Molecular cloning of Y-Box binding protein-1 from mandarin fish and its roles in stress-response and antiviral immunity.

Mandarin fish (Siniperca chuatsi) is a universally farmed fish species in China and has a large farming scale and economic value. With the high-density cultural mode in mandarin fish, viral diseases, such as infectious spleen and kidney necrosis virus (ISKNV) and Siniperca chuatsi rhabdovirus (SCRV), have increased loss, which has seriously restricted the development of aquaculture. Y-Box binding protein 1 (YB-1) is a member of cold shock protein family that regulates multiple cellular processes. The roles of mammalian YB-1 protein in environmental stress and innate immunity have been studied well, but its roles in teleost fishes remain unknown. In the present study, the characteristic of S. chuatsi YB-1 (scYB-1) and its roles in cold stress and virus infection were investigated. The scYB-1 obtained an 1541 bp cDNA that contains a 903 bp open reading frame encoding a protein of 300 amino acids. Tissue distribution results showed that the scYB-1 is a ubiquitously expressed gene found among tissues from mandarin fish. Overexpression of scYB-1 can increase the expression levels of cold shock-responsive genes, such as scHsc70a, scHsc70b, and scp53. Furthermore, the role of scYB-1 in innate immunity was also investigated in mandarin fish fry (MFF-1) cells. The expression level of scYB-1 was significant change in response to poly (I:C), poly (dG:dC), PMA, ISKNV, or SCRV stimulation. The overexpression of scYB-1 can significantly increase the expression levels of NF-κB-responsive genes, including scIL-8, scTNF-α, and scIFN-h. The NF-κB-luciferase report assay results showed that the relative expression of luciferin was significantly increased in the cells overexpressed with scYB-1 compared with those in cells overexpressed with control plasmid. These results indicate that scYB-1 can induce the NF-κB signaling pathway in MFF-1 cells. Overexpressed scYB-1 can downregulate the expression of ISKNV viral major capsid protein (mcp) gene but upregulates the expression of SCRV mcp gene. Moreover, knockdown of scYB-1 using siRNA can upregulate the expression of ISKNV mcp gene but downregulates the expression of SCRV mcp gene. These results indicate that scYB-1 suppresses ISKNV infection while enhancing SCRV infection. The above observations suggest that scYB-1 is involved in cold stress and virus infection. Our study will provide an insight into the roles of teleost fish YB-1 protein in stress response and innate immunity.

Targeting the YB-1/PD-L1 Axis to Enhance Chemotherapy and Antitumor Immunity.

Tumor cells can escape immune destruction in tumor chemoresistance, but the mechanism for this phenomenon remains unclear. Y-box binding protein 1 (YB-1), which is upregulated in chemoresistant tumor cells, plays a role in the acquisition of multidrug resistance. Here, we demonstrate that chemotherapy induced an immunosuppressive microenvironment in the tumor and induced immune evasion through YB-1-mediated programmed death-1 ligand 1 (PD-L1) upregulation. Examination of the YB-1 protein and mRNA showed an increase in YB-1 expression in hepatocellular carcinoma (HCC). High YB-1 expression negatively correlated with the overall survival of HCC patients. YB-1 expression positively correlated with PD-L1, and YB-1 induced PD-L1 expression by binding a PD-L1 promoter motif. YB-1 expression was upregulated in chemoresistant HCC cells, and YB-1 knockdown reversed chemoresistance via T-cell activation in the tumor microenvironment due to blocked PD-L1 expression. We also found that inhibition of the tumor immunosuppressive environment and immune evasion was accompanied by proliferation of functional cytotoxic CD8+ T cells and inhibition of myeloid-derived suppressor cells and regulatory T cells in the tumor environment. Our data indicate that targeting the YB-1 signaling axis, which simultaneously reverses both tumor immune evasion and multidrug resistance, may improve the antitumor response. This finding suggests a treatment modality against tumor chemoresistance.

High prevalence of Y-box protein-1/p18 fragment in plasma of patients with malignancies of different origin.

BACKGROUND: Expression of the cold shock protein Y-box protein 1 (YB-1) is associated with deleterious outcome in various malignant diseases. Our group recently showed that the detection of an 18 kDa YB-1 fragment (YB-1/p18) in human plasma identifies patients with malignant diseases. We now tested the prevalence, clinical, and diagnostic value of YB-1/p18 detection in common tumors. METHODS: A newly established monoclonal YB-1 antibody was used to detect YB-1/p18 by immunoblotting in plasma samples from 151 unselected tumor patients, alongside established tumor markers and various diagnostic measures, during evaluation for a cancerous disease and in follow-up studies after therapeutic interventions. RESULTS: Circulating YB-1/p18 was detected in 78% of patients having a tumor disease. YB-1/p18 positivity was highly prevalent in all examined malignancies, including lung cancer (32/37; 87%), breast cancer (7/10; 70%), cancer of unknown primary (CUP; 5/5, 100%) or hematological malignancies (42/62; 68%). Positivity for YB-1/p18 was independent of other routine laboratory parameters, tumor stage, or histology. In comparison to 13 established tumor markers (cancer antigens 15-3, 19-9, 72-4, and 125; carcinoembryonic antigen; cytokeratin fragments 21-1; neuron-specific enolase; alpha-fetoprotein; beta-2-microglobulin; squamous cell carcinoma antigen; thymidine kinase; tissue polypeptide antigen; pro-gastrin-releasing peptide), YB-1/p18 detection within serum samples was the most sensitive general parameter identifying malignant disorders. YB-1/p18 concentrations altered during therapeutic interventions, but did not predict prognosis. CONCLUSIONS: Plasma YB-1/p18 detection has a high specific prevalence in malignancies, thereby providing a novel tool for cancer screening independent of the tumor origin.

Autism-specific maternal autoantibodies recognize critical proteins in developing brain.

Autism spectrum disorders (ASDs) are neurodevelopmental in origin, affecting an estimated 1 in 88 children in the United States. We previously described ASD-specific maternal autoantibodies that recognize fetal brain antigens. Herein, we demonstrate that lactate dehydrogenase A and B (LDH), cypin, stress-induced phosphoprotein 1 (STIP1), collapsin response mediator proteins 1 and 2 (CRMP1, CRMP2) and Y-box-binding protein to comprise the seven primary antigens of maternal autoantibody-related (MAR) autism. Exclusive reactivity to specific antigen combinations was noted in 23% of mothers of ASD children and only 1% of controls. ASD children from mothers with specific reactivity to LDH, STIP1 and CRMP1 and/or cypin (7% vs 0% in controls; P<0.0002; odds ratios of 24.2 (95% confidence interval: 1.45-405)) had elevated stereotypical behaviors compared with ASD children from mothers lacking these antibodies. We describe the first panel of clinically significant biomarkers with over 99% specificity for autism risk thereby advancing our understanding of the etiologic mechanisms and therapeutic possibilities for MAR autism.

GW/P-bodies and autoimmune disease.

Human autoantibodies have performed admirably in the service of characterizing GW/P-bodies. These antibodies have provided a critical point of reference by which other proteins have been shown to be components of GW/P-bodies. In addition,autoantibodies have been used to identify new GW/P-body components, including Ge-l, GW182, RAP55, and YB-1. Using new, high-throughput screening assays, it is likely that additional, novel GW/P-body components will be identified. Human auto antibodies have also raised the possibility of a functional link between two apparently unrelated cellular structures, PML-SplOO nuclear bodies and GW/P-bodies.A key unanswered question remains: What is the role of GW/P-bodies in the pathogenesis of autoimmune disease? Over the next 10 years, as more is learned about the function of GW/P-bodies, it is hoped that molecular and cellular biologists will further consider this question and remember the important contributions of patients with autoimmune disease to the early characterization of these cellular structures.

Cloning, characterization and expression analysis of a cold shock domain family member YB-1 in turbot Scophthalmus maximus.

The Y-box proteins are a family of highly conserved nucleic acid binding proteins. In this report we have identified a new member, YB-1 from turbot (Scophthalmus maximus) spleen cDNA library. The full-length cDNA sequence of turbot YB-1 was obtained and then the expression at transcriptional level was researched by qRT-PCR. In normal organs, the expression of YB-1 was higher in liver, brain, gill and heart, respectively. YB-1 had the highest expression level at gastrula stage during the early stages of embryo development. In the liver, kidney and spleen, the turbot YB-1 expression level was the highest at 72 h after challenge with lymphocystis disease virus (LCDV) and the highest at 12 h after challenge with Vibrio anguillarum (V. anguillarum). Furthermore, the expression of turbot YB-1 also distinctly increased in turbot kidney cells (TK) at 24 h after challenge with V. anguillarum and LCDV. These results indicated that the turbot YB-1 protein may play a significant role in the immune response of turbot.

Y-box binding protein 1 and RNase UK114 mediate monocyte chemoattractant protein 1 mRNA stability in vascular smooth muscle cells.

Monocyte chemoattractant protein 1 (MCP-1) plays a pivotal role in many inflammatory processes, including the progression of atherosclerosis and the response of the arterial wall to injury. We previously demonstrated that dexamethasone (Dex) inhibits MCP-1 mRNA accumulation in smooth muscle cells by decreasing its half-life. The effect of Dex was dependent upon the glucocorticoid receptor (GR) and independent of new transcription. Using RNA affinity and column chromatography, we have identified two proteins involved in regulating MCP-1 mRNA stability: Y-box binding protein 1 (YB-1), a multifunctional DNA/RNA-binding protein, and endoribonuclease UK114 (UK). By immunoprecipitation, YB and GR formed a complex present in equal amounts in extracts from untreated and Dex-treated cells. YB-1, UK, and GR small interfering RNA (siRNA) substantially inhibited the effect of Dex on MCP-1 mRNA accumulation. In addition, YB-1 antibody blocked the degradation of MCP-1 mRNA by cytoplasmic extracts from the Dex-treated cells. The degradative activity of extracts immunoprecipitated with antibodies to either YB-1 or GR was blocked with UK antibody. UK did not degrade MCP-1 mRNA; however, upon addition to nondegrading control extracts, it rapidly degraded MCP-1 mRNA. These studies define new roles for GR, YB-1, and UK in the formation of a molecular complex that degrades MCP-1 mRNA.

Role of cold shock Y-box protein-1 in inflammation, atherosclerosis and organ transplant rejection.

Chemokines (chemoattractant cytokines) are crucial regulators of immune cell extravasation from the bloodstream into inflamed tissue. Dysfunctional regulation and perpetuated chemokine gene expression are linked to progressive chronic inflammatory diseases and, in respect to transplanted organs, may trigger graft rejection. RANTES (regulated upon activation, normal T cell expressed and secreted (also known as CCL5)) is a model chemokine with relevance in numerous inflammatory diseases where the innate immune response predominates. Transcription factor Y-box binding protein-1 (YB-1) serves as a trans-regulator of CCL5 gene transcription in vascular smooth muscle cells and leucocytes. This review provides an update on YB-1 as a mediator of inflammatory processes and focuses on the role of YB-1 in CCL5 expression in diseases with monocytic cell infiltrates, albeit acute or chronic. Paradigms of such diseases encompass atherosclerosis and transplant rejection where cold shock protein YB-1 takes a dominant role in transcriptional regulation.

Differential regulation of chemokine CCL5 expression in monocytes/macrophages and renal cells by Y-box protein-1.

The Y-box protein-1 (YB-1) belongs to the family of cold shock proteins that have pleiotropic functions such as gene transcription, RNA splicing, and mRNA translation. YB-1 has a critical role in atherogenesis due to its regulatory effects on chemokine CCL5 (RANTES) gene transcription in vascular smooth muscle cells. Since CCL5 is a key mediator of kidney transplant rejection, we determined whether YB-1 is involved in allograft rejection by manipulating its expression. In human kidney biopsies, YB-1 transcripts were amplified 17-fold in acute and 21-fold in chronic allograft rejection with a close correlation between CCL5 and YB-1 mRNA expression in both conditions. Among three possible YB-1 binding sites in the CCL5 promoter, a critical element was mapped at -28/-10 bps. This site allowed up-regulation of CCL5 transcription in monocytic THP-1 and HUT78 T-cells and in human primary monocytes; however, it repressed transcription in differentiated macrophages. Conversely, YB-1 knockdown led to decreased CCL5 transcription and secretion in monocytic cells. We show that YB-1 is a cell-type specific regulator of CCL5 expression in infiltrating T-cells and monocytes/macrophages and acts as an adaptive controller of inflammation during kidney allograft rejection.

The up-regulation of Y-box binding proteins (DNA binding protein A and Y-box binding protein-1) as prognostic markers of hepatocellular carcinoma.

PURPOSE: The development of hepatocellular carcinoma is associated with the chronic inflammation of the liver caused by various factors such as hepatitis B or C virus infection. Previously, we reported DNA binding protein A (dbpA) as a candidate molecule that can accelerate inflammation-induced hepatocarcinogenesis. DbpA belongs to the Y-box binding protein family, and Y-box binding protein-1 (YB-1), the prototype member of this family, is reported to be a prognostic marker of malignant diseases other than hepatocellular carcinoma. The purpose of this study is to examine the significance of the expression of dbpA or of the T-to-G transversion in the dbpA promoter region, which enhances the promoter activity in vitro, for the progression of hepatocellular carcinoma. EXPERIMENTAL DESIGN: We studied the expression of dbpA (as well as of YB-1) in 82 formalin-fixed hepatocellular carcinoma tissues by immunohistochemistry and determined the sequence of the dbpA promoter region in 42 frozen hepatocellular carcinoma tissues. We examined the relationship between these findings and the clinicopathologic factors of hepatocellular carcinoma patients. RESULTS: DbpA expression was associated with the advanced stages of hepatocellular carcinoma, and the cases with the nuclear dbpA expression had a poor prognosis. DbpA contributed more significantly to this association than YB-1. Furthermore, the T-to-G transversion in the dbpA promoter region was related to the nuclear localization of dbpA. CONCLUSION: DbpA was a more significant prognostic marker of hepatocellular carcinoma than YB-1. The T-to-G transversion in the dbpA promoter region was suggested to be a predisposing factor for the progression of hepatocellular carcinoma.