Quiescin Sulfhydryl Oxidase Research Articles

QSOX1 Modulates Glioblastoma Cell Proliferation and Migration In Vitro and Invasion In Vivo

Background: Quiescin Sulfhydryl Oxidase 1 (QSOX1) is an enzyme that catalyzes the oxidation of free thiols to generate disulfide bonds in a variety of proteins, including the cell surface and extracellular matrix. QSOX1 has been reported to be upregulated in a number of cancers, and the overexpression of QSOX1 has been correlated with aggressive cancers and poor patient prognosis. Glioblastoma (GBM) brain cancer has been practically impossible to treat effectively, with cells that rapidly invade normal brain tissue and escape surgery and other treatment. Thus, there is a crucial need to understand the multiple mechanisms that facilitate GBM cell invasion and to determine if QSOX1 is involved. Methods and Results: Here, we investigated the function of QSOX1 in human glioblastoma cells using two cell lines derived from T98G cells, whose proliferation, motility, and invasiveness has been shown by us to be dependent on disulfide bond-containing adhesion and receptor proteins, such as L1CAM and the FGFR. We lentivirally introduced shRNA to attenuate the QSOX1 protein expression in one cell line, and a Western blot analysis confirmed the decreased QSOX1 expression. A DNA content/cell cycle analysis using flow cytometry revealed 27% fewer knockdown cells in the S-phase of the cell cycle, indicating a reduced proliferation. A cell motility analysis utilizing our highly quantitative SuperScratch time-lapse microscopy assay revealed that knockdown cells migrated more slowly, with a 45% decrease in migration velocity. Motility was partly rescued by the co-culture of knockdown cells with control cells, indicating a paracrine effect. Surprisingly, knockdown cells exhibited increased motility when assayed using a Transwell migration assay. Our novel chick embryo orthotopic xenograft model was used to assess the in vivo invasiveness of knockdown vs. control cells, and tumors developed from both cell types. However, fewer invasive knockdown cells were observed after about a week. Conclusions: Our results indicate that an experimental reduction in QSOX1 expression in GBM cells leads to decreased cell proliferation, altered in vitro migration, and decreased in vivo invasion.

QSOX1 facilitates dormant esophageal cancer stem cells to evade immune elimination via PD-L1 upregulation and CD8 T cell exclusion

Dormant cancer stem cells (DCSCs) exhibit characteristics of chemotherapy resistance and immune escape, and they are a crucial source of tumor recurrence and metastasis. However, the underlying mechanisms remain unrevealed. We demonstrate that enriched Gzmk+ CD8+ T cells within the niche of esophageal DCSCs restrict the outgrowth of tumor mass. Nonetheless, DCSCs can escape immune elimination by enhancing PD-L1 signaling, thereby maintaining immune equilibrium. Quiescent fibroblast-derived quiescin sulfhydryl oxidase 1 (QSOX1) promotes the expression of PD-L1 and its own expression in DCSCs by elevating the level of reactive oxygen species. Additionally, high QSOX1 in the dormant tumor niche contributes to the exclusion of CD8+ T cells. Conversely, blocking QSOX1 with Ebselen in combination with anti-PD-1 and chemotherapy can effectively eradicate residual DCSCs by reducing PD-L1 expression and promoting CD8+ T cell infiltration. Clinically, high expression of QSOX1 predicts a poor response to anti-PD-1 treatment in patients with esophageal cancer. Thus, our findings reveal a mechanism whereby QSOX1 promotes PD-L1 upregulation and T cell exclusion, facilitating the immune escape of DCSCs, and QSOX1 inhibition, combined with immunotherapy and chemotherapy, represents a promising therapeutic approach for eliminating DCSCs and preventing recurrence.

Plasma-based proteomic and metabolomic characterization of lung and lymph node metastases in cervical cancer patients

Metastasis is the leading cause of mortality in cervical cancer (CC), with a particular prevalence of lymph node and lung metastases. Patients with CC who have developed distant metastases typically face a poor prognosis, and there is a scarcity of non-invasive strategies for predicting CC metastasis. In this study, we utilized label-free proteomics and untargeted metabolomics to analyze plasma samples from 25 non-metastatic, 14 with lung metastasis, and 15 with lymph node metastasis CC patients. Pathway enrichment analysis revealed a shared inflammatory process between the two metastatic groups, while the central carbon metabolism in cancer showed distinct features in the lung metastasis cohort. Additionally, cholesterol metabolism, hypoxia-inducible factor 1, and ferroptosis signaling pathways were specifically altered in the lymph node metastasis group. Utilizing the receiver operating characteristic curve analysis and Random Forest algorithm, we identified two distinct biomarker panels for the prediction of lung metastasis and lymph node metastasis, respectively. The lung metastasis panel includes properdin, neural cell adhesion molecule 1, and keratin 6 A, whereas the lymph node metastasis panel consists of quiescin sulfhydryl oxidase 1, paraoxonase 1, and keratin 6 A. Each panel exhibited significant diagnostic potential, with high area under the curve (AUC) values for lung metastasis (training set: 0.989, testing set: 0.789) and lymph node metastasis (training set: 0.973, testing set: 0.900). This study conducted an integrated proteomic and metabolomic analysis to clarify the factors contributing to lung and lymph node metastases in CC and has successfully established two biomarker panels for their prediction.

Immunogenicity and transmission-blocking potential of quiescin sulfhydryl oxidase in Plasmodium vivax.

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 P. vivax. Quiescin-sulfhydryl oxidase (QSOX) is a sexual stage protein in the rodent malaria parasite Plasmodium berghei 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 P. vivax. The full-length recombinant PvQSOX protein (rPvQSOX) was expressed in the Escherichia coli expression system. The anti-rPvQSOX antibodies were generated following immunization with the rPvQSOX in rabbits. A parasite integration of the pvqsox gene into the P. berghei pbqsox gene knockout genome was developed to express full-length PvQSOX protein in P. berghei (Pv-Tr-PbQSOX). In western blot, the anti-rPvQSOX antibodies recognized the native PvQSOX protein expressed in transgenic P. berghei 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 in vivo and in vitro using transgenic parasites. Direct membrane feeding assays of anti-rPvQSOX antibodies were conducted using four field P. vivax 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 P.vivax Salvador (Sal) I strain sequences, the pvqsox 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 P. vivax TBVs candidate, which warrants further evaluation and optimization.

Cre-LoxP and tamoxifen-induced deletion of ovarian quiescin sulfhydryl oxidase 2 showed disruption of ovulatory activity in mice

BackgroundQuiescin sulfhydryl oxidase 2 (QSOX2) is a flavin adenine dinucleotide-dependent sulfhydryl oxidase that is known to be involved in protein folding, cell growth regulation, and redox state modification through oxidative activities. Earlier studies demonstrated the tissue and cellular localization of QSOX2 in the male reproductive tract, as well as the highly-regulated mechanism of QSOX2 protein synthesis and expression through the coordinated action of testosterone and epididymal-enriched amino acid, glutamate. However, the presence and the functions of QSOX2 in female reproduction are unknown. In this study, we applied the Cre-loxP gene manipulation system to generate the heterozygous and homozygous Qsox2 knockout mice and examined its effects on ovarian function.ResultsWe demonstrated that QSOX2 was detected in the follicle-supporting cells (granulosa and cumulus cells) of ovarian follicles of all stages but was absent in the corpus luteum, suggesting its supportive role in folliculogenesis. In comparison with reproductive organogenesis in wild-type mice, there was no difference in testicular and epididymal structure in male Qsox2 knockout; however, Qsox2 knockout disrupted the regular ovulation process in female mice as a drastic decrease in the formation of the corpus luteum was detected, and no pregnancy was achieved when mating males with homozygous Qsox2 knockout females. RNAseq analyses further revealed that Qsox2 knockout altered critical signaling pathways and genes that are responsible for maintaining ovarian functions.ConclusionOur data demonstrated for the first time that Qsox2 is critical for ovarian function in mice.

S-nitrosylation switches the Arabidopsis redox sensor protein, QSOX1, from an oxidoreductase to a molecular chaperone under heat stress

The Arabidopsis quiescin sulfhydryl oxidase 1 (QSOX1) thiol-based redox sensor has been identified as a negative regulator of plant immunity. Here, we have found that small molecular weight proteins of QSOX1 were converted to high molecular weight (HMW) complexes upon exposure to heat stress and that this was accompanied by a switch in QSOX1 function from a thiol-reductase to a molecular chaperone. Plant treatment with S-nitrosoglutathione (GSNO), which causes nitrosylation of cysteine residues (S-nitrosylation), but not with H2O2, induced HMW QSOX1 complexes. Thus, functional switching of QSOX1 is induced by GSNO treatment. Accordingly, simultaneous treatment of plants with heat shock and GSNO led to a significant increase in QSOX1 chaperone activity by increasing its oligomerization. Consequently, transgenic Arabidopsis overexpressing QSOX1 (QSOX1OE) showed strong resistance to heat shock, whereas qsox1 knockout plants exhibited high sensitivity to heat stress. Plant treatment with GSNO under heat stress conditions increased their resistance to heat shock. We conclude that S-nitrosylation allows the thiol-based redox sensor, QSOX1, to respond to various external stresses in multiple ways.

Evaluation of novel biomarkers for early pregnancy outcome prediction†.

To assess performance and discriminatory capacity of commercially available enzyme-linked immunosorbent assays of biomarkers for predicting first trimester pregnancy outcome in a multi-center cohort. In a case-control study at three academic centers of women with pain and bleeding in early pregnancy, enzyme-linked immunosorbent assays of biomarkers were screened for assay performance. Performance was assessed via functional sensitivity, assay reportable range, recovery/linearity, and intra-assay precision (%Coefficient of Variation). Top candidates were analyzed for discriminatory capacity for viability and location among 210 women with tubal ectopic pregnancy, viable intrauterine pregnancy, or miscarriage. Assay discrimination was assessed by visual plots, area under the curve with 95% confidence intervals, and measures of central tendency with two-sample t-tests. Of 25 biomarkers evaluated, 22 demonstrated good or acceptable assay performance. Transgelin-2, oviductal glycoprotein, and integrin-linked kinase were rejected due to poor performance. The best biomarkers for discrimination of pregnancy location were pregnancy-specific beta-1-glycoprotein 9, pregnancy-specific beta-1-glycoprotein 1, insulin-like growth factor binding protein 1, kisspeptin (KISS1), pregnancy-specific beta-1-glycoprotein 3, and beta parvin (PARVB). The best biomarkers for discrimination of pregnancy viability were pregnancy-specific beta-1-glycoprotein 9, pregnancy-specific beta-1-glycoprotein 3, EH domain-containing protein 3, KISS1, WAP four-disulfide core domain protein 2 (HE4), quiescin sulfhydryl oxidase 2, and pregnancy-specific beta-1-glycoprotein 1. Performance of commercially available enzyme-linked immunosorbent assays was acceptable for a panel of novel biomarkers to predict early pregnancy outcome. Of these, six and seven candidates demonstrated good discriminatory capacity of pregnancy location and viability, respectively, when validated in a distinct external population. Four markers demonstrated good discrimination for both location and viability.

Abstract 14734: Circulating Sulfhydryl Oxidase and Risk of Cardiovascular Disease

Introduction: Whether circulating quiescin sulfhydryl oxidase (QSOX) family enzymes level is causally associated with an increased risk of cardiovascular disease remains unestablished. In this study, we applied Mendelian randomization (MR) analysis to investigate the potential causal relationship between circulating QSOX family enzymes and a wide range of cardiovascular disease outcomes in European ancestry. Methods: Independent single nucleotide polymorphisms associated with QSOX1 and QSOX2 at the genome-wide significance level were selected as instrumental variables. Summary-level data for 9 cardiovascular endpoints were obtained from genetic consortia, the UK Biobank study, the FinnGen consortium, HERMES and MEGASTROKE. Results: Higher genetically predicted circulating QSOX2 levels were associated with an increased risk of coronary artery disease. For each standard deviation increase in genetically predicted QSOX2 levels, the odds ratio was 1.05 (95% confidence interval, 1.02, 1.07; p = 0.0001) for coronary artery disease. Significant causal association of genetically determined QSOX2 level with heart failure, ischemic stroke and peripheral artery disease was also found with the inverse-variance weighted MR method. However, no potential causal relationships between circulating QSOX2 and atrial fibrillation, intracerebral hemorrhage, pulmonary embolism, transient ischemic attack and venous thromboembolism were observed. There was no association between genetically predicted QSOX1 and cardiovascular disease. Conclusions: This study reveals suggestive evidence that lowering circulating QSOX2 levels may reduce the risk of coronary artery disease, heart failure, ischemic stroke and peripheral artery disease.

Proteomic Profiling in Patients With Peripartum Cardiomyopathy: A Biomarker Study of the ESC EORP PPCM Registry

BackgroundPeripartum cardiomyopathy (PPCM) remains an important cause of maternal morbidity and mortality globally. The pathophysiology remains incompletely understood, and the diagnosis is often missed or delayed. ObjectivesThis study explored the serum proteome profile of patients with newly diagnosed PPCM, as compared with matched healthy postpartum mothers, to unravel novel protein biomarkers that would further an understanding of the pathogenesis of PPCM and improve diagnostic precision. MethodsStudy investigators performed untargeted serum proteome profiling using data-independent acquisition–based label-free quantitative liquid chromatography–tandem mass spectrometry on 84 patients with PPCM, as compared with 29 postpartum healthy controls (HCs). Significant changes in protein intensities were determined with nonpaired Student’s t-tests and were further classified by using the Boruta algorithm. The proteins’ diagnostic performance was evaluated by area under the curve (AUC) and validated using the 10-fold cross-validation. ResultsPatients with PPCM presented with a mean left ventricular ejection fraction of 33.5% ± 9.3% vs 57.0% ± 8.8% in HCs (P < 0.001). Study investigators identified 15 differentially up-regulated and 14 down-regulated proteins in patients with PPCM compared with HCs. Seven of these proteins were recognized as significant by the Boruta algorithm. The combination of adiponectin, quiescin sulfhydryl oxidase 1, inter-α-trypsin inhibitor heavy chain, and N-terminal pro–B-type natriuretic peptide had the best diagnostic precision (AUC: 0.90; 95% CI: 0.84-0.96) to distinguish patients with PPCM from HCs. ConclusionsSalient biologic themes related to immune response proteins, inflammation, fibrosis, angiogenesis, apoptosis, and coagulation were predominant in patients with PPCM compared with HCs. These newly identified proteins warrant further evaluation to establish their role in the pathogenesis of PPCM and potential use as diagnostic markers.

Aberrant m5C hypermethylation mediates intrinsic resistance to gefitinib through NSUN2/YBX1/QSOX1 axis in EGFR-mutant non-small-cell lung cancer

BackgroundRNA 5-methylcytosine (m5C) modification plays critical roles in the pathogenesis of various tumors. However, the function and molecular mechanism of RNA m5C modification in tumor drug resistance remain unclear.MethodsThe correlation between RNA m5C methylation, m5C 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 m5C 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.ResultsRNA m5C 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 m5C-BisSeq analyses identified quiescin sulfhydryl oxidase 1 (QSOX1) as a potential target of aberrant m5C modification. NSUN2 methylated QSOX1 coding sequence region, leading to enhanced QSOX1 translation through m5C reader Y-box binding protein 1 (YBX1).ConclusionsOur study reveals a critical function of aberrant RNA m5C modification via the NSUN2-YBX1-QSOX1 axis in mediating intrinsic resistance to gefitinib in EGFR-mutant NSCLC.

Effects of redox modulation on quiescin/sulfhydryl oxidase activity of melanoma cells.

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. Ourpresent 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.

A Golgi oxygen sensor controls intestinal mucin glycosylation.

Intestinal mucin glycosylation is important for mucus-bacterial homeostasis and is altered in disease. In this issue of The EMBO Journal, Ilani etal (2022) identify the Golgi enzyme quiescin sulfhydryl oxidase 1 (QSOX1) as a novel mucus regulator by controlling mucin sialylation.

Secretion of the disulfide bond generating catalyst QSOX1 from pancreatic tumor cells into the extracellular matrix: association with extracellular vesicles and matrix proteins.

Quiescin sulfhydryl oxidase 1 (QSOX1) is a disulfide bond generating catalyst that is overexpressed in solid tumors. Expression of QSOX1 is linked to cancer cell invasion, tumor grade, and 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 separated conditioned media based on size and density. We discovered that the majority of secreted QSOX1 resides in the EV-depleted fraction 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 disulfides 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 disulfides. These observations offer significant insight into how enzymes, such as QSOX1, can facilitate matrix remodeling events in solid tumor progression.

Recombinant Rice Quiescin Sulfhydryl Oxidase Strengthens the Gluten Structure through Thiol/Disulfide Exchange and Hydrogen Peroxide Oxidation.

Recombinant rice quiescin sulfhydryl oxidase (rQSOX) has the potential to improve the flour processing quality, but the mechanisms remain unclear. The effects of rQSOX on bread quality, dough rheology, and gluten structure and composition, with glucose oxidase as a positive control, were investigated. rQSOX addition could improve the dough processing quality, as proved by enhanced viscoelastic properties of dough as well as a softer crumb, higher specific volume, and lower moisture loss of bread. These beneficial effects were attributed to gluten protein polymerization and gluten network strengthening, evidenced by the improved concentration of SDS-insoluble gluten and formation of large gluten aggregates and the increased α-helix and β-turn conformation. Furthermore, decreased free sulfhydryl and increased dityrosine in gluten as well as improved H2O2 content in dough suggested that the rQSOX dough strengthening mechanism was mainly based on the formation of disulfide bonds and dityrosine cross-links in gluten by both thiol/disulfide direct exchange and hydrogen peroxide indirect oxidation pathways.

Transcriptome-Wide Mapping of Small-Molecule RNA-Binding Sites in Cells Informs an Isoform-Specific Degrader of QSOX1 mRNA.

The interactions between cellular RNAs in MDA-MB-231 triple negative breast cancer cells and a panel of small molecules appended with a diazirine cross-linking moiety and an alkyne tag were probed transcriptome-wide in live cells. The alkyne tag allows for facile pull-down of cellular RNAs bound by each small molecule, and the enrichment of each RNA target defines the compound's molecular footprint. Among the 34 chemically diverse small molecules studied, six bound and enriched cellular RNAs. The most highly enriched interaction occurs between the novel RNA-binding compound F1 and a structured region in the 5' untranslated region of quiescin sulfhydryl oxidase 1 isoform a (QSOX1-a), not present in isoform b. Additional studies show that F1 specifically bound RNA over DNA and protein; that is, we studied the entire DNA, RNA, and protein interactome. This interaction was used to design a ribonuclease targeting chimera (RIBOTAC) to locally recruit Ribonuclease L to degrade QSOX1 mRNA in an isoform-specific manner, as QSOX1-a, but not QSOX1-b, mRNA and protein levels were reduced. The RIBOTAC alleviated QSOX1-mediated phenotypes in cancer cells. This approach can be broadly applied to discover ligands that bind RNA in cells, which could be bioactive themselves or augmented with functionality such as targeted degradation.

Polarized epithelium-sperm co-culture system reveals stimulatory factors for the secretion of mouse epididymal quiescin sulfhydryl oxidase 1.

Spermatozoa acquire fertilization ability through post-translational modifications. These membrane surface alterations occur in various segments of the epididymis. Quiescin sulfhydryl oxidases, which catalyze thiol-oxidation reactions, are involved in disulfide bond formation, which is essential for sperm maturation, upon transition and migration in the epididymis. Using castration and azoospermia transgenic mouse models, in the present study, we showed that quiescin sulfhydryl oxidase 1 (QSOX1) protein expression and secretion are positively correlated with the presence of testosterone and sperm cells. A two-dimensional in vitro epithelium-sperm co-culture system provided further evidence in support of the notion that both testosterone and its dominant metabolite, 5α-dihydrotestosterone, promote epididymal QSOX1 secretion. We also demonstrated that immature caput spermatozoa, but not mature cauda sperm cells, exhibited great potential to stimulate QSOX1 secretion in vitro, suggesting that sperm maturation is a key regulatory factor for mouse epididymal QSOX1 secretion. Proteomic analysis identified 582 secretory proteins from the co-culture supernatant, of which 258 were sperm-specific and 154 were of epididymal epithelium-origin. Gene Ontology analysis indicated that these secreted proteins exhibit functions known to facilitate sperm membrane organization, cellular activity, and sperm-egg recognition. Taken together, our data demonstrated that testosterone and sperm maturation status are key regulators of mouse epididymal QSOX1 protein expression and secretion.

The Spliced Leader RNA Silencing (SLS) Pathway in Trypanosoma brucei Is Induced by Perturbations of Endoplasmic Reticulum, Golgi Complex, or Mitochondrial Protein Factors: Functional Analysis of SLS-Inducing Kinase PK3.

ABSTRACTIn the parasite Trypanosoma brucei, the causative agent of human African sleeping sickness, all mRNAs are trans-spliced to generate a common 5′ exon derived from the spliced leader (SL) RNA. Perturbations of protein translocation across the endoplasmic reticulum (ER) induce the spliced leader RNA silencing (SLS) pathway. SLS activation is mediated by a serine-threonine kinase, PK3, which translocates from the cytosolic face of the ER to the nucleus, where it phosphorylates the TATA-binding protein TRF4, leading to the shutoff of SL RNA transcription, followed by induction of programmed cell death. Here, we demonstrate that SLS is also induced by depletion of the essential ER-resident chaperones BiP and calreticulin, ER oxidoreductin 1 (ERO1), and the Golgi complex-localized quiescin sulfhydryl oxidase (QSOX). Most strikingly, silencing of Rhomboid-like 1 (TIMRHOM1), involved in mitochondrial protein import, also induces SLS. The PK3 kinase, which integrates SLS signals, is modified by phosphorylation on multiple sites. To determine which of the phosphorylation events activate PK3, several individual mutations or their combination were generated. These mutations failed to completely eliminate the phosphorylation or translocation of the kinase to the nucleus. The structures of PK3 kinase and its ATP binding domain were therefore modeled. A conserved phenylalanine at position 771 was proposed to interact with ATP, and the PK3F771L mutation completely eliminated phosphorylation under SLS, suggesting that the activation involves most if not all of the phosphorylation sites. The study suggests that the SLS occurs broadly in response to failures in protein sorting, folding, or modification across multiple compartments.

Quiescin Sulfhydryl Oxidase 2 Overexpression Predicts Poor Prognosis and Tumor Progression in Patients With Colorectal Cancer: A Study Based on Data Mining and Clinical Verification.

Background: As a member of the atypical thiol oxidase family, quiescin sulfhydryl oxidase 2 (QSOX2) has been reported to play an important role in several biological processes, but the expression and function of QSOX2 in colorectal cancer (CRC) remains elusive.Methods: The difference of QSOX2 expression, and its relationship with clinicopathological features and prognosis in CRC, was analyzed by bioinformatic analysis and validated by clinical CRC specimen cohort. The functional characterization of QSOX2 was detected via in vitro and vivo experiments in CRC cell lines, while the potential signaling pathways were predicted by Gene Set Enrichment Analysis (GSEA).Results: Our data based on bioinformatical analysis and clinical validation demonstrated that the expression of QSOX2 in CRC tissues was significantly upregulated. Additionally, the chi-square test, logistic regression analysis, and Fisher’s exact test showed that QSOX2 overexpression was significantly correlated with advanced clinicopathological parameters, such as pathological stage and lymph node metastasis. The Kaplan–Meier curves and univariate Cox regression model showed that QSOX2 overexpression predicts poor overall survival (OS) and disease-free survival (DFS) in CRC patients. More importantly, multivariate Cox regression model showed that QSOX2 overexpression could serve as an independent factor for CRC patients. In vitro and vivo data showed that the proliferation and metastasis ability of CRC cells were suppressed on condition of QSOX2 inhibition. In addition, GSEA showed that the QSOX2 high expression phenotype has enriched multiple potential cancer-related signaling pathways.Conclusion: QSOX2 overexpression is strongly associated with malignant progression and poor oncological outcomes in CRC. QSOX2 might act as a novel biomarker for prognosis prediction and a new target for biotherapy in CRC.

A negative feedback loop controls ROS production in plant immunity

A negative feedback loop controls ROS production in plant immunity

Conformational switches and redox properties of the colon cancer-associated human lectin ZG16.

Zymogen granule membrane protein 16 (ZG16) is produced in organs that secrete large quantities of enzymes and other proteins into the digestive tract. ZG16 binds microbial pathogens, and lower ZG16 expression levels correlate with colorectal cancer, but the physiological function of the protein is poorly understood. One prominent attribute of ZG16 is its ability to bind glycans, but other aspects of the protein may also contribute to activity. An intriguing feature of ZG16 is a CXXC motif at the carboxy terminus. Here, we describe crystal structures and biochemical studies showing that the CXXC motif is on a flexible tail, where it contributes little to structure or stability but is available to engage in redox reactions. Specifically, we demonstrate that the ZG16 cysteine thiols can be oxidized to a disulfide by quiescin sulfhydryl oxidase 1, which is a sulfhydryl oxidase present together with ZG16 in the Golgi apparatus and in mucus, as well as by protein disulfide isomerase. ZG16 crystal structures also draw attention to a nonproline cis peptide bond that can isomerize within the protein and to the mobility of glycine‐rich loops in the glycan‐binding site. An understanding of the properties of the ZG16 CXXC motif and the discovery of internal conformational switches extend existing knowledge relating to the glycan‐binding activity of the protein.