Description: Publication date: Available online 30 October 2014 Source: FEBS Open Bio Author(s): Ingo Lange , Dana-Lynn T. Koomoa Neuroblastoma is an extra-cranial solid cancer in children. MYCN gene amplification is a prognostic indicator of poor outcome in neuroblastoma. Recent studies have shown that the multiple steps involved in cell migration are dependent on the availability of intracellular calcium (Ca 2+ ). Although significant advances have been made in understanding the role of Ca 2+ during migration, little has been achieved towards understanding its impact on the progression of diseases such as cancer. Interestingly, previous studies showed that cancer cell migration is regulated by TRPM7, a calcium-permeable ion channel. The objective of the current study was to elucidate the mechanism by which MycN promotes NB cell migration and the mechanism regulating TRPM7 expression. The results showed that MycN increased TRPM7 expression, induced TRPM7 channel activity, increased intracellular Ca 2+ signaling, and promoted cell migration in NB cells. The results also showed that inhibition or down-regulation of ornithine decarboxylase (ODC) inhibited TRPM7 expression, a process that was reversed by spermidine. Overall, this study provides evidence that MycN promotes TRPM7 expression and cell migration through a mechanism that involves ODC synthesis of polyamines.

Description: Publication date: Available online 16 October 2014 Source: FEBS Open Bio Author(s): Amrita Banerjee , Sulagna Sanyal , Kirti K. Kulkarni , Kuladip Jana , Siddhartha Roy , Chandrima Das , Dipak Dasgupta Mithramycin (MTR) is a clinically approved DNA-binding antitumor antibiotic currently in Phase 2 clinical trials at National Institutes of Health for treatment of osteosarcoma. In view of the resurgence in the studies of this generic antibiotic as a human medicine, we have examined the binding properties of MTR with the integral component of chromatin – histone proteins – as a part of our broad objective to classify DNA-binding molecules in terms of their ability to bind chromosomal DNA alone (single binding mode) or both histones and chromosomal DNA (dual binding mode). The present report shows that besides DNA, MTR also binds to core histones present in chromatin and thus possesses the property of dual binding in the chromatin context. In contrast to the MTR–DNA interaction, association of MTR with histones does not require obligatory presence of bivalent metal ion like Mg 2+ . As a consequence of its ability to interact with core histones, MTR inhibits histone H3 acetylation at lysine 18, an important signature of active chromatin, in vitro and ex vivo . Reanalysis of microarray data of Ewing sarcoma cell lines shows that upon MTR treatment there is a significant down regulation of genes, possibly implicating a repression of H3K18Ac-enriched genes apart from DNA-binding transcription factors. Association of MTR with core histones and its ability to alter post-translational modification of histone H3 clearly indicates an additional mode of action of this anticancer drug that could be implicated in novel therapeutic strategies.

Description: Publication date: Available online 31 October 2014 Source: FEBS Open Bio Author(s): Hiroyuki Okano , Masashi Ozaki , Eiko Kanaya , Joong-Jae Kim , Clement Angkawidjaja , Yuichi Koga , Shigenori Kanaya Ten genes encoding novel cellulases with putative signal peptides at the N-terminus, termed pre-LC-CelA–J, were isolated from a fosmid library of a leaf–branch compost metagenome by functional screening using agar plates containing carboxymethyl cellulose and trypan blue. All the cellulases except pre-LC-CelG have a 14–29 residue long flexible linker (FL) between the signal peptide and the catalytic domain. LC-CelA without a signal peptide (residues 20–261), which shows 76% amino acid sequence identity to Cel12A from Rhodothermus marinus ( Rm Cel12A), was overproduced in Escherichia coli , purified and characterized. LC-CelA exhibited its highest activity across a broad pH range (pH 5–9) and at 90 °C, indicating that LC-CelA is a highly thermostable cellulase, like Rm Cel12A. The crystal structure of LC-CelA was determined at 1.85 Å resolution and is nearly identical to that of Rm Cel12A determined in a form without the FL. Both proteins contain two disulfide bonds. LC-CelA has a 16-residue FL (residues 20–35), most of which is not visible in the electron density map, probably due to structural disorder. However, Glu34 and Pro35 form hydrogen bonds with the central region of the protein. ΔFL-LC-CelA (residues 36–261) and E34A-LC-CelA with a single Glu34 → Ala mutation were therefore constructed and characterized. ΔFL-LC-CelA and E34A-LC-CelA had lower melting temperatures ( T m ) than LC-CelA by 14.7 and 12.0 °C respectively. The T m of LC-CelA was also decreased by 28.0 °C in the presence of dithiothreitol. These results suggest that Glu34-mediated hydrogen bonds and the two disulfide bonds contribute to the stabilization of LC-CelA.

Description: Publication date: Available online 31 October 2014 Source: FEBS Open Bio Author(s): Huijun Qin , Xiaolin Zhang , Fei Ye , Liangwei Zhong High-fat diet (HFD) can induce oxidative stress. Thioredoxin (Trx) and thioredoxin reductase (TrxR) are critical antioxidant proteins but how they are affected by HFD remains unclear. Using HFD-induced insulin-resistant mouse model, we show here that liver Trx and TrxR are significantly decreased, but, remarkably, the degree of their S-acylation is increased after consuming HFD. These HFD-induced changes in Trx/TrxR may reflect abnormalities of lipid metabolism and insulin signaling transduction. HFD-driven accumulation of 4-hydroxynonenal is another potential mechanism behind inactivation and decreased expression of Trx/TrxR. Thus, we propose HFD-induced impairment of liver Trx/TrxR as major contributor to oxidative stress and as a novel feature of insulin resistance.

Description: Publication date: Available online 8 November 2014 Source: FEBS Open Bio Author(s): Sara Vicente-Muñoz , Paco Romero , Lorena Magraner-Pardo , Celia P. Martinez-Jimenez , Vicente Tordera , Mercè Pamblanco Histone acetylation affects several aspects of gene regulation, from chromatin remodelling to gene expression, by modulating the interplay between chromatin and key transcriptional regulators. The exact molecular mechanism underlying acetylation patterns and crosstalk with other epigenetic modifications requires further investigation. In budding yeast, these epigenetic markers are produced partly by histone acetyltransferase enzymes, which act as multi-protein complexes. The Sas3-dependent NuA3 complex has received less attention than other histone acetyltransferases (HAT), such as Gcn5-dependent complexes. Here, we report our analysis of Sas3p-interacting proteins using tandem affinity purification (TAP), coupled with mass spectrometry. This analysis revealed Pdp3p, a recently described component of NuA3, to be one of the most abundant Sas3p-nteracting proteins. The PDP3 gene, was TAP-tagged and protein complex purification confirmed that Pdp3p co-purified with the NuA3 protein complex, histones, and several transcription-related and chromatin remodelling proteins. Our results also revealed that the protein complexes associated with Sas3p presented HAT activity even in the absence of Gcn5p and vice versa . We also provide evidence that Sas3p cannot substitute Gcn5p in acetylation of lysine 9 in histone H3 in vivo . Genome-wide occupancy of Sas3p using ChIP-on-Chip tiled microarrays showed that Sas3p was located preferentially within the 5’-half of the coding regions of target genes, indicating its probable involvement in the transcriptional elongation process. Hence, this work further characterises the function and regulation of the NuA3 complex by identifying novel post-translational modifications in Pdp3p, additional Pdp3p-co-purifying chromatin regulatory proteins involved in chromatin-modifying complex dynamics and gene regulation, and a subset of genes whose transcriptional elongation is controlled by this complex.

Description: Publication date: Available online 28 October 2014 Source: FEBS Open Bio Author(s): Tamara P. Martin , Maria P. Hortigon-Vinagre , Jane E. Findlay , Christina Elliott , Susan Currie , George S. Baillie Phosphorylated heat shock protein 20 (HSP20) is cardioprotective. Using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and a mouse model of pressure overload mediated hypertrophy, we show that peptide disruption of the HSP20–phosphodiesterase 4D (PDE4D) complex results in attenuation of action potential prolongation and protection against adverse cardiac remodelling. The later was evidenced by improved contractility, decreased heart weight to body weight ratio, and reduced interstitial and perivascular fibrosis. This study demonstrates that disruption of the specific HSP20–PDE4D interaction leads to attenuation of pathological cardiac remodelling.

Description: Publication date: Available online 12 November 2014 Source: FEBS Open Bio Author(s): Jelle B. Bultema , Bas J.H. Kuipers , Lubbert Dijkhuizen The Bacillus circulans ATCC 31382 β-galactosidase (BgaD) is a retaining-type glycosidase of glycoside hydrolase family 2 (GH2). Its commercial enzyme preparation, Biolacta N5, is used for commercial-scale production of galacto-oligosaccharides (GOS). The BgaD active site and catalytic amino acid residues have not been studied. Using bioinformatic routines we identified two putative catalytic glutamates and two highly conserved active site histidines. The site-directed mutants E447N, E532Q, and H345F, H379F had lost (almost) all catalytic activity. This confirmed their essential role in catalysis, as general acid/base catalyst (E447) and nucleophile (E532), and as transition state stabilizers (H345, H379), respectively.

Description: Publication date: Available online 4 November 2014 Source: FEBS Open Bio Author(s): Alessandra Mangolini , Anna Bonon , Stefano Volinia , Giovanni Lanza , Roberto Gambari , Paolo Pinton , Gian Rosario Russo , Laura del Senno , Lucio Dell’Atti , Gianluca Aguiari Renal cell carcinoma is a common neoplasia of the adult kidney that accounts for about 3% of adult malignancies. Clear cell renal carcinoma is the most frequent subtype of kidney cancer and 20–40% of patients develop metastases. The absence of appropriate biomarkers complicates diagnosis and prognosis of this disease. In this regard, small noncoding RNAs (microRNAs), which are mutated in several neoplastic diseases including kidney carcinoma, may be optimal candidates as biomarkers for diagnosis and prognosis of this kind of cancer. Here we show that patients with clear cell kidney carcinoma that express low levels of miR501-5p exhibited a good prognosis compared with patients with unchanged or high levels of this microRNA. Consistently, in kidney carcinoma cells the downregulation of miR501-5p induced an increased caspase-3 activity, p53 expression as well as decreased mTOR activation, leading to stimulation of the apoptotic pathway. Conversely, miR501-5p upregulation enhanced the activity of mTOR and promoted both cell proliferation and survival. These biological processes occurred through p53 inactivation by proteasome degradation in a mechanism involving MDM2-mediated p53 ubiquitination. Our results support a role for miR501-5p in balancing apoptosis and cell survival in clear cell renal carcinoma. In particular, the downregulation of microRNA501-5p promotes a good prognosis, while its upregulation contributes to a poor prognosis, in particular, if associated with p53 and MDM2 overexpression and mTOR activation. Thus, the expression of miR501-5p is a possible biomarker for the prognosis of clear cell renal carcinoma.

Description: Publication date: Available online 7 October 2014 Source: FEBS Open Bio Author(s): Mehrdad Sobhkhez , Astrid Skjesol , Ernst Thomassen , Linn Greiner Tollersrud , Dimitar B. Iliev , Baojian Sun , Børre Robertsen , Jorunn B. Jørgensen Mammalian IRF9 and STAT2, together with STAT1, form the ISGF3 transcription factor complex, which is critical for type I interferon (IFN)-induced signaling, while IFNγ stimulation is mediated by homodimeric STAT1 protein. Teleost fish are known to possess most JAK and STAT family members, however, description of their functional activity in lower vertebrates is still scarce. In the present study we have identified two different STAT2 homologs and one IRF9 homolog from Atlantic salmon ( Salmo salar ). Both proteins have domain-like structures with functional motifs that are similar to higher vertebrates, suggesting that they are orthologs to mammalian STAT2 and IRF9. The two identified salmon STAT2s, named STAT2a and STAT2b, showed high sequence identity but were divergent in their transactivation domain (TAD). Like STAT1, ectopically expressed STAT2a and b were shown to be tyrosine phosphorylated by type I IFNs and, interestingly, also by IFNγ. Microscopy analyses demonstrated that STAT2 co-localized with STAT1a in the cytoplasm of unstimulated cells, while IFNa1 and IFNγ stimulation seemed to favor their nuclear localization. Overexpression of STAT2a or STAT2b together with STAT1a activated a GAS-containing reporter gene construct in IFNγ-stimulated cells. The highest induction of GAS promoter activation was found in IFNγ-stimulated cells transfected with IRF9 alone. Taken together, these data suggest that salmon STAT2 and IRF9 may have a role in IFNγ-induced signaling and promote the expression of GAS-driven genes in bony fish. Since mammalian STAT2 is primarily an ISGF3 component and not involved in IFNγ signaling, our finding features a novel role for STAT2 in fish.

Description: Publication date: Available online 7 October 2014 Source: FEBS Open Bio Author(s): Knud Larsen , Jamal Momeni , Leila Farajzadeh , Christian Bendixen The membrane protein SLITRK1 functions as a developmentally regulated stimulator of neurite outgrowth and variants in this gene have been implicated in Tourette syndrome. In the current study we have cloned and characterized the porcine SLITRK1 gene. The genomic organization of SLITRK1 lacks introns, as does its human and mouse counterparts. RT-PCR cloning revealed two SLITRK1 transcripts: a full-length mRNA and a transcript variant that results in a truncated protein. The encoded SLITRK1 protein, consisting of 695 amino acids, displays a very high homology to human SLITRK1 (99%). The porcine SLITRK1 gene is expressed exclusively in brain tissues.