Cold Shock Protein Family Research Articles

DNA-binding protein-A promotes kidney ischemia/reperfusion injury and participates in mitochondrial function

DNA-binding protein-A (DbpA; gene: Ybx3) belongs to the cold shock protein family with known functions in cell cycling, transcription, translation, and tight junction communication. In chronic nephritis, DbpA is upregulated. However, its activities in acute injury models, such as kidney ischemia/reperfusion injury (IRI), are unclear. To study this, mice harboring Ybx3+/+, Ybx3+/- or the Ybx3-/- genotype were characterized over 24 months and following experimental kidney IRI. Mitochondrial function, number and integrity were analyzed by mitochondrial stress tests, MitoTracker staining and electron microscopy. Western Blot, immunohistochemistry and flow cytometry were performed to quantify tubular cell damage and immune cell infiltration. DbpA was found to be dispensable for kidney development and tissue homeostasis under healthy conditions. Furthermore, endogenous DbpA protein localizes within mitochondria in primary tubular epithelial cells. Genetic deletion of Ybx3 elevates the mitochondrial membrane potential, lipid uptake and metabolism, oxygen consumption rates and glycolytic activities of tubular epithelial cells. Ybx3-/- mice demonstrated protection from IRI with less immune cell infiltration, endoplasmic reticulum stress and tubular cell damage. A presumed protective mechanism was identified via upregulated antioxidant activities and reduced ferroptosis, when Ybx3 was deleted. Thus, our studies reveal DbpA acts as a mitochondrial protein with profound adverse effects on cell metabolism and highlights a protective effect against IRI when Ybx3 is genetically deleted. Hence, preemptive DbpA targeting in situations with expected IRI, such as kidney transplantation or cardiac surgery, may preserve post-procedure kidney function.

Genome-Wide Identification and Expression Analysis Unveil the Involvement of the Cold Shock Protein (CSP) Gene Family in Cotton Hypothermia Stress.

Cold shock proteins (CSPs) are DNA/RNA binding proteins with crucial regulatory roles in plant growth, development, and stress responses. In this study, we employed bioinformatics tools to identify and analyze the physicochemical properties, conserved domains, gene structure, phylogenetic relationships, cis-acting elements, subcellular localization, and expression patterns of the cotton CSP gene family. A total of 62 CSP proteins were identified across four cotton varieties (Gossypium arboreum, Gossypium raimondii, Gossypium barbadense, Gossypium hirsutum) and five plant varieties (Arabidopsis thaliana, Brassica chinensis, Camellia sinensis, Triticum aestivum, and Oryza sativa). Phylogenetic analysis categorized cotton CSP proteins into three evolutionary branches, revealing similar gene structures and motif distributions within each branch. Analysis of gene structural domains highlighted the conserved CSD and CCHC domains across all cotton CSP families. Subcellular localization predictions indicated predominant nuclear localization for CSPs. Examination of cis-elements in gene promoters revealed a variety of elements responsive to growth, development, light response, hormones, and abiotic stresses, suggesting the potential regulation of the cotton CSP family by different hormones and their involvement in diverse stress responses. RT-qPCR results suggested that GhCSP.A1, GhCSP.A2, GhCSP.A3, and GhCSP.A7 may play roles in cotton's response to low-temperature stress. In conclusion, our findings underscore the significant role of the CSP gene family in cotton's response to low-temperature stress, providing a foundational basis for further investigations into the functional aspects and molecular mechanisms of cotton's response to low temperatures.

Evaluation of serum and tissue levels of cold-inducible RNA-binding protein in non-segmental Vitiligo.

Damage-associated molecular patterns (DAMPs) play a role in the pathogenesis of vitiligo. It has been established that the cold-inducible RNA-binding protein (CIRP), a member of the family of cold-shock proteins that respond to stress, is a DAMP molecule that promotes inflammation. The objective was to evaluate the serum and tissue CIRP expression in non-segmental vitiligo (NSV) patients. A sample of 40 participants, 20 NSV patients and 20 control groups of age- and sex-matched healthy individuals were included in this case-control study where the enzyme-linked immunosorbent assay was used in detecting the serum and tissue CIRP levels in participants. The serum and tissue CIRP levels significantly increased in NSV patients compared with the healthy controls, (165.35 ± 24.42, 226.29 ± 24.00 versus 59.81 ± 12.10, 105.86 ± 11.27pg/ml, respectively) (P < 0.01). Serum and tissue CIRP are significantly correlated with each other (r = 0.641, P = 0.002). Except for a statistically significant positive correlation between CIRP tissue level and VASI (r = 0.539, P = 0.014), the CIRP Serum and tissue did not show any statistically significant correlations with different clinical parameters in patients. ROC curve shows that the cut-off point for serum and tissue CIRP level to differentiate between patients and controls was 86.5, 124.3pg/ml, respectively, with 100.0% sensitivity, 100.0% specificity and 1.000 AUC for each of them. It is concluded that CIRP may have a crucial role in the pathogenesis of NSV and could be used as a marker for vitiligo and its extent with the need for further large-scale study.

YB-1 Expression Is Associated with Lymph Node Metastasis and Drug Resistance to Adriamycin in Breast Cancer.

Breast cancer (BC) is the most common malignant tumor among females. Although there are multiple treatments for breast cancer, many patients still face the dilemma of drug resistance after multiline treatment. It would be greatly helpful for clinical work to identify additional and improved prognostic predictors. Y-box binding protein-1 (YB-1) is a member of the cold shock protein family, and patients with overexpression of YB-1 have a worse prognosis. This study collected 48 specimens from 48 patients with breast cancer and analyzed the clinicopathological characteristics of the patients. Immunohistochemistry, immunofluorescence, cell viability analysis, tumor spheroid formation and cell morphology, cell invasion, cycle analysis, qRT-PCR, Western blot, and tumorigenicity in BALB/c nude mice were performed to verify the results. We found that patients with overexpression of YB-1 were related to lymph node metastasis and the patients' age tended to be young. Because of the short follow-up time, a survival analysis could not be performed. Based on the results of in vitro and in vivo experiments, this study indicated that breast cancer cells with overexpression of YB-1 had stronger proliferation, migration, and invasion abilities than cells with low expression of YB-1. Compared with cells with low expression of YB-1, the proliferation, migration, and invasion abilities of YB-1 overexpressed cells were not significantly affected by adriamycin. This suggested that breast cancer cells with overexpression of YB-1 were resistant to adriamycin. Therefore, YB-1 is associated with lymph node metastasis of breast cancer cell. YB-1 could be a prognostic, predictive factor and a novel therapeutic target of BC.

Clinical relevance for circulating cold-inducible RNA-binding protein (CIRP) in patients with adult-onset Still's disease.

Adult-onset Still's disease (AOSD) is a systemic autoinflammatory disease in which danger-associated molecular patterns (DAMPs)-mediated inflammasome activation seems to be involved in the disease pathogenesis. Cold-inducible RNA-binding protein (CIRP) belongs to a family of cold-shock proteins that respond to cellular stress and has been identified as a DAMP that triggers the inflammatory response. The aim of this study is to investigate the clinical significance of serum CIRP levels in AOSD. Serum samples were obtained from 44 patients with active AOSD or 50 patients with rheumatoid arthritis (RA), 20 patients with systemic lupus erythematosus (SLE), and 15 healthy control patients (HCs). Serum levels of CIRP and IL-18 were determined using enzyme-linked immunosorbent assay. Results were compared among AOSD patients, RA patients, SLE patients and HCs. Results were also analyzed according to the clinical features of AOSD. Serum CIRP levels were significantly higher in AOSD patients compared with RA patients (median: 9.6 ng/mL, IQR [5.7-14.4] versus 3.2 ng/mL, IQR [1.9-3.8]; p < 0.001) and with HCs (2.8 ng/mL, [IQR; 1.4-4.9], p < 0.001). There was a significant positive correlation between serum CIRP levels and AOSD disease activity score (Pouchot's score r = 0.45, p = 0.003) as well as between AOSD-specific biomarkers ferritin and IL-18. However, there was no significant difference in the serum CIRP levels among AOSD patients with three different disease phenotypes. These results suggest that CIRP may play a significant role in the pathophysiology of AOSD and could be a potential biomarker for monitoring the disease activity of AOSD.

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.

Multiple functions of cold-inducible RNA-binding protein in biological systems

Cold-inducible RNA-binding protein (CIRP) is an 18-kDa nuclear protein belonging to the family of cold-shock proteins. It is widely expressed in various tissues and upregulated in response to various cellular stresses. Originally, CIRP was found to stabilize specific mRNAs to facilitate their translation, which results in a survival advantage for cells under stress. Hypothermia-induced CIRP has been shown to protect the neuronal system. Recently, CIRP was found to be a multifunctional protein. In addition to acting as a damage-associated molecular pattern molecule to promote inflammation, it was also found to affect tumorigenesis. CIRP was also found to regulate telomerase activity and cardiac repolarization. Therefore, biological activities of CIRP in novel systems should receive more attention. This review will provide a brief introduction to the recent studies of CIRP with the aim of inspiring future research of novel CIRP functions.

Clinical Relevance for Serum Cold-Inducible RNA-Binding Protein Level in Alopecia Areata.

BackgroundAlopecia areata (AA), a chronic, relapsing hair-loss disorder, is considered to be a T-cell-mediated autoimmune disease. Cold-inducible RNA-binding protein (CIRP) belongs to a family of cold-shock proteins that respond to cold stress, and has been identified as a damage-associated molecular pattern (DAMP) molecule that triggers the inflammatory response. Recent studies have shown that high-mobility group box 1, another DAMP molecule, is elevated in serum and scalp tissue of AA patients, suggesting a relationship between DAMP molecules and the pathogenesis of AA.ObjectiveTo investigate the clinical significance of serum CIRP levels in AA.MethodsThe serum levels of CIRP were compared between 68 patients with AA and 20 healthy controls. Additionally, the correlation between CIRP level and various clinical parameters was evaluated.ResultsThe serum CIRP levels were significantly higher in AA patients compared to healthy subjects. Moreover, there was an association between the serum CIRP level and clinical characteristics, such as disease duration and disease activity. However, there was no significant difference in the serum CIRP level among the clinical types of AA (AA multiplex, alopecia totalis, and alopecia universalis).ConclusionThese results suggest that CIRP may play a significant role in the pathogenesis of AA and could be a potential biologic marker for monitoring the disease activity of AA.

Computational Analysis of Evolutionary Relationship of a Family of Cold Shock Proteins in Ten Mammalian Species

Computational Analysis of Evolutionary Relationship of a Family of Cold Shock Proteins in Ten Mammalian Species

The Cold-Shock Protein Ybx1 Is Required for Development and Maintenance of Acute Myeloid Leukemia (AML) in Vitro and In Vivo

The family of cold shock proteins (CSPs) is highly conserved and consists of 8 members, including Ybx1-3, Csde1 and Lin28. Ybx1 is a multifunctional DNA/RNA binding protein that modulates gene transcription and translation during inflammation and malignant transformation. Recently, our group identified Ybx1 as a mediator of Jak2 signaling in MPN that protects Jak2-mutated cells from Jak-inhibitor induced apoptosis.In a recently published genome wide CRISPR-Cas9 dropout screen in AML cell lines, depletion of Ybx1 resulted in the highest dropout indices compared to other CSP members, with strongest dependencies in cell lines harboring MLL-rearrangements. Protein expression of Ybx1 in healthy individuals (n=10), primary MDS (n=54) and AML (n=58) bone marrow (BM) biopsies, revealed high protein expression in the majority of AML and MDS cases. Consistently, gene expression data revealed high mRNA expression of Ybx1 in AML samples compared to normal controls.Genetic inactivation of Ybx1 in human AML cell lines by RNAi resulted in reduced proliferative capacity. Therefore, we sought to investigate the requirement for Ybx1 in malignant transformation. We used BM cells from a previously published conventional knockout (ko) mouse model (Lu et al., 2005) in which homozygous deletion is embryonically lethal due to brain malformation. We sorted Lineage-Sca1+Kit+ (LSK-) cells from the BM of heterozygous (Ybx1+/-) and wildtype (Ybx1+/+) mice. Cells were retrovirally infected with either MLL-AF9 (MA9) or HoxA9 and Meis1a (HA9M1) to assess for disease development by serial plating in methylcellulose. Haploinsufficiency for Ybx1 in MA9- or HA9M1 transformed cells limited re-plating capacity to 2-4 rounds. When we injected 2,5x 104 MA9-infected LSK cells into sublethally irradiated recipient mice, recipients of MA9-Ybx1+/- cells (n=8) and MA9-Ybx1+/+ (n=10) showed development of AML. However, recipients of MA9-Ybx1+/- cells had a significant delay in AML development (median survival 67.5 days for Ybx1+/+ versus 101.5 days for Ybx1+/- animals, p=0.0078**). This effect appeared even more pronounced when 1x 106 whole BM cells were transplanted into sublethally irradiated secondary recipients. Besides a significant delay in AML development (median survival 37.5 days for recipients of MA9-Ybx1+/+ versus 79 days for MA9-Ybx1+/- BM, p=0.0042**), disease penetrance was reduced by 40%, indicating that haploinsufficiency for Ybx1 impairs development of MA9 driven AML. In contrast, immunophenotypic abundance of stem- and progenitor cells in Ybx1+/+ versus Ybx1+/- animals revealed comparable numbers in all relevant subpopulations. Serial competitive transplantation of Ybx1+/+ and Ybx1+/- BM into primary and secondary recipient animals showed no competitive disadvantage or lack of self-renewal capacity of Ybx1+/- cells.To address the question whether Ybx1 may also be essential for maintenance of AML, we used RNAi to deplete Ybx1 in already established MA9 driven AML. LSK cells from BL/6 mice transformed with MA9 were injected into primary recipient mice. After AML onset, MA9-LSK cells were sorted and infected with either one of 3 shRNAs against Ybx1 or non-targeting (NT-) control. Lentiviral knockdown of 40% reduced colony formation by more than 50% but did not limit the re-plating capacity in vitro. When injected into sub-lethally irradiated recipient mice, lentiviral knockdown (kd) of Ybx1 resulted in a significant delay in AML development (median survival 39.5 days for NT-control versus 53 days for Ybx1 kd, p=0.0446*).To validate our findings, we used a newly generated conditional ko mouse model for Ybx1, in which exon 3 coding for the cold-shock domain is deleted by activation of an Mx1-Cre-recombinase following pIpC administration. Preliminary results provide first evidence that genetic deletion of Ybx1 after onset of MA9 driven leukemia resulted in improved survival of primary recipient (median survival 73 versus 83 days) and a reduced penetrance in secondary recipient mice.Taken together our results may provide first evidence for a functional role of Ybx1 in MLL-AF9 driven AML. As Ybx1 seems to be dispensable for normal hematopoietic cells, these findings may offer a potential therapeutic index. Experiments to assess for the requirement for Ybx1 in maintenance of murine and human AML as well as analysis on proteomic and transcriptional changes following Ybx1 deletion are currently under way. DisclosuresNo relevant conflicts of interest to declare.

CspB of an arctic bacterium, Polaribacter irgensii KOPRI 22228, confers extraordinary freeze-tolerance.

Freezing temperatures are a major challenge for life at the poles. Decreased membrane fluidity, uninvited secondary structure formation in nucleic acids, and protein cold-denaturation all occur at cold temperatures. Organisms adapted to polar regions possess distinct mechanisms that enable them to survive in extremely cold environments. Among the cold-induced proteins, cold shock protein (Csp) family proteins are the most prominent. A gene coding for a Csp-family protein, cspB, was cloned from an arctic bacterium, Polaribacter irgensii KOPRI 22228, and overexpression of cspB greatly increased the freeze-survival rates of Escherichia coli hosts, to a greater level than any previously reported Csp. It also suppressed the cold-sensitivity of an E. coli csp-quadruple deletion strain, BX04. Sequence analysis showed that this protein consists of a unique domain at its N-terminal end and a well conserved cold shock domain at its C-terminal end. The most common mechanism of Csp function in cold adaption is melting of the secondary structures in RNA and DNA molecules, thus facilitating transcription and translation at low temperatures. P. irgensii CspB bound to oligo(dT)-cellulose resins, suggesting single-stranded nucleic acid-binding activity. The unprecedented level of freeze-tolerance conferred by P. irgensii CspB suggests a crucial role for this protein in survival in polar environments.

Inflammatory cell infiltration and resolution of kidney inflammation is orchestrated by the cold-shock protein Y-box binding protein-1

Tubular cells recruit monocytic cells in inflammatory tubulointerstitial kidney diseases. The cell-cell communication that establishes pro- or anti-inflammatory activities is mainly influenced by cytokines, reactive oxygen species, nitric oxide, and phagocytosis. Key proteins orchestrating these processes such as cold-shock proteins linked with chemoattraction and cell maturation have been identified. The prototypic member of the cold-shock protein family, Y-box binding protein (YB)-1, governs specific phenotypic alterations in monocytic cells and was explored in the present study. Following tubulointerstitial injury by unilateral ureteral obstruction, increased inflammatory cell infiltration and tubular cell CCL5 expression was found in conditional Ybx1 knockout animalswith specific depletion in monocytes/macrophages (YB-1ΔLysM). Furthermore, YB-1ΔLysM mice exhibit enhanced tissue damage, myofibroblast activation, and fibrosis. To investigate relevant molecular mechanism(s), we utilized bone marrow-derived macrophage cultures and found that YB-1-deficient macrophages display defects in cell polarization and function, including reduced proliferation and nitric oxide production, loss of phagocytic activity, and failure to upregulate IL-10 and CCL5 expression in response to inflammatory stimuli. Co-culture with primary tubular cells confirmed these findings. Thus, monocytic YB-1 has prominent and distinct roles for cellular feed-forward crosstalk and resolution of inflammatory processes by its ability to regulate cell differentiation and cytokine/chemokine synthesis.

A deficiency in cold-inducible RNA-binding protein accelerates the inflammation phase and improves wound healing.

Chronic or non-healing wounds are a major concern in clinical practice and these wounds are mostly associated with diabetes, and venous and pressure ulcers. Wound healing is a complex process involving overlapping phases and the primary phase in this complex cascade is the inflammatory state. While inflammation is necessary for wound healing, a prolonged inflammatory phase leads to impaired healing. Cold-inducible RNA-binding protein (CIRP) belongs to a family of cold-shock proteins that are expressed in high levels under stress conditions. Recently, we demonstrated that a deficiency in CIRP led to decreased inflammation and mortality in an experimental model of hemorrhagic shock. Thus, we hypothesized that a deficiency in CIRP would accelerate the inflammatory phase and lead to an improvement in cutaneous wound healing. In this study, to examine this hypothesis, a full-thickness wound was created on the dorsum of wild-type (WT) and CIRP-/- mice. The wound size was measured every other day for 14 days. The wound area was significantly decreased in the CIRP-/- mice by day 9 and continued to decrease until day 14 compared to the WT mice. In a separate cohort, mice were sacrificed on days 3 and 7 after wounding and the skin tissues were harvested for histological analysis and RNA measurements. On day 3, the mRNA expression of tumor necrossis factor (TNF)-α in the skin tissues was increased by 16-fold in the WT mice, whereas these levels were increased by 65-fold in the CIRP-/- mice. Of note on day 7, while the levels of TNF-α remained high in the WT mice, these levels were significantly decreased in the CIRP-/- mice. The histological analysis of the wounded skin tissue indicated an improvement as early as day 3 in the CIRP-/- mice, whereas in the WT mice, infiltrated immune cells were still present on day 7. On day 7 in the CIRP-/- mice, Gr-1 expression was low and CD31 expression was high, whereas in the WT mice, Gr-1 expression was high and CD31 expression was low, indicating that the CIRP-/- mice have already moved into the angiogenesis and tissue formation phase, whereas the WT mice were still in the inflammatory state. These data collectively suggest that a deficiency in CIRP accelerates the wound healing process.

CspC regulates the expression of the glyoxylate cycle genes at stationary phase in Caulobacter.

BackgroundThe Cold Shock proteins are RNA binding proteins involved in various cellular processes, including adaptation to low temperature, nutritional stress, cell growth and stationary phase. They may have an impact on gene expression by interfering with RNA stability and acting as transcription antiterminators. Caulobacter crescentus cspC is an essential gene encoding a stationary phase-induced protein of the Cold Shock Protein family and this work had as goal investigating the basis for the requirement of this gene for survival at this phase. In this work we investigate the role of CspC in C. crescentus stationary phase and discuss the molecular mechanisms that could be involved.ResultsThe expression of cspC increased significantly at stationary phase in complex media and in glucose depletion, indicating a putative role in responding to carbon starvation. Global transcriptional profiling experiments comparing cspC and the wild type strain both at exponential and stationary phases as well as comparing exponential and stationary phase in wild type strain were carried out by DNA microarray analysis. The results showed that the absence of cspC affected the transcription of 11 genes at exponential phase and 60 genes at stationary phase. Among the differentially expressed genes it is worth noting those encoding respiratory enzymes and genes for sulfur metabolism, which were upregulated, and those encoding enzymes of the glyoxylate cycle, which were severely downregulated in the mutant at stationary phase. mRNA decay experiments showed that the aceA mRNA, encoding isocitrate lyase, was less stable in the cspC mutant, indicating that this effect was at least partially due to posttranscriptional regulation. These observations were supported by the observed arrested growth phenotype of the cspC strain when grown in acetate as the sole carbon source, and by the upregulation of genes for assimilatory sulfate reduction and methionine biosynthesis.ConclusionsThe stationary phase-induced RNA binding protein CspC has an important role in gene expression at this phase, and is necessary for maximal expression of the glyoxylate cycle genes. In the case of aceA, its downregulation may be attributed to the shorter half-life of the mRNA in the cspC mutant, indicating that one of the possible regulatory mechanisms is via altering RNA stabilization.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1845-1) contains supplementary material, which is available to authorized users.

Constitutive and functional expression of YB-1 in microglial cells

Y-box-binding protein (YB-1) is a member of the cold-shock protein family and participates in a wide variety of DNA/RNA-dependent cellular processes including DNA repair, transcription, mRNA splicing, packaging, and translation. At the cellular level, YB-1 is involved in cell proliferation and differentiation, stress responses, and malignant cell transformation. A general role for YB-1 during inflammation has also been well described; however, there are minimal data concerning YB-1 expression in microglia, which are the immune cells of the brain. Therefore, we studied the expression of YB-1 in a clinically relevant global ischemia model for neurological injury following cardiac arrest. This model is characterized by massive neurodegeneration of the hippocampal CA1 region and the subsequent long-lasting activation of microglia. In addition, we studied YB-1 expression in BV-2 cells, which are an accepted microglia culture model. BV-2 cells were stressed by oxygen/glucose deprivation (OGD), OGD-relevant mediators, lipopolysaccharide (LPS), and phagocytosis-inducing cell debris and nanoparticles. Using quantitative polymerase chain reaction (PCR), we show constitutive expression of YB-1 transcripts in unstressed BV-2 cells. The functional upregulation of the YB-1 protein was demonstrated in microglia in vivo and in BV-2 cells in vitro. All stressors except for LPS were potent enhancers of the level of YB-1 protein, which appears to be regulated primarily by proteasomal degradation and, to a lesser extent, by the activation (phosphorylation) of the translation initiation factor eIF4E. The proteasome of BV-2 cells is impaired by OGD, which results in decreased protein degradation and therefore increased levels of YB-1 protein. LPS induces proteasome activity, which enables the level of YB-1 protein to remain at control levels despite enhanced protein ubiquitination. The proteasome inhibitor MG-132 was able to increase YB-1 protein levels in control and LPS-treated cultures. YB-1 upregulation was not accompanied by its translocation from the cytoplasm to the nucleus. YB-1 induction appeared to be related to microglial proliferation because it was partially co-regulated with Ki67. In addition, YB-1 protein levels correlated with microglia phagocytic activity because its upregulation could also be induced by inert NPs.

Cyclic AMP receptor protein (CRP) regulates the expression of cspA, cspB, cspG and cspI, members of cspA family, in Escherichia coli.

Escherichia coli K-12 contains nine paralogs of CspA, CspA-CspI, collectively known as CspA family of cold-shock proteins (CSPs). In spite of the high degree of similarity among themselves, only five (cspA, B, E, G and I) are induced during cold-stress. In the present study, we show that cspB, cspG and cspI, the members of cspA family, known to be induced in response to cold shock, are regulated by cyclic AMP receptor protein (CRP) , a global regulator involved in sugar metabolism, during growth at 37°C as well as at 15°C, as seen by green fluorescent protein (gfp) promoter fusions assays. Interestingly, cspA is selectively regulated by CRP during growth at 15°C but not at 37°C. The regulation of cspA, cspB, cspG and cspI by CRP was found to be through an indirect mechanism as determined by electrophoretic mobility shift assay (EMSA). These results substantiate our earlier study demonstrating a role for CRP during growth at low temperature.

Nuclear Y-box-binding protein-1 is a poor prognostic marker and related to epidermal growth factor receptor in uterine cervical cancer

ObjectiveY-box binding protein-1 (YB-1) is a member of the cold shock protein family and functions in transcription and translation. Many studies indicate that YB-1 is strongly expressed in tumor cells and is considered a marker of tumor aggressiveness and clinical prognosis. Overexpression of epidermal growth factor receptor (EGFR) has been associated with poor outcomes in cervical cancer. Clinical trials of EGFR family-base therapy are currently being initiated in cervical cancer. Nuclear YB-1 expression correlates with EGFR expression in various types of cancer. However, the clinical significance of nuclear YB-1 expression in different settings, the correlation with EGFR, and the prognostic implications of YB-1 expression in cervical cancer remain elusive. Patients and methodsNuclear YB-1 expression was immunohistochemically analyzed in tissue specimens obtained from 204 patients with cervical cancer who underwent surgery. Associations of nuclear YB-1 expression with clinicopathological factors such as survival, EGFR expression, and human epidermal growth factor receptor 2 (HER2) expression were investigated. ResultsNuclear YB-1 expression was found in 41 (20.2%) of 204 cases of cervical cancer and correlated with disease stage, tumor diameter, stromal invasion, and lymph-node metastasis. Nuclear YB-1 expression also correlated with EGFR expression (P=0.0114) as well as HER2 expression (P=0.0053). Kaplan–Meier survival analysis showed that nuclear YB-1 expression was significantly associated with poor progression-free survival (P=0.0033) and overall survival (P=0.0003), respectively. ConclusionNuclear YB-1 expression is a prognostic marker and correlates with EGFR expression in cervical cancer.

Links between the Oncoprotein YB-1 and Small Non-Coding RNAs in Breast Cancer

BackgroundThe nucleic acid-binding protein YB-1, a member of the cold-shock domain protein family, has been implicated in the progression of breast cancer and is associated with poor patient survival. YB-1 has sequence similarity to LIN28, another cold-shock protein family member, which has a role in the regulation of small noncoding RNAs (sncRNAs) including microRNAs (miRNAs). Therefore, to investigate whether there is an association between YB-1 and sncRNAs in breast cancer, we investigated whether sncRNAs were bound by YB-1 in two breast cancer cell lines (luminal A-like and basal cell-like), and whether the abundance of sncRNAs and mRNAs changed in response to experimental reduction of YB-1 expression.ResultsRNA-immunoprecipitation with an anti-YB-1 antibody showed that several sncRNAs are bound by YB-1. Some of these were bound by YB-1 in both breast cancer cell lines; others were cell-line specific. The small RNAs bound by YB-1 were derived from various sncRNA families including miRNAs such as let-7 and miR-320, transfer RNAs, ribosomal RNAs and small nucleolar RNAs (snoRNA). Reducing YB-1 expression altered the abundance of a number of transcripts encoding miRNA biogenesis and processing proteins but did not alter the abundance of mature or precursor miRNAs.ConclusionsYB-1 binds to specific miRNAs, snoRNAs and tRNA-derived fragments and appears to regulate the expression of miRNA biogenesis and processing machinery. We propose that some of the oncogenic effects of YB-1 in breast cancer may be mediated through its interactions with sncRNAs.

CspR, a Cold Shock RNA-Binding Protein Involved in the Long-Term Survival and the Virulence of Enterococcus faecalis

By coprecipitation, we identified RNA-binding proteins in the Gram-positive opportunistic pathogen Enterococcus faecalis known to be deficient of the RNA chaperone Hfq. In particular, we characterized one belonging to the cold shock protein (Csp) family (Ef2925) renamed CspR for cold shock protein RNA binding. Compared to the wild-type strain, the ΔcspR mutant was less virulent in an insect infection model (Galleria mellonella) and exhibited a decreased persistence in mouse kidneys and a low survival rate in peritoneal macrophages. As expected, we found that the ΔcspR mutant strain was more impaired in its growth than the parental strain under cold conditions and in its long-term survival under nutrient starvation. All these phenotypes were restored after complementation of the ΔcspR mutant. In addition, Western blot analysis showed that CspR was overexpressed under cold shock conditions and in the stationary phase. Since CspR may act as an RNA chaperone, putative targets were identified using a global proteomic approach completed with transcriptomic assays. This study revealed that 19 proteins were differentially expressed in the ΔcspR strain (9 upregulated, 10 downregulated) and that CspR mainly acted at the posttranscriptional level. These data highlight for the first time the role of the RNA-binding protein CspR as a regulator in E. faecalis and its requirement in stress response and virulence in this important human pathogen.

Expression of ferritin-like protein in Listeria monocytogenes after cold and freezing stress

The cold shock protein family consists of the transfer of the foodborne pathogen Listeria monocytogenes from 37 to 4 and −20 °C and was characterized by the sharp induction of a low molecular mass protein. This major cold shock protein ferritin-like protein (Flp) has an important role in regulation of various microbial physiological processes. Flp have a molecular mass of about 18 kDa, as observed on SDS–PAGE. The purification procedure including ammonium sulfate fractionation was used. Monospecific polyclonal antibodies raised in rabbits against the purified new Flp immunostained a single 18-kDa Flp band in extracts from different cytoplasmic proteins blotted onto nitrocellulose. A 411-bp cDNA fragment that corresponds to an internal region of an flp gene was obtained by RT-PCR. Our result indicated a surexpression of major cold shock protein and an important increase in flp mRNA amount after a downshift temperature especially at −20 °C.