Structural properties of the peroxiredoxin AhpC2 from the hyperthermophilic eubacterium Aquifex aeolicus.

Peroxiredoxins (Prxs) are thiol peroxidases that scavenge various peroxide substrates such as hydrogen peroxide (H2O2), alkyl hydroperoxides and peroxinitrite. They also function as chaperones and are involved in signal transduction by H2O2 in eukaryotic cells. The genome of Aquifex aeolicus, a microaerophilic, hyperthermophilic eubacterium, encodes four Prxs, among them an alkyl hydroperoxide reductase AhpC2 which was found to be closely related to archaeal 1-Cys peroxiredoxins. We determined the crystal structure of AhpC2 at 1.8 Šresolution and investigated its oligomeric state in solution by electron microscopy. AhpC2 is arranged as a toroid-shaped dodecamer instead of the typically observed decamer. The basic folding topology and the active site structure are conserved and possess a high structural similarity to other 1-Cys Prxs. However, the C-terminal region adopts an opposite orientation. AhpC2 contains three cysteines, Cys49, Cys212, and Cys218. The peroxidatic cysteine CP49 was found to be hyperoxidized to the sulfonic acid (SO3H) form, while Cys212 forms an intra-monomer disulfide bond with Cys218. Mutagenesis experiments indicate that Cys212 and Cys218 play important roles in the oligomerization of AhpC2. Based on these structural characteristics, we proposed the catalytic mechanism of AhpC2. This study provides novel insights into the structure and reaction mechanism of 1-Cys peroxiredoxins.

Proteomic differences between focal and diffuse traumatic brain injury in human brain tissue.

The early molecular response to severe traumatic brain injury (TBI) was evaluated using biopsies of structurally normal-appearing cortex, obtained at location for intracranial pressure (ICP) monitoring, from 16 severe TBI patients. Mass spectrometry (MS; label free and stable isotope dimethyl labeling) quantitation proteomics showed a strikingly different molecular pattern in TBI in comparison to cortical biopsies from 11 idiopathic normal pressure hydrocephalus patients. Diffuse TBI showed increased expression of peptides related to neurodegeneration (Tau and Fascin, p < 0.05), reduced expression related to antioxidant defense (Glutathione S-transferase Mu 3, Peroxiredoxin-6, Thioredoxin-dependent peroxide reductase; p < 0.05) and increased expression of potential biomarkers (e.g. Neurogranin, Fatty acid-binding protein, heart p < 0.05) compared to focal TBI. Proteomics of human brain biopsies displayed considerable molecular heterogeneity among the different TBI subtypes with consequences for the pathophysiology and development of targeted treatments for TBI.

Isolation and characterization of peroxiredoxin 1 gene of Dunaliella salina.

Peroxiredoxin 1 (Prdx1) is a ubiquitously expressed protein in eukaryotic cells, and plays an important role in cell proliferation, differentiation, apoptosis, and redox signaling. Although Prdx1 has been better studied in yeasts and humans, only few Prdx1 genes have been cloned in green algae. The microalga Dunaliella salina (D. salina) is a model for the study of a variety of human cilia-related diseases. In this study, a suppression subtractive hybridization cDNA library of D. salina was constructed, and 6 flagellum-associated genes including D. salina Prdx1 (DsPrdx1) were isolated and identified. A 956bp full-length cDNA of DsPrdx1 was cloned using rapid amplification of cDNA end (RACE). The open reading frame (ORF) of this DNA sequence encodes a polypeptide of 201 amino acids with a predicted molecular weight of 22kDa and a theoretical isoelectric point (pI) of 5.27. Sequence comparison showed that Prdx1 is highly evolutionarily conserved from the unicellular green alga D. salina to human. To our knowledge, this is the first reported full-length sequence of Prdx1 in D. salina. Interestingly, the protein expression of DsPrdx1 was obviously increased during flagellar disassembly in D. salina. Additionally, a yeast two-hybrid assay showed interaction between Prdx1 and RNA, and suggested that DsPrdx1 can protect RNA from degradation by RNase. Taken together, DsPrdx1 not only participates in flagellar disassembly, but also protects RNA from degradation.

Characterization of a bacterioferritin comigratory protein family 1-Cys peroxiredoxin from Candidatus Liberibacter asiaticus.

To defend against the lethality of the reactive oxygen species (ROS), nature has armed microorganisms with a range of antioxidant proteins. These include peroxiredoxin (Prx) super family proteins which are ubiquitous cysteine-based non-heme peroxidases. The phytopathogenic bacterium Candidatus Liberibacter asiaticus (CLA), an etiological agent of citrus plants diseases, posses many genes for defense against oxidative stress. The bacterioferritin comigratory protein (BCP), a member of Prxs, is part of an oxidative stress defense system of CLA. The key residue of these enzymes is peroxidatic Cys (termed CPSH) which is contained within an absolutely conserved PXXX (T/S) XXC motif. In the present study, a 1-Cys Prx enzyme (CLa-BCP), having CPSH/sulfenic acid cysteine (C-46) but lacking the resolving cysteine (CRSH), was characterized from CLA. The peroxidase activity was demonstrated using a non-physiological electron donor DTT against varied substrates. The protein was shown to have the defensive role against peroxide-mediated cell killing and an antioxidant activity. In vitro DNA-binding studies showed that this protein can protect supercoiled DNA from oxidative damage. To the best of our knowledge, this is the first report on a 1-Cys BCPs to have an intracellular reactive oxygen species scavenging activity.

The identification of goat peroxiredoxin-5 and the evaluation and enhancement of its stability by nanoparticle formation.

An anticancer bioactive peptide (ACBP), goat peroxiredoxin-5 (gPRDX5), was identified from goat-spleen extract after immunizing the goat with gastric cancer-cell lysate. Its amino acid sequence was determined by employing 2D nano-LC-ESI-LTQ-Orbitrap MS/MS combined with Mascot database search in the goat subset of the Uniprot database. The recombinant gPRDX5 protein was acquired by heterogeneous expression in Escherichia coli. Subsequently, the anti-cancer bioactivity of the peptide was measured by several kinds of tumor cells. The results indicated that the gPRDX5 was a good anti-cancer candidate, especially for killing B16 cells. However, the peptide was found to be unstable without modification with pharmaceutical excipients, which would be a hurdle for future medicinal application. In order to overcome this problem and find an effective way to evaluate the gPRDX5, nanoparticle formation, which has been widely used in drug delivery because of its steadiness in application, less side-effects and enhancement of drug accumulation in target issues, was used here to address the issues. In this work, the gPRDX5 was dispersed into nanoparticles before delivered to B16 cells. By the nanotechnological method, the gPRDX5 was stabilized by a fast and accurate procedure, which suggests a promising way for screening the peptide for further possible medicinal applications.

Comparative analysis of cyanobacterial and plant peroxiredoxins and their electron donors: peroxidase activity and susceptibility to overoxidation.

Peroxiredoxins (Prxs) are peroxidases that use thiol-based catalytic mechanisms implying redox-active cysteines. The different Prx families have homologs in all photosynthetic organisms, including plants, algae, and cyanobacteria. However, recent studies show that the physiological reduction systems that provide Prxs with reducing equivalents to sustain their activities differ considerably between cyanobacterial strains. Thus, for example, the filamentous cyanobacterium Anabaena sp. PCC 7120 is similar to the chloroplast in that it possesses an abundant 2-Cys Prx, which receives electrons from the NADPH-dependent thioredoxin reductase C (NTRC). In contrast, the unicellular cyanobacterium Synechocystis sp. PCC 6803, which lacks NTRC, has little 2-Cys Prx but high amounts of PrxII and 1-Cys Prx. The characterization of cyanobacterial Prxs and their electron donors relies on straightforward enzymatic assays and tools to study the physiological relevance of these systems. Here, we present methods to measure peroxidase activities in vitro and peroxide decomposition in vivo. Several approaches to detect overoxidation of the active site cysteine in cyanobacterial 2-Cys Prxs are also described.

Biochemical, physicochemical and molecular characterization of a genuine 2-Cys-peroxiredoxin purified from cowpea [Vigna unguiculata (L.) Walpers] leaves.

BACKGROUND: Peroxiredoxins have diverse functions in cellular defense-signaling pathways. 2-Cys-peroxiredoxins (2-Cys-Prx) reduce H2O2 and alkyl-hydroperoxide. This study describes the purification and characterization of a genuine 2-Cys-Prx from Vigna unguiculata (Vu-2-Cys-Prx). METHODS: Vu-2-Cys-Prx was purified from leaves by ammonium sulfate fractionation, chitin affinity and ion exchange chromatography. RESULTS: Vu-2-Cys-Prx reduces H2O2 using NADPH and DTT. Vu-2-Cys-Prx is a 44 kDa (SDS-PAGE)/46 kDa (exclusion chromatography) protein that appears as a 22 kDa molecule under reducing conditions, indicating that it is a homodimer linked intermolecularly by disulfide bonds and has a pI range of 4.56­4.72; its NH2-terminal sequence was similar to 2-Cys-Prx from Phaseolus vulgaris (96%) and Populus tricocarpa (96%). Analysis by ESI-Q-TOF MS/MS showed a molecular mass/pI of 28.622 kDa/5.18. Vu-2-Cys-Prx has 8% α-helix, 39% ß-sheet, 22% of turns and 31% of unordered forms. Vu-2-Cys-Prx was heat stable, has optimal activity at pH 7.0, and prevented plasmid DNA degradation. Atomic force microscopy shows that Vu-2-Cys-Prx oligomerized in decamers which might be associated with its molecular chaperone activity that prevented denaturation of insulin and citrate synthase. Its cDNA analysis showed that the redox-active Cys52 residue and the amino acids Pro45, Thr49 and Arg128 are conserved as in other 2-Cys-Prx. GENERAL SIGNIFICANCE: The biochemical and molecular features of Vu-2-Cys-Prx are similar to other members of 2-Cys-Prx family. To date, only one publication reported on the purification of native 2-Cys-Prx from leaves and the subsequent analysis by N-terminal Edman sequencing, which is crucial for construction of stromal recombinant 2-Cys-Prx proteins.

Extraordinary µs-ms backbone dynamics in Arabidopsis thaliana peroxiredoxin Q.

Peroxiredoxin Q (PrxQ) isolated from Arabidopsis thaliana belongs to a family of redox enzymes called peroxiredoxins, which are thioredoxin- or glutaredoxin-dependent peroxidases acting to reduce peroxides and in particular hydrogen peroxide. PrxQ cycles between an active reduced state and an inactive oxidized state during its catalytic cycle. The catalytic mechanism involves a nucleophilic attack of the catalytic cysteine on hydrogen peroxide to generate a sulfonic acid intermediate with a concerted release of a water molecule. This intermediate is subsequently relaxed by the reaction of a second cysteine, denoted the resolving cysteine, generating an intramolecular disulfide bond and release of a second water molecule. PrxQ is recycled to the active state by a thioredoxin-dependent reduction. Previous structural studies of PrxQ homologues have provided the structural basis for the switch between reduced and oxidized conformations. Here, we have performed a detailed study of the activity, structure and dynamics of PrxQ in both the oxidized and reduced states. Reliable and experimentally validated structural models of PrxQ in both oxidation states were generated using homology based modeling. Analysis of NMR spin relaxation rates shows that PrxQ is monomeric in both oxidized and reduced states. As evident from R(2) relaxation rates the reduced form of PrxQ undergoes unprecedented dynamics on the slow µs-ms timescale. The ground state of this conformational dynamics is likely the stably folded reduced state as implied by circular dichroism spectroscopy. We speculate that the extensive dynamics is intimately related to the catalytic function of PrxQ.

Identification of a highly immunoreactive epitope of Brugia malayi TPx recognized by the endemic sera.

Filarial thiordoxin peroxidase is a major antioxidant that plays a crucial role in parasite survival. Although Brugia malayi TPx has been shown to be a potential vaccine candidate, it shares 63% homology with its mammalian counterpart, limiting its use as a vaccine or drug target. In silico analysis of TPx sequence revealed a linear B epitope in the host's nonhomologous region. The peptide sequence (TPx peptide(27-48)) was synthesized, and its reactivity with clinical sera from an endemic region was analyzed. The peptide showed significantly high reactivity (P < 0.05) against the sera of putatively immune individuals compared to the nonendemic control sera. It also showed high reactivity against the sera of patients with chronic pathology and patent infection. The high reactivity of the peptide with endemic immune sera equivalent to that of whole protein shows that it forms a dominant B epitope of TPx protein and thus could be utilized for incorporation into a multiepitope vaccine construct for filariasis.

Removal of amino acid, peptide and protein hydroperoxides by reaction with peroxiredoxins 2 and 3.

Prxs (peroxiredoxins) are a ubiquitous family of cysteine-dependent peroxidases that react rapidly with H2O2 and alkyl hydroperoxides and provide defence against these reactive oxidants. Hydroperoxides are also formed on amino acids and proteins during oxidative stress, and they too are a potential cause of biological damage. We have investigated whether Prxs react with amino acid, peptide and protein hydroperoxides, and whether the reactions are sufficiently rapid for these enzymes to provide antioxidant protection against these oxidants. Isolated Prx2, which is a cytosolic protein, and Prx3, which resides within mitochondria, were reacted with a selection of hydroperoxides generated by γ-radiolysis or singlet oxygen, on free amino acids, peptides and proteins. Reactions were followed by measuring the accumulation of disulfide-linked Prx dimers, via non-reducing SDS/PAGE, or the loss of the corresponding hydroperoxide, using quench-flow and LC (liquid chromatography)/MS. All the hydroperoxides induced rapid oxidation, with little difference in reactivity between Prx2 and Prx3. N-acetyl leucine hydroperoxides reacted with Prx2 with a rate constant of 4 × 10(4) M-1 · s-1. Hydroperoxides present on leucine, isoleucine or tyrosine reacted at a comparable rate, whereas histidine hydroperoxides were ~10-fold less reactive. Hydroperoxides present on lysozyme and BSA reacted with rate constants of ~100 M-1 · s-1. Addition of an uncharged derivative of leucine hydroperoxide to intact erythrocytes caused Prx2 oxidation with no concomitant loss in GSH, as did BSA hydroperoxide when added to concentrated erythrocyte lysate. Prxs are therefore favoured intracellular targets for peptide/protein hydroperoxides and have the potential to detoxify these species in vivo.

Molecular cloning and characterization of 1-Cys and 2-Cys peroxiredoxins from the bumblebee Bombus ignitus.

We cloned and characterized two peroxiredoxins (Prxs), BiPrx1 (a 1-Cys Prx) and BiTPx1 (a 2-Cys Prx) from the bumblebee Bombus ignitus. The BiPrx1 gene consists of 5 exons, encoding 220 amino acid residues with one conserved cysteine residue. The BiTPx1 gene consists of three exons, encoding 195 amino acid residues with 2 conserved cysteine residues. Recombinant BiPrx1 (27 kDa) and BiTPx1 (25 kDa), expressed in baculovirus-infected insect Sf9 cells, reduced H2O2 in the presence of electrons donated by dithiothreitol. Unlike BiTPx1, however, BiPrx1 did not show reduction activity when thioredoxin was used as the electron donor. Both BiPrx1 and BiTPx1 protected super-coiled DNA from damage by metal-catalyzed oxidation (MCO) in vitro. Tissue distribution analyses showed the presence of BiPrx1 and BiTPx1 in the fat body, midgut, muscle and epidermis, but not in the hemolymph, suggesting that BiPrx1 and BiTPx1 are not secretable. When H2O2 was injected into B. ignitus bees, BiPrx1 and BiTPx1 transcripts were acutely up-regulated in the fat body tissues. We also demonstrated the regulation of BiPrx1 and BiTPx1 expression via reduction of transcript levels in the fat body with RNA interference (RNAi). Under H2O2 overload, the RNAi-induced BiPrx1 knock-down B. ignitus worker bees showed up-regulated expression of BiTPx1. Reciprocally, BiTPx1 RNAi knockdowns showed up-regulated BiPrx1 expression in the fat body. These results indicate that the loss of expression of BiPrx1 or BiTPx1 is compensated by the up-regulation of expression of the other peroxidase in response to H2O2 overload.

Cloning, overexpression, purification, crystallization and preliminary X-ray diffraction analysis of an atypical two-cysteine peroxiredoxin (SAOUHSC_01822) from Staphylococcus aureus NCTC 8325.

An atypical two-cysteine peroxidase, SAOUHSC_01822, from the virulent Staphylococcus aureus strain NCTC 8325 plays a major role in the response of the bacterium to oxidative stress. The protein was cloned, expressed, purified to homogeneity and crystallized. The protein was crystallized from 2 M ammonium sulfate, 0.1 M Na HEPES pH 7, 2%(v/v) PEG 400. A complete diffraction data set was collected to 2.3 angstrom resolution using a Rigaku MicroMax HF007 Cu K alpha X-ray generator and a Rigaku R-AXIS IV(+)(+) detector. The crystals belonged to space group P2(1), with unit-cell parameters a = 43.50, b = 149.35, c = 73.73 angstrom, beta = 104.4 degrees, and contained four molecules in the asymmetric unit.

A comprehensive analysis of the peroxiredoxin reduction system in the Cyanobacterium Synechocystis sp. strain PCC 6803 reveals that all five peroxiredoxins are thioredoxin dependent.

Cyanobacteria perform oxygenic photosynthesis, which gives rise to the continuous production of reactive oxygen species, such as superoxide anion radicals and hydrogen peroxide, particularly under unfavorable growth conditions. Peroxiredoxins, which are present in both chloroplasts and cyanobacteria, constitute a class of thiol-dependent peroxidases capable of reducing hydrogen peroxide as well as alkyl hydroperoxides. Chloroplast peroxiredoxins have been studied extensively and have been found to use a variety of endogenous electron donors, such as thioredoxins, glutaredoxins, or cyclophilin, to sustain their activities. To date, however, the endogenous reduction systems for cyanobacterial peroxiredoxins have not been systematically studied. We have expressed and purified all five Synechocystis sp. strain PCC 6803 peroxiredoxins, which belong to the classes 1-Cys Prx, 2-Cys Prx, type II Prx (PrxII), and Prx Q, and we have examined their capacities to interact with and receive electrons from the m-, x-, and y-type thioredoxins from the same organism, which are called TrxA, TrxB, and TrxQ, respectively. Assays for peroxidase activity demonstrated that all five enzymes could use thioredoxins as electron donors, whereas glutathione and Synechocystis sp. strain PCC 6803 glutaredoxins were inefficient. The highest catalytic efficiency was obtained for the couple consisting of PrxII and TrxQ thioredoxin. Studies of transcript levels for the peroxiredoxins and thioredoxins under different stress conditions highlighted the similarity between the PrxII and TrxQ thioredoxin expression patterns.

Identification of the Vibrio vulnificus ahpCl gene and its influence on survival under oxidative stress and virulence.

Pathogens have evolved sophisticated mechanisms to survive oxidative stresses imposed by host defense systems, and the mechanisms are closely linked to their virulence. In the present study, ahpCl, a homologue of Escherichia coli ahpC encoding a peroxiredoxin, was identified among the Vibrio vulnificus genes specifically induced by exposure to H(2)O(2). In order to analyze the role of AhpCl in the pathogenesis of V. vulnificus, a mutant, in which the ahpCl gene was disrupted, was constructed by allelic exchanges. The ahpCl mutant was hypersusceptable to killing by reactive oxygen species (ROS) such as H(2)O(2) and t-BOOH, which is one of the most commonly used hydroperoxides in vitro. The purified AhpCl reduced H(2)O(2) in the presence of AhpF and NADH as a hydrogen donor, indicating that V. vulnificus AhpCl is a NADH-dependent peroxiredoxin and constitutes a peroxide reductase system with AhpF. Compared to wild type, the ahpCl mutant exhibited less cytotoxicity toward INT-407 epithelial cells in vitro and reduced virulence in a mouse model. In addition, the ahpCl mutant was significantly diminished in growth with INT-407 epithelial cells, reflecting that the ability of the mutant to grow, survive, and persist during infection is also impaired. Consequently, the combined results suggest that AhpCl and the capability of resistance to oxidative stresses contribute to the virulence of V. vulnificus by assuring growth and survival during infection.

The peroxidase and peroxynitrite reductase activity of human erythrocyte peroxiredoxin 2.

Peroxiredoxin 2 (Prx2) is a 2-Cys peroxiredoxin extremely abundant in the erythrocyte. The peroxidase activity was studied in a steady-state approach yielding an apparent K(M) of 2.4 microM for human thioredoxin and a very low K(M) for H2O2 (0.7 microM). Rate constants for the reaction of peroxidatic cysteine with the peroxide substrate, H2O2 or peroxynitrite, were determined by competition kinetics, k(2) = 1.0 x 10(8) and 1.4 x 10(7) M(-1) s(-1) at 25 degrees C and pH 7.4, respectively. Excess of both oxidants inactivated the enzyme by overoxidation and also tyrosine nitration and dityrosine were observed with peroxynitrite treatment. Prx2 associates into decamers (5 homodimers) and we estimated a dissociation constant K(d) < 10(-23) M(4) which confirms the enzyme exists as a decamer in vivo. Our kinetic results indicate Prx2 is a key antioxidant enzyme for the erythrocyte and reveal red blood cells as active oxidant scrubbers in the bloodstream.

Characterization of the antioxidant enzyme, thioredoxin peroxidase, from the carcinogenic human liver fluke, Opisthorchis viverrini.

The human liver fluke, Opisthorchis viverrini, induces inflammation of the hepatobiliary system. Despite being constantly exposed to inimical oxygen radicals released from inflammatory cells, the parasite survives for many years. The mechanisms by which it avoids oxidative damage are unknown. In this study, thioredoxin peroxidase (TPx), a member of the peroxiredoxin superfamily, was cloned from an O. viverrini cDNA library. O. viverrini TPx cDNA encoded a polypeptide of 212 amino acid residues, of molecular mass 23.57kDa. The putative amino acid sequence shared 60-70% identity with TPXs from other helminths and from mammals, and phylogenetic analysis revealed a close relationship between TPxs from O. viverrini and other trematodes. Recombinant O. viverrini TPx was expressed as soluble protein in Escherichia coli. The recombinant protein dimerized, and its antioxidant activity was deduced by observing protection of nicking of supercoiled plasmid DNA by hydroxyl radicals. Antiserum raised against O. viverrini TPx recognized native proteins from egg, metacercaria and adult developmental stages of the liver fluke and excretory-secretory products released by adult O. viverrini. Immunolocalization studies revealed ubiquitous expression of TPx in O. viverrini organs and tissues. TPx was also detected in bile fluid and bile duct epithelial cells surrounding the flukes 2 weeks after infection of hamsters with O. viverrini. In addition, TPx was observed in the secondary (small) bile ducts where flukes cannot reach due to their large size. These results suggested that O. viverrini TPx plays a significant role in protecting the parasite against damage induced by reactive oxygen species from inflammation.

Proteome analysis of aflatoxin B1-induced hepatocarcinogenesis in tree shrew (Tupaia belangeri chinensis) and functional identification of candidate protein peroxiredoxin II.

In order to explore the proteins responsible for hepatocellular carcinoma (HCC), aflatoxin B(1)-induced hepatocarcinogenesis in tree shrew (Tupaia belangeri chinensis) was analyzed with 2-DE and MS. By comparing HCC samples with their own precancerous biopsies and HCC-surrounding tissues, a group of candidate proteins that differentially expressed in HCC were obtained. Peroxiredoxin (Prx) II, one of the candidates with distinct alteration, was further investigated and validated. Western blot and RT-PCR assays confirmed the overexpression of Prx II in both tree shrew and human HCC tissues. RNA interference for silencing Prx II was employed subsequently to explore the function and underlying mechanism of Prx II on liver cancer cell line Hep3B. Results showed the cell proliferation and clone formation decreased obviously when Prx II expression was inhibited, while the flow cytometer analysis showed the percentage of cell apoptosis enhanced. Inhibition of Prx II expression also obviously increased the generation of ROS and malondialdehyde, both are the products from peroxidation. These results imply the important role of Prx II in hepatocarcinogenesis, possibly through its function in regulating peroxidation and hereby to provide a favorable microenvironment for cancer cell surviving and progressing.

Identification of candidate prostate cancer biomarkers in prostate needle biopsy specimens using proteomic analysis.

Although serum prostate specific antigen (PSA) is a well-established diagnostic tool for prostate cancer (PCa) detection, the definitive diagnosis of PCa is based on the information contained in prostate needle biopsy (PNBX) specimens. To define the proteomic features of PNBX specimens to identify candidate biomarkers for PCa, PNBX specimens from patients with PCa or benign prostatic hyperplasia (BPH) were subjected to comparative proteomic analysis. 2-DE revealed that 52 protein spots exhibited statistically significantly changes among PCa and BPH groups. Interesting spots were identified by MALDI-TOF-MS/MS. The 2 most notable groups of proteins identified included latent androgen receptor coregulators [FLNA(7-15) and FKBP4] and enzymes involved in mitochondrial fatty acid beta-oxidation (DCI and ECHS1). An imbalance in the expression of peroxiredoxin subtypes was noted in PCa specimens. Furthermore, different post-translationally modified isoforms of HSP27 and HSP70.1 were identified. Importantly, changes in FLNA(7-15), FKBP4, and PRDX4 expression were confirmed by immunoblot analyses. Our results suggest that a proteomics-based approach is useful for developing a more complete picture of the protein profile of PNBX specimen. The proteins identified by this approach may be useful molecular targets for PCa diagnostics and therapeutics.