Protein Disulfide Research Articles

A novel disulfidptosis-related prognostic gene signature and experimental validation identify ACTN4 as a novel therapeutic target in lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is a prevalent form of malignancy globally. Disulfidptosis is novel programmed cell death pathway based on disulfide proteins, may have a positive impact on the development of LUAD treatment strategies. To investigate the impact of disulfidptosis-related genes (DRGs) on the prognosis of LUAD, developed a risk model to facilitate the diagnosis and prognostication of patients. We also explored ACTN4 (DRGs) as a new therapeutic biomarker for LUAD. We investigated the expression patterns of DRGs in both LUAD and noncancerous tissues. To assess the prognostic value of the DRGs, we developed risk models through univariate Cox analysis and lasso regression. The expression and function of ACTN4 was evaluated by qRT-PCR, immunohistochemistry and in vitro experiments. The TIMER examined the association between ACTN4 expression and immune infiltration in LUAD. Ten differentially expressed DRGs were identified. And ACTN4 was identified as potential risk factors through univariate Cox regression analysis (P< 0.05). ACTN4 expression and riskscore were used to construct a risk model to predict overall survival in LUAD, and high-risk demonstrated a significantly higher mortality rate compared to the low-risk cohort. qRT-PCR and immunohistochemistry assays indicated ACTN4 was upregulated in LUAD, and the upregulation was associated with clinicopathologic features. In vitro experiments showed the knockdown of ACTN4 expression inhibited the proliferation in LUAD cells. The TIMER analysis demonstrated a correlation between the expression of ACTN4 and the infiltration of diverse immune cells. Elevated ACTN4 expression was associated with a reduction in memory B cell count. Additionally, the ACTN4 expression was associated with m6A modification genes. Our study introduced a prognostic model based on DRGs, which could forecast the prognosis of patients with LUAD. The biomarker ACTN4 exhibits promise for the diagnosis and management of LUAD, given its correlation with tumor immune infiltration and m6A modification.

Pan-cancer genetic analysis of disulfidptosis-related gene set

BackgroundA recent study has identified a novel programmed cell death pathway, termed disulfidptosis, which is based on disulfide proteins. This discovery provides new insight into the mechanisms of cell death and may have implications for therapeutic strategies targeting cell death pathways. This study aimed to evaluate the pan-cancer genomics and clinical association of disulfidptosis and disulfidptosis-related cell death genes, including SLC7A11, INF2, CD2AP, PDLIM1, ACTN4, MYH9, MYH10, IQGAP1, FLNA, FLNB, TLN1, MYL6, ACTB, DSTN, and CAPZB. MethodsUsing multi-omics profiling data, this study provides a comprehensive and systematic characterization of disulfidptosis genes across more than 9000 samples of over 30 types of cancer. ResultsFLNA and FLNB were the two most frequently mutated disulfidptosis cell death genes in cancer. UCEC and SKCM were the two cancer types that have the highest mutation rates while the mutation of ACTN4 was associated with worse survival of CESC and ESCA. Breast cancer was potentially affected by disulfidptosis because its subtypes are different in disulfidptosis gene expression. Similarly, KIRC might also be associated with disulfidptosis. Additionally, the association of disulfidptosis-related cell death genes with survival was analyzed, with MESO and LGG as the top cancer types with survival associated with disulfidptosis cell death genes. The correlation between CNV and survival across multiple cancer types found that UCEC, KIRP, LGG, and KIRC were the top cancer types where the CNV level was associated with survival. There was a negative correlation between expression and methylation for most of the genes and there was only a slight correlation between methylation levels and survival of cancer in LGG. About half of the disulfidptosis-related cell death proteins were associated with the activation of EMT. Disulfidptosis genes were correlated to immune cell infiltration levels in cancers. Multiple compounds were identified as potential drugs that might be affected by disulfidptosis-related cell death for future study. ConclusionDisulfidptosis cell death genes are potentially involved in many cancer types and can be developed as candidates for cancer diagnosis, prognosis, and therapeutic biomarkers.

Cysteinyl and methionyl redox switches: Structural prerequisites and consequences

Redox modifications of specific cysteinyl and methionyl residues regulate key enzymes and signal-transducing proteins in various pathways. Here, we analyzed the effect of redox modifications on protein structure screening the RCSB protein data bank for oxidative modifications of proteins, i.e. protein disulfides, mixed disulfides with glutathione, cysteinyl sulfenic acids, cysteinyl S-nitrosylation, and methionyl sulfoxide residues. When available, these structures were compared to the structures of the same proteins in the reduced state with respect to both pre-requirements for the oxidative modifications as well as the structural consequences of the modifications. In general, the conformational changes induced by the redox modification are small, i.e. within the range of normal fluctuations. Some redox modifications, disulfides in particular, induces alterations in the electrostatic properties of the proteins. Solvent accessibility does not seem to be a strict pre-requirement for the redox modification of a particular residue. We identified an enrichment of certain other amino acid residues in the vicinity of the susceptible residues, for disulfide and sulfenic acid modifications, for instance, histidyl and tyrosyl residues. These motifs, as well as the specific features of the susceptible sulfur-containing amino acids, may become helpful for the prediction of redox modifications.

Topological twists in nature: Proteins with a pierced lasso topology.

The correlation between structural motifs and their 3D-structure has previously been established to influence biology. However, less is known about the biological implications of protein topology, i.e., motifs that can act as a structural switch in response to environmental changes. To answer biological questions regarding proteins subject to topological constraints that can be controlled by the chemical environment, we study proteins with a pierced lasso topology (PLT) involved in cell regulation, growth, and cell migration. PLTs are threaded “knot-like” topologies, where part of the chain pierces through a covalent loop. Interestingly, PLTs exist in 18% of all disulfide-containing proteins found in all kingdoms of life. PLTs can further be divided into 14 different classes according to their mechanism of action. They are mainly found in the extracellular matrix and organelles with an oxidizing environment. This discovery of PLTs emphasizes the topological importance of loops and links for biological activity, adding a new putative role of disulfides in proteins. Despite this large number, a connection between topology and biological function has not yet been determined. Our results show that the threaded topology initiates conformational dynamics affecting protein-protein interaction and biological function controlled by the chemical environment. Therefore, we associate the threaded disulfide-linked topology with biological function. Furthermore, we hypothesize that the introduction of PLTs in proteins may not be a stochastic event in evolution but represent a new role of disulfides in vivo.

DMSO-Induced Unfolding of the Antifungal Disulfide Protein PAF and Its Inactive Variant: A Combined NMR and DSC Study.

PAF and related antifungal proteins are promising antimicrobial agents. They have highly stable folds around room temperature due to the presence of 3-4 disulfide bonds. However, unfolded states persist and contribute to the thermal equilibrium in aqueous solution, and low-populated states might influence their biological impact. To explore such equilibria during dimethyl sulfoxide (DMSO)-induced chemical unfolding, we studied PAF and its inactive variant PAFD19S using nuclear magnetic resonance (NMR) and differential scanning calorimetry (DSC). According to the NMR monitoring at 310 K, the folded structures disappear above 80 v/v% DMSO concentration, while the unfolding is completely reversible. Evaluation of a few resolved peaks from viscosity-compensated 15N-1H HSQC spectra of PAF yielded ∆G = 23 ± 7 kJ/M as the average value for NMR unfolding enthalpy. The NMR-based structures of PAF and the mutant in 50 v/v% DMSO/H2O mixtures were more similar in the mixed solvents then they were in water. The 15N NMR relaxation dynamics in the same mixtures verified the rigid backbones of the NMR-visible fractions of the proteins; still, enhanced dynamics around the termini and some loops were observed. DSC monitoring of the Tm melting point showed parabolic dependence on the DMSO molar fraction and suggested that PAF is more stable than the inactive PAFD19S. The DSC experiments were irreversible due to the applied broad temperature range, but still suggestive of the endothermic unfolding of PAF.

Looking for the mechanism of arsenate respiration of Fusibacter sp. strain 3D3, independent of ArrAB.

The literature has reported the isolation of arsenate-dependent growing microorganisms which lack a canonical homolog for respiratory arsenate reductase, ArrAB. We recently isolated an arsenate-dependent growing bacterium from volcanic arsenic-bearing environments in Northern Chile, Fusibacter sp. strain 3D3 (Fas) and studied the arsenic metabolism in this Gram-positive isolate. Features of Fas deduced from genome analysis and comparative analysis with other arsenate-reducing microorganisms revealed the lack of ArrAB coding genes and the occurrence of two arsC genes encoding for putative cytoplasmic arsenate reductases named ArsC-1 and ArsC-2. Interestingly, ArsC-1 and ArsC-2 belong to the thioredoxin-coupled family (because of the redox-active disulfide protein used as reductant), but they conferred differential arsenate resistance to the E. coli WC3110 ΔarsC strain. PCR experiments confirmed the absence of arrAB genes and results obtained using uncouplers revealed that Fas growth is linked to the proton gradient. In addition, Fas harbors ferredoxin-NAD+ oxidoreductase (Rnf) and electron transfer flavoprotein (etf) coding genes. These are key molecular markers of a recently discovered flavin-based electron bifurcation mechanism involved in energy conservation, mainly in anaerobic metabolisms regulated by the cellular redox state and mostly associated with cytoplasmic enzyme complexes. At least three electron-bifurcating flavoenzyme complexes were evidenced in Fas, some of them shared in conserved genomic regions by other members of the Fusibacter genus. These physiological and genomic findings permit us to hypothesize the existence of an uncharacterized arsenate-dependent growth metabolism regulated by the cellular redox state in the Fusibacter genus.

N-Acetylcysteine Inhibits Platelet Function through the Regeneration of the Non-Oxidative Form of Albumin.

N-acetylcysteine (NAC) is able to break down protein disulfides, generating free thiols. This mechanism occurs on mixed disulfides of albumin (HSA) to form mercaptoalbumin (HMA), the main antioxidant species in the plasma. Circulating HSA exists in two main forms: the reduced form (HMA), and the oxidized forms, whose predominant modification is cystenylation (HSA-Cys). Increased levels of oxidized HSA have been detected in several diseases associated with oxidative stress. This study showed that NAC inhibits platelet aggregation by restoring HMA. In addition, the regeneration of HMA by NAC inhibits platelet functions such as intracellular calcium mobilization, reactive oxygen species generation, arachidonic acid metabolites synthesis, and adhesion to the collagen matrix. In our conditions, the exposure of platelets to NAC did not increase GSH levels. However, the inhibition of platelet aggregation was also detected following treatment of platelet-rich plasma with GSH, which, similarly to NAC, reduced HSA-Cys levels. Furthermore, this study showed that cysteine, another compound able to restore HMA by reducing the HSA-Cys content, inhibited platelet aggregation to a similar extent as NAC. The results obtained in this study suggest a new mechanism by which NAC can modulate platelet activation and suggest its possible use as an antiplatelet drug in conditions associated with oxidative stress.

Ligand-induced transition in conformations of vicinal cysteine disulfides in proteins.

Vicinal cysteine disulfides are thought to be associated with specific conformations of cysteine disulfides due to the restricted rotation of single bonds in an eight-membered cyclic disulfide loop. Conformations of vicinal cysteine disulfides are analyzed using χ1 , χ2 , χ3 , χ2 ', χ1 ' torsion angles in the crystal structures of proteins retrieved from Protein Data Bank (PDB). 85% of vicinal disulfides have (+, -)LHStaple conformation with trans configuration of the peptide bond and 9% have (-, -)RHStaple conformation with cis configured peptide bond. Conformational analysis of dipeptide Cys-Cys vicinal disulfide by density functional theory (DFT) further supported (+, -)LHStaple, (-, -)RHStaple, and (+, +)RHStaple as the preferred conformations of vicinal disulfides. Interestingly, the rare conformations of vicinal disulfides are observed in the ligand-bound forms of proteins and have higher disulfide strain energy. Conformations of vicinal disulfides in palmitoyl protein thioesterase 1, AChBP, and α7 nicotinic receptor are changed from preferred (+, -)LHStaple to rare (+, -)AntiLHHook/(+, -)AntiRHHook/(+, +)RHStaple conformation due to binding of ligands. Surprisingly, ligands are proximal to the vicinal disulfides in protein complexes that exhibited rare conformations of vicinal disulfides. The report has identified (+, -) LHStaple/(-, -) RHStaple as the hallmark conformations of vicinal disulfides and unraveled ligand-induced transition in conformations of vicinal cysteine disulfides in proteins.

Fluorescent Probes for Imaging Protein Disulfides in Live Organisms.

Cellular redox homeostasis is predominantly controlled by the ratio of thiols and disulfides, and reversible thiol-disulfide exchange reactions are fundamental of the biological redox regulation. However, due to the dynamic exchanges of thiols and disulfides, the detection, especially the in situ detection, of protein disulfides (PDS) is challenging. We employ the strategy, i.e., the increase of emission upon an environment-sensitive dye binding to proteins, to design PDS probes and discover a two-photon probe PDSTP590 (S6) that selectively recognizes PDS in live organisms. With the aid of the probe, we further disclose the elevation of PDS in brains of the mouse stroke model.

Mechanochemical disulfide reduction reveals imprints of noncovalent sulfuroxygen chalcogen bonds in protein-inspired mimics in aqueous solution.

The surprisingly rich chemistry of mechanically activated cleavage of disulfide bonds has been uncovered only recently. Using a disulfide protein mimic together with Cleland's reagent (DTT) as the attacking nucleophile in aqueous solution, our isotensional ab initio simulations add another surprise to the list. They unveil that noncovalent chalcogen-chalcogen 1,5-type SO interactions involving the S-S bridge and γ-carbonyl O are controlling the mechanochemical reactivity of disulfides at very low forces, thus adding a third reactivity regime to the hitherto known ones. In stark contrast to what is found in aqueous solution, no such chalcogen bonding arrangements are observed in the gas phase, which supports the conclusion that water plays a crucial role in stabilizing preferred conformations that support noncovalent SO bonds. These findings open the door to investigate chalcogen bonding in the realm of proteins using single-molecule force spectroscopy.

Thioredoxin and protein-disulfide isomerase selectivity for redox regulation of proteins in Corynebacterium glutamicum.

Thioredoxins (Trxs) and protein-disulfide isomerases (PDIs) are believed to play a pivotal role in ensuring the proper folding of proteins, facilitating appropriate functioning of proteins, and maintaining intracellular redox homeostasis in bacteria. Two thioredoxins (Trxs) and three thiol-disulfide isomerases (PDIs) have been annotated in Corynebacterium glutamicum. However, nothing is known about their functional diversity in the redox regulation of proteins. Thus, we here analyzed the Trx- and PDI-dependent redox shifts of ribonucleotide reductase (RNR), insulin, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), and several thiol-dependent peroxidases by measuring enzyme activity and thiol status in vitro. We found that the two Trxs and the three PDIs had activities in the cleavage of the disulfidebond, whereas the PDIs had a lower efficiency than the two Trxs. Trx2 could activate thiol-dependent peroxidases with an efficiency comparable with that of Trx1, but the PDIs were inefficient. The redox-active Cys-X-X-Cys motif harbored in both Trxs and PDIs was essential to supply efficiently the donor of reducing equivalents for protein disulfides. In addition, stress-responsive extracytoplasmic function (ECF)-sigma factor H (SigH)-dependent Trxs and PDIs expressions were observed. These results contributed importantly to our overall understanding of reducing functionality of the Trx and PDI systems, and also highlighted the complexity and plasticity of the intracellular redox network.

Inhibitory effect of protein Y3 from Coprinus comatus on tobacco mosaic virus

The antiviral protein Y3 produced by the edible mushroom Coprinus comatus disrupts the tobacco mosaic virus (TMV) and inhibits the multiplication of TMV in Nicotiana tabacum; however, the detailed mechanism of its activity remains unclear. In this study, Y3 was demonstrated to interact with TMV coat protein (TMV-CP) in vitro as well as in tobacco plants by using a yeast two-hybrid system and bimolecular fluorescence complementation (BiFC). To explore the interaction site between Y3 and TMV-CP, the phenylalanine (Phe) at the 43rd and arginine (Arg) at the 55th amino acid of Y3 were individually converted to cysteine (Cys) and serine (Ser), named Y3T1 and Y3T2, respectively, and were then used in BiFC assays. Based on the information obtained about disulfide bonds in the protein structure, the two mutations were predicted to change the protein's disulfide bonds. The results showed Y3T1 lost the ability to interact with TMV-CP, suggesting that a specific Phe of Y3 is critical for the interaction between Y3 and CP in plants. Furthermore, a prokaryotic expression system was used to test the antiviral activities of protein Y3 (PY3) and two other mutated proteins (P-Y3T1, P-Y3T2). The results showed that recombinant protein P-Y3 had a slightly lower inhibitory effect against TMV than Y3 extracted directly from mushrooms; further, P-Y3T1 decreased antiviral activity in the tobacco plant significantly compared with P-Y3, suggesting that the anti-TMV effect of Y3 was directly related to the Y3-CP interaction. In contrast, P-Y3T2 was able to still interact with TMV-CP in the tobacco plant, and it increased the ability of the plant to resist TMV compared with PY3, indicating that PY3-T2 could be a candidate peptide for plant protection against TMV and that Y3 may have other inhibitory mechanisms against TMV in addition to its interaction with TMV-CP.

The Effect of Acid Treatment and Freeze-Thaw Cycle on Textural Properties and Microstructure of Cooked Aged Rice

This research aims to improve frozen cooked aged rice's textural properties using lime juice, citric acid, and ascorbic acid solutions. Aged (12 months) rice is cooked and freeze-thawed up to 3 cycles. Cooked fresh rice (0.7 months) is used as a reference. Addition of acid significantly decreases hardness but significantly increases stickiness for freeze-thawed, cooked, aged rice (p ≤ 0.05). Scanning electron micrographs show a rough surface on cooked rice after repeated freeze-thaw cycles, especially for cooked aged rice. However, the microstructure of acid treated cooked aged rice has a smoother surface than untreated. These results correlate with decreased starch retrogradation in acid treated cooked aged rice. Gel electrophoresis shows disulfide bond formation in aged rice protein which restricted granular swelling and gelatinization. Addition of acid did not cleave aged rice protein's disulfide bonds but reduces protein content. These results demonstrate addition of acid improves frozen cooked aged rice's texture.

Formation of Protein Disulfide Bonds Catalyzed by OsPDIL1;1 is Mediated by MicroRNA5144-3p in Rice.

Correct folding of proteins in the endoplasmic reticulum is important for their stability and function under stress. The protein disulfide isomerase (PDI) OsPDIL1;1 is a key protein-folding catalyst in rice (Oryza sativa L.). Here, microRNA5144 (osa-miR5144-3p) is reported to mediate the formation of protein disulfide bonds via targeting OsPDIL1;1 mRNA in rice seeds and seedlings during development and under conditions of abiotic stress, respectively. Expression analysis of transgenic rice and identification of cleavage sites showed that OsPDIL1;1 mRNA is a target of osa-miR5144-3p. Expression of osa-miR5144-3p and OsPDIL1;1 was shown to be inversely regulated in developing organs and under abiotic stress. The down-regulation of osa-miR5144-3p or overexpression of OsPDIL1;1 in transgenic rice showed increased total protein-disulfide bond content, compared with the wild type. This indicates that protein-disulfide bond formation is enhanced by down-regulation of osa-miR5144-3p or overexpression of OsPDIL1;1. These transgenic rice plants also displayed strong resistance to salinity and mercury stress, in comparison with the wild type. In contrast, the transgenic rice plants overexpressing osa-miR5144-3p or down-regulating OsPDIL1;1 had a lower protein-disulfide bond content; they were susceptible to abiotic stress and produced abnormal grains with small and loosely packed starch granules. These results indicate that protein-disulfide bond formation catalyzed by OsPDIL1;1 is modulated by osa-miR5144-3p in rice during development and is involved in resistance to abiotic stress.

93 - Protein S-Nitrosylation: Possible Links between Psychophysiological Stress and Neurodegeneration

Psychophysiological stress has long been recognized as a risk factor for diverse pathologies including cardiovascular disease, psychiatric illnesses, and aging-related neurodegenerative disorders but molecular events linking stress to disease remain ill-defined. Mounting evidence supports a role for nitro-oxidative modifications of protein thiols in cellular signaling and toxicity. In light of numerous reports that acute stress can increase the activities and/or expression levels of multiple nitric oxide synthase isoforms in the brains of rodent models, we investigated the possibility that subjecting rats to acute restraint stress may promote S-nitrosylation of proteins from the brain measured by the classical biotin-switch method. Our results indeed show that restraint stress increased S-nitrosylation of brain proteins albeit transiently. S-nitrosylation was highest following 2 hours of restraint but returned to levels at or below the control by six hours. Among the S-nitrosylated proteins identified by LC-MS/MS were, from highest to lowest abundance, glyceraldehyde-3-phosphate dehydrogenase, peroxiredoxin-2, protein phosphatase 2B, and the ADP/ATP translocase all of which have immediate implications for cellular pathology. As protein nitrosthiols are generally unstable and readily convert to disulfides, efforts were also made to measure the impacts of restraint stress on disulfides involving proteins. Levels of protein-glutathione mixed disulfides appeared to be inversely related to S-nitrosylation but changes across groups were not statistically-significant. In addition, unique challenges to the trapping of the in vivo redox states of protein vicinal thiols and the determination of the effects of restraint stress on oxidations of these, resulting in protein disulfides, were noted and linked to the very high reactivity of the catalytic thiols of peroxiredoxins. These findings provide initial support for the notion that protein S-nitrosylation may link psychophysiological stress to neural insults and open the door for further investigations of nitro-oxidative modifications of protein thiols as potential links between life stresses and disease.

Membrane-enriched proteome changes and prion protein expression during neural differentiation and in neuroblastoma cells

BackgroundThe function of the prion protein, involved in the so-called prion diseases, remains a subject of intense debate and the possibility that it works as a pleiotropic protein through the interaction with multiple membrane proteins is somehow supported by recent reports. Therefore, the use of proteomic and bioinformatics combined to uncover cellular processes occurring together with changes in the expression of the prion protein may provide further insight into the putative pleiotropic role of the prion protein.ResultsThis study assessed the membrane-enriched proteome changes accompanying alterations in the expression of the prion protein. A 2D-DIGE approach was applied to two cell lines after prefractionation towards the membrane protein subset: an embryonic stem cell line and the PK1 subline of neuroblastoma cells which efficiently propagates prion infection. Several proteins were differentially abundant with the increased expression of the prion protein during neural differentiation of embryonic stem cells and with the knockdown of the prion protein in PK1 cells. The identity of around 20% of the differentially abundant proteins was obtained by tandem MS. The catalytic subunit A of succinate dehydrogenase, a key enzyme for the aerobic energy metabolism and redox homeostasis, showed a similar abundance trend as the prion protein in both proteomic experiments. A gene ontology analysis revealed “myelin sheath”, “organelle membrane” and “focal adhesion” associated proteins as the main cellular components, and “protein folding” and “ATPase activity” as the biological processes enriched in the first set of differentially abundant proteins. The known interactome of these differentially abundant proteins was customized to reveal four interactors with the prion protein, including two heat shock proteins and a protein disulfide isomerase.ConclusionsOverall, our study shows that expression of the prion protein occurs concomitantly with changes in chaperone activity and cell-redox homeostasis, emphasizing the functional link between these cellular processes and the prion protein.

Endoplasmic reticulum protein ERp46 in prostate adenocarcinoma.

Endoplasmic reticulum (ER) protein ERp46 is a member of the protein disulfide isomerase family of oxidoreductases, which facilitates the reduction of disulfides in proteins and their folding. Accumulation of misfolded proteins has been implicated in cancer. The objectives of the present study were to investigate the role of ERp46 in prostate cancer, its expression and its effects on prostate cancer growth. A tissue microarray with human prostate cancer and normal prostate tissue samples was stained for ERp46 followed by image analysis. Human prostate adenocarcinoma 22Rv1 cells were stably transfected with short hairpin RNA (shRNA) specific for ERp46, a non-effective scrambled control or a plasmid containing full-length human ERp46 cDNA, and cell growth was determined. Subcloned cells were treated with thapsigargin or tunicamycin to induce ER stress and lysates were subjected to western blot analysis for ER stress proteins. Subcutaneous xenografts of parental 22Rv1, ERp46-overexpressing (ERp46+), shERp46 or scrambled control cells were established in male inbred BALB/c nude mice (n=10/group). Tumor growth curves of the xenografts were constructed over a period of 30 days and subsequently the mice were sacrificed and the amount of serum prostate-specific antigen was determined. The results demonstrated increased ERp46 expression levels in prostate cancer tissue samples of Gleason ≥7 compared with normal prostate tissue samples. When ERp46 was stably knocked down using shRNA or overexpressed in prostate carcinoma 22Rv1 cells, tumor growth in vitro and in BALB/c nude mice was inhibited and accelerated, respectively. ERp46 overexpression led to reduced sensitivity to ER stress as indicated by higher half maximal inhibitory concentrations for tunicamycin and thapsigargin in ERp46+ cells. The shERp46 cells lost the ability to upregulate protein disulfide isomerase following tunicamycin-induced ER stress. The present study suggests a role for ERp46 as a therapeutic target in prostate cancer, given its expression profile in human prostate cancer, and its effect on prostate cancer cell growth.

PreImplantation factor (PIF) protects cultured embryos against oxidative stress: relevance for recurrent pregnancy loss (RPL) therapy.

Recurrent pregnancy loss (RPL) affects 2-3% of couples. Despite a detailed work-up, the etiology is frequently undefined, leading to non-targeted therapy. Viable embryos and placentae express PreImplantation Factor (PIF). Maternal circulating PIF regulates systemic immunity and reduces circulating natural killer cells cytotoxicity in RPL patients. PIF promotes singly cultured embryos’ development while anti-PIF antibody abrogates it. RPL serum induced embryo toxicity is negated by PIF. We report that PIF rescues delayed embryo development caused by <3 kDa RPL serum fraction likely by reducing reactive oxygen species (ROS). We reveal that protein disulfide isomerase/thioredoxin (PDI/TRX) is a prime PIF target in the embryo, rendering it an important ROS scavenger. The 16F16-PDI/TRX inhibitor drastically reduced blastocyst development while exogenous PIF increased >2 fold the number of embryos reaching the blastocyst stage. Mechanistically, PDI-inhibitor preferentially binds covalently to oxidized PDI over its reduced form where PIF avidly binds. PIF by targeting PDI/TRX at a distinct site limits the inhibitor’s pro-oxidative effects. The >3kDa RPL serum increased embryo demise by three-fold, an effect negated by PIF. However, embryo toxicity was not associated with the presence of putative anti-PIF antibodies. Collectively, PIF protects cultured embryos both against ROS, and higher molecular weight toxins. Using PIF for optimizing in vitro fertilization embryos development and reducing RPL is warranted.

The impact of different mutations at Arg54 on structure, chaperone-like activity and oligomerization state of human αA-crystallin: The pathomechanism underlying congenital cataract-causing mutations R54L, R54P and R54C

A major part of cataractogenic mutations in human αA-Crystallin (αA-Cry) occurs at Arg residues. While Arg54 is highly conserved within different species, the cataractogenic mutations R54L, R54P and R54C have been recently identified in CRYAA gene, encoding human αA-Cry. The detailed structural and functional aspects, stability and amyloidogenic properties of αA-Cry were determined upon the above-mentioned missense mutations, using various spectroscopic techniques, gel electrophoresis, electron microscopy, size exclusion chromatography analyses, and chaperone-like activity assay. The different mutations at Arg54 result in diverse structural alterations among mutant proteins. In addition, the mutant proteins displayed reduced thermal stability, increased amyloidogenic properties and attenuated chaperone-like activity against aggregation of γ-Cry, catalase and lysozyme. The mutant proteins were also capable of forming larger oligomeric complexes with γ-Cry which is the natural partner of α-Cry in the eye lenses. The most significant structural and functional damages were observed upon R54L mutation which was also accompanied with increased oligomeric size distribution of the mutant protein. The cataractogenic nature of R54P mutation can be explained with its detrimental effect on chaperone-like activity, conformational stability and proteolytic digestibility of the mutant protein. Also, R54C αA-Cry displayed an important intrinsic propensity for disulfide protein cross-linking with significantly reduced chaperone-like activity against all client proteins. These mutations revealed a range of detrimental effects on the structure, stability and functional properties of αA-Cry which all together can explain the pathomechanisms underlying development of the associated congenital cataract disorders.

Reactivity of disulfide bonds is markedly affected by structure and environment: implications for protein modification and stability.

Disulfide bonds play a key role in stabilizing protein structures, with disruption strongly associated with loss of protein function and activity. Previous data have suggested that disulfides show only modest reactivity with oxidants. In the current study, we report kinetic data indicating that selected disulfides react extremely rapidly, with a variation of 104 in rate constants. Five-membered ring disulfides are particularly reactive compared with acyclic (linear) disulfides or six-membered rings. Particular disulfides in proteins also show enhanced reactivity. This variation occurs with multiple oxidants and is shown to arise from favorable electrostatic stabilization of the incipient positive charge on the sulfur reaction center by remote groups, or by the neighboring sulfur for conformations in which the orbitals are suitably aligned. Controlling these factors should allow the design of efficient scavengers and high-stability proteins. These data are consistent with selective oxidative damage to particular disulfides, including those in some proteins.