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The magnetic field is the most common element in the universe, and high static magnetic field (HiSMF) has been reported to act as an inhibited factor for osteoclasts differentiation. Although many studies have indicated the negative role of HiSMF on osteoclastogenesis of RANKL-induced RAW264.7 cells, the molecular mechanism is still elusive. In this study, the HiSMF-retarded cycle and weakened differentiation of RAW264.7 cells was identified. Through RNA-seq analysis, RANKL-induced RAW264.7 cells under HiSMF were analysed, and a total number of 197 differentially expressed genes (DEGs) were discovered. Gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that regulators of cell cycle and cell division such as Bub1b, Rbl1, Ube2c, Kif11, and Nusap1 were highly expressed, and CtsK, the marker gene of osteoclastogenesis was downregulated in HiSMF group. In addition, pathways related to DNA replication, cell cycle, and metabolic pathways were significantly inhibited in the HiSMF group compared to the Control group. Collectively, this study describes the negative changes occurring throughout osteoclastogenesis under 16 T HiSMF treatment from the morphological and molecular perspectives. Our study provides information that may be utilized in improving magnetotherapy on bone disease.

Cell cycle gene expression programs fuel proliferation and are universally dysregulated in cancer. The retinoblastoma (RB)-family of proteins, RB1, RBL1/p107, and RBL2/p130, coordinately represses cell cycle gene expression, inhibiting proliferation, and suppressing tumorigenesis. Phosphorylation of RB-family proteins by cyclin-dependent kinases is firmly established. Like phosphorylation, ubiquitination is essential to cell cycle control, and numerous proliferative regulators, tumor suppressors, and oncoproteins are ubiquitinated. However, little is known about the role of ubiquitin signaling in controlling RB-family proteins. A systems genetics analysis of CRISPR/Cas9 screens suggested the potential regulation of the RB-network by cyclin F, a substrate recognition receptor for the SCF family of E3 ligases. We demonstrate that RBL2/p130 is a direct substrate of SCFcyclin F. We map a cyclin F regulatory site to a flexible linker in the p130 pocket domain, and show that this site mediates binding, stability, and ubiquitination. Expression of a mutant version of p130, which cannot be ubiquitinated, severely impaired proliferative capacity and cell cycle progression. Consistently, we observed reduced expression of cell cycle gene transcripts, as well a reduced abundance of cell cycle proteins, analyzed by quantitative, iterative immunofluorescent imaging. These data suggest a key role for SCFcyclin Fin the CDK-RB network and raise the possibility that aberrant p130 degradation could dysregulate the cell cycle in human cancers.

Cell cycle gene expression programs fuel proliferation and are dysregulated in many cancers. The retinoblastoma-family proteins, RB, p130/RBL2 and p107/RBL1, coordinately repress cell cycle gene expression, inhibiting proliferation and suppressing tumorigenesis. Ubiquitin-dependent protein degradation is essential to cell cycle control, and numerous proliferative regulators, tumor suppressors, and oncoproteins are ubiquitinated. However, little is known about the role of ubiquitin signaling in controlling RB-family proteins. A systems genetics analysis of several hundred CRISPR/Cas9 loss-of-function screens suggested the potential regulation of the RB-network by cyclin F, a substrate recognition receptor for the SCF family of E3 ligases. We demonstrate that RBL2/p130 is a direct substrate of SCFcyclin F. We map a cyclin F regulatory site to a flexible linker in the p130 pocket domain, and show that this site mediates binding, stability, and ubiquitination. Expression of a non-degradable p130 represses cell cycle gene expression and strongly reduces proliferation. These data suggest that SCFcyclin Fplays a key role in the CDK-RB network and raises the possibility that aberrant p130 degradation could dysregulate the cell cycle in human cancers.

High-grade neuroendocrine lung carcinomas (LCNEC, SCLC) are recalcitrant cancers for which no optimal management has been achieved. We have recently described two models of LCNEC and SCLC developed upon inactivation of 4 tumor suppressors genes (Rb1 (RB transcriptional corepressor 1), Rbl1 (RB transcriptional corepressor like 1), Pten (phosphatase and tensin homolog), Trp53 (transformation-related protein 53), which provide a suitable frame for preclinical intervention. A defined model for LCNEC had not been previously reported.

The RACK1 protein interacts with numerous proteins involved in signal transduction, the cytoskeleton, and mRNA splicing and translation. We used the 2-hybrid system to identify additional proteins interacting with RACK1 and isolated the RNA-binding protein SERBP1. SERPB1 shares amino acid sequence homology with HABP4 (also known as Ki-1/57), a component of the RNA spicing machinery that has been shown previously to interact with RACK1. Several different isoforms of SERBP1, generated by alternative mRNA splicing, interacted with RACK1 with indistinguishable interaction strength, as determined by a 2-hybrid beta-galactosidase assay. Analysis of deletion constructs of SERBP1 showed that the C-terminal third of the SERBP1 protein, which contains one of its two substrate sites for protein arginine N-methyltransferase 1 (PRMT1), is necessary and sufficient for it to interact with RACK1. Analysis of single amino acid substitutions in RACK1, identified in a reverse 2-hybrid screen, showed very substantial overlap with those implicated in the interaction of RACK1 with the cAMP-selective phosphodiesterase PDE4D5. These data are consistent with SERBP1 interacting selectively with RACK1, mediated by an extensive interaction surface on both proteins.

Reversible protein phosphorylation catalyzed by protein kinases and phosphatases represents the most prolific and well-characterized posttranslational modification known. Here, we demonstrate that Arabidopsis (Arabidopsis thaliana) Shewanella-like protein phosphatase 2 (AtSLP2) is a bona fide Ser/Thr protein phosphatase that is targeted to the mitochondrial intermembrane space (IMS) where it interacts with the mitochondrial oxidoreductase import and assembly protein 40 (AtMIA40), forming a protein complex. Interaction with AtMIA40 is necessary for the phosphatase activity of AtSLP2 and is dependent on the formation of disulfide bridges on AtSLP2. Furthermore, by utilizing atslp2 null mutant, AtSLP2 complemented and AtSLP2 overexpressing plants, we identify a function for the AtSLP2-AtMIA40 complex in negatively regulating gibberellic acid-related processes during seed germination. Results presented here characterize a mitochondrial IMS-localized protein phosphatase identified in photosynthetic eukaryotes as well as a protein phosphatase target of the highly conserved eukaryotic MIA40 IMS oxidoreductase.

Alterations of the retinoblastoma and/or the p53 signaling network are associated with specific cancers such as high-grade astrocytoma/glioblastoma, small-cell lung cancer (SCLC), choroid plexus tumors, and small-cell pancreatic neuroendocrine carcinoma (SC-PaNEC). However, the intricate functional redundancy between RB1 and the related pocket proteins RBL1/p107 and RBL2/p130 in suppressing tumorigenesis remains poorly understood. Here we performed lineage-restricted parallel inactivation of rb1 and rbl1 by multiplex CRISPR/Cas9 genome editing in the true diploid Xenopus tropicalis to gain insight into this in vivo redundancy. We show that while rb1 inactivation is sufficient to induce choroid plexus papilloma, combined rb1 and rbl1 inactivation is required and sufficient to drive SC-PaNEC, retinoblastoma and astrocytoma. Further, using a novel Li-Fraumeni syndrome-mimicking tp53 mutant X. tropicalis line, we demonstrate increased malignancy of rb1/rbl1-mutant glioma towards glioblastoma upon concomitant inactivation of tp53. Interestingly, although clinical SC-PaNEC samples are characterized by abnormal p53 expression or localization, in the current experimental models, the tp53 status had little effect on the establishment and growth of SC-PaNEC, but may rather be essential for maintaining chromosomal stability. SCLC was only rarely observed in our experimental setup, indicating requirement of additional or alternative oncogenic insults. In conclusion, we used CRISPR/Cas9 to delineate the tumor suppressor properties of Rbl1, generating new insights in the functional redundancy within the retinoblastoma protein family in suppressing neuroendocrine pancreatic cancer and glioma/glioblastoma.

Alterations of the retinoblastoma and/or the p53 signaling network are associated with specific cancers such as high-grade astrocytoma/glioblastoma, small cell lung cancer (SCLC), choroid plexus tumors and small-cell pancreatic neuroendocrine carcinoma (SC-PaNEC). However, the intricate functional compensation between RB1 and the related pocket proteins RBL1/p107 and RBL2/p130 in suppressing tumorigenesis remains poorly understood. Here we performed lineage-restricted parallel inactivation ofrb1andrbl1by multiplex CRISPR/Cas9 genome editing in the true diploidXenopus tropicalisto gain insight into thesein vivocompensatory mechanisms. We show that whilerb1inactivation is sufficient to induce choroid plexus papilloma, combinedrb1andrbl1inactivation is required and sufficient to drive SC-PaNEC, retinoblastoma and astrocytoma. Further, using a novel Li-Fraumeni syndrome-mimickingtp53mutantX. tropicalisline, we demonstrate increased malignancy of retinoblastoma-mutant neural malignancies upon concomitant inactivation oftp53. Interestingly, although clinical SC-PaNEC samples are characterized by abnormal p53 expression or localization, in the current experimental models, thetp53status had little effect on the establishment and growth of SC-PaNEC, but may rather be essential for maintaining chromosomal stability. SCLC was only rarely observed in our experimental set-up, indicating requirement of additional or alternative oncogenic insults. In conclusion, we used CRISPR/Cas9 to delineate the tumor suppressor properties of Rbl1 and generate new insights in functional compensation within the retinoblastoma protein family in suppressing pancreatic and specific neural cancers.

High mammographic density (MD) is associated with a 4–6 times increase in breast cancer risk. For post-menopausal women,MD often decreases over time, but little is known about the underlying biological mechanisms. MD reflects breast tissuecomposition, and may be associated with microenvironment subtypes previously identified in tumor-adjacent normal tissue. Currently, these subtypes have not been explored in normal breast tissue. We obtained biopsies from breasts of healthy women at two different time points several years apart and performed microarray gene expression analysis. At time point 1, 65 samples with both MD and gene expression were available. At time point 2, gene expression and MD data were available from 17 women, of which 11 also had gene expression data available from the first time point. We validated findings from our previous study; negative correlation between RBL1 and MD in post-menopausal women, indicating involvement of the TGFβ pathway. We also found that breast tissue samples from women with a large decrease in MD sustained higher expression of genes in the histone family H4. In addition, we explored the previously defined active and inactive microenvironment subtypes and demonstrated that normal breast samples of the active subtype had characteristics similar to the claudin-low breast cancer subtype. Breast biopsies from healthy women are challenging to obtain, but despite a limited sample size, we have identified possible mechanisms relevant for changes in breast biology and MD over time that may be of importance for breast cancer risk and tumor initiation. publishedVersion

Hair follicle (HF) development is characterized by periodic growth cycles regulated by numerous factors. We previously showed that SMAD2 might be involved in the HF growth cycle in Angora rabbits. However, its extra role in the HF growth and development remains obscure. In this study, we cloned the complete coding sequence (CDS) of the Angora rabbit SMAD2 gene. Within SMAD2 CDS, we identified the open reading frame (ORF) had a length of 1314 bp and encoding 437 amino acids. Bioinformatics analyses revealed that the SMAD2 protein is unstable and hydrophilic, and predominately localizes in the cell nucleus. We identified that SMAD2 expression was elevated in the telogen phase of the during HF cycle. The knockdown and overexpression of SMAD2 could regulate HF growth and development related genes, such as WNT2, FGF2, and LEF1.Furthermore, SMAD2 may upregulate TGF-β signaling pathway-related genes, including TFDP1, E2F4, and RBL1. In conclusion, our results indicate that SMAD2 plays a vital role in HF development by regulating the TGF-β signaling pathway.

Since Green Fluorescent Protein (GFP) was first successfully expressed in heterologous systems in 1994, many genes encoding other natural autofluorescent proteins (AFPs) have been cloned and subsequently modified by protein engineering to improve their physicochemical properties. Throughout this twenty-two-year period, glycine 67 (Gly67) has been regarded as the only amino acid in the entire protein family that is essential for the formation of the different reported chromophores. In this work, we demonstrate that a synthetic gene encoding LanFP10-A, a natural protein encoded in the genome of the lancelet Branchiostoma floridae containing the G67A mutation, produces a heterologous, functional yellow fluorescent protein when expressed in E. coli. In contrast to LanFP10-A, LanFP6-A, a second GFP-like protein found in the lancelet genome that also contains the natural G67A mutation, was non-fluorescent.

// Clotilde Costa 1, 3 , Mirentxu Santos 1, 2 , Monica Martinez-Fernandez 1, 2 , Corina Lorz 1, 2 , Sara Lazaro 1 , Jesus M. Paramio 1, 2 1 Unidad de Oncologia Molecular, CIEMAT (ed70A), 28040 Madrid, Spain 2 Unidad de Oncologia Molecular y Celular, Instituto de Investigaciones Biomed, Hospital Universitario 12 de Octubre, 28041 Madrid, Spain 3 Present address: Unidad Mixta Roche-Chus, Hospital Universitario, 15706 Santiago de Compostela, Spain Correspondence to: Jesus M. Paramio, email: jesusm.paramio@ciemat.es Mirentxu Santos, email: mirentxu.santos@ciemat.es Keywords: epidermis, Rb, E2F4, mouse, cancer Received: July 16, 2016 Accepted: September 19, 2016 Published: September 30, 2016 ABSTRACT E2F/RB activity is altered in most human tumors. The retinoblastoma family of proteins plays a key role in regulating the progression of the cell cycle from the G1 to S phases. This is achieved through negative regulation of E2F transcription factors, important positive regulators of cell cycle entry. E2F family members are divided into two groups: activators (E2F1-E2F3a) and repressors (E2F3b-E2F8). E2F4 accounts for a large part of the E2F activity and is a main E2F repressor member in vivo . Perturbations in the balance from quiescence towards proliferation contribute to increased mitotic gene expression levels frequently observed in cancer. We have previously reported that combined Rb1 - Rbl1 or Rb1 - E2f1 ablation in epidermis produces important alterations in epidermal proliferation and differentiation, leading to tumor development. However, the possible roles of E2F4 in this context are still to be determined. Here, we show the absence of any discernible phenotype in the skin of mice lacking of E2f4 . In contrast, the inducible loss of Rb1 in the epidermis of E2F4-null mice produced multiple skin abnormalities including altered differentiation and proliferation, spontaneous wounds, carcinoma in situ development and stem cell perturbations. All these phenotypic alterations are associated with extensive gene expression changes, the induction of c-myc and the Akt activation. Moreover the whole transcriptome analyses in comparison with previous models generated also revealed extensive changes in multiple repressive complexes and in transcription factor activity. These results point to E2F4 as a master regulator in multiple steps of epidermal homeostasis in Rb1 absence.

Viral proteins mimic host protein structure and function to redirect cellular processes and subvert innate defenses. Small basic proteins compact and regulate both viral and cellular DNA genomes. Nucleosomes are the repeating units of cellular chromatin and play an important part in innate immune responses. Viral-encoded core basic proteins compact viral genomes, but their impact on host chromatin structure and function remains unexplored. Adenoviruses encode a highly basic protein called protein VII that resembles cellular histones. Although protein VII binds viral DNA and is incorporated with viral genomes into virus particles, it is unknown whether protein VII affects cellular chromatin. Here we show that protein VII alters cellular chromatin, leading us to hypothesize that this has an impact on antiviral responses during adenovirus infection in human cells. We find that protein VII forms complexes with nucleosomes and limits DNA accessibility. We identified post-translational modifications on protein VII that are responsible for chromatin localization. Furthermore, proteomic analysis demonstrated that protein VII is sufficient to alter the protein composition of host chromatin. We found that protein VII is necessary and sufficient for retention in the chromatin of members of the high-mobility-group protein B family (HMGB1, HMGB2 and HMGB3). HMGB1 is actively released in response to inflammatory stimuli and functions as a danger signal to activate immune responses. We showed that protein VII can directly bind HMGB1 in vitro and further demonstrated that protein VII expression in mouse lungs is sufficient to decrease inflammation-induced HMGB1 content and neutrophil recruitment in the bronchoalveolar lavage fluid. Together, our in vitro and in vivo results show that protein VII sequesters HMGB1 and can prevent its release. This study uncovers a viral strategy in which nucleosome binding is exploited to control extracellular immune signaling.

Tumor suppressor protein p53 plays important roles in initiating cell cycle arrest and promoting tumor cell apoptosis. Previous studies have shown that p53 is either mutated or defective in approximately 50% of human cancers; therefore restoring normal p53 activity in cancer cells might be an effective anticancer therapeutic approach. Herein, we designed a chimeric p53 protein flanked with the MyoD N-terminal transcriptional activation domain (amino acids 1-62, called M3) and a poly-arginine (R12) cell penetrating signal in its N-and C-termini respectively. This chimeric protein, M3-p53-R12, can be expressed in E. coli and purified using immobilized metal ion chromatography followed by serial refolding dialysis. The purified M3-p53-R12 protein retains DNA-binding activity and gains of cell penetrating ability. Using MTT assay, we demonstrated that M3-p53-R12 inhibited the growth of K562, Jurkat as well as HL-60 leukemia cells carrying mutant p53 genes. Results from FACS analysis also demonstrated that transduction of M3-p53-R12 protein induced cell cycle arrest of these leukemia cells. Of special note, M3-p53-R12 has no apoptotic effect on normal mesenchymal stem cells (MSC) and leukocytes, highlighting its differential effects on normal and tumor cells. To sum up, our results reveal that purified recombinant M3-p53-R12 protein has functions of suppressing the leukemia cell lines' proliferation and launching cell apoptosis, suggesting the feasibility of using M3-p53-R12 protein as an anticancer drug. In the future we will test whether this chimeric protein can preferentially trigger the death of malignant cancer cells without affecting normal cells in animals carrying endogenous or xenographic tumors.

It is now known that multicomponent protein assemblies strictly regulate many protein functions. The S100 protein family is known to play various physiological roles, which are associated with alternative complex formations. To prepare sufficient amounts of heterodimeric S100A8 and S100A9 proteins, we developed a method for bicistronic coexpression from a single-vector system using Escherichia coli cells as a host. The complex formation between S100A8 and S100A9 appears to be dependent on the thermodynamic stability of the protein during expression. The stable S100A8/A9 heterodimer complex spontaneously formed during coexpression, and biologically active samples were purified by cation-exchange chromatography. Semi-stable homodimers of S100A8 and S100A9 were also formed when expressed individually. These results suggest that the assembly of S100 protein complexes might be regulated by expression levels of partner proteins in vivo. Because protein assembly occurs rapidly after protein synthesis, coexpression of relevant proteins is crucial for the design of multicomponent recombinant protein expression systems., Graphical abstract fx1, Highlights • S100A8/A9 heterodimer efficiently formed by coexpression in E. coli. • S100A8/A9 heterodimer preferentially assembled as compared to homodimer. • Simplified purification procedure for recombinant S100A8/A9 without fusion of affinity tag. • Convenient recombination method for construction of coexpression vector.

Cancer is a dreadful disease constituting abnormal growth and proliferation of malignant cells in the body. Next to lung cancer, breast cancer is the most common form of cancer affecting women. The apoptotic pathway regulators, B cell lymphoma family of protein, play a key role in various malignancies defining cancer and their constitutive expression plays an integral role in breast cancer chemotherapy. The research work discusses the identification and molecular cloning of a B cell lymphoma like gene from human breast cancer cell line. The open reading frame of the gene consisted of 965 nucleotides, encoding a protein of 380 amino acids with a predicted molecular weight of 42.5 kilodalton. The predicted physiochemical properties of the gene were as follows: Isoelectric point - 9.49, molecular formula - C1893H3004N534O548S16, total number of negatively charged residues, (Aspartate+Glutamate) - 26, total number of positively charged residues, (Arginine+Lysine)-39, instability index-42.08 (unstable protein) and grand average of hydropathicity is -0.202. Additionally, phobius prediction suggested non-cytoplasmic localization of the putative protein. The presence of secondary structure in the protein was determined by Memsat program. A 3 dimensional protein homology model was generated using threading based method of protein modeling for structural and functional annotation of the putative protein. Future prospects accounts for the biochemical characterization of the enzyme including in vitro assays on breast cancer cell line would establish the functional characteristics of the protein and its physiological mechanisms in breast cancer development and its therapeutic-target role in future.

T-complex protein 10A homolog 2 (TCP10L) was previously demonstrated to be a potential tumor suppressor in human hepatocellular carcinoma (HCC). However, little is known about the molecular mechanism. MAX dimerization protein 1 (MAD1) is a key transcription suppressor that is involved in regulating cell cycle progression and Myc-mediated cell transformation. In this study, we identified MAD1 as a novel TCP10L-interacting protein. The interaction depends on the leucine zipper domain of both TCP10L and MAD1. TCP10L, but not the interaction-deficient TCP10L mutant, synergizes with MAD1 in transcriptional repression, cell cycle G1 arrest and cell growth suppression. Mechanistic exploration further revealed that TCP10L is able to stabilize intracellular MAD1 protein level. Consistently, the MAD1-interaction-deficient TCP10L mutant exerts no effect on stabilizing the MAD1 protein. Taken together, our results strongly indicate that TCP10L stabilizes MAD1 protein level through direct interaction, and they cooperatively regulate cell cycle progression. [BMB Reports 2016; 49(6): 325-330].

DNA damage binding protein 2 (DDB2) is a protein involved in the early step of DNA damage recognition of the nucleotide excision repair (NER) process. Recently, it has been suggested that DDB2 may play a role in DNA replication, based on its ability to promote cell proliferation. We have previously shown that DDB2 binds PCNA during NER, but also in the absence of DNA damage; however, whether and how this interaction influences cell proliferation is not known. In this study, we have addressed this question by using HEK293 cell clones stably expressing DDB2(Wt) protein, or a mutant form (DDB2(Mut)) unable to interact with PCNA. We report that overexpression of the DDB2(Mut) protein provides a proliferative advantage over the wild type form, by influencing cell cycle progression. In particular, an increase in the number of S-phase cells, together with a reduction in p21(CDKN1A) protein level, and a shorter cell cycle length, has been observed in the DDB2(Mut) cells. These results suggest that DDB2 influences cell cycle progression thanks to its interaction with PCNA.

Human papillomavirus (HPV) is a necessary but insufficient cause of a subset of oral squamous cell carcinomas (OSCCs) that is increasing markedly in frequency. To identify contributory, secondary genetic alterations in these cancers, we used comprehensive genomics methods to compare 149 HPV-positive and 335 HPV-negative OSCC tumor/normal pairs. Different behavioral risk factors underlying the two OSCC types were reflected in distinctive genomic mutational signatures. In HPV-positive OSCCs, the signatures of APOBEC cytosine deaminase editing, associated with anti-viral immunity, were strongly linked to overall mutational burden. In contrast, in HPV-negative OSCCs, T>C substitutions in the sequence context 5′-ATN-3′ correlated with tobacco exposure. Universal expression of HPV E6*1 and E7 oncogenes was a sine qua non of HPV-positive OSCCs. Significant enrichment of somatic mutations was confirmed or newly identified in PIK3CA, KMT2D, FGFR3, FBXW7, DDX3X, PTEN, TRAF3, RB1, CYLD, RIPK4, ZNF750, EP300, CASZ1, TAF5, RBL1, IFNGR1, and NFKBIA. Of these, many affect host pathways already targeted by HPV oncoproteins, including the p53 and pRB pathways, or disrupt host defenses against viral infections, including interferon (IFN) and nuclear factor kappa B signaling. Frequent copy number changes were associated with concordant changes in gene expression. Chr 11q (including CCND1) and 14q (including DICER1 and AKT1) were recurrently lost in HPV-positive OSCCs, in contrast to their gains in HPV-negative OSCCs. High-ranking variant allele fractions implicated ZNF750, PIK3CA, and EP300 mutations as candidate driver events in HPV-positive cancers. We conclude that virus-host interactions cooperatively shape the unique genetic features of these cancers, distinguishing them from their HPV-negative counterparts.

The retinoblastoma (RB) protein family includes RB1/p105, RBL1/p107, and RBL2/p130, which are key factors in cell-cycle regulation and stand at the crossroads of multiple pathways dictating cell fate decisions. The role of RB proteins in apoptosis is controversial because they can inhibit or promote apoptosis depending on the context, on the apoptotic stimuli and on their intrinsic status, impacting on the response to antitumoral treatments. Here we identified RBL2/p130 as a direct substrate of the AKT kinase, a key antiapoptotic factor hyperactive in multiple cancer types. We showed that RBL2/p130 and AKT1 physically interact and AKT phosphorylates RBL2/p130 Ser941, located in the pocket domain, but not when this residue is mutated into Ala. We found that pharmacological inhibition of AKT, through the highly selective AKT inhibitor VIII (AKTiVIII), impairs RBL2/p130 Ser941 phosphorylation and increases RBL2/p130 stability, mRNA expression and nuclear levels in both lung cancer and mesothelioma cell lines, mirroring the more extensively studied effects on the p27 cell-cycle inhibitor. Consistently, AKT inhibition reduced cell viability, induced cell accumulation in G0/G1, and triggered apoptosis, which proved to be largely dependent on RBL2/p130 itself, as shown upon RBL2/p130 silencing. AKT inhibition induced RBL2/p130-dependent apoptosis also in HEK-293 cells, in which re-expression of a short hairpin-resistant RBL2/p130 was able to rescue AKTiVIII-induced apoptosis upon RBL2/p130 silencing. Our data also showed that the combination of AKT and cyclin-dependent kinases (CDK) inhibitors, which converge on the re-activation of RBL2/p130 antitumoral potential, could be a promising anticancer strategy.

Tumor necrosis factor alpha (TNFα)-induced protein 1 (TNFAIP1) was originally identified as a protein involved in DNA replication, DNA damage repair, apoptosis and the progression of certain diseases, such as Alzheimer's disease. In the present study, forskolin, a stimulant of cyclic adenosine monophosphate (cAMP), was found to significantly reduce human TNFAIP1 mRNA levels and TNFAIP1 promoter activity in the SKNSH human neuroblastoma cell line as indicated by polymerase chain reaction analysis and a luciferase reporter assay. The association between transcription factor cAMP response element‑binding protein (CREB) and TNFAIP1 was further investigated using loss- and gain of function-studies with western blot analysis and luciferase reporter assays. The CREB-specific inhibitor KG‑501 significantly increased TNFAIP1 protein levels, while overexpression of wild‑type CREB, but not CREB mutated at ser133a or its DNA-binding site, significantly decreased human TNFAIP1 protein levels and TNFAIP1 promoter activity in SKNSH cells. Furthermore, two CRE sites located at ‑285 and ‑425 bp of the human TNFAIP1 promoter were identified to be responsible for CREB‑induced inhibition of human TNFAIP1 promoter activity. Chromatin immunoprecipitation assays confirmed that CREB bound to the TNFAIP1 promoter region harboring these two CRE sites. A further luciferase reporter assay demonstrated that CREB phosphorylation on ser133 was responsible for forskolin‑induced inhibition of TNFAIP1 expression. In conclusion, the present study suggested that CREB is a negative regulator of the TNFAIP1 gene.

Porcine circovirus type 2 (PCV2) is the primary infectious agent of PCV-associated disease (PCVAD) in swine. ORF4 protein is a newly identified viral protein of PCV2 and is involved in virus-induced apoptosis. However, the molecular mechanisms of ORF4 protein regulation of apoptosis remain unclear, especially given there is no information regarding any cellular partners of the ORF4 protein. Here, we have utilized the yeast two-hybrid assay and identified four host proteins (FHC, SNRPN, COX8A and Lamin C) interacting with the ORF4 protein. Specially, FHC was chosen for further characterization due to its important role in apoptosis. GST pull-down, subcellular co-location and co-immunoprecipitation assays confirmed that the PCV2 ORF4 protein indeed interacted with the heavy-chain ferritin, which is an interesting clue that will allow us to determine the role of the ORF4 protein in apoptosis.

BRCA1 is a well established tumor suppressor gene, which is involved in many cellular processes, including DNA damage repair, cell cycle control, apoptosis, as well as transcriptional control. In this work, we have found that BRCA1 is involved in regulating TGF-β1/Smad pathway. The loss of endogenous BRCA1 greatly attenuated TGF-β1-induced growth inhibition and cell cycle G1 arrest. BRCA1 greatly maintains stability of Smad4 protein, and the loss of BRCA1 results in Smad4 down-regulation, which is likely related to its downstream gene Gadd45a. Gadd45a is able to interact with β-Trcp1, a-F-box protein of SCF E3 ligase, and consequently suppresses the ubiquitin-degradation of Smad4 by SCF(β-trcp1), as reflected by the observations that the induction of Gadd45a substantially stabilizes Smad4 protein. In addition, exogenous expression of Gadd45a can largely rescue the protein level of Smad4 in BRCA1 deficient cells. These results further demonstrate that BRCA1 may act as an important negative regulator in cell cycle progression and tumorigenesis through regulating the stability of Smad4, and define a novel link that connects BRCA1 to TGF-β1/Smad pathway.

The myotubularins are a family of phosphatases that dephosphorylate the phosphatidylinositols phosphatidylinositol-3-phosphate and phosphatidylinositol-3,5-phosphate. Several family members are mutated in disease, yet the biological functions of the majority of myotubularins remain unknown. To gain insight into the roles of the individual enzymes, we have used affinity purification coupled to mass spectrometry to identify protein–protein interactions for the myotubularins. The myotubularin interactome comprises 66 high confidence (false discovery rate ≤1%) interactions, including 18 pairwise interactions between individual myotubularins. The results reveal a number of potential signaling contexts for this family of enzymes, including an intriguing, novel role for myotubularin-related protein 3 and myotubularin-related protein 4 in the regulation of abscission, the final step of mitosis in which the membrane bridge remaining between two daughter cells is cleaved. Both depletion and overexpression of either myotubularin-related protein 3 or myotubularin-related protein 4 result in abnormal midbody morphology and cytokinesis failure. Interestingly, myotubularin-related protein 3 and myotubularin-related protein 4 do not exert their effects through lipid regulation at the midbody, but regulate abscission during early mitosis, by interacting with the mitotic kinase polo-like kinase 1, and with centrosomal protein of 55 kDa (CEP55), an important regulator of abscission. Structure-function analysis reveals that, consistent with known intramyotubularin interactions, myotubularin-related protein 3 and myotubularin-related protein 4 interact through their respective coiled coil domains. The interaction between myotubularin-related protein 3 and polo-like kinase 1 relies on the divergent, nonlipid binding Fab1, YOTB, Vac1, and EEA1 domain of myotubularin-related protein 3, and myotubularin-related protein 4 interacts with CEP55 through a short GPPXXXY motif, analogous to endosomal sorting complex required for transport-I components. Disruption of any of these interactions results in abscission failure, by disrupting the proper recruitment of CEP55, and subsequently, of endosomal sorting complex required for transport-I, to the midbody. Our data suggest that myotubularin-related protein 3 and myotubularin-related protein 4 may act as a bridge between CEP55 and polo-like kinase 1, ensuring proper CEP55 phosphorylation and regulating CEP55 recruitment to the midbody. This work provides a novel role for myotubularin-related protein 3/4 heterodimers, and highlights the temporal and spatial complexity of the regulation of cytokinesis.

Summary Copy-number variations (CNVs) are ubiquitous in cancer and often act as driver events, but the effects of CNVs on the proteome of tumors are poorly understood. Here, we analyze recently published genomics, transcriptomics, and proteomics datasets made available by CPTAC and TCGA consortia on 282 breast, ovarian, and colorectal tumor samples to investigate the impact of CNVs in the proteomes of these cells. We found that CNVs are buffered by post-transcriptional regulation in 23%–33% of proteins that are significantly enriched in protein complex members. Our analyses show that complex subunits are highly co-regulated, and some act as rate-limiting steps of complex assembly, as their depletion induces decreased abundance of other complex members. We identified 48 such rate-limiting interactions and experimentally confirmed our predictions on the interactions of AP3B1 with AP3M1 and GTF2E2 with GTF2E1. This study highlights the importance of post-transcriptional mechanisms in cancer that allow cells to cope with their altered genomes., Graphical Abstract, Highlights • 23%–33% of the proteins have copy-number changes attenuated, likely via degradation • Protein complex members are more co-regulated at protein level than mRNA level • Systematic identification of rate-limiting members for the assembly of the complex • AP3B1, GTF2E2, and GTF2E1 are limiting members of their complexes, Post-transcriptional regulation, likely via control of protein degradation, can attenuate copy-number alterations in tumors for at least 23% of measured proteins. This effect is enriched in protein complexes, with some complex members acting as rate-limiting factors for complex formation.

Cell cycle reactivation in adult neurons is an early hallmark of neurodegeneration. The lipopolysaccharide (LPS) is a well-known pro-inflammatory factor that provokes neuronal cell death via glial cells activation. The retinoblastoma (RB) family includes RB1/p105, retinoblastoma-like 1 (RBL1/p107), and retinoblastoma-like 2 (Rb2/p130). Several studies have indicated that RB proteins exhibit tumor suppressor activities, and play a central role in cell cycle regulation. In this study, we assessed LPS-mediated inflammatory effect on cell cycle reactivation and apoptosis of neuronally differentiated cells. Also, we investigated whether the LPS-mediated inflammatory response can influence the function and expression of RB proteins. Our results showed that LPS challenges triggered cell cycle reactivation of differentiated neuronal cells, indicated by an accumulation of cells in S and G2/M phase. Furthermore, we found that LPS treatment also induced apoptotic death of neurons. Interestingly, we observed that LPS-mediated inflammatory effect on cell cycle re-entry and apoptosis was concomitant with the aberrant expression of RBL1/p107 and RB1/p105. To the best of our knowledge, our study is the first to indicate a role of LPS in inducing cell cycle re-entry and/or apoptosis of differentiated neuronal cells, perhaps through mechanisms altering the expression of specific members of RB family proteins. This study provides novel information on the biology of post-mitotic neurons and could help in identifying novel therapeutic targets to prevent de novo cell cycle reactivation and/or apoptosis of neurons undergoing neurodegenerative processes.

Heat shock protein 70 (Hsp70) is an evolutionarily well-conserved molecular chaperone involved in several cellular processes such as folding of proteins, modulating protein-protein interactions, and transport of proteins across the membrane. Binding partners of Hsp70 (known as "clients") are identified on an individual basis as researchers discover their particular protein of interest binds to Hsp70. A full complement of Hsp70 interactors under multiple stress conditions remains to be determined. A promising approach to characterizing the Hsp70 "interactome" is the use of protein epitope tagging and then affinity purification followed by mass spectrometry (AP-MS/MS). AP-MS analysis is a widely used method to decipher protein-protein interaction networks and identifying protein functions. Conventionally, the proteins are overexpressed ectopically which interferes with protein complex stoichiometry, skewing AP-MS/MS data. In an attempt to solve this issue, we used CRISPR/Cas9-mediated gene editing to integrate a tandem-affinity (TAP) epitope tag into the genomic locus of HSC70. This system offers several benefits over existing expression systems including native expression, no requirement for selection, and homogeneity between cells. This cell line, freely available to chaperone researchers, will aid in small and large-scale protein interaction studies as well as the study of biochemical activities and structure-function relationships of the Hsc70 protein.

Cucumber mosaic virus (CMV) is a model virus for plant-virus protein interaction and mechanism research because of its wide distribution, high-level of replication and simple genome structure. The 2b protein is a multifunctional protein encoded by CMV that suppresses RNA silencing-based antiviral defense and contributes to CMV virulence in host plants. In this report, 12 host proteins were identified as CMV LS2b binding partners using the yeast two-hybrid screen system from the Arabidopsis thaliana cDNA library. Among the host proteins, 30S ribosomal subunit protein S11 (RPS11) was selected for further studies. The interaction between LS2b and full-length RPS11 was confirmed using the yeast two-hybrid system. Bimolecular fluorescence complementation (BIFC) assays observed by confocal laser microscopy and Glutathione S-transferase (GST) pull-down assays were used to verify the interaction between endogenous NbRPS11 and viral CMVLS2b both in vivo and in vitro. TRV-based gene silencing vector was used to knockdown NbRPS11 transcription, and immunoblot analysis revealed a decline in infectious viral RNA replication and a decrease in CMV infection in RPS11 down-regulated Nicotiana benthamiana plants. Thus, the knockdown of RPS11 likely inhibited CMV replication and accumulation. The gene silencing suppressor activity of CMV2b protein was reduced by the RPS11 knockdown. This study demonstrated that the function of viral LS2b protein was remarkably affected by the interaction with host RPS11 protein.

Nucleophosmin (NPM1) is a multifunctional nucleolar protein implicated in ribogenesis, centrosome duplication, cell cycle control, regulation of DNA repair and apoptotic response to stress stimuli. The majority of these functions are played through the interactions with a variety of protein partners. NPM1 is frequently overexpressed in solid tumors of different histological origin. Furthermore NPM1 is the most frequently mutated protein in acute myeloid leukemia (AML) patients. Mutations map to the C-terminal domain and lead to the aberrant and stable localization of the protein in the cytoplasm of leukemic blasts. Among NPM1 protein partners, a pivotal role is played by the tumor suppressor Fbw7γ, an E3-ubiquitin ligase that degrades oncoproteins like c-MYC, cyclin E, Notch and c-jun. In AML with NPM1 mutations, Fbw7γ is degraded following its abnormal cytosolic delocalization by mutated NPM1. This mechanism also applies to other tumor suppressors and it has been suggested that it may play a key role in leukemogenesis. Here we analyse the interaction between NPM1 and Fbw7γ, by identifying the protein surfaces implicated in recognition and key aminoacids involved. Based on the results of computational methods, we propose a structural model for the interaction, which is substantiated by experimental findings on several site-directed mutants. We also extend the analysis to two other NPM1 partners (HIV Tat and CENP-W) and conclude that NPM1 uses the same molecular surface as a platform for recognizing different protein partners. We suggest that this region of NPM1 may be targeted for cancer treatment.

Protein 4.1B/DAL-1 is a membrane skeletal protein that belongs to the protein 4.1 family. Protein 4.1B/DAL-1 is localized to sites of cell-cell contact and functions as an adapter protein, linking the plasma membrane to the cytoskeleton or associated cytoplasmic signaling effectors and facilitating their activities in various pathways. Protein 4.1B/DAL-1 is involved in various cytoskeleton-associated processes, such as cell motility and adhesion. Moreover, protein 4.1B/DAL-1 also plays a regulatory role in cell growth, differentiation, and the establishment of epithelial-like cell structures. Protein 4.1B/DAL-1 is normally expressed in multiple human tissues, but loss of its expression or prominent down-regulation of its expression is frequently observed in corresponding tumor tissues and tumor cell lines, suggesting that protein 4.1B/DAL-1 is involved in the molecular pathogenesis of these tumors and acts as a potential tumor suppressor. This review will focus on the structure of protein 4.1B/DAL-1, 4.1B/DAL-1-interacting molecules, 4.1B/DAL-1 inactivation and tumor progression, and anti-tumor activity of the 4.1B/DAL-1.

Human mucin 1 (MUC1) is a target for immunotherapy. The major problem associated with MUC1‑based cancer vaccines is the weakness of the immunogenicity of MUC1. The present study aimed to develop an efficient cancer vaccine through generating a recombinant fusion protein consisting of MUC1 and maltose‑binding protein (MBP) by inserting seven tandem repeats encoding the human MUC1 gene into the pMAL‑c2 expression vector. Bacillus Calmette‑Guerin (BCG) was used as an adjuvant. MUC1 was found to predominantly induce T helper type 2 (Th2) cell responses. MUC1/BCG and MUC1‑MBP were found to generate T helper (Th) type 1 and 2 responses, while MUC1‑MBP/BCG induced a Th1 immune profile and stimulated MUC1‑specific cytotoxic T lymphocyte killing activity. MUC1‑MBP, as well as MBP and BCG alone were found to induce natural killer (NK) cell activity, with MUC1‑MBP/BCG observed to synergistically induce NK cell activity. Furthermore, MUC1‑MBP/BCG significantly inhibited MUC1+ B16 cell growth in mice. These findings show that MBP augments the immunogenicity of MUC1 and that BCG enhances the efficacy of the MUC1‑MBP vaccine. Thus, MUC1‑MBP/BCG may have potential as a cancer vaccine for clinical application.

TIM-1 (also known as KIM-1 and HAVcr-1) is a type I transmembrane glycoprotein member of the TIM family that may play important roles in innate and adaptive immune responses. The overexpression of proteins associated with membrane proteins is a major obstacle to overcome in studies of membrane protein structures and functions. In this study, we successfully coupled the overexpression of the TIM-1 protein with a C-terminal enhanced green fluorescent protein (GFP) tag in Escherichia coli. To the best of our knowledge, this report is the first to describe the overexpression of human TIM-1 in E. coli. The purified TIM-1-EGFP fusion protein recognized and bound directly to apoptotic cells and did not to bind to viable cells. Furthermore, we confirmed that the interactions of TIM-1-EGFP with apoptotic cells were blocked by TIM-1-Fc fusion proteins. This fusion protein represents a readily obtainable source of biologically active TIM-1 that may prove useful in future studies of human TIM-1.

Aim. To gain more insights into properties of the human translation elongation factor eEF1Bγ and its interaction with partners we intended to produce the full-length protein and its truncated forms. Methods. cDNAs encoding truncated forms of eEF1Bγ were generated by PCR amplification with respective primers and cloned into vectors providing polyhistidine, glutathione S-transferase or maltose binding protein tags. The recombinant proteins were expressed in Escherichia coli and purified by affinity chromatography. An aggregation state of the proteins was analyzed by analytical gel filtration. Results. The expression, purification and storage conditions for the full-length recombinant His-eEF1Bγ were optimized. Several truncated forms of eEF1Bγ were also expressed and purified to homogeneity. Two short variants of C-terminal domain comprising amino acids 263–437 or 228–437 were obtained in monomeric state. Two short variants of N-terminal domain comprising amino acids 1–33 or 1–230, fused with glutathione S-transferase, were obtained and estimated to be dimers by gel filtration. The mutants of N-terminal domain comprising amino acids 1–93 or 1–165, fused with maltose binding protein, were obtained as soluble high molecular weight aggregates only. Conclusions. The purified recombinant His-eEF1Bγ and several truncated forms of the protein were obtained and characterized. These protein variants will be used for further studies on the protein-protein interaction. Мета. Для детального вивчення властивостей фактора елонгації трансляції eEF1Bγ людини і його взаємодії з партнерами оптимізувати експресію кДНК повнорозмірного білка та його вкорочених форм. Методи. кДНК, які кодують вкорочені форми eEF1Bγ, синтезували методом ПЛР-ампліфікації з відповідними праймерами і клонували у вектори, що містять які афінну мітку полігістидинову послідовність, глутатіон S-трансферазу або білок, який зв’язує мальтозу. Рекомбінантні білки експресували в бактеріях і очищували афінною хроматографією. Агрегатний стан отриманих білків аналізували з використанням аналітичної гель-фільтрації. Результати. Оптимізовано експресію, очищення та умови зберігання повнорозмірного рекомбінантного His-eEF1Bγ. Також експресовано і очищено до гомогенного стану кілька вкорочених форм eEF1Bγ. Дві вкорочені форми С-кінцевого домену, які містять амінокислотні залишки 263–437 і 228–437, отримано у вигляді розчинних мономерних білків. Дві вкорочені форми N-кінцевого домену, що містять амінокислотні залишки 1–33 і 1–230, злиті з глутатіон S-трансферазою, одержано у вигляді димерів згідно з результатами гель-фільтрації. Інші делеційні мутанти, які містять амінокислотні залишки 1–93 і 1–165 N-кінцевого домену та злиті з білком, що зв’язує мальтозу, можуть бути отримані лише у вигляді розчинних високомолекулярних агрегатів. Висновки. Отримано і охарактеризовано рекомбінантний білок His-eEF1Bγ та його чотири вкорочені форми. Ці форми в подальшому буде використано для вивчення білково-білкових взаємодій. Цель. Для более детального изучения свойств человеческого фактора элонгации трансляции eEF1Bγ и его взаимодействия с партнерами оптимизировать експрессию кДНК полноразмерного белка, а также его усеченных форм. Методы. кДНК, кодирующие укороченные формы eEF1Bγ, генерировали методом ПЦР-амплификации с соответствующими праймерами и клонировали в векторы, содержащие в качестве аффинной метки полигистидиновую последовательность, глутатион S-трансферазу или белок, связывающий мальтозу. Рекомбинантные белки экспрессировали в бактериях и очищали аффинной хроматографией. Агрегатное состояние полученных белков анализировали с помощью аналитической гель-фильтрации. Результаты. Оптимизированы экспрессия, очистка и условия хранения полноразмерного рекомби- нантного His-eEF1Bγ. Также експрессированы и очищены до гомогенного состояния несколько усеченных форм eEF1Bγ. Две укороченные формы С-концевого домена, содержащие аминокис- лотные остатки 263–437 и 228–437, получены в виде растворимых мономерных белков. Две укороченные формы N-концевого домена, содержащие аминокислотные остатки 1–33 и 1–230, слитые с глутатион S-трансферазой, получены в виде димеров по результатам гель-фильтрации. Другие делеционные мутанты, содержащие аминокислотные остатки 1–93 и 1–165 N-концевого домена и слиты с белком, связывающим мальтозу, могут быть получены только в виде растворимых высокомолекулярных агрегатов. Выводы. Получены и охарактеризованы рекомбинантный фактор элонгации трансляции His-eEF1B и его четыре усеченные формы, которые в дальнейшем будут использованы для изу- чения белково-белковых взаимодействий.

CHES1/FOXN3 inhibits cell proliferation and protein biosynthesis in tumor cell lines but not in normal fibroblasts. CHES1 directly represses the expression of the gene coding for the protein kinase PIM2, and PIM2 or eIF4E counteracts the antiproliferative effect of CHES1. The levels of CHES1 and PIM2 are inversely correlated in several human cancers., The expression of the forkhead transcription factor checkpoint suppressor 1 (CHES1), also known as FOXN3, is reduced in many types of cancers. We show here that CHES1 decreases protein synthesis and cell proliferation in tumor cell lines but not in normal fibroblasts. Conversely, short hairpin RNA–mediated depletion of CHES1 increases tumor cell proliferation. Growth suppression depends on the CHES1 forkhead DNA-binding domain and correlates with the nuclear localization of CHES1. CHES1 represses the expression of multiple genes, including the kinases PIM2 and DYRK3, which regulate protein biosynthesis, and a number of genes in cilium biogenesis. CHES1 binds directly to the promoter of PIM2, and in cells expressing CHES1 the levels of PIM2 are reduced, as well as the phosphorylation of the PIM2 target 4EBP1. Overexpression of PIM2 or eIF4E partially reverses the antiproliferative effect of CHES1, indicating that PIM2 and protein biosynthesis are important targets of the antiproliferative effect of CHES1. In several human hematopoietic cancers, CHES1 and PIM2 expressions are inversely correlated, suggesting that repression of PIM2 by CHES1 is clinically relevant.

The aim of the present study was to explore the pro-apoptotic effect and specific mechanism of action of melittin (MEL) in humans. The effects of MEL on apoptosis in osteosarcoma and fetal osteoblast cells were investigated, and the mechanism that induced MG63 cell growth was also explored. The effects of MEL on cell proliferation were detected by a 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide analysis. Apoptosis was detected by flow cytometric analysis. MEL protein, inositol-requiring protein-1 (IRE-α), phosphorylated-protein kinase R-like endoplasmic reticulum (ER) kinase, spliced X-box-1 (XBP1), eukaryotic translation initiation factor-2α, cleaved activating transcription factor-6, caspase-12 and C/EBP homology protein (CHOP) were detected in three groups and two cell lines by western blot analysis. The results indicated that the expression or incubation of MEL in the MG63 cells triggered apoptosis and the inhibition of proliferation. One protein from the ER stress unfolded protein response pathway, IRE-α, was involved in the MEL-induced apoptosis in MG63 cells. Furthermore, spliced XBP1 protein was significantly increased in the MEL peptide incubated and MEL expressing groups of MG63 cells. Furthermore, CHOP protein expression was activated in MG63 cells following being incubated with or expressing MEL. In conclusion, MEL serves as an effective factor that inhibits the proliferation of MG63 cells via activating the ER stress-mediated apoptosis pathway. This activation is triggered by the IRE-α pathway mediated by inducing CHOP protein expression.

Equine herpesvirus type 1 (EHV-1) ORF59 is predicted to encode a protein consisting of 180 amino acids. To determine whether ORF59 in fact encodes a protein, sequences encoding an HA epitope (YPYDVPDYA) was inserted at the carboxyterminus of the ORF59 protein in EHV-1 strain Ab4. Using anti-HA monoclonal antibodies, a 21-kDa band was specifically detected by western blot analysis in lysates of cells infected with a recombinant EHV-1 from strain Ab4 that carries the pORF59-HA but not in cells infected with parental Ab4. Further characterization of the protein using immunofluorescence and fractionation studies showed that pORF59 is an early protein that localizes to the cytosol in virus-infected cells. Recombinant EHV-1 lacking ORF59 (rAb4∆59) exhibited a small-plaque phenotype and could not be propagated. Our findings suggest that the ORF59 protein plays a major role in EHV-1 replication in vitro and likely in vivo.

NF-κB is a well-known transcription factor in regulation of multiple gene transcription and biological processes, and most of them are relied on its transcriptional activity of the p65/RelA subunit, while biological function of another ubiquitously expressed subunit NF-κB1 (p50) remains largely unknown due to lack transcriptional activation domain. Here we discovered a novel biological function of p50 as a regulator of oncogenic c-Myc protein degradation upon arsenite treatment in a NF-κB transcriptional-independent mechanism. Our results found that p50 was crucial for c-Myc protein induction following arsenite treatment by using specific knockdown and deletion of p50 in its normal expressed cells as well as reconstituting expression of p50 in its deficient cells. Subsequently we showed that p50 upregulated c-Myc protein expression mainly through inhibiting its degradation. We also identified that p50 exhibited this novel property by suppression of FBW7 expression. FBW7 was profoundly upregulated in p50-deficient cells in comparison to that in p50 intact cells, whereas knockdown of FBW7 in p50-/- cells restored arsenite-induced c-Myc protein accumulation, assuring that FBW7 up-regulation was responsible for defect of c-Myc protein expression in p50-/- cells. In addition, we discovered that p50 suppressed fbw7 gene transcription via inhibiting transcription factor E2F1 transactivation. Collectively, our studies demonstrated a novel function of p50 as a regulator of c-Myc protein degradation, contributing to our notion that p50-regulated protein expression through multiple levels at protein translation and degradation, further providing a significant insight into the understanding of biomedical significance of p50 protein.

μ-Crystallin (CRYM) is also known as NADPH-dependent cytosolic T3-binding protein. A study using CRYM-null mice suggested that CRYM stores triiodothyronine (T3) in tissues. We previously established CRYM-expressing cells derived from parental GH3 cells. To examine the precise regulation of T3-responsive genes in the presence of CRYM, we evaluated serial alterations of T3-responsive gene expression by changing pericellular T3 concentrations in the media. We estimated the constitutive expression of three T3-responsive genes, growth hormone (GH), deiodinase 1 (Dio1), and deiodinase 2 (Dio2), in two cell lines. Subsequently, we measured the responsiveness of these three genes at 4, 8, 16, and 24 h after adding various concentrations of T3. We also estimated the levels of these mRNAs 24 and 48 h after removing T3. The levels of constitutive expression of GH and Dio1 were low and high in C8 cells, respectively, while Dio2 expression was not significantly different between GH3 and C8 cells. When treated with T3, Dio2 expression was significantly enhanced in C8 cells, while there were no differences in GH or Dio1 expression between GH3 and C8 cell lines. In contrast, removal of T3 retained the mRNA expression of GH and Dio2 in C8 cells. These results suggest that CRYM expression increases and sustains the T3 responsiveness of genes in cells, especially with alteration of the pericellular T3 concentration. The heterogeneity of T3-related gene expression is dependent on cellular CRYM expression in cases of dynamic changes in pericellular T3 concentration.

Lysosome-associated protein transmembrane 4β (LAPTM4B) is a gene that has been indicated to be involved in cancer. It is located at chromosome 8q22 and is composed of seven exons and six introns. LAPTM4B encodes two protein isoforms: LAPTM4B-35 and LAPTM4B-24. LAPTM4B-35 is markedly upregulated and LAPTM4B-24 is downregulated in several types of cancer. LAPTM4B-35 is 91 amino acids (N91) longer than LAPTM4B-24 at the N-terminus. In the present study, western blotting, enzyme-linked immunosorbent spot analysis and the B16F10-N91 tumor bearing-mice experiments were used to evaluate whether the overexpression of N91 indicates its potential as a candidate tumor-associated antigen. The results revealed that N91 was expressed in a wide range of normal mouse tissues and human peripheral blood mononuclear cells, with varying expression levels. The weak immunogenicity of N91 protein suggested it was a weak candidate antigen; however, the N91 protein was associated with cell proliferation.

microRNAs (miRNAs) are a growing class of small non-coding RNAs that exhibit widespread dysregulation in prostate cancer. We profiled miRNA expression in syngeneic human prostate cancer cell lines that differed in their metastatic potential in order to determine their role in aggressive prostate cancer. miR-888 was the most differentially expressed miRNA observed in human metastatic PC3-ML cells relative to non-invasive PC3-N cells, and its levels were higher in primary prostate tumors from cancer patients, particularly those with seminal vesicle invasion. We also examined a novel miRNA-based biomarker source called expressed prostatic secretions in urine (EPS urine) for miR-888 expression and found that its levels were preferentially elevated in prostate cancer patients with high-grade disease. These expression studies indicated a correlation for miR-888 in disease progression. We next tested how miR-888 regulated cancer-related pathways in vitro using human prostate cancer cell lines. Overexpression of miR-888 increased proliferation and migration, and conversely inhibition of miR-888 activity blocked these processes. miR-888 also increased colony formation in PC3-N and LNCaP cells, supporting an oncogenic role for this miRNA in the prostate. Our data indicates that miR-888 functions to promote prostate cancer progression and can suppress protein levels of the tumor suppressor genes RBL1 and SMAD4. This miRNA holds promise as a diagnostic tool using an innovative prostatic fluid source as well as a therapeutic target for aggressive prostate cancer.

Aberrant expression of microRNAs (miRNAs) has been implicated in cancer initiation and progression. However, little is known about the potential role of miRNAs in glioma tumorigenesis. In this study, we found that miRNA-106b-5p was significantly upregulated in glioma tumor samples and cell lines compared with normal brain tissues, and its expression level correlated with the pathological grading. Overexpression of miR-106b-5p in glioma tumor cells significantly promoted cell proliferation, although inhibited cell apoptosis in vitro and in vivo. In contrast, knockdown of miR-106b-5p significantly inhibited cell proliferation, although enhanced cell apoptosis. Mechanistic study revealed that two target genes, retinoblastoma-like 1 (RBL1) and RBL2, were involved in miR-106b-5p's regulation of cell proliferation and one target gene, caspase-8 (CASP8), mediated miR-106b-5p's regulation of apoptosis. We also investigated the function of the three targets in glioma tumorigenesis by RNA interference manipulation and demonstrated that knockdown of these target genes led to cell proliferation enhancement or cell apoptosis inhibition in vitro. More interestingly, the expression levels of these targets were significantly downregulated in glioma samples and knockdown of these targets in glioma cells inhibited the xenograft tumor formation in vivo. Moreover, we verified the regulation function of miR-106b-5p and its targets on cell proliferation and apoptosis of the primary cultured astrocytes isolated from glioma tumor samples and healthy controls. Collectively, our findings show the critical roles of miR-106b-5p and its targets, RBL1, RBL2 and CASP8, in glioma tumorigenesis and provide potential candidates for malignant glioma therapy.

Protein engineering offers a robust platform for the design and production in cell factories of a plethora of protein-based drugs, including nonviral gene therapy vehicles. We have determined here that a protein nanoparticle, formed by highly cationic protein monomers, fails to bind exogenous DNA and to promote detectable gene expression in target cells despite recruiting all the needed functions. Removal of DNA and RNA with nucleases previous to forming complexes with exogenous DNA dramatically enhances the ability of the protein to bind and transfer DNA to target cell nuclei. These data point out contaminant nucleic acids deriving from the cell factory as a major factor impairing the performance of protein-based artificial viruses and stress the need of a nuclease step in the downstream of proteins whose function is based on cationic domains. (c) 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1458-1463, 2013

Summary The cucumber mosaic virus (CMV) 2b protein is an RNA silencing suppressor protein that can also play direct and indirect roles in symptom induction. Previous work has shown that a hybrid virus, FRad352b-CMV (renamed here as CMV-FRad2b-Pro), generated by replacement of the 2b gene of strain Fny-CMV with that from Rad35-CMV, displays markedly lower pathogenicity than Fny-CMV on Nicotiana species. However, the replacement of proline with leucine at position 55 of the 2b protein of CMV-FRad2b-Pro (protein Rad2b-Pro) created a virus (CMV-FRad2b-Leu) that induced severe symptoms. Infection of Arabidopsis thaliana mutants defective in the expression of DICER-like (DCL) endoribonucleases 2 and 4, which mediate antiviral RNA silencing, as well as of dcl3 and dcl2/3/4 triple-mutant plants, indicated that Rad2b-Pro was a weaker RNA silencing suppressor than the protein Rad2b-Leu. This was confirmed in Nicotiana benthamiana using agroinfiltration assays, showing that, compared with either Rad2b-Leu or the Fny2b protein, Rad2b-Pro was ineffective at inhibiting local or systemic silencing of expression of a green fluorescent protein reporter gene. Transgenic expression of Rad2b-Leu, but not of Rad2b-Pro, in Arabidopsis induced symptom-like phenotypes and rescued the accumulation of the 2b-deletion mutant Fny-CMVΔ2b. Bimolecular fluorescent complementation indicated that, in planta, Rad2b-Leu, but not Rad2b-Pro, self-interacts. Thus, self-interaction is crucial to the ability of the 2b protein to suppress silencing and induce a symptom-like phenotype, and is dependent on the properties of the residue at position 55.

In humans, the gene encoding the BRCA1 C terminus-repeat inhibitor of human telomerase expression 1 (BRIT1) protein is located on chromosome 8p23.1, a region implicated in the development of several malignancies, including breast cancer. Previous studies by our group and others suggested that BRIT1 might function as a novel tumor suppressor. Thus, identifying the molecular mechanisms that underlie BRIT1's tumor suppressive function is important to understand cancer etiology and to identify effective therapeutic strategies for BRIT1-deficient tumors. We thus investigated the role of BRIT1 as a tumor suppressor in breast cancer by using genetic approaches. We discovered that BRIT1 functions as a post-transcriptional regulator of p53 expression. BRIT1 regulates p53 protein stability through blocking murine double minute 2-mediated p53 ubiquitination. To fully demonstrate the role of BRIT1 as a tumor suppressor, we depleted BRIT1 in normal breast epithelial cells. We found that knockdown of BRIT1 caused the oncogenic transformation of normal mammary epithelial cells. Furthermore, ectopic expression of BRIT1 effectively suppressed breast cancer cell proliferation and colony formation in vitro and tumor growth in vivo. Taken together, our study provides new insights into the biological functions of BRIT1 as a tumor suppressor in human breast cancer.

Loss of RB1 gene is considered either a causal or an accelerating event in retinoblastoma. A variety of mechanisms inactivates RB1 gene, including intragenic mutations, loss of expression by methylation and chromosomal deletions, with effects which are species-and cell type-specific. RB1 deletion can even lead to aneuploidy thus greatly increasing cancer risk. The RB1gene is part of a larger gene family that includes RBL1 and RBL2, each of the three encoding structurally related proteins indicated as pRb, p107, and p130, respectively. The great interest in these genes and proteins springs from their ability to slow down neoplastic growth. pRb can associate with various proteins by which it can regulate a great number of cellular activities. In particular, its association with the E2F transcription factor family allows the control of the main pRb functions, while the loss of these interactions greatly enhances cancer development. As RB1 gene, also pRb can be functionally inactivated through disparate mechanisms which are often tissue specific and dependent on the scenario of the involved tumor suppressors and oncogenes. The critical role of the context is complicated by the different functions played by the RB proteins and the E2F family members. In this review, we want to emphasize the importance of the mechanisms of RB1/pRb inactivation in inducing cancer cell development. The review is divided in three chapters describing in succession the mechanisms of RB1 inactivation in cancer cells, the alterations of pRb pathway in tumorigenesis and the RB protein and E2F family in cancer.

Highlights ► Overexpression of HCV p7 protein induces apoptosis in Huh7.5 cells. ► p7-induced apoptosis involves both the intrinsic and extrinsic pathways. ► p7-induced apoptosis is independent of its ion channel activity. ► p7 is functionally similar to the well-characterized M2 protein of influenza A virus but they differ in their activation of autophagic cell-death., Most viruses encode proteins that modulate cell-death signaling by the host. For hepatitis C virus (HCV) infection, apoptosis and other forms of cell-death have been observed in vitro and in vivo but the detailed understanding of this intricate viral-host interplay is unclear. This study examined the role played by the HCV p7 protein in the induction of cell-death. By measuring caspase-3/7 activation and cleavage of endogenous PARP, two hallmarks of apoptosis, the overexpression of p7 protein was shown to induce apoptosis in Huh7.5 cells. Furthermore, p7-induced apoptosis is caspase-dependent and involves both the intrinsic and extrinsic pathways. Similar to the M2 protein of influenza A virus, p7-induced apoptosis is independent of its ion channel activity. Coimmunoprecipitation experiments further showed that both M2 and p7 interact with the essential autophagy protein Beclin-1. However, only the M2 protein could cause an increase in the level of LC3-II, which is an indicator of autophagic activity. Thus, although the p7 protein is functionally similar to the well-characterized M2 protein, they differ in their activation of autophagic cell-death. Taken together, these results shed more light on the relationship between the HCV p7 ion channel protein and cell-death induction in host cells.

The biological function of NF-κB1 (p50) in the regulation of protein expression is far from well understood owing to the lack of a transcriptional domain. Here, we report a novel function of p50 in its regulation of p53 protein translation under stress conditions. We found that the deletion of p50 (p50–/–) impaired arsenite-induced p53 protein expression, which could be restored after reconstitutive expression of HA-p50 in p50–/– cells, p50–/– (Ad-HA-p50). Further studies indicated that the amounts of p53 mRNA, p53 promoter-driven transcription activity and p53 protein degradation were comparable between wild-type and p50–/– cells. Moreover, we found that p50 was crucial for Akt/S6 ribosomal protein activation via inhibition of the translation of the PH domain and leucine-rich repeat protein phosphatases 1 (PHLPP1), a phosphatase of Akt. Further studies showed that p50-mediated upregulation of miR-190 was responsible for the inhibition of PHLPP1 translation by targeting the 3′-untranslated region of its mRNA. Collectively, we have identified a novel function of p50 in modulating p53 protein translation via regulation of the miR-190/PHLPP1/Akt-S6 ribosomal protein pathway.

Retinoblastoma (Rb) protein is a tumor suppressor that is dysregulated in a majority of human cancers. Rb functions to inhibit cell cycle progression in part by directly disabling the E2F family of cell cycle-promoting transcription factors. Because the de novo synthesis of multiple glutamine-derived anabolic precursors is required for cell cycle progression, we hypothesized that Rb also may directly regulate proteins involved in glutamine metabolism. We examined glutamine metabolism in mouse embryonic fibroblasts (MEFs) isolated from mice that have triple knock-outs (TKO) of all three Rb family members (Rb-1, Rbl1 and Rbl2) and found that loss of global Rb function caused a marked increase in (13)C-glutamine uptake and incorporation into glutamate and tricarboxylic acid cycle (TCA) intermediates in part via upregulated expression of the glutamine transporter ASCT2 and the activity of glutaminase 1 (GLS1). The Rb-controlled transcription factor E2F-3 altered glutamine uptake by direct regulation of ASCT2 mRNA and protein expression, and E2F-3 was observed to associate with the ASCT2 promoter. We next examined the functional consequences of the observed increase in glutamine uptake and utilization and found that glutamine exposure potently increased oxygen consumption, whereas glutamine deprivation selectively decreased ATP concentration in the Rb TKO MEFs but not the wild-type (WT) MEFs. In addition, TKO MEFs exhibited elevated production of glutathione from exogenous glutamine and had increased expression of gamma-glutamylcysteine ligase relative to WT MEFs. Importantly, this metabolic shift towards glutamine utilization was required for the proliferation of Rb TKO MEFs but not for the proliferation of the WT MEFs. Last, addition of the TCA cycle intermediate α-ketoglutarate to the Rb TKO MEFs reversed the inhibitory effects of glutamine deprivation on ATP, GSH levels and viability. Taken together, these studies demonstrate that the Rb/E2F cascade directly regulates a major energetic and anabolic pathway that is required for neoplastic growth.

Detection of nonstructural protein (NS1) is an important diagnostic marker during acute phase of dengue infection. Not only for diagnostic purpose, the protein had important role in vaccine design as well, as a candidate for studying virus assembly and maturation. Various researchers employed different expression systems and strategies for recombinant NS1 protein production. Attempts to express NS1 protein in prokaryotic and yeast expression system result in formation of insoluble protein which needs to undergo refolding to attain native structural and functional forms. Here, we report the production of soluble NS1 protein inE. coliby using appropriate vector and employing suitable culture conditions to maximize protein production. Proteins were purified using metal affinity chromatography. SDS-PAGE and western blot analysis reveal the native structure of NS1 protein. Solid phase ELISA using the recombinantly expressed antigen with positive and negative dengue samples showed that the expressed protein retains its antigenic and immunological properties. To our knowledge, this is the first report on the successful production of functionally active recombinant dengue-2 NS1 protein production without undergoing anyin vitroposttranslational modification process.

S100A6 protein belongs to the A group of the S100 protein family of Ca2+-binding proteins. It is expressed in a limited number of cell types in adult normal tissues and in several tumor cell types. As an intracellular protein, S100A6 has been implicated in the regulation of several cellular functions, such as proliferation, apoptosis, the cytoskeleton dynamics, and the cellular response to different stress factors. S100A6 can be secreted/released by certain cell types which points to extracellular effects of the protein. RAGE (receptor for advanced glycation endproducts) and integrin β1 transduce some extracellular S100A6's effects. Dosage of serum S100A6 might aid in diagnosis in oncology.