Towards the mechanism(s) of YB-3 synthesis regulation by YB-1.

Y-box binding proteins are members of the family of proteins containing the evolutionarily conserved cold shock domain. Their cellular functions are quite diverse, including transcription and translation regulation, participation in pre-mRNA splicing, mRNA stabilization and packaging into mRNPs, involvement in DNA repair, and some others. To date, we know little about the plausible functional interchangeability of Y-box binding proteins. Our previous finding was that in YB-1-null HEK293T cells the synthesis of YB-3 is enhanced, thus enabling YB-3 to interact with a larger set of mRNAs and compensate for the YB-1 absence. We suggested the existence of a mechanism of YB-3 synthesis regulation by its paralog, YB-1. Here we demonstrate that YB-1 participates in the translational control and stabilization of YB-3 mRNA through untranslated regions of YB-3 mRNA.

Cold Shock Domain Proteins: Structure and Interaction with Nucleic Acids.

This review summarizes the features of cold shock domain (CSD) proteins in the context of their interactions with nucleic acids and describes similarities and differences in the structure of cold shock proteins of prokaryotes and CSD proteins of eukaryotes with special emphasis on the functions related to the RNA/DNA-binding ability of these proteins. The mechanisms and specificity of their interaction with nucleic acids in relation to the growing complexity of protein domain structure are described, as well as various complexes of the mammalian Y-box binding protein 1 (YB-1) with nucleic acids (filaments, globules, toroids). The role of particular amino acid residues in the binding of nitrogenous bases and the sugar-phosphate backbone of nucleic acids is emphasized. The data on the nucleic acid sequences recognized by the Y-box binding proteins are systematized. Post-translational modifications of YB-1, especially its phosphorylation, affect the recognition of specific sequences in the promoter regions of various groups of genes by YB-1 protein. The data on the interaction of Lin28 protein with let-7 miRNAs are summarized. The features of the domain structure of plant CSD proteins and their effect on the interaction with nucleic acids are discussed.

YB-3 substitutes YB-1 in global mRNA binding.

Y-box binding proteins are DNA- and RNA-binding proteins with an evolutionarily ancient and conserved cold shock domain. The Y-box binding protein 1 (YB-1) is the most studied due to its abundance in somatic cells. YB-1 is involved in a variety of cellular processes, including proliferation, differentiation and stress response. Here, using Ribo-Seq and RIP-Seq we confirm that YB-1 binds a wide range of mRNAs and globally acts as a translation inhibitor. Surprisingly, YBX1 knockout results in only minor alterations in the expression of other genes, mostly caused by changes in RNA abundance. But YB-3 mRNA is an exception: it is better translated in the absence of YB-1, thereby producing an increased amount of YB-3 and thus suggesting that its synthesis is under YB-1 negative control. We have shown that the set of mRNAs bound to YB-3 is strikingly similar to that of YB-1, and that the mRNA-binding by YB-3 is enhanced in the absence of YB-1, resulting in a similar global reduction of translation of bound mRNAs in YB-1-null cells. Thus, YB-3 acts as a substitute for YB-1 in mRNA binding and, probably, in global translational control.

Selective regulation of YB-1 mRNA translation by the mTOR signaling pathway is not mediated by 4E-binding protein.

The Y-box binding protein 1 (YB-1) is a key regulator of gene expression at the level of both translation and transcription. The mode of its action on cellular events depends on its subcellular distribution and the amount in the cell. So far, the regulatory mechanisms of YB-1 synthesis have not been adequately studied. Our previous finding was that selective inhibition of YB-1 mRNA translation was caused by suppression of activity of the mTOR signaling pathway. It was suggested that this event may be mediated by phosphorylation of the 4E-binding protein (4E-BP). Here, we report that 4E-BP alone can only slightly inhibit YB-1 synthesis both in the cell and in vitro, although it essentially decreases binding of the 4F-group translation initiation factors to mRNA. With inhibited mTOR kinase, the level of mRNA binding to the eIF4F-group factors was decreased, while that to 4E-BP1 was increased, as was observed for both mTOR kinase-sensitive mRNAs and those showing low sensitivity. This suggests that selective inhibition of translation of YB-1 mRNA, and probably some other mRNAs as well, by mTOR kinase inhibitors is not mediated by the action of the 4E-binding protein upon functions of the 4F-group translation initiation factors.

The Y-Box Binding Protein 1 Suppresses Alzheimer's Disease Progression in Two Animal Models.

The Y-box binding protein 1 (YB-1) is a member of the family of DNA- and RNA binding proteins. It is involved in a wide variety of DNA/RNA-dependent events including cell proliferation and differentiation, stress response, and malignant cell transformation. Previously, YB-1 was detected in neurons of the neocortex and hippocampus, but its precise role in the brain remains undefined. Here we show that subchronic intranasal injections of recombinant YB-1, as well as its fragment YB-11-219, suppress impairment of spatial memory in olfactory bulbectomized (OBX) mice with Alzheimer's type degeneration and improve learning in transgenic 5XFAD mice used as a model of cerebral amyloidosis. YB-1-treated OBX and 5XFAD mice showed a decreased level of brain ß-amyloid. In OBX animals, an improved morphological state of neurons was revealed in the neocortex and hippocampus; in 5XFAD mice, a delay in amyloid plaque progression was observed. Intranasally administered YB-1 penetrated into the brain and could enter neurons. In vitro co-incubation of YB-1 with monomeric ß-amyloid (1-42) inhibited formation of ß-amyloid fibrils, as confirmed by electron microscopy. This suggests that YB-1 interaction with ß-amyloid prevents formation of filaments that are responsible for neurotoxicity and neuronal death. Our data are the first evidence for a potential therapeutic benefit of YB-1 for treatment of Alzheimer's disease.

Innate immunity: Bacterial cell-wall muramyl peptide targets the conserved transcription factor YB-1.

The bacterial cell wall muramyl dipeptides MDP and glucosaminyl-MDP (GMDP) are powerful immunostimulators but their binding target remains controversial. We previously reported expression cloning of GMDP-binding polypeptides and identification of Y-box protein 1 (YB-1) as their sole target. Here we show specific binding of GMDP to recombinant YB-1 protein and subcellular colocalization of YB-1 and GMDP. GMDP binding to YB-1 upregulated gene expression levels of NF-κB2, a mediator of innate immunity. Furthermore, YB-1 knockdown abolished GMDP-induced Nfkb2 expression. GMDP/YB-1 stimulation led to NF-κB2 cleavage, transport of activated NF-κB2 p52 to the nucleus, and upregulation of NF-κB2-dependent chemokine Cxcr4 gene expression. Therefore, our findings identify YB-1 as new target for muramyl peptide signaling.

Y-box binding protein 1 (YB-1) promotes detection of DNA bulky lesions by XPC-HR23B factor.

The nucleotide excision repair system (NER) is one of the main mechanisms protecting cellular DNA from lesions caused by such significant environmental factors as UV radiation, the influence of polycyclic aromatic hydrocarbons, and medical treatment by several antitumor drugs, e.g. cisplatin. One of the major NER components is XPC-HR23B, the key factor during the damage recognition step of repair. Binding of XPC-HR23B to DNA that contains different bulky lesions impairing the structure of DNA is the basis for the wide substrate specificity of this DNA repair pathway. The multifunctional protein YB-1 among other protein factors has high affinity towards damaged DNA. Involvement of YB-1 in the cellular response to genotoxic stress and its ability to interact with damaged DNA harboring lesions of various origins pinpoint its putative involvement as a modulatory factor in DNA damage recognition and verification steps of NER. In the present work, we assayed functional interactions of protein factors XPC-HR23B and YB-1 upon binding to DNA structures mimicking damaged DNA containing single bulky lesions, as substrates of NER, and bulky lesions combined with abasic sites as an example of clustered lesions. The results indicate that YB-1 and XPC-HR23B stimulate each other in binding to DNA containing a bulky or clustered lesion, which suggests the involvement of YB-1 in the regulation of DNA repair by the NER mechanism.

Immunocytochemical study of YB-1 nuclear distribution in different cell types.

In the present work we studied the distribution of YB-1 in the nuclei of mouse hepatocytes, early embryos and human skin fibroblasts with the use of light and electron microscopy. To reveal YB-1, we applied rat polyclonal antibody against the C-terminal fragment of YB-1 molecule and rabbit polyclonal antibody against full-length YB-1 molecule. YB-1 distribution patterns varied significantly in different cell types. YB-1 was found to be colocalized with RNA polymerase I in mouse hepatocytes and embryos. Besides, YB-1 was revealed in a population of Cajal bodies in 2-cell mouse embryos but not in other cells studied.

[Connection of intracellular protein YB-1 localization in cell cultures of human tumors with multidrug resistance].

In this study, we investigated how the protein YB-1 influenced on the expression of genes coding ABC transporters and on drug resistance in several cell lines, in which originally gene MDR1, coding P-glycoprotein, was not expressed. These populations were significantly different in the presence of mRNA YB-1 and the nature of the intracellular localization of the protein YB-1. However incubation of cells in all studied populations in the culture medium with serum after starvation led to translocation of YB-1 in the cell nucleus. The increase of the number of cells with nuclear localization of YB-1 correlated with increased amount of mRNA YB-1. Processing of cells with drug LY-294,002 by PI3K/Akt inhibitor prevented the translocation of the protein YB-1 into the nuclei of cells, and the cells became more sensitive to the toxic action. Thus, we observed that the signaling pathways involved in control of cell proliferation, in particular a signaling cascade PI3K/Akt were involved in the control of the intracellular localization of YB-1 in cell populations of ovarian cancer, melanoma and human prostate cancer. In these cells the nuclear localization of YB-1 correlated with an expression of MDR and MRP1 DCRP genes and with a sensitivity of cells to a number of drugs.

Identification of proteins specifically interacting with YB-1 mRNA 3' UTR and the effect of hnRNP Q on YB-1 mRNA translation.

In this study, proteins specifically interacting with the 3' untranslated region (UTR) of mRNA of the multifunctional Y-box-binding protein 1 (YB-1) were identified. One of these, hnRNP Q, was shown to specifically interact with the regulatory element (RE) in YB-1 mRNA 3' UTR and to inhibit translation of this mRNA. Its binding to the RE was accompanied by displacement from this element of the poly(A)-binding protein (PABP), a positive regulator of YB-1 mRNA translation, and by enhanced binding of the negative YB-1 mRNA translation regulator - YB-1 itself.

Poly(A)-binding proteins: structure, domain organization, and activity regulation.

RNA-binding proteins are of vital importance for mRNA functioning. Among these, poly(A)-binding proteins (PABPs) are of special interest due to their participation in virtually all mRNA-dependent events that is caused by their high affinity for A-rich mRNA sequences. Apart from mRNAs, PABPs interact with many proteins, thus promoting their involvement in cellular events. In the nucleus, PABPs play a role in polyadenylation, determine the length of the poly(A) tail, and may be involved in mRNA export. In the cytoplasm, they participate in regulation of translation initiation and either protect mRNAs from decay through binding to their poly(A) tails or stimulate this decay by promoting mRNA interactions with deadenylase complex proteins. This review presents modern notions of the role of PABPs in mRNA-dependent events; peculiarities of regulation of PABP amount in the cell and activities are also discussed.

Y-box-binding protein 1 (YB-1) and its functions.

This review describes the structure and functions of Y-box binding protein 1 (YB-1) and its homologs. Interactions of YB-1 with DNA, mRNAs, and proteins are considered. Data on the participation of YB-1 in DNA reparation and transcription, mRNA splicing and translation are systematized. Results on interactions of YB-1 with cytoskeleton components and its possible role in mRNA localization are discussed. Data on intracellular distribution of YB-1, its redistribution between the nucleus and the cytoplasm, and its secretion and extracellular functions are summarized. The effect of YB-1 on cell differentiation, its involvement in extra- and intracellular signaling pathways, and its role in early embryogenesis are described. The mechanisms of regulation of YB-1 expression in the cell are presented. Special attention is paid to the involvement of YB-1 in oncogenic cell transformation, multiple drug resistance, and dissemination of tumors. Both the oncogenic and antioncogenic activities of YB-1 are reviewed. The potential use of YB-1 in diagnostics and therapy as an early cancer marker and a molecular target is discussed.

YB-1 is capable of forming extended nanofibrils.

Here we are the first to report that multifunctional Y-box binding protein 1 (YB-1) forms extended fibrils with a diameter of 15-20 nm. The YB-1 fibrils were visualized by atomic force and electron microscopy after 1-h incubation in solution with 2 M LiCl. Their length grew with incubation time and could exceed 10 microm; their shape is helical or zigzag-like. They possess polarity and tend to associate with one another to give structures of a higher order, like ribbons or bundles. The YB-1 fibrillar architecture has a distinct periodicity with a repeat unit of about 52 nm.

[Studies of YB-1 protein in breast tumors].

The multifunctional mammalian protein YB-1 is involved in multiple DNA- and mRNA-dependent events in the cell and regulates gene expression at different levels. The intracellular localization and relative mRNA content of YB-1 in the breast tumors were studied. The presence of cells with nuclear YB-1 localization in the tumor cell population is a poor predictor that correlates with larger tumors (more than 5 cm). The high YB-1 mRNA content in the breast tumors promotes metastasis of small neoplasms and patients with breast cancer who have high tumor tissue YB-1 mRNA levels may referred to as an early distant metastasis risk group. The findings suggest that combined determination of YB-1 intercellular localization and mRNA levels can ensure a more reliable prognosis of breast cancer.

Proteasome system of protein degradation and processing.

In eukaryotic cells, degradation of most intracellular proteins is realized by proteasomes. The substrates for proteolysis are selected by the fact that the gate to the proteolytic chamber of the proteasome is usually closed, and only proteins carrying a special "label" can get into it. A polyubiquitin chain plays the role of the "label": degradation affects proteins conjugated with a ubiquitin (Ub) chain that consists at minimum of four molecules. Upon entering the proteasome channel, the polypeptide chain of the protein unfolds and stretches along it, being hydrolyzed to short peptides. Ubiquitin per se does not get into the proteasome, but, after destruction of the "labeled" molecule, it is released and labels another molecule. This process has been named "Ub-dependent protein degradation". In this review we systematize current data on the Ub-proteasome system, describe in detail proteasome structure, the ubiquitination system, and the classical ATP/Ub-dependent mechanism of protein degradation, as well as try to focus readers' attention on the existence of alternative mechanisms of proteasomal degradation and processing of proteins. Data on damages of the proteasome system that lead to the development of different diseases are given separately.

Nucleocytoplasmic transport of proteins.

In eukaryotic cells, the movement of macromolecules between the nucleus and cytoplasm occurs through the nuclear pore complex (NPC)--a large protein complex spanning the nuclear envelope. The nuclear transport of proteins is usually mediated by a family of transport receptors known as karyopherins. Karyopherins bind to their cargoes via recognition of nuclear localization signal (NLS) for nuclear import or nuclear export signal (NES) for export to form a transport complex. Its transport through NPC is facilitated by transient interactions between the karyopherins and NPC components. The interactions of karyopherins with their cargoes are regulated by GTPase Ran. In the current review, we describe the NPC structure, NLS, and NES, as well as the model of classic Ran-dependent transport, with special emphasis on existing alternative mechanisms; we also propose a classification of the basic mechanisms of protein transport regulation.

Development of drug resistance in the population of colon cancer cells under the effect of multifunctional protein YB-1.

The effects of YB-1 gene on the expression level of P-glycoprotein and drug resistance of tumor cells were studied in cultured HCT116 colon cancer cells. Transitory transfection of chimeric YB-1/GFP gene rendered HCT116 cells a selective advantage in a medium with vinblastine, which caused translocation of the chimeric protein into cell nuclei. This was paralleled by an increase in the expression of P-glycoprotein (multiple drug resistance protein).

[Nonspecific and specific interaction of Y-box binding protein 1 (YB-1) with mRNA and posttranscriptional regulation of protein synthesis in animal cells].

YB-1 is a multifunctional DNA/RNA-binding protein of animal cells that participates in reproduction, storing and expression of genetic information. This protein displays high nonspecific and specific affinity for RNA and accompanies mRNA during its entire lifetime, from synthesis to decay. In the nucleus, YB-1 regulates mRNA processing. In the cytoplasm, it is responsible for the global and selective regulation of protein synthesis and for mRNA life span. This review is mostly focused on the role played by YB-1 in regulation of translation, and specifically, on possible mechanisms of its positive and negative effects. Evidence based on our recent results is provided for YB-1 acting as the major structural component of mRNPs and for its participating, together with mRNA, in formation of these particles. Also, we first present an overall detailed description of specific interaction between YB-1 and RNA.

Intracellular localization and content of YB-1 protein in multidrug resistant tumor cells.

The multifunctional mammalian protein YB-1 is a member of the large DNA- and RNA-binding protein family with an evolutionarily ancient cold-shock domain. YB-1 is involved in multiple DNA- and mRNA-dependent events and regulates gene expression at various levels. It can be found both in the nucleus and the cytoplasm. Bound to DNA in the cell nucleus, YB-1 functions as a transcription factor interacting with inverted CCAAT-box (Y-box) in promoters and enhancers of multiple genes. In particular, YB-1 regulates activity of the multidrug resistance (MDR) genes MDR1 and LRP. In tumors, YB-1 has been suggested to be an early and global marker of MDR. In this study, we compared amounts of YB-1 mRNAs and intracellular localization of YB-1 protein in six pairs of drug sensitive and drug resistant sublines of diverse tumors. We have shown that neither great increase in the level of YB-1 mRNA nor substantial increase in the number of cells with nuclear localization of YB-1 are obligatory traits of drug resistant tumor cell populations. However, the cells with highest amounts of YB-1 mRNA also demonstrated increased quantities of MDR1, MRP1, BCRP, and LRP mRNAs encoding different MDR proteins. Transfection of two different populations of drug-sensitive cells with YB-1 cDNA led to increase in the amount of YB-1 mRNA. The quantities of MRP1 and LRP mRNAs increased in both populations. Introduction of YB-1 small hairpin RNA (shRNA) resulted in decreased amounts of YB-1 mRNA, as well as MRP1, LRP, and MDR1 mRNAs (in three different cell lines). Our data suggest that although YB-1 regulates several MDR genes, it could not be regarded as a global marker of already formed drug resistant tumor cell populations. It is most likely that at the first steps of MDR development YB-1 activity is necessary for propagation of resistant cell populations rather than for maintenance of drug resistance.