Thioredoxin Family Research Articles

P2556TXNDC5 is a novel therapeutic target of atrial fibrosis and fibrillation

Abstract Background Atrial fibrillation (AF), one of the most common cardiac arrhythmias, increases the risk of stroke, systemic embolization and cardiovascular mortality. Atrial fibrosis, a hallmark of chronic AF, provides substrates to initiate/propagate fibrillation waves in the atria. There, however, lacks effective and specific therapeutics targeting atrial fibrosis. We have recently identified an endoplasmic reticulum (ER) protein thioredoxin domain containing 5 (TXNDC5) as a critical mediator of cardiac ventricular fibrosis. We hypothesized that TXNDC5 could also play an important role in the pathogenesis of atrial fibrosis and fibrillation. Purpose To determine the role of TXNDC5 in atrial fibrosis and fibrillation. Methods and results TXNDC5 transcript and protein levels were both significantly upregulated in the atrial tissue from patients with AF. In addition, TXNDC5 mRNA expression levels were positively correlated with those of transcripts encoding transforming growth factor β1 (TGFβ1) and extracellular matrix (ECM) proteins in human atrial tissue. Knockdown of TXNDC5 in human atrial fibroblasts (hAF) attenuated TGFβ1–induced hAF activation, proliferation and ECM protein upregulation, whereas overexpression of TXNDC5 was sufficient to trigger hAF activation, proliferation and ECM protein production. Further experiments revealed that the fibrogenic effects of TXNDC5 were dependent on c-Jun N-terminal kinase (JNK) signaling. Furthermore, using α-MHC-TGFβcys33ser mice, a transgenic mouse model with cardiac-specific overexpression of constitutively active TGFβ, which develop extensive atrial fibrosis and inducible AF, we showed that TXNDC5 was strongly upregulated in the fibrotic atria of α-MHC-TGFβcys33ser mice and specifically enriched in collagen-secreting atrial fibroblasts. Targeted deletion of TXNDC5 (Txndc5−/−) in α-MHC-TGFβcys33ser mice considerably mitigated the extent of atrial fibrosis. In addition, transesophageal atrial burst pacing induced AF in 75% (3 out of 4) α-MHC-TGFβcys33ser mice, whereas knockout of Txndc5 markedly reduced the inducibility of AF (25%, 3 out of 12) in α-MHC-TGFβcys33ser mice (Figure). TXNDC5 KO Reduces AF Inducibility Conclusion The present study revealed that ER protein TXNDC5 augments atrial fibrosis by promoting cardiac fibroblast proliferation and ECM protein production via JNK signaling activation. Targeted deletion of Txndc5 protects against TGFβ induced atrial fibrosis and AF. Targeting TXNDC5, therefore, could be a promising new therapeutic approach to treat or prevent atrial fibrosis and AF.

Intracellular free radical scavenging activity and protective role of mammalian cells by antioxidant peptide from thioredoxin disulfide reductase of Arthrospira platensis

Abstract In this study, we have analyzed the antioxidant role of thioredoxin disulfide reductase (TR) identified from Arthrospira platensis (Ap). A short peptide (VH12) derived from thioredoxin domain between Val368 and His379 of ApTR and it named as VH12. The peptide expressed the antioxidant activity against hydroxyl radical and superoxide radical at the concentration of 6.25 µM. Cytotoxic activity was performed on human leucocytes which showed that VH12 is not cytotoxic to any of the cellular population of human leucocytes. In addition, we determined the intracellular ROS level in cardiac myocytes to know the ROS scavenging level of VH12 peptide; the level of ROS scavenging was nearly 55% by the peptide at a concentration of 12.5 µM. Finally, the ROS scavenging of the VH12 peptide was tested against H2O2 stress induced zebrafish embryo, in which the intracellular ROS was reduced by the peptide at a concentration of 20 µM.

The Edwardsiella piscicida thioredoxin-like protein inhibits ASK1-MAPKs signaling cascades to promote pathogenesis during infection.

It is important that bacterium can coordinately deliver several effectors into host cells to disturb the cellular progress during infection, however, the precise role of effectors in host cell cytosol remains to be resolved. In this study, we identified a new bacterial virulence effector from pathogenic Edwardsiella piscicida, which presents conserved crystal structure to thioredoxin family members and is defined as a thioredoxin-like protein (Trxlp). Unlike the classical bacterial thioredoxins, Trxlp can be translocated into host cells, mimicking endogenous thioredoxin to abrogate ASK1 homophilic interaction and phosphorylation, then suppressing the phosphorylation of downstream Erk1/2- and p38-MAPK signaling cascades. Moreover, Trxlp-mediated inhibition of ASK1-Erk/p38-MAPK axis promotes the pathogenesis of E. piscicida in zebrafish larvae infection model. Taken together, these data provide insights into the mechanism underlying the bacterial thioredoxin as a virulence effector in downmodulating the innate immune responses during E. piscicida infection.

Discovery and Qualification of Serum Protein Biomarker Candidates for Cholangiocarcinoma Diagnosis.

Cholangiocarcinoma (CCA) is a major health problem in northeastern Thailand. The majority of CCA cases are clinically silent and difficult to detect at an early stage. Although abdominal ultrasonography (US) can detect premalignant periductal fibrosis (PDF), this method is not suitable for screening populations in remote areas. With the goal of developing a blood test for detecting CCA in the at-risk population, we carried out serum protein biomarker discovery and qualification. Label-free shotgun proteomics was performed on depleted serum samples from 30 participants (n = 10 for US-normal, US-PDF, and CCA groups). Of 40 protein candidates selected using multiple reaction monitoring on 90 additional serum samples (n = 30 per group), 11 discriminatory proteins were obtained using supervised multivariate statistical analysis. We further evaluated 3 candidates using ELISA and immunohistochemistry (IHC). S100A9, thioredoxin (TRX), and cadherin-related family member 2 (CDHR2) were significantly different between CCA and normal, and CCA and PDF groups when measured in an additional 247 serum samples (P < 0.0001). By IHC, TRX and CDHR2 were detected in the cytoplasm and nucleus of CCA and inflammatory cells. S100A9 was detected in the infiltrating tumor stroma immune cells. Proteomics discovery and qualification in depleted sera revealed promising biomarker candidates for CCA diagnosis.

Molecular characterization and functional analysis of glutathione S-transferase kappa 1 (GSTκ1) from the big belly seahorse (Hippocampus abdominalis): Elucidation of its involvement in innate immune responses

Glutathione S-transferases (GSTs) are essential enzymes for the bioactivation of xenobiotics through the conjugation of the thiol group of glutathione (GSH). In this study, a kappa class of GST was identified from the big belly seahorse (Hippocampus abdominalis) (HaGSTκ1) and its biochemical and functional properties were analyzed. HaGSTκ1 has 231 amino acids encoded by a 696 bp open reading frame (ORF). The protein has a predicted molecular mass of 26.04 kDa and theoretical isoelectric point (pI) of 8.28. It comprised a thioredoxin domain, disulfide bond formation protein A (DsbA) general fold, and Ser15 catalytic site as well as GSH-binding and polypeptide-binding sites. Phylogenetic analysis revealed that HaGSTκ1 is closely clustered with the kappa class of GSTs from teleost fishes. The recombinant (rHaGSTκ1) protein exhibited activity toward 1-chloro-2,4-dinitrobenzene (CDNB), 4-nitrobenzyl (4-NBC), and 4-nitrophenethyl bromide (4-NPB) but not 1,2-dichloro-4-nitrobenzene (DCNB). The optimum pH and temperature were 8 and 30 °C, respectively, for the catalysis of CDNB and the universal substrate of GSTs. The rHaGSTκ1 activity was efficiently inhibited in the presence of Cibacron blue (CB) as compared with hematin. Most prominent expression of HaGSTκ1 was observed in the liver and kidney among the fourteen different tissues of normal seahorse. After challenge with lipopolysaccharide (LPS), polyinosinic-polycytidylic (poly I:C), gram-negative Edwardsiella tarda, and gram-positive Streptococcus iniae, HaGSTκ1 expression was significantly modulated in the liver and blood tissues. Altogether, our study proposes the plausible important role of HaGSTκ1 in innate immunity and detoxification of harmful xenobiotics.

PICOT (GLRX3) is a positive regulator of stress-induced DNA-damage response

Protein kinase C (PKC)-interacting cousin of thioredoxin (PICOT; also termed glutaredoxin 3 (Glrx3)) is a ubiquitously expressed protein that possesses an N-terminal monothiol thioredoxin (Trx) domain and two C-terminal tandem copies of a monothiol Glrx domain. It has an overall highly conserved amino acid sequence and is encoded by a unique gene, both in humans and mice, without having other functional gene homologs in the entire genome. Despite being discovered almost two decades ago, the biological function of PICOT remains largely ill-defined and its ramifications are underestimated considering the fact that PICOT-deficiency in mice results in embryonic lethality.Since classical Glrxs are important regulators of the cellular redox homeostasis, we tested whether PICOT participate in the stress-induced DNA-damage response, focusing on nuclear proteins that function as integral components of the DNA repair machinery. Using wild type versus PICOT-deficient (PICOT-KD) Jurkat T cells we found that the anti-oxidant mechanism in PICOT-deficient cells is impaired, and that these cells respond to genotoxic drugs, such as etoposide and camptothecin, by increased caspase-3 activity, a reduced survival and a slower and diminished phosphorylation of the histone protein, H2AX. Nevertheless, the effect of PICOT on the drug-induced phosphorylation of H2AX was independent of the cellular levels of reactive oxygen species. PICOT-deficient cells also demonstrated reduced and slower γH2AX foci formation in response to radiation. Furthermore, immunofluorescence staining using PICOT- and γH2AX-specific Abs followed by confocal microscopy demonstrated partial localization of PICOT at the γH2AX-containing foci at the site of the DNA double strand breaks. In addition, PICOT knockdown resulted in inhibition of phosphorylation of ATR, Chk1 and Chk2 kinases, which play an essential role in the DNA-damage response and serve as upstream regulators of γH2AX. The present data suggest that PICOT protects cells from DNA damage-inducing agents by operating as an upstream positive regulator of ATR-dependent signaling pathways. By promoting the activity of ATR, PICOT indirectly regulates the phosphorylation and activation of Chk1, Chk2, and γH2AX, which are critical components of the DNA damage repair mechanism and thereby attenuate the stress- and replication-induced genome instability.

Functional, Structural and Biochemical Features of Plant Serinyl-Glutathione Transferases.

Glutathione transferases (GSTs) belong to a ubiquitous multigenic family of enzymes involved in diverse biological processes including xenobiotic detoxification and secondary metabolism. A canonical GST is formed by two domains, the N-terminal one adopting a thioredoxin (TRX) fold and the C-terminal one an all-helical structure. The most recent genomic and phylogenetic analysis based on this domain organization allowed the classification of the GST family into 14 classes in terrestrial plants. These GSTs are further distinguished based on the presence of the ancestral cysteine (Cys-GSTs) present in TRX family proteins or on its substitution by a serine (Ser-GSTs). Cys-GSTs catalyze the reduction of dehydroascorbate and deglutathionylation reactions whereas Ser-GSTs catalyze glutathione conjugation reactions and eventually have peroxidase activity, both activities being important for stress tolerance or herbicide detoxification. Through non-catalytic, so-called ligandin properties, numerous plant GSTs also participate in the binding and transport of small heterocyclic ligands such as flavonoids including anthocyanins, and polyphenols. So far, this function has likely been underestimated compared to the other documented roles of GSTs. In this review, we compiled data concerning the known enzymatic and structural properties as well as the biochemical and physiological functions associated to plant GSTs having a conserved serine in their active site.

Evaluation of proteome profiles of Salix spp. pollen and relationship between glucose oxidase activity and pollen content in willow honey

Proteins from hand- and bee- collected pollen were isolated and separated in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis (2DE) system as well by direct gel-free mass spectrometry (MS) analysis. Total thirty six plant proteins with known functions and three uncharacterized were identified using mass spectrometry (MS) techniques from fourteen protein spots separated in 2D gel electrophoresis. Our study revealed two forms of profilin proteins with their slight different mass 14.19 kDa and 14.09 kDa and beta actin fragment was 33.25 kDa both in hand and bee collected pollen as well proteins of thioredoxin family with molecular weight - 47.29 kDa. The antioxidant enzyme glucose oxidase (GOX) activity was investigated in twelve samples of monofloral willow honey, having Salix spp. pollen from 44.7 % to 80.2 %. Data of paired regression analysis between Salix spp. pollen content and GOX activity show moderate and negative correlation coefficient (r= - 0.56).&#x0D; Keywords: willow pollen; honey; protein expression; separation; two-dimensional electrophoresis; mass spectrometry

The redox regulator sulfiredoxin forms a complex with thioredoxin domain–containing 5 protein in response to ER stress in lung cancer cells

Sulfiredoxin (Srx) reduces hyperoxidized 2-cysteine-containing peroxiredoxins (Prxs) and protects cells against oxidative stress. Previous studies have shown that Srx is highly expressed in primary specimens of lung cancer patients and plays a pivotal role in lung tumorigenesis and cancer progression. However, the oncogenic mechanisms of Srx in cancer are incompletely understood. In this study, we found that Srx knockdown sensitizes lung cancer cells to endoplasmic reticulum (ER) stress-induced cell death. Through MS analysis, we determined that Srx forms a complex with the ER-resident protein thioredoxin domain-containing protein 5 (TXNDC5). Using reciprocal co-immunoprecipitation, immunofluorescence imaging, subcellular fractionation, and domain-mapping assays with site-specific mutagenesis and purified recombinant proteins, we further characterized the Srx-TXNDC5 interaction. In response to ER stress but not to oxidative stress, Srx exhibits an increased association with TXNDC5, facilitating the retention of Srx in the ER. Of note, TXNDC5 knockdown in lung cancer cells inhibited cell proliferation and repressed anchorage-independent colony formation and migration, but increased cell invasion and activation of mitogen-activated protein kinases. Using immunohistochemical staining, we demonstrate that TXNDC5 is highly expressed in patient-derived lung cancer specimens. Bioinformatics analysis of publicly available data sets revealed that those with high Srx levels have significantly shorter survival and that those with high TXNDC5 levels have longer survival. We conclude that the cellular levels of Srx and TXNDC5 may be useful as biomarkers to predict the survival of individuals with lung cancer.

Structural Basis for YjbH Adaptor-Mediated Recognition of Transcription Factor Spx

YjbH is a bacterial adaptor protein required for efficient proteolysis of the RNA polymerase-binding transcription factor Spx by the ClpXP protease. We report the structure of YjbH in complex with Spx. YjbH comprises a DsbA-like thioredoxin domain connected via a linker to a C-terminal domain reminiscent of the winged helix-turn-helix fold. The interaction between YjbH and Spx involves a large surface area. Binding to YjbH stabilizes the C-terminal ClpX recognition region of Spx. We show that mutation of critical YjbH contact residues abrogates Spx recognition. Small-angle X-ray scattering and hydrogen-deuterium exchange mass spectrometry analyses determined the existence of a stable heterodimeric complex in solution and provide evidence that binding of Spx to YjbH reduces the overall conformational flexibility of Spx. Our findings provide insights into the molecular basis for Spx recognition and suggest a model for how YjbH stabilizes Spx and displays the C terminus of Spx for engagement by ClpXP.

Bacterial Glutathione S‐Transferases (GSTs) Involved in Breaking the β‐Aryl Ether Bond of Lignin Reveal Novel Catalytic Abilities and Reaction Mechanisms within the GST Superfamily

The glutathione S‐transferase (GST) protein superfamily contains thousands of known or predicted members, all of which contain an N‐terminal thioredoxin domain that binds a glutathione (GSH) molecule and activates it for catalysis. GSTs are important in many organisms for conjugating GSH to toxic molecules, making them more soluble and easier to excrete from cells. However, GSTs are also capable of catalyzing a wide and continually expanding range of other reactions using GSH as cofactor.Sphingomonad bacteria are capable of metabolizing low molecular weight oligomers and monomers derived from lignin, a heterogeneous polymer of aromatic subunits that is a major component of plant biomass. These bacteria and their enzymes have potential applications in converting naturally abundant lignin into valuable commodities. Sphingomonads use GSTs (called β‐etherases and GSH lyases) to catalyze two consecutive steps in their pathway for breaking the β‐aryl ether bond commonly found between aromatic subunits in the lignin polymer: β‐etherases stereospecifically break the β‐aryl ether bond by replacing it with a thioether bond involving GSH, and GSH lyases remove the GSH moiety from the resulting β‐thioether GSH conjugate.We have identified a heterodimeric β‐etherase (called BaeAB) that provides insight into the diversity of GSTs capable of acting as β‐etherases and into the evolution of the GST ability to break the β‐aryl ether bond. In addition, BaeAB has properties that are unique amongst characterized GSTs in general, including the majority of its catalytic activity apparently residing in only one of its subunits, and using an active site asparagine for catalysis (whereas most characterized GSTs use an amino acid residue with a side chain hydroxyl for catalysis).We have also identified a GSH lyase (called NaGSTNu), which has close homologues in many different organisms, though the physiological roles of these homologues are largely unknown in other organisms besides sphingomonads. We found that the two homologues from Escherichia coli were able to catalyze the GSH lyase reaction in the pathway for breaking the β‐aryl ether bond, despite the fact that E. coli is not known to be able to metabolize lignin‐derived oligomers. This suggests that GSH lyase activity may be a common feature of these homologues (and that they likely act on GSH‐conjugated compounds naturally found within their respective organisms). In addition, the reaction mechanism of NaGSTNu involves binding two GSH molecules (whereas most characterized GSTs can only bind one GSH), as well as amino acid residues located in a channel leading from the active site to the protein exterior.Altogether, besides helping to identify novel GSTs that could potentially be used to generate value from lignin, our work is helping to uncover unique features of the large and widely studied, but relatively uncharacterized, GST superfamily.Support or Funding InformationThis work was supported by U.S. Department of Energy (DOE) Great Lakes Bioenergy Research Center grants (DOE Office of Science BER DE‐FC02‐07ER64494 and DE‐SC0018409).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Protein Disulfide Isomerase Inhibits and Reverses Alpha‐Synuclein Fibrillization

Protein disulfide isomerase (PDI) is a ubiquitously expressed member of the thioredoxin family with related but independent oxidoreductase and chaperone properties. As a chaperone, PDI plays a crucial role in preventing the aggregation of misfolded proteins. Notably, PDI is known to inhibit alpha‐synuclein (SYN) fibrillization, a pathological hallmark of Parkinson's disease, but the mechanism by which PDI blocks the formation of SYN fibrils is unknown. Here, we report for the first time that PDI not only prevents SYN aggregation but is able to reverse aggregation both in vitro and in cultured cells. Assays using Thioflavin‐T fluorescence and transmission electron microscopy revealed that PDI is able to break down nascent but not mature fibrils. Furthermore, PDI was able to inhibit and reverse cellular fibrillization in HEK293T cells as assessed by protein‐fragment complementation and non‐reducing SDS‐PAGE. These data suggest a novel disaggregase function for PDI that disrupts the SYN fibrils most commonly found in Parkinson's disease. Current treatments for PD are limited to palliative care, but strategies focused on targeted therapeutic intervention are becoming popular. Thus, our work suggests the disaggregase property of PDI may lend itself to therapeutic development for Parkinson's disease.Support or Funding InformationThis work was supported, in part, by a LIFE at UCF Richard Tucker Gerontology Applied Research Award and a UCF College of Medicine competitive research grant.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Agr2-interacting Prod1-like protein Tfp4 from Xenopus laevis is necessary for early forebrain and eye development as well as for the tadpole appendage regeneration.

The Agr family genes, Ag1, Agr2, and Agr3, encode for the thioredoxin domain containing secreted proteins and are specific only for vertebrates. These proteins are attracting increasing attention due to their involvement in many physiological and pathological processes, including exocrine secretion, cancer, regeneration of the body appendages, and the early brain development. At the same time, the mode by which Agrs regulate intracellular processes are poorly understood. Despite that the receptor to Agr2, the membrane anchored protein Prod1, has been firstly discovered in Urodeles, and it has been shown to interact with Agr2 in the regenerating limb, no functional homologs of Prod1 were identified in other vertebrates. This raises the question of the mechanisms by which Agrs can regulate regeneration in other lower vertebrates. Recently, we have identified that Tfp4 (three-fingers Protein 4), the structural and functional homolog of Prod1 in Anurans, interacts with Agr2 in Xenopus laevis embryos. In the present work we show by several methods that the activity of Tfp4 is essential for the tadpole tail regeneration as well as for the early eye and forebrain development during embryogenesis. These data show for the first time the common molecular mechanism of regeneration regulation in amphibians by interaction of Prod1 and Agr2 proteins.

Molecular characterization, host defense mechanisms, and functional analysis of ERp44 from big-belly seahorse: A novel member of the teleost thioredoxin family present in the endoplasmic reticulum.

Endoplasmic reticulum resident protein 44 (ERp44) is a protein disulfide isomerase (PDI) and a member of thioredoxin family, which is involved in several functions such as oxidative folding and polymerization of molecules, carrier protein activity, regulation of the Ca2+ ion levels in the endoplasmic reticulum (ER), cellular signaling, and maintenance of lumen redox homeostasis. In this study, ERp44 from Hippocampus abdominalis, commonly known as the big belly seahorse, was characterized. ERp44 possessed three PDI-like domains and one thioredoxin fold with a CXXC conserved motif. The open reading frame consisted of 1233 bp encoding 410 amino acids. Additionally, it contained a C-terminal RDEL motif, which suggests a localization of ERp44 to the endoplasmic reticulum. ShERp44 showed highest mRNA expression in the ovary, brain, and gills. Temporal expression of ShERp44 in blood showed significant upregulation against bacterial, LPS, and PolyI:C stimuli at 24 and 72 h. Trunk kidney tissue exhibited upregulated ShERp44 expression at 24 h in response to lipopolysaccharides and Streptococcus iniae and at 72 h in response to Edwardsiella tarda and poly I:C. NADPH turnover was observed as 0.06122 ± 0.0075 μmol/s protein/μg through the HED assay. Insulin aggregation assay showed a significant reduction ability of rShERp44 by precipitating insulin rapidly, beginning at 5 min. Moreover, rShERp44-treated fathead minnow cells showed significant cell survival against 2-hydroxyethyl disulfide and thus exhibited capability to resist oxidative stress. Taken together, these findings provide insight into teleost defense mechanisms and functional properties of ERp44 in controlling redox homeostasis at the molecular level.

Profiling of apoptosis- and autophagy-associated molecules in human lung cancer A549 cells in response to cisplatin treatment using stable isotope labeling with amino acids in cell culture.

Cis‑diammine‑dichloro‑platinum II‑based adjuvant chemotherapy provides an alternative therapy to improve the survival of patients with lung tumors, especially those with non‑small cell lung cancer (NSCLC). However, drug resistance is a large clinical problem and its underlying mechanism remains unclear. In the present study, NSCLC A549 cells were treated with a low concentration of cisplatin in order to observe and determine the development of chemoresistance, via growth curves, colony forming assays and apoptosis assays. Then the induction of autophagy was examined in the cisplatin‑treated A549 cells with a fluorescence reporter. Profiled proteins in the cisplatin‑treated A549 cells were also assessed using the stable isotope labeling by amino acids in cell culture (SILAC) method, and then the differentially expressed molecules were verified. The results demonstrated that A549 cells became less sensitive to cisplatin [resistant A549 cells (A549R)] following 20passages in the medium containing a low concentration of cisplatin, with less apoptotic cells post‑cisplatin treatment. A549R cells grew more efficiently in the cisplatin medium, with more colony formation and more cells migrating across the baseline. In addition, NSCLC results demonstrated that more autophagy‑related proteins (ATGs) were expressed in the A549R cells. Furthermore, the western blotting results confirmed this upregulation of ATGs in A549R cells. In addition, two repeated SILAC screening experiments recognized 15proteins [glucose‑regulated protein, 78kDa (GRP78), heat shock protein71, pre‑mRNA processing factor19, polypyrimidine tract binding protein1, translationally controlled tumor protein, CathepsinD, Cytochromec, thioredoxin domain containing5, MutS homolog (MSH)6, AnnexinA2 (ANXA2), BRCA2 and Cyclin dependent kinase inhibitor1A interacting protein, MSH2, protein phosphatase2A 55kDa regulatory subunitBα, Rho glyceraldehyde‑3‑phosphate‑dissociation inhibitor1 and ANXA4] that were upregulated by >1.5‑fold in heavy(H)‑ and light(L)‑labeled A549R cells. In addition, 16 and 14proteins were downregulated by >1.5‑fold in the H‑ and L‑labeled A549R cells, respectively. The majority of the downregulated proteins were associated with apoptosis. In conclusion, the present study isolated a cisplatin‑resistant human lung cancer A549 cell clone, with reduced apoptosis and high levels of autophagy, in response to cisplatin treatment. In cisplatin‑resistant A549R cells, SILAC proteomics recognized the high expression of GRP78 and other proteins that are associated with anti‑apoptosis and/or autophagy promotion.

Characterization of a protozoan Phosducin-like protein-3 (PhLP-3) reveals conserved redox activity.

We recently identified three novel thioredoxin-like genes in the genome of the protozoan parasite Plasmodium that belong to the Phosducin-like family of proteins (PhLP). PhLPs are small cytosolic proteins hypothesized to function in G-protein signaling and protein folding. Although PhLPs are highly conserved in eukaryotes from yeast to mammals, only a few representatives have been experimentally characterized to date. In addition, while PhLPs contain a thioredoxin domain, they lack a CXXC motif, a strong indicator for redox activity, and it is unclear whether members of the PhLP family are enzymatically active. Here, we describe PbPhLP-3 as the first phosducin-like protein of a protozoan organism, Plasmodium berghei. Initial transcription analysis revealed continuous low-level expression of pbphlp-3 throughout the complex Plasmodium life cycle. Attempts to knockout pbphlp-3 in P. berghei did not yield live parasites, suggesting an essential role for the gene in Plasmodium. We cloned, expressed and purified PbPhLP-3 and determined that the recombinant protein is redox active in vitro in a thioredoxin-coupled redox assay. It also has the capacity to reduce the organic compound tert-Butyl hydroperoxide (TBHP) in vitro, albeit at low efficiency. Sequence analysis, structural modeling, and site-directed mutagenesis revealed a conserved cysteine in the thioredoxin domain to be the redox active residue. Lastly, we provide evidence that recombinant human PhLP-3 exhibits redox activity similar to that of PbPhLP-3 and suggest that redox activity may be conserved in PhLP-3 homologs of other species. Our data provide new insight into the function of PhLP-3, which is hypothesized to act as co-chaperones in the folding and regulation of cytoskeletal proteins. We discuss the potential implications of PhLP-3 as a thioredoxin-target protein and possible links between the cellular redox network and the eukaryotic protein folding machinery.

Bacterial expression, purification and biophysical characterization of wheat germ agglutinin and its four hevein-like domains.

Wheat germ agglutinin (WGA), a chitin binding lectin, has attracted increasing interest because of its unique characteristics such as conformational stability, binding specificity and transcytosis capacity. To pave the way for the study of the molecular basis of WGA's structural stability and binding capacity, as well as to facilitate its use in biomedical and biotechnological developments, we produced recombinant WGA and its 4 isolated hevein-like domains in a bacterial system. All the proteins were expressed as fusion constructs linked to a thioredoxin domain, which was enzymatically or chemically released. The structural and ligand-binding properties of recombinant WGA were similar to the wild lectin. The 4 isolated domains folded and were ligand-binding competent, indicating that each domain constitutes an independent folding unity. The biophysical characterization of the recombinant domains sheds new light on the intricate folding and binding behavior of this emblematic lectin.

Structural and mechanistic aspects of S-S bonds in the thioredoxin-like family of proteins.

Disulfide bonds play a critical role in a variety of structural and mechanistic processes associated with proteins inside the cells and in the extracellular environment. The thioredoxin family of proteins like thioredoxin (Trx), glutaredoxin (Grx) and protein disulfide isomerase, are involved in the formation, transfer or isomerization of disulfide bonds through a characteristic thiol-disulfide exchange reaction. Here, we review the structural and mechanistic determinants behind the thiol-disulfide exchange reactions for the different enzyme types within this family, rationalizing the known experimental data in light of the results from computational studies. The analysis sheds new atomic-level insight into the structural and mechanistic variations that characterize the different enzymes in the family, helping to explain the associated functional diversity. Furthermore, we review here a pattern of stabilization/destabilization of the conserved active-site cysteine residues presented beforehand, which is fully consistent with the observed roles played by the thioredoxin family of enzymes.

TXNDC9 promotes hepatocellular carcinoma progression by positive regulation of MYC-mediated transcriptional network

The thioredoxin domain containing proteins are a group of proteins involved in redox regulation and have been recently reported to be associated with tumor progression. However, the role of thioredoxin proteins in hepatocellular carcinoma (HCC) remains largely unknown. Here in our study, we demonstrated that thioredoxin domain containing protein 9 (TXNDC9) was over-expressed in HCC and promoted HCC progression. We found that TXNDC9 expression was amplified in HCC tissues and associated with an advanced grade of HCC. And, we demonstrated that overexpression of TXNDC9 was correlated with poor prognosis of HCC. Furthermore, by using CRISPR-Cas9 mediated TXNDC9 knockout and RNA-seq analysis, we found that TXNDC9 accelerated HCC proliferation regulation. Moreover, we demonstrated that TXNDC9 directly interacted with MYC and knockout/knockdown of TXNDC9 decreased the protein levels of MYC and inhibited MYC-mediated transcriptional activation of its targets. Besides, we identified that TXNDC9 was trans-activated by FOXA1, JUND, and FOSL2 in HCC. Taken together, our study unveiled an oncogenic role of TXNDC9 in HCC and provided a mechanistic insight into the TXNDC9 mediated gene regulation network during HCC development.

Stress-immune responses and DNA protection function of thioredoxin domain containing 12 in zebrafish (Danio rerio)

Proteins with dithiol-disulfide oxidoreductase catalytic domain are well known for their capacity in the cellular redox homeostasis. In this study, we characterized the zebrafish thioredoxin domain containing 12 (Zftxndc12) gene, analyzed the transcriptional responses and studied the functional properties of its recombinant protein. Full-length cDNA of Zftxndc12 consists 519 bp coding region encoding 172 amino acids (AA) including the signal peptide. Highly consensus active motif (65WCGAC69) and probable ER retrieval motif (169GDEL172) were identified. Ubiquitous expression of Zftxndc12 mRNA was observed from one cell to juvenile stage as well as different organs of adult zebrafish. Moreover, whole mount in situ hybridization (WISH) results showed a higher expression of Zftxndc12 in primordial gills, central nerves system and eye. The tissue specific expression analysis (by qRT-PCR) also showed the highest expression in gills followed by brain in adult zebrafish. In larvae, up-regulated Zftxndc12 mRNA expression upon exposure to H2O2,Edwardsiella tarda and Saprolegnia parasitica suggests that it may involve in both stress and immune responses. Moreover, transcriptional expression of Zftxndc12 was up-regulated upon Streptococcus iniae challenge in gills of adult zebrafish. The recombinant ZfTxndc12 (rZfTxndc12) was overexpressed, purified and tested for its biological activities. Results revealed that rZfTxndc12 is able to reduce the DNA damage and detoxify the H2O2 toxicity in concentration dependent manner. Overall results suggest that Zftxndc12 is important antioxidant and immune functional member of the host defense system in zebrafish.