER homologues (Mpd1p, Mpd2p, Eug1p, Eps1p) are required for efficient carboxypeptidase Y maturation. Consistent with its function as a disulfide isomerase in vivo, the active sites of Pdi1p are partially reduced (32 +/- 8%) in vivo. These results suggest that PDI and its ER homologues contribute both oxidase and isomerase activities to the yeast ER. The isomerase activity of PDI can be compromised without affecting growth and viability, implying that yeast proteins that are essential under laboratory conditions may not require efficient disulfide isomerization.     

Analysis of myotilin turnover provides mechanistic insight into the role of myotilinopathy-causing mutations

In eukaryotes, disulfide bonds are formed in the endoplasmic reticulum, facilitated by the Ero1 (endoplasmic reticulum oxidoreductin 1) oxidase/PDI (protein disulfide-isomerase) system. Mammals have two ERO1 genes, encoding Ero1α and Ero1β proteins. Ero1β is constitutively expressed in professional secretory tissues and induced during the unfolded protein response. In the present work, we show that recombinant human Ero1β is twice as active as Ero1α in enzymatic assays. Ero1β oxidizes PDI more efficiently than other PDI family members and drives oxidative protein folding preferentially via the active site in the á domain of PDI. Our results reveal that Ero1β oxidase activity is regulated by long-range disulfide bonds and that Cys130 plays a critical role in feedback regulation. Compared with Ero1α, however, Ero1β is loosely regulated, consistent with its role as a more active oxidase when massive oxidative power is required.     

Dynamic Regulation of Ero1α and Peroxiredoxin 4 Localization in the Secretory Pathway

In the early secretory compartment (ESC), a network of chaperones and enzymes assists oxidative folding of nascent proteins. Ero1 flavoproteins oxidize protein disulfide isomerase (PDI), generating H₂O₂ as a byproduct. Peroxiredoxin 4 (Prx4) can utilize luminal H₂O₂ to oxidize PDI, thus favoring oxidative folding while limiting oxidative stress. Interestingly, neither ER oxidase contains known ER retention signal(s), raising the question of how cells prevent their secretion. Here we show that the two proteins share similar intracellular localization mechanisms. Their secretion is prevented by sequential interactions with PDI and ERp44, two resident proteins of the ESC-bearing KDEL-like motifs. PDI binds preferentially Ero1α, whereas ERp44 equally retains Ero1α and Prx4. The different binding properties of Ero1α and Prx4 increase the robustness of ER redox homeostasis.     

Small-molecule catalysts of oxidative protein folding

Oxidative protein folding occurs both in vivo and in vitro and involves the formation and rearrangement of protein disulfide bonds (SS bonds). In vivo these reactions are catalyzed by enzymes, including the eukaryotic enzyme protein disulfide isomerase (PDI). Using the physical properties of PDI as a guide, several small-molecule catalysts of oxidative protein folding have been designed, synthesized, and tested. These small molecules can improve the folding rate of the model substrate ribonuclease A by a factor of over 10 and improve the yield by up to a factor of 3 over traditional conditions. The molecules have also been demonstrated to significantly improve the in vivo folding of proteins as well.     

Oxidative protein folding in the plant endoplasmic reticulum

For most of the proteins synthesized in the endoplasmic reticulum (ER), disulfide bond formation accompanies protein folding in a process called oxidative folding. Oxidative folding is catalyzed by a number of enzymes, including the family of protein disulfide isomerases (PDIs), as well as other proteins that supply oxidizing equivalents to PDI family proteins, like ER oxidoreductin 1 (Ero1). Oxidative protein folding in the ER is a basic vital function, and understanding its molecular mechanism is critical for the application of plants as protein production tools. Here, I review the recent research and progress related to the enzymes involved in oxidative folding in the plant ER. Firstly, nine groups of plant PDI family proteins are introduced. Next, the enzymatic properties of plant Ero1 are described. Finally, the cooperative folding by multiple PDI family proteins and Ero1 is described.     

Modulation of Conotoxin Structure and Function Is Achieved through a Multienzyme Complex in the Venom Glands of Cone Snails

The oxidative folding of large polypeptides has been investigated in detail; however, comparatively little is known about the enzyme-assisted folding of small, disulfide-containing peptide substrates. To investigate the concerted effect of multiple enzymes on the folding of small disulfide-rich peptides, we sequenced and expressed protein-disulfide isomerase (PDI), peptidyl-prolyl cis-trans isomerase, and immunoglobulin-binding protein (BiP) from Conus venom glands. Conus PDI was shown to catalyze the oxidation and reduction of disulfide bonds in two conotoxins, α-GI and α-ImI. Oxidative folding rates were further increased in the presence of Conus PPI with the maximum effect observed in the presence of both enzymes. In contrast, Conus BiP was only observed to assist folding in the presence of microsomes, suggesting that additional co-factors were involved. The identification of a complex between BiP, PDI, and nascent conotoxins further suggests that the folding and assembly of conotoxins is a highly regulated multienzyme-assisted process. Unexpectedly, all three enzymes contributed to the folding of the ribbon isomer of α-ImI. Here, we identify this alternative disulfide-linked species in the venom of Conus imperialis, providing the first evidence for the existence of a “non-native” peptide isomer in the venom of cone snails. Thus, ER-resident enzymes act in concert to accelerate the oxidative folding of conotoxins and modulate their conformation and function by reconfiguring disulfide connectivities. This study has evaluated the role of a number of ER-resident enzymes in the folding of conotoxins, providing novel insights into the enzyme-guided assembly of these small, disulfide-rich peptides.     

Novel Roles of the Non-catalytic Elements of Yeast Protein-disulfide Isomerase in Its Interplay with Endoplasmic Reticulum Oxidoreductin 1

The formation of disulfide bonds in the endoplasmic reticulum (ER) of eukaryotic cells is catalyzed by the sulfhydryl oxidase, ER oxidoreductin 1 (Ero1), and protein-disulfide isomerase (PDI). PDI is oxidized by Ero1 to continuously introduce disulfides into substrates, and feedback regulates Ero1 activity by manipulating the regulatory disulfides of Ero1. In this study we find that yeast Ero1p is enzymatically active even with its regulatory disulfides intact, and further activation of Ero1p by reduction of the regulatory disulfides requires the reduction of non-catalytic Cys⁹⁰-Cys⁹⁷ disulfide in Pdi1p. The principal client-binding site in the Pdi1p b′ domain is necessary not only for the functional Ero1p-Pdi1p disulfide relay but also for the activation of Ero1p. We also demonstrate by complementary activation assays that the regulatory disulfides in Ero1p are much more stable than those in human Ero1α. These new findings on yeast Ero1p-Pdi1p interplay reveal significant differences from our previously identified mode of human Ero1α-PDI interplay and provide insights into the evolution of the eukaryotic oxidative protein folding pathway.     

Expression and purification of recombinant rat protein disulfide isomerase from Escherichia coli.

Rat liver protein disulfide isomerase (PDI) catalyzes the oxidative folding of proteins containing disulfide bonds. We have developed an efficient method for its overproduction in Escherichia coli. Using a T7 RNA polymerase expression system, isolated yields of 15-30 mg/liter of recombinant rat PDI are readily obtained. Convenient purification of the enzyme from E. coli lysates involves ion-exchange (DEAE) chromatography combined with zinc chelate chromatography. The recombinant PDI shows catalytic activity identical to that of PDI isolated from bovine liver in both the reduction of insulin and the oxidative folding of ribonuclease A. The enzyme is expressed in E. coli as a soluble, cytoplasmic protein. After complete reduction and denaturation in 6 M guanidinium hydrochloride, PDI regains complete activity within 3 min after removal of the denaturant, implying that disulfide bonds are not essential for the maintenance of PDI tertiary structure. Both the protein isolated from E. coli and the protein isolated from liver contained free cysteine residues (1.8 +/- 0.2 and 1.4 +/- 0.3 SH/monomer, respectively).     

Protein disulfide isomerase homolog TrPDI2 contributing to cellobiohydrolase production in Trichoderma reesei

The majority of the cysteine residues in the secreted proteins form disulfide bonds via protein disulfide isomerase (PDI)-mediated catalysis, stabilizing the enzyme activity. The role of PDI in cellulase production is speculative, as well as the possibility of PDI as a target for improving enzyme production efficiency of Trichoderma reesei, a widely used producer of enzyme for the production of lignocellulose-based biofuels and biochemicals. Here, we report that a PDI homolog, TrPDI2 in T. reesei exhibited a 36.94% and an 11.81% similarity to Aspergillus niger TIGA and T. reesei PDI1, respectively. The capability of TrPDI2 to recover the activity of reduced and denatured RNase by promoting refolding verified its protein disulfide isomerase activity. The overexpression of Trpdi2 increased the secretion and the activity of CBH1 at the early stage of cellulase induction. In addition, both the expression level and redox state of TrPDI2 responded to cellulase induction in T. reesei, providing sustainable oxidative power to ensure cellobiohydrolase maturation and production. The results suggest that TrPDI2 may contribute to cellobiohydrolase secretion by enhancing the capability of disulfide bond formation, which is essential for protein folding and maturation.     

A Novel Reaction of Peroxiredoxin 4 towards Substrates in Oxidative Protein Folding

Peroxiredoxin 4 (Prx4) is the only endoplasmic reticulum localized peroxiredoxin. It functions not only to eliminate peroxide but also to promote oxidative protein folding via oxidizing protein disulfide isomerase (PDI). In Prx4-mediated oxidative protein folding we discovered a new reaction that the sulfenic acid form of Prx4 can directly react with thiols in folding substrates, resulting in non-native disulfide cross-linking and aggregation. We also found that PDI can inhibit this reaction by exerting its reductase and chaperone activities. This discovery discloses an off-pathway reaction in the Prx4-mediated oxidative protein folding and the quality control role of PDI.     

Cyclotide proteins and precursors from the genus Gloeospermum: Filling a blank spot in the cyclotide map of Violaceae

Cyclotides are disulfide-rich plant proteins that are exceptional in their cyclic structure; their N and C termini are joined by a peptide bond, forming a continuous circular backbone, which is reinforced by three interlocked disulfide bonds. Cyclotides have been found mainly in the coffee (Rubiaceae) and violet (Violaceae) plant families. Within the Violaceae, cyclotides seem to be widely distributed, but the cyclotide complements of the vast majority of Violaceae species have not yet been explored. This study provides insight into cyclotide occurrence, diversity and biosynthesis in the Violaceae, by identifying mature cyclotide proteins, their precursors and enzymes putatively involved in their biosynthesis in the tribe Rinoreeae and the genus Gloeospermum. Twelve cyclotides from two Panamanian species, Gloeospermum pauciflorum Hekking and Gloeospermum blakeanum (Standl.) Hekking (designated Glopa A-E and Globa A-G, respectively) were characterised through cDNA screening and protein isolation. Screening of cDNA for the oxidative folding enzymes protein-disulfide isomerase (PDI) and thioredoxin (TRX) resulted in positive hits in both species. These enzymes have demonstrated roles in oxidative folding of cyclotides in Rubiaceae, and results presented here indicate that Violaceae plants have evolved similar mechanisms of cyclotide biosynthesis. We also describe PDI and TRX sequences from a third cyclotide-expressing Violaceae species, Viola biflora L., which further support this hypothesis.     

Functional Relationship between Protein Disulfide Isomerase Family Members during the Oxidative Folding of Human Secretory Proteins

To examine the relationship between protein disulfide isomerase family members within the mammalian endoplasmic reticulum, PDI, ERp57, ERp72, and P5 were depleted with high efficiency in human hepatoma cells, either singly or in combination. The impact was assessed on the oxidative folding of several well-characterized secretory proteins. We show that PDI plays a predominant role in oxidative folding because its depletion delayed disulfide formation in all secretory proteins tested. However, the phenotype was surprisingly modest suggesting that other family members are able to compensate for PDI depletion, albeit with reduced efficacy. ERp57 also exhibited broad specificity, overlapping with that of PDI, but with preference for glycosylated substrates. Depletion of both PDI and ERp57 revealed that some substrates require both enzymes for optimal folding and, furthermore, led to generalized protein misfolding, impaired export from the ER, and degradation. In contrast, depletion of ERp72 or P5, either alone or in combination with PDI or ERp57 had minimal impact, revealing a narrow substrate specificity for ERp72 and no detectable role for P5 in oxidative protein folding.     

Oxidized NADH Oxidase Inhibits Activity of an ATP/NAD Kinase from a Thermophilic Archaeon

NADH oxidases (NOXs) are important enzymes in detoxifying oxidative stress and regenerating oxidized pyridine nucleotides. In the present study, a NOX from Thermococcus kodakarensis KOD1 (NOXtk) was recombinantly expressed in Escherichia coli and purified to homogeneity. NOXtk displayed NADH oxidase activity that was inhibited by oxidization. Under physiological conditions, unoxidized and oxidized NOXtk formed dimers and hexamers, respectively. Mutating the single cysteine residue Cys45 to alanine (NOXtkC45A) decreased NADH oxidase activity without affecting dimerization or hexamerization, suggesting that oligomerization does not occur through disulfide bond formation. Pull-down assay results indicated that an ATP/NAD kinase from T. kodakarensis KOD1 (ANKtk) binds to NOXtk. Use of several assays revealed that ANKtk can only bind to oxidized hexameric NOXtk, through which it inhibits ANKtk activity. Because ANKtk converts NADH to NADPH (an important factor in oxidative stress protection), a model based on in vitro result was proposed in which NOXtk hexamerization under oxic conditions inhibits both NOXtk and ANKtk activities, thereby sensitizing cells to oxidative stress-induced death.     

Molecular biology of plasma membrane redox enzymes: A survey of current knowledge

Summary Despite a large body of evidence for enzymatic activities and physiological functions of plasma membrane redox function, few of these enzymes have been characterized in terms of molecular biology. Examples for these with at least some molecular data up to complete sequences, membrane topology and binding sites for substrates and coenzymes or prosthetic groups are NADH-ferricyanide reductase of Ehrlich ascites membranes, NADH-coenzyme Q reductase of liver, NADH oxidase ectoenzyme of liver and HeLa (and possibly other) cells, protein disulfide isomerase which is widespread, and relatives thereof, as well as cytochromes P-450 andb ₅₅₈, NADPH oxidase of fat and thyroid cells and fat cell amine oxidase. Ferricyanide reductase and coenzyme O reductase may be identical, but NADH oxidase ectoenzyme is distinct and possibly functions also as a disulfide and a copper reductase. On the other hand, the plasma-membrane-located protein disulfide isomerase (PDI), despite its similar enzymatic activity, is completely different from the ectooxidase. The latter is shed from the membrane into the surrounding medium by proteolysis, whereas PDI is not an integral membrane protein and is secreted intact. Another disulfide reductase has been demonstrated in THP-1 cells, which again is totally different from the former two. It turns out that enzymatic activities are insufficient to describe redox enzymes. Special forms of cytochrome P-450 can be induced to expression at the cell membrane of liver, where they are transported by the cytoskeleton-associated secretory pathway. Why some isoforms are expressed at the surface and some are not is not yet clear. Cytochromeb ₅₅₈, the flavocytochrome of neutrophils, is described in other cells too, but there are different isoforms, which are genetically distinct. A relative has also been identified in duodenal cells, where it functions as a ferric reductase involved in iron absorption. NADPH oxidase of fat cells has very similar properties, but the identity is unproved, whereas thyroid oxidase is a non-heme protein which is calcium-sensitive and does not need assembly of subunits for activation. Finally, fat cell membranes also possess a quinone-containing amine-oxidase which may be involved in signaling of glucose-transport regulation, as it is also found in GLUT4-containing vesicles. However, the physiological connection has yet to be demonstrated.