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Target proteins in biotechnological applications are highly diverse. Therefore, versatile flexible expression systems for their functional overproduction are required. In order to find the right heterologous gene expression strategy, suitable host-vector systems, which combine different genetic circuits, are useful. In this study, we designed a novel Bacillus subtilis expression toolbox, which allows the overproduction and secretion of potentially toxic enzymes. This toolbox comprises a set of 60 expression vectors, which combine two promoter variants, four strong secretion signals, a translation-enhancing downstream box, and three plasmid backbones. This B. subtilis toolbox is based on a tailor-made, clean deletion mutant strain, which is protease and sporulation deficient and exhibits reduced autolysis and secondary metabolism. The appropriateness of this alternative expression platform was tested for the overproduction of two difficult-to-produce eukaryotic model proteins. These included the sulfhydryl oxidase Sox from Saccharomyces cerevisiae , which forms reactive hydrogen peroxide and undesired cross-linking of functional proteins, and the human interleukin-1β, a pro-inflammatory cytokine. For the best performing Sox and interleukin, overproducing and secreting variants of these new B. subtilis toolbox fermentation strategies were developed and tested. This study demonstrates the suitability of the prokaryotic B. subtilis host-vector system for the extracellular production of two eukaryotic proteins with biotechnological relevance. Key points • Construction of a versatile Bacillus subtilis gene expression toolbox. • Verification of the toolbox by the secretory overproduction of two difficult-to-express proteins. • Fermentation strategy for an acetoin-controlled overproduction of heterologous proteins.

Transmission-blocking vaccines (TBVs) can effectively prevent the community's spread of malaria by targeting the antigens of mosquito sexual stage parasites. At present, only a few candidate antigens have demonstrated transmission-blocking activity (TBA) potential in . Quiescin-sulfhydryl oxidase (QSOX) is a sexual stage protein in the rodent malaria parasite and is associated with a critical role in protein folding by introducing disulfides into unfolded reduced proteins. Here, we reported the immunogenicity and transmission-blocking potency of the PvQSOX in . The full-length recombinant PvQSOX protein (rPvQSOX) was expressed in the expression system. The anti-rPvQSOX antibodies were generated following immunization with the rPvQSOX in rabbits. A parasite integration of the gene into the gene knockout genome was developed to express full-length PvQSOX protein in (Pv-Tr-PbQSOX). In western blot, the anti-rPvQSOX antibodies recognized the native PvQSOX protein expressed in transgenic gametocyte and ookinete. In indirect immunofluorescence assays, the fluorescence signal was detected in the sexual stages, including gametocyte, gamete, zygote, and ookinete. Anti-rPvQSOX IgGs obviously inhibited the ookinetes and oocysts development both and using transgenic parasites. Direct membrane feeding assays of anti-rPvQSOX antibodies were conducted using four field isolates (named isolates #1-4) in Thailand. Oocyst density in mosquitoes was significantly reduced by 32.00, 85.96, 43.52, and 66.03% with rabbit anti-rPvQSOX antibodies, respectively. The anti-rPvQSOX antibodies also showed a modest reduction of infection prevalence by 15, 15, 20, and 22.22%, respectively, as compared to the control, while the effect was insignificant. The variation in the DMFA results may be unrelated to the genetic polymorphisms. Compared to the Salvador (Sal) I strain sequences, the in isolate #1 showed no amino acid substitution, whereas isolates #2, #3, and #4 all had the M361I substitution. Our results suggest that PvQSOX could serve as a potential TBVs candidate, which warrants further evaluation and optimization.

A thiol group plays an essential role in sperm metabolism and the antioxidative defense state. Zinc is the second most abundant element in the human body, following iron. The present study was conducted to study the effect of zinc supplementation on the characteristics of semen along with thiol and thiol-related enzymes in semen of asthenospermic patients. Semen samples were obtained from 60 fertile and 60 asthenospermic men, from couples who had consulted the infertility clinic of Babil Hospital (Hillah city, Iraq). The subfertile group was treated with zinc; every participant took two 220 mg capsules of zinc sulfate per day for 3 months. Semen samples were obtained (before and after zinc supplementation). The levels of reduced thiol, oxidized thiol, thiol oxido-reductive index, and thiol-related enzymes activities were determined in spermatozoa and seminal plasma of patients and healthy groups. Oxidized thiol levels were significantly higher in the infertile patients compared to that in the fertile group. Conversely, reduced thiol level, sulfhydryl oxidase activity, and glutathione peroxidase activity significantly decreased in the infertile patients compared to that in the fertile group. Oxidized thiol levels, reduced thiol levels, and thiol-related enzymes activities of the infertile patients were restored to normal values after treatment with zinc. However, reduced and oxidized thiol levels in spermatozoa did not change significantly in the group treated with zinc. The quantitative values for RSH/RSSR and thiol-related enzymes may provide useful means to qualitatively express the oxidant/antioxidant balance in clinical and epidemiologic studies. ClinicalTrials.gov Identifier: NCT02985905

Quiescin sulfhydryl oxidase 1 (QSOX1) is a disulphide bond generating catalyst that is overexpressed in solid tumours. Expression of QSOX1 is linked to cancer cell invasion, tumour grade, and aberrant extracellular matrix (ECM) protein deposition. While the secreted version of QSOX1 is known to be present in various fluids and secretory tissues, its presence in the ECM of cancer is less understood. To characterize secreted QSOX1, we isolated extracellular vesicles (and particles) (EV(P)s) from conditioned media using ultracentrifugation and separated the supernatant using tangential flow filtration. We discovered that most of the secreted QSOX1 resides in the EVP‐depleted supernatant and in the soluble protein fraction. Very little QSOX1 could be detected in the EVP fraction. We used immunofluorescence to image subpopulations of EVs and found QSOX1 in Golgi‐derived vesicles and medium/large vesicles, but in general, most extracellular QSOX1 was not attributed to these vesicles. Next, we quantified QSOX1 co‐localization with the EV marker Alix. For the medium/large EVs, ∼98% contained QSOX1 when fibronectin was used as a coating. However, on collagen coatings, only ∼60% of these vesicles contained QSOX1, suggesting differences in EV cargo based on ECM coated surfaces. About 10% of small EVs co‐localized with QSOX1 on every ECM protein surface except for collagen (0.64%). We next investigated adhesion of QSOX1 to ECM proteins in vitro and in situ and found that QSOX1 preferentially adheres to fibronectin, laminins, and Matrigel compared to gelatin and collagen. This mechanism was found to be, in part, mediated by the formation of mixed disulphides between QSOX1 and cysteine‐rich ECM proteins. In summary, we found that QSOX1 (1) is in subpopulations of medium/large EVs, (2) seems to interact with small Alix+ EVs, and (3) adheres to cysteine‐rich ECM proteins, potentially through the formation of intermediate disulphides. These observations offer significant insight into how enzymes, such as QSOX1, can facilitate matrix remodelling events in solid tumour progression.

Quiescin sulfhydryl oxidase (QSOX), present in a wide variety of eukaryotic species, catalyzes the insertion of disulfide bonds into unfolded, reduced proteins. Here we characterized the QSOX protein from the rodent malaria parasite (PbQSOX), which is conserved in all sequenced malaria parasite species. The PbQSOX protein was not expressed in asexual erythrocytic stages, but was most abundantly expressed in ookinetes. Indirect immunofluorescence assays revealed PbQSOX was not only localized in cytoplasm of gametocytes, gametes and ookinetes, but also expressed on the surface of gametes and ookinetes. Western blot identified extracellular presence of PbQSOX in the culture medium of ookinetes suggestive of secretion. deletion (Δ ) did not affect asexual intraerythrocytic development, but reduced exflagellation of male gametocytes as well as formation and maturation of ookinetes. deletion also led to a significant increase in the reduced thiol groups of ookinete surface proteins, suggesting that it may play a role in maintaining the integrity of disulfide bonds of surface proteins, which might be needed for ookinete development. Mosquitoes that fed on Δ -infected mice showed a significant reduction in ookinete and oocyst numbers compared to those fed on wild-type parasite-infected mice. Further, both polyclonal mouse antisera and a monoclonal antibody against the recombinant PbQSOX exhibited substantial transmission-blocking activities in and mosquito feeding assays, suggesting QSOX is a potential target for blocking parasite transmission.

Background The infectious prion protein (PrP Sc or prion) is derived from its cellular form (PrP C ) through a conformational transition in animal and human prion diseases. Studies have shown that the interspecies conversion of PrP C to PrP Sc is largely swayed by species barriers, which is mainly deciphered by the sequence and conformation of the proteins among species. However, the bank vole PrP C (BVPrP) is highly susceptible to PrP Sc from different species. Transgenic mice expressing BVPrP with the polymorphic isoleucine (109I) but methionine (109M) at residue 109 spontaneously develop prion disease. Results To explore the mechanism underlying the unique susceptibility and convertibility, we generated soluble BVPrP by co-expression of BVPrP with Quiescin sulfhydryl oxidase (QSOX) in Escherichia coli . Interestingly, rBVPrP-109M and rBVPrP-109I exhibited distinct seeded aggregation pathways and aggregate morphologies upon seeding of mouse recombinant PrP fibrils, as monitored by thioflavin T fluorescence and electron microscopy. Moreover, they displayed different aggregation behaviors induced by seeding of hamster and mouse prion strains under real-time quaking-induced conversion. Conclusions Our results suggest that QSOX facilitates the formation of soluble prion protein and provide further evidence that the polymorphism at residue 109 of QSOX-induced BVPrP may be a determinant in mediating its distinct convertibility and susceptibility.

Background RNA 5-methylcytosine (m 5 C) modification plays critical roles in the pathogenesis of various tumors. However, the function and molecular mechanism of RNA m 5 C modification in tumor drug resistance remain unclear. Methods The correlation between RNA m 5 C methylation, m 5 C writer NOP2/Sun RNA methyltransferase family member 2 (NSUN2) and EGFR-TKIs resistance was determined in non-small-cell lung cancer (NSCLC) cell lines and patient samples. The effects of NSUN2 on EGFR-TKIs resistance were investigated by gain- and loss-of-function assays in vitro and in vivo . RNA-sequencing (RNA-seq), RNA bisulfite sequencing (RNA-BisSeq) and m 5 C methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) were performed to identify the target gene of NSUN2 involved in EGFR-TKIs resistance. Furthermore, the regulatory mechanism of NSUN2 modulating the target gene expression was investigated by functional rescue and puromycin incorporation assays. Results RNA m 5 C hypermethylation and NSUN2 were significantly correlated with intrinsic resistance to EGFR-TKIs. Overexpression of NSUN2 resulted in gefitinib resistance and tumor recurrence, while genetic inhibition of NSUN2 led to tumor regression and overcame intrinsic resistance to gefitinib in vitro and in vivo . Integrated RNA-seq and m 5 C-BisSeq analyses identified quiescin sulfhydryl oxidase 1 (QSOX1) as a potential target of aberrant m 5 C modification. NSUN2 methylated QSOX1 coding sequence region, leading to enhanced QSOX1 translation through m 5 C reader Y-box binding protein 1 (YBX1). Conclusions Our study reveals a critical function of aberrant RNA m 5 C modification via the NSUN2-YBX1-QSOX1 axis in mediating intrinsic resistance to gefitinib in EGFR-mutant NSCLC.

African swine fever virus (ASFV), the causative pathogen of the recent ASF epidemic, is a highly contagious double-stranded DNA virus. Its genome is in the range of 170~193 kbp and encodes 68 structural proteins and over 100 non-structural proteins. Its high pathogenicity strains cause nearly 100% mortality in swine. Consisting of four layers of protein shells and an inner genome, its structure is obviously more complicated than many other viruses, and its multi-layered structures play different kinds of roles in ASFV replication and survival. Each layer possesses many proteins, but very few of the proteins have been investigated at a structural level. Here, we concluded all the ASFV proteins whose structures were unveiled, and explained their functions from the view of structures. Those structures include ASFV AP endonuclease, dUTPases (E165R), pS273R protease, core shell proteins p15 and p35, non-structural proteins pA151R, pNP868R (RNA guanylyltransferase), major capsid protein p72 (gene B646L), Bcl-2-like protein A179L, histone-like protein pA104R, sulfhydryl oxidase pB119L, polymerase X and ligase. These novel structural features, diverse functions, and complex molecular mechanisms promote ASFV to escape the host immune system easily and make this large virus difficult to control.

Male fertility is the ability of sperm to fertilize the egg and sustain embryo development. Several factors determine the fertilizing capacity of mammalian sperm, including those intrinsic to sperm and components of the seminal plasma. The present study analyzed the seminal fluid proteome of Bos taurus and potential associations between proteins and fertility scores. Mass spectrometry coupled with nano HPLC allowed the identification of 1,159 proteins in the dairy bull seminal plasma. There were 50 and 29 seminal proteins more abundant in high (HF) low fertility (LF) bulls, respectively. Based on multivariate analysis, C-type natriuretic peptide, TIMP-2, BSP5 and sulfhydryl oxidase indicated relationship with HF bulls. Clusterin, tissue factor pathway inhibitor 2, galectin-3-binding protein and 5′-nucleotidase were associated with LF bulls. Abundance of NAD(P)(+)-arginine ADP-ribosyltransferase, prosaposin and transmembrane protein 2 proteins had the highest positive correlations with fertility ranking. Quantities of vitamin D-binding protein, nucleotide exchange factor SIL1 and galectin-3-binding protein showed the highest negative correlations with fertility ranking. A fertility ranking score was calculated and the relationship with these proteins was significant (Spearman’s rho = 0.94). The present findings represent a major and novel contribution to the study of bovine seminal proteins. Indicators of fertility can be used to improve reproductive biotechnologies.

Secreted quiescin/sulfhydryl oxidase (QSOX) is overexpressed in many tumor cell lines, including melanoma, and is usually associated with a pro-invasive phenotype. Our previous work described that B16-F10 cells enter in a quiescent state as a protective mechanism against damage generated by reactive oxygen species (ROS) during melanogenesis stimulation. Our present results show that QSOX activity was two-fold higher in cells with stimulated melanogenesis when compared to control cells. Considering that glutathione (GSH) is one of the main factor responsible for controlling redox homeostasis in cells, this work also aimed to investigate the relationship between QSOX activity, GSH levels and melanogenesis stimulation in B16-F10 murine melanoma cell line. The redox homeostasis was impaired by treating cells with GSH in excess or depleting its intracellular levels through BSO treatment. Interestingly, GSH-depleted cells without stimulation of melanogenesis kept high levels of viability, suggesting a possible adaptive mechanism of survival even under low GSH levels. They also showed lower extracellular activity of QSOX, and higher QSOX intracellular immunostaining, suggesting that this enzyme was less excreted from cells and corroborating with a diminished extracellular QSOX activity. On the other hand, cells under melanogenesis stimulation showed a lower GSH/GSSG ratio (8:1) in comparison with control (non-stimulated) cells (20:1), indicating a pro-oxidative state after stimulation. This was accompanied by decreased cell viability after GSH-depletion, no alterations in QSOX extracellular activity, but higher QSOX nucleic immunostaining. We suggest that melanogenesis stimulation and redox impairment caused by GSH-depletion enhanced the oxidative stress in these cells, contributing to additional alterations of its metabolic adaptive response.