BBA - General Subjects (v.1861, #7)

The “good-cop bad-cop” TGF-beta role in breast cancer modulated by non-coding RNAs by Diana Gulei; Nikolay Mehterov; Hui Ling; Giorgio Stanta; Cornelia Braicu; Ioana Berindan-Neagoe (1661-1675).
Lack of early diagnosis methods and the development of drug resistance are among the main reasons for increased mortality rates within breast cancer patients. These two aspects are governed by specific pro-carcinogenic modifications, where TGBβ-induced EMT is one of the leading actors. Endowment of the epithelial cells with mesenchymal characteristics allows them to migrate and invade secondary tissues in order to form malignant sites and also confers chemoresistance. TGFβ which role switches from the tumor suppressor cytokine to the oncogenic one favoring the tumor microenvironment regulates this process.This review aims to comprehensively present the updated TGFβ-induced EMT in breast cancer, including the regulatory role of the non-coding RNAs with focus on the miR-200 family and newly discovered lncRNAs such as HOTAIRM1. Additionally, a new phenotype, P-EMT, also modulated by miR-200 and miR-34 families that form complex feedback loops with TGFβ, SNAI1 and ZEB1/2 is presented under an updated form.The hallmarks of EMT are becoming increasingly associated with aggressive forms of breast cancer and low survival rates among patients. Considering that this phenotypical switch can trigger drug resistance, invasion and metastasis, inhibition of EMT could represent an important milestone in mammary cancer treatment.The present review assembles the most recent data regarding TGFβ induced EMT, including the input of non-coding RNAs, contributing to the possible development of new targeted treatment strategies for cancer patients.
Keywords: Breast cancer; EMT; TGF-β; miRNA; lncRNA;

Analysis of the in vitro and in vivo effects of photodynamic therapy on prostate cancer by using new photosensitizers, protoporphyrin IX-polyamine derivatives by Chloë Fidanzi-Dugas; Bertrand Liagre; Guillaume Chemin; Aurélie Perraud; Claire Carrion; Claude-Yves Couquet; Robert Granet; Vincent Sol; David Yannick Léger (1676-1690).
Photodynamic therapy, using porphyrins as photosensitizers (PS), has been approved in treatment of several solid tumors. However, commonly used PS induce death but also resistance pathways in cancer cells and an alteration of surrounding normal tissues. Because polyamines (PA) are actively accumulated in cancer cells by the Polyamine Transport System (PTS), they may enable PS to specifically target cancer cells. Here, we investigated whether new protoporphyrin IX-polyamine derivatives were effective PS against prostate cancer and whether PA increased PDT specificity after 630 nm irradiation.CHO and CHO-MG cells (differing in their PTS activity) were used to assess efficacy of polyamine vectorization. MTT assays were performed on human prostate non-malignant (RWPE-1) and malignant (PC-3, DU 145 and LNCaP) cell lines to test PS phototoxicity. ROS generation, DNA fragmentation and cell signalling were assessed by ELISA/EIA, western-blots and gel shift assays. Finally, PS effects were studied on tumor growth in nude mice.Our PS were more effective on cancer cells compared to non-malignant cells and more effective than PpIX alone. PpIX-PA generated ROS production involved in induction of apoptotic intrinsic pathways. Different pathways involved in apoptosis resistance were studied: PS inhibited Bcl-2, Akt, and NF-κB but activated p38/COX-2/PGE2 pathways which were not implicated in apoptosis resistance in our model. In vivo experiments showed PpIX-PA efficacy was greater than results obtained with PpIX.All together, our results showed that PpIX-PA exerted its maximum effects without activating resistance pathways and appears to be a good candidate for prostate cancer PDT treatment.Display Omitted
Keywords: Prostate cancer; Porphyrin; Photodynamic therapy; Apoptosis; Cyclooxygenase;

GATA5 SUMOylation is indispensable for zebrafish cardiac development by Bin Wen; Hao Yuan; Xiaohui Liu; Haihong Wang; Saijuan Chen; Zhu Chen; Hugues de The; Jun Zhou; Jun Zhu (1691-1701).
SUMOylation is a critical regulatory protein modification in eukaryotic cells and plays a pivotal role in cardiac development and disease. Several cardiac transcription factors are modified by SUMO, but little is known about the impact of SUMOylation on their function during cardiac development.We used a zebrafish model to address the impact of SUMOylation on GATA5, an essential transcription factor in zebrafish cardiac development. GATA5 SUMOylation was probed by western blot, the subcellular localization and transcriptional activity of GATA5 mutants were examined by immunostaining and luciferase reporter assay. The in vivo function of GATA5 SUMOylation was evaluated by gata5 mutants mRNA microinjection and in situ hybridization in gata5 morphants and ubc9 mutants.Firstly, we identified GATA5 as a SUMO substrate, and lysine 324 (K324) and lysine 360 (K360) as two major modification sites. Conversion of lysine to arginine at these two sites did not affect subcellular localization, but did affect the transcriptional activity of GATA5. Secondly, in vivo experiments demonstrated that the wild type (WT) and K324R mutant of gata5 could rescue impaired cardiac precursor differentiation, while the K360R mutant of gata5 drastically lost this potency in gata5 morphant. Furthermore, in SUMOylation-deficient ubc9 mutants, the abnormal expression pattern displayed by the early markers of cardiac development (nkx2.5 and mef2cb) could be restored using a sumo-gata5 fusion, but not with a WT gata5.GATA5 SUMOylation is indispensable for early zebrafish cardiac development.Our studies highlight the potential importance of transcription factor SUMOylation in cardiac development.
Keywords: Protein modification; SUMOylation; Transcription factor; GATA5; Heart development; ubc9;

Integrated mass spectrometry imaging and omics workflows on the same tissue section using grid-aided, parafilm-assisted microdissection by Jusal Quanico; Julien Franck; Maxence Wisztorski; Michel Salzet; Isabelle Fournier (1702-1714).
In spite of the number of applications describing the use of MALDI MSI, one of its major drawbacks is the limited capability of identifying multiple compound classes directly on the same tissue section.We demonstrate the use of grid-aided, parafilm-assisted microdissection to perform MALDI MS imaging and shotgun proteomics and metabolomics in a combined workflow and using only a single tissue section. The grid is generated by microspotting acid dye 25 using a piezoelectric microspotter, and this grid was used as a guide to locate regions of interest and as an aid during manual microdissection. Subjecting the dissected pieces to the modified Folch method allows to separate the metabolites from proteins. The proteins can then be subjected to digestion under controlled conditions to improve protein identification yields.The proof of concept experiment on rat brain generated 162 and 140 metabolite assignments from three ROIs (cerebellum, hippocampus and midbrain/hypothalamus) in positive and negative modes, respectively, and 890, 1303 and 1059 unique proteins. Integrated metabolite and protein overrepresentation analysis identified pathways associated with the biological functions of each ROI, most of which were not identified when looking at the protein and metabolite lists individually.This combined MALDI MS imaging and multi-omics approach further extends the amount of information that can be generated from single tissue sections.To the best of our knowledge, this is the first report combining both imaging and multi-omics analyses in the same workflow and on the same tissue section.Integrated MSI and OMICS workflow scheme. The parafilm covering of the glass slide serves as a support that allows the dissection of entire sections or ROIs after MALDI MS image acquisition. The sections can then be subjected to downstream metabolite and protein identification by LC MS, and the data generated by both methods are used for integrated analyses. Lipid/metabolite identifications can also be correlated back to the imaging dataset to provide peak assignments.Display Omitted
Keywords: Parafilm-assisted microdissection; MALDI MS imaging; Metabolomics; Proteomics; Brain regions; Multiomics;

Astaxanthin attenuated pressure overload-induced cardiac dysfunction and myocardial fibrosis: Partially by activating SIRT1 by Jun Zhang; Quan-zhen Wang; Shao-hua Zhao; Xiang Ji; Jie Qiu; Jian Wang; Yi Zhou; Qian Cai; Jie Zhang; Hai-qing Gao (1715-1728).
Myocardial fibrosis contributes to cardiac dysfunction. Astaxanthin (AST), a member of the carotenoid family, is a well-known antioxidant, but its effect on and underlying mechanisms in myocardial fibrosis are poorly understood. In vivo, myocardial fibrosis and cardiac dysfunction were induced using transverse aortic constriction (TAC). AST was administered to mice for 12 weeks post-surgery. In vitro, transforming growth factor β1 (TGF-β1) was used to stimulate human cardiac fibroblasts (HCFs). EX-527 (6-chloro-2, 3, 4, 9-tetrahydro-1H-carbazole-1-carboxamide) and SIRT1 siRNA were used to inhibit SIRT1 in vivo and in vitro, respectively. The effects of AST on cardiac function and fibrosis were determined. SIRT1 expression and activity were measured to explore the mechanisms underlying its effects.AST improved cardiac function and attenuated fibrosis. Receptor activated-SMADs (R-SMADs), including SMAD2 and SMAD3, played important roles in these processes. The TAC surgery-induced increases in the expression of phosphorylated and acetylated R-SMADs were attenuated by treatment with AST, the translocation and transcriptional activity of R-SMADs were also restrained. These effects were accompanied by an increase in the expression and activity of SIRT1. Inhibiting SIRT1 attenuated the acetylation and transcriptional activity of R-SMADs, but not their phosphorylation and translocation.Our data demonstrate that AST improves cardiac function and attenuates fibrosis by decreasing phosphorylation and deacetylation of R-SMADs. SIRT1 contributes to AST’s protective function by reducing acetylation of R-SMADs.These data suggest that AST may be useful as a preventive/therapeutic agent for cardiac dysfunction and myocardial fibrosis.
Keywords: Astaxanthin; Fibrosis; Cardiac function; SIRT1; R-SMADs;

Akr1 attenuates methylmercury toxicity through the palmitoylation of Meh1 as a subunit of the yeast EGO complex by Zhi-Ting Zhang; Yousuke Ogiwara; Yuichi Ito; Akinori Hikida; Nobuhiko Miura; Shusuke Kuge; Akira Naganuma; Gi-Wook Hwang (1729-1736).
We previously reported that palmitoyltransferase activity of Akr1 is required for alleviation of methylmercury toxicity in yeast. In this study, we identified a factor that alleviates methylmercury toxicity among the substrate proteins palmitoylated by Akr1, and investigated the role of this factor in methylmercury toxicity.Gene disruption and site-directed mutagenesis were used to examine the relationship of methylmercury toxicity and vacuole function. Palmitoylation was investigated using the acyl-biotinyl exchange method. Vacuoles were stained with the fluorescent probe FM4-64.We found that Meh1 (alias Ego1), a substrate protein of Akr1, participates in the alleviation of methylmercury toxicity. Moreover, almost no palmitoylation of Meh1 when Akr1 was knocked out, and mutant Meh1, which is not palmitoylated, did not show alleviation of methylmercury toxicity. The palmitoylated Meh1 was involved in the alleviation of methylmercury toxicity as a constituent of EGO complex which suppresses autophagy. Methylmercury caused vacuole deformation, and this was greater in the yeasts knocking out the EGO complex subunits. 3-Methyladenine, an autophagy inhibitor, suppresses vacuole deformation and cytotoxicity caused by methylmercury. The elevated methylmercury sensitivity by Meh1 knockout almost completely disappeared in the presence of 3-methyladenine.Akr1 reduces methylmercury toxicity through palmitoylation of Meh1. Furthermore, the EGO complex including Meh1 reduces methylmercury toxicity by suppressing the induction of vacuole deformation caused by methylmercury.These findings propose that Meh1 palmitoylated by Akr1 may act as a constituent of the EGO complex when contributing to the decreased cytotoxicity by negatively controlling the induction of autophagy by methylmercury.
Keywords: Methylmercury; Toxicity; Akr1; Palmitoyltransferase; EGO complex; Yeast;

Biophysics and protein corona analysis of Janus cyclodextrin-DNA nanocomplexes. Efficient cellular transfection on cancer cells by M. Martínez-Negro; G. Caracciolo; S. Palchetti; D. Pozzi; A.L. Capriotti; C. Cavaliere; A. Laganà; C. Ortiz Mellet; J.M. Benito; J.M. García Fernández; E. Aicart; E. Junquera (1737-1749).
The self-assembling processes underlining the capabilities of facially differentiated (“Janus”) polycationic amphiphilic cyclodextrins (paCDs) as non-viral gene nanocarriers have been investigated by a pluridisciplinary approach. Three representative Janus paCDs bearing a common tetradecahexanoyl multitail domain at the secondary face and differing in the topology of the cluster of amino groups at the primary side were selected for this study. All of them compact pEGFP-C3 plasmid DNA and promote transfection in HeLa and MCF-7 cells, both in absence and in presence of human serum. The electrochemical and structural characteristics of the paCD-pDNA complexes (CDplexes) have been studied by using zeta potential, DLS, SAXS, and cryo-TEM. paCDs and pDNA, when assembled in CDplexes, render effective charges that are lower than the nominal ones. The CDplexes show a self-assembling pattern corresponding to multilamellar lyotropic liquid crystal phases, characterized by a lamellar stacking of bilayers of the CD-based vectors with anionic pDNA sandwiched among them. When exposed to human serum, either in the absence or in the presence of pDNA, the surface of the cationic CD-based vector becomes coated by a protein corona (PC) whose composition has been analyzed by nanoLC-MS/MS. Some of the CDplexes herein studied showed moderate-to-high transfection levels in HeLa and MCF-7 cancer cells combined with moderate-to-high cell viabilities, as determined by FACS and MTT reduction assays. The ensemble of data provides a detail picture of the paCD-pDNA-PC association processes and a rational base to exploit the protein corona for targeted gene delivery on future in vivo applications.Display Omitted
Keywords: CDplexes; Effective charge ratio; Multilamellar phases; Cellular transfection; Cytotoxicity; Protein corona;

The regulation of lipid metabolism by a hypothetical P-loop NTPase and its impact on fecundity of the brown planthopper by Rui Pang; Jieqi Qiu; Tengchao Li; Pan Yang; Lei Yue; Yixin Pan; Wenqing Zhang (1750-1758).
Insect fecundity can be regulated by multiple genes in several important signaling pathways which form an extremely complicated regulatory network. However, there are still many genes that have significant impact on insect fecundity but their action mode are still unknown.Quantitative real-time PCR (qRT-PCR), immunofluorescence and western blot were used to study the expression profile of Nl23867 in the brown planthopper, Nilaparvata lugens. RNA interference (RNAi), RNA-seq and isobaric tags for relative and absolute quantification (iTRAQ) were performed to investigate the action mode of Nl23867 in the regulation of fecundity. High performance liquid chromatography (HPLC) analysis was performed to detect the fatty acid contents.We show that knockdown of Nl23867, a gene encoding a hypothetical P-loop NTPase, significantly decreased fecundity of N. lugens. Underdeveloped ovaries, fewer eggs laid and reduction in vitellogenin (Vg) protein expression were observed after RNAi knockdown of Nl23867, and most of the affected genes and pathways are fatty acid metabolism-related. We further determined that Nl23867 directly impacts the palmitic acid biosynthesis by regulating the expression of palmitoyl-protein thioesterase (PPT), subsequently affecting the content of total lipids in N. lugens. Nl23867 regulates the fecundity of N. lugens by modulating the biosynthetic pathway of palmitic acid and affecting lipid metabolism during vitellogenesis and oocyte development.The presented study pioneers the exploration into how a function-unknown gene takes part in the regulation of fecundity in an insect, and will contribute to the construction of gene regulatory network for insect fecundity.Display Omitted

DJ-1, a small ubiquitously expressed protein implicated in several pathways associated with Parkinson's disease pathogenesis, has been found to interact with α-synuclein and modulate its aggregation, yet the exact mechanisms remain unclear.The stability and aggregation properties of wild-type DJ-1 under denaturing conditions, such as low pH, high temperature, presence of denaturants were investigated. The interaction between DJ-1 and α-synuclein was tested by SDS-PAGE gel and native gel electrophoresis and by size-exclusion HPLC. Fibrillization was monitored by thioflavin T fluorescence assays and amorphous aggregation was followed by light scattering measurements. The morphology of aggregated species was observed by transmission electron microscopy and atomic force microscopy. Protein secondary structures were characterized by far-UV circular dichroism.DJ-1 fibrillization was first observed at low pH or by adding denaturants. Amorphous aggregates formed at neutral pH, and the aggregation was dramatically accelerated by elevated temperature and the presence of α-synuclein. Aggregation of DJ-1 were enhanced by heating and perturbed by the co-occurrence of α-synuclein but strong interactions between the two proteins were not found.Varying environmental factors led to different aggregation pathways of DJ-1 although a simulated physiological condition would not lead to fibrillization. DJ-1 co-aggregating with α-synuclein may result from weak hydrophobic interaction and DJ-1 exhibited chaperon-like activity in the initial time of α-synuclein aggregation at high temperature.This research on DJ-1 presented its aggregation behavior under denaturing conditions and interaction mechanism with α-synuclein that may help to decipher its potential neuroprotective or neurotoxic role in Parkinson's disease.
Keywords: Parkinson's disease; DJ-1; α-Synuclein; Fibrillization; Aggregation; Denaturing conditions;

Impairment of the class IIa bacteriocin receptor function and membrane structural changes are associated to enterocin CRL35 high resistance in Listeria monocytogenes by Emilse Masias; Fernando G. Dupuy; Paulo Ricardo da Silva Sanches; Juan Vicente Farizano; Eduardo Cilli; Augusto Bellomio; Lucila Saavedra; Carlos Minahk (1770-1776).
Enterocin CRL35 is a class IIa bacteriocin with anti-Listeria activity. Resistance to these peptides has been associated with either the downregulation of the receptor expression or changes in the membrane and cell walls. The scope of the present work was to characterize enterocin CRL35 resistant Listeria strains with MICs more than 10,000 times higher than the MIC of the WT sensitive strain. Listeria monocytogenes INS7 resistant isolates R2 and R3 were characterized by 16S RNA gene sequencing and rep-PCR. Bacterial growth kinetic was studied in different culture media. Plasma membranes of sensitive and resistant bacteria were characterized by FTIR and Langmuir monolayer techniques.The growth kinetic of the resistant isolates was slower as compared to the parental strain in TSB medium. Moreover, the resistant isolates barely grew in a glucose-based synthetic medium, suggesting that these cells had a major alteration in glucose transport. Resistant bacteria also had alterations in their cell wall and, most importantly, membrane lipids. In fact, even though enterocin CRL35 was able to bind to the membrane-water interface of both resistant and parental sensitive strains, this peptide was only able to get inserted into the latter membranes.These results indicate that bacteriocin receptor is altered in combination with membrane structural modifications in enterocin CRL35-resistant L. monocytogenes strains.Highly enterocin CRL35-resistant isolates derived from Listeria monocytogenes INS7 have not only an impaired glucose transport but also display structural changes in the hydrophobic core of their plasma membranes.Display Omitted
Keywords: Bacteriocins; Enterocin CRL35; Synthetic peptides; Listeria;

Polo-like kinase 1 expression is suppressed by CCAAT/enhancer-binding protein α to mediate colon carcinoma cell differentiation and apoptosis by Nirmalya Dasgupta; Bhupesh Kumar Thakur; Atri Ta; Sayan Das; George Banik; Santasabuj Das (1777-1787).
Human polo-like kinase 1 (PLK1), a highly conserved serine/threonine kinase is a key player in several essential cell-cycle events. PLK1 is considered an oncogene and its overexpression often correlates with poor prognosis of cancers, including colorectal cancer (CRC). However, regulation of PLK1 expression in colorectal cells was never studied earlier and it is currently unknown if PLK1 regulates differentiation and apoptosis of CRC.PLK1 expression was analyzed by real-time PCR and western blotting. Transcriptional regulation was studied by reporter assay, gene knock-down, EMSA and ChIP.PLK1 expression was down-regulated during butyrate-induced differentiation of HT-29 and other CRC cells. Also, PLK1 down-regulation mediated the role of butyrate in CRC differentiation and apoptosis. We report here a novel transcriptional regulation of PLK1 by butyrate. Transcription factors CCAAT/enhancer-binding protein α (C/EBPα) and Oct-1 share an overlapping binding site over the PLK1 promoter. Elevated levels of C/EBPα by butyrate treatment of CRC cells competed out the activator protein Oct-1 from binding to the PLK1 promoter and sequestered it. Binding of C/EBPα was associated with increased deacetylation near the transcription start site (TSS) of the PLK1 promoter, which abrogated transcription through reduced recruitment of RNA polymerase II. We also found a synergistic role between the synthetic PLK1-inhibitor SBE13 and butyrate on the apoptosis of CRC cells.This study offered a novel p53-independent regulation of PLK1 during CRC differentiation and apoptosis.Down-regulation of PLK1 is one of the mechanisms underlying the anti-cancer role of dietary fibre-derived butyrate in CRC.Display Omitted
Keywords: Polo-like kinase 1; Sodium butyrate; SBE13; Colorectal cancer; C/EBPα;

RUVBL1-ITFG1 interaction is required for collective invasion in breast cancer by Wenjun Fan; Jiajun Xie; Jianglong Xia; Yan Zhang; Mengying Yang; Hefei Wang; Yujia Pan; Mengjuan Zhang; Baochun Han; Baitong Wu; Zhijie Hou; Dapeng Liang; Chunli Wang; Jie Xu; Lijuan Song; Quentin Liu (1788-1800).
The mechanisms of breast cancer collective invasion are poorly understood limiting the metastasis therapy. The ATPase RUVBL1 is frequently overexpressed in various cancers and plays a crucial role in oncogenic process. We further investigated the role of RUVBL1 in promoting collective invasion and uncovered that targeting RUVBL1 could inhibit metastatic progression.The expression levels of RUVBL1 and ITFG1 were examined by Western blot and qRT-PCR. Co-localization and interaction of RUVBL1 and ITFG1 were determined by immunofluorescence and co-immunoprecipitation. The invasive ability was examined by transwell assay and microfluidic assay. The metastatic and tumorigenic abilities of breast cancer cells were revealed in BALB/c nude mice by xenograft and tail vein injection.ATPase RUVBL1 is highly expressed in breast cancer and predicts the poor prognosis. Elevated expression of RUVBL1 is found in high metastatic breast cancer cells. Silencing RUVBL1 suppresses cancer cell expansion and invasion in vitro and in vivo. RUVBL1 interacts with a conserved transmembrane protein ITFG1 in cytoplasm and plasma membrane to promote the collective invasion. Using a microfluidic model, we demonstrated that silencing RUVBL1 or ITFG1 individually compromises collective invasion of breast cancer cells.RUVBL1 is a vital regulator for collective invasion. The interaction between RUVBL1 and ITFG1 is required for breast cancer cell collective invasion and progression.Targeting collective invasion promoted by RUVBL1-ITFG1 complex provides a novel therapeutic strategy to improve the prognosis of invasive breast cancer.
Keywords: Breast cancer; RUVBL1; ITFG1; Microfluidic; Collective invasion;

Modulation of gut microbiota contributes to curcumin-mediated attenuation of hepatic steatosis in rats by Wenhuan Feng; Hongdong Wang; Pengzi Zhang; Caixia Gao; Junxian Tao; Zhijuan Ge; Dalong Zhu; Yan Bi (1801-1812).
Structural disruption of gut microbiota contributes to the development of non-alcoholic fatty liver disease (NAFLD) and modulating the gut microbiota represents a novel strategy for NAFLD prevention. Although previous studies have demonstrated that curcumin alleviates hepatic steatosis, its effect on the gut microbiota modulation has not been investigated.Next generation sequencing and multivariate analysis were utilized to evaluate the structural changes of gut microbiota in a NAFLD rat model induced by high fat-diet (HFD) feeding.We found that curcumin attenuated hepatic ectopic fat deposition, improved intestinal barrier integrity, and alleviated metabolic endotoxemia in HFD-fed rats. More importantly, curcumin dramatically shifted the overall structure of the HFD-disrupted gut microbiota toward that of lean rats fed a normal diet and altered the gut microbial composition. The abundances of 110 operational taxonomic units (OTUs) were altered by curcumin. Seventy-six altered OTUs were significantly correlated with one or more hepatic steatosis associated parameters and designated ‘functionally relevant phylotypes’. Thirty-six of the 47 functionally relevant OTUs that were positively correlated with hepatic steatosis associated parameters were reduced by curcumin.These results indicate that curcumin alleviates hepatic steatosis in part through stain-specific impacts on hepatic steatosis associated phylotypes of gut microbiota in rats.Compounds with antimicrobial activities should be further investigated as novel adjunctive therapies for NAFLD.
Keywords: Curcumin; Hepatic steatosis; Gut microbiota;

Potential role of heme metabolism in the inducible expression of heme oxygenase-1 by Taka-aki Takeda; Machiko Sasai; Yuka Adachi; Keiko Ohnishi; Jun-ichi Fujisawa; Shingo Izawa; Shigeru Taketani (1813-1824).
The degradation of heme significantly contributes to cytoprotective effects against oxidative stress and inflammation. The enzyme heme oxygenase-1 (HO-1), involved in the degradation of heme, forms carbon monoxide (CO), ferrous iron, and bilirubin in conjunction with biliverdin reductase, and is induced by various stimuli including oxidative stress and heavy metals. We examined the involvement of heme metabolism in the induction of HO-1 by the inducers sulforaphane and sodium arsenite.We examined the expression of HO-1 in sulforaphane-, sodium arsenite- and CORM3-treated HEK293T cells, by measuring the transcriptional activity and levels of mRNA and protein.The blockade of heme biosynthesis by succinylacetone and N-methyl protoporphyrin, which are inhibitors of heme biosynthesis, markedly decreased the induction of HO-1. The knockdown of the first enzyme in the biosynthesis of heme, 5-aminolevulinic acid synthase, also decreased the induction of HO-1. The cessation of HO-1 induction occurred at the transcriptional and translational levels, and was mediated by the activation of the heme-binding transcriptional repressor Bach1 and translational factor HRI. CO appeared to improve the expression of HO-1 at the transcriptional and translational levels.We demonstrated the importance of heme metabolism in the stress-inducible expression of HO-1, and also that heme and its degradation products are protective factors for self-defense responses.The key role of heme metabolism in the stress-inducible expression of HO-1 may promote further studies on heme and its degradation products as protective factors of cellular stresses and iron homeostasis in specialized cells, organs, and whole animal systems.Display Omitted
Keywords: HO-1; Heme metabolism; Sulforaphane; CORM3; Bach1; HRI;

Structural optimization of an aptamer generated from Ligand-Guided Selection (LIGS) resulted in high affinity variant toward mIgM expressed on Burkitt's lymphoma cell lines by Hasan E. Zümrüt; Sana Batool; Nabeela Van; Shanell George; Sanam Bhandari; Prabodhika Mallikaratchy (1825-1832).
Aptamers are synthetic, short nucleic acid molecules capable of specific target recognition. Aptamers are selected using a screening method termed Systematic Evolution of Ligands by Exponential enrichment (SELEX). We recently have introduced a variant of SELEX called “Ligand-Guided-Selection” (LIGS) that allows the identification of specific aptamers against known cell-surface proteins. Utilizing LIGS, we introduced three specific aptamers against membrane-bound IgM (mIgM), which is the hallmark of B cells. Out of the three aptamers selected against mIgM, an aptamer termed R1, in particular, was found to be interesting due to its ability to recognize mIgM on target cells and then block anti-IgM antibodies binding their antigen. We systematically truncated parent aptamer R1 to design shorter variants with enhanced affinity. Importantly, herein we show that the specificity of the most optimized variant of R1 aptamer is similar to that of anti-IgM antibody, indicating that the specificity of the ligand utilized in selective elution of the aptamer determines the specificity of the LIGS-generated aptamer. Furthermore, we report that truncated variants of R1 are able to recognize mIgM-positive human B lymphoma BJAB cells at physiological temperature, demonstrating that LIGS-generated aptamers could be re-optimized into higher affinity variants. Collectively, these findings show the significance of LIGS in generating highly specific aptamers with potential applications in biomedicine.
Keywords: Aptamer; SELEX; B-Cell Receptor; truncation; LIGS;

Cellular effects of the microtubule-targeting agent peloruside A in hypoxia-conditioned colorectal carcinoma cells by Jiří Řehulka; Narendran Annadurai; Ivo Frydrych; Pawel Znojek; Petr Džubák; Peter Northcote; John H. Miller; Marián Hajdúch; Viswanath Das (1833-1843).
Hypoxia is a prominent feature of solid tumors, dramatically remodeling microtubule structures and cellular pathways and contributing to paclitaxel resistance. Peloruside A (PLA), a microtubule-targeting agent, has shown promising anti-tumor effects in preclinical studies. Although it has a similar mode of action to paclitaxel, it binds to a distinct site on β-tubulin that differs from the classical taxane site. In this study, we examined the unexplored effects of PLA in hypoxia-conditioned colorectal HCT116 cancer cells.Cytotoxicity of PLA was determined by cell proliferation assay. The effects of a pre-exposure to hypoxia on PLA-induced cell cycle alterations and apoptosis were examined by flow cytometry, time-lapse imaging, and western blot analysis of selected markers. The hypoxia effect on stabilization of microtubules by PLA was monitored by an intracellular tubulin polymerization assay.Our findings show that the cytotoxicity of PLA is not altered in hypoxia-conditioned cells compared to paclitaxel and vincristine. Furthermore, hypoxia does not alter PLA-induced microtubule stabilization nor the multinucleation of cells. PLA causes cyclin B1 and G2/M accumulation followed by apoptosis.The cellular and molecular effects of PLA have been determined in normoxic conditions, but there are no reports of PLA effects in hypoxic cells. Our findings reveal that hypoxia preconditioning does not alter the sensitivity of HCT116 to PLA.These data report on the cellular and molecular effects of PLA in hypoxia-conditioned cells for the first time, and will encourage further exploration of PLA as a promising anti-tumor agent.Display Omitted
Keywords: Apoptosis; Hypoxia; Microtubule; Multi-drug resistance; Paclitaxel; Peloruside A;

Modulating short tryptophan- and arginine-rich peptides activity by substitution with histidine by Mihaela Bacalum; Lorant Janosi; Florina Zorila; Ana-Maria Tepes; Cristina Ionescu; Elena Bogdan; Niculina Hadade; Liviu Craciun; Ion Grosu; Ioan Turcu; Mihai Radu (1844-1854).
High antimicrobial efficacy of short tryptophan-and arginine-rich peptides makes them good candidates in the fight against pathogens. Substitution of tryptophan and arginine by histidine could be used to modulate the peptides efficacy by optimizing their structures.The peptide (RRWWRWWRR), reported to showed good antimicrobial efficacy, was used as template, seven new analogs being designed substituting tryptophan or arginine with histidine. The peptides' efficacy was tested against E. coli, B. subtilis and S. aureus. The cytotoxicity and hemolytic effect were evaluated and the therapeutic index was inferred for each peptide. Atomic force microscopy and molecular simulation were used to analyze the effects of peptides on bacterial membrane.The substitution of tryptophan by histidine proved to strongly modulate the antimicrobial activity, mainly by changing the peptide-to-membrane binding energy. The substitution of arginine has low effect on the antimicrobial efficacy. The presence of histidine residue reduced the cytotoxic and hemolytic activity of the peptides in some cases maintaining the same efficacy against bacteria. The peptides' antimicrobial activity was correlated to the 3D-hydrophobic moment and to a simple structure-based packing parameter.The results show that some of these peptides have the potential to become good candidates to fight against bacteria. The substitution by histidine proved to fine tune the therapeutic index allowing the optimization of the peptide structure mainly by changing its binding energy and 3D-hydrophobic moment.The short tryptophan reach peptides therapeutic index can be maximized using the histidine substitution to optimize their structure.
Keywords: Antimicrobial peptides; Therapeutic index; Tryptophan; Arginine; Cytotoxicity; Molecular simulations;

The analysis of the thermodynamic driving forces of ligand-protein binding has been suggested to be a key component for the selection and optimization of active compounds into drug candidates. The binding enthalpy as deduced from isothermal titration calorimetry (ITC) is usually interpreted assuming single-step binding of a ligand to one conformation of the target protein. Although successful in many cases, these assumptions are oversimplified approximations of the reality with flexible proteins and complicated binding mechanism in many if not most cases. The relationship between protein flexibility and thermodynamic signature of ligand binding is largely understudied.Directed mutagenesis, X-ray crystallography, enzyme kinetics and ITC methods were combined to dissect the influence of loop flexibility on the thermodynamics and mechanism of ligand binding to histone deacetylase (HDAC)-like amidohydrolases.The general ligand-protein binding mechanism comprises an energetically demanding gate opening step followed by physical binding. Increased flexibility of the L2-loop in HDAC-like amidohydrolases facilitates access of ligands to the binding pocket resulting in predominantly enthalpy-driven complex formation.The study provides evidence for the great importance of flexibility adjacent to the active site channel for the mechanism and observed thermodynamic driving forces of molecular recognition in HDAC like enzymes.The flexibility or malleability in regions adjacent to binding pockets should be given more attention when designing better drug candidates. The presented case study also suggests that the observed binding enthalpy of protein-ligand systems should be interpreted with caution, since more complicated binding mechanisms may obscure the significance regarding potential drug likeness.
Keywords: Thermodynamic signatures; Protein-ligand binding; Histone deacetylases; Binding mechanism; Conformational flexibility;

Recently, aptamers have been extensively researched for therapy and diagnostic applications. Thrombin-binding aptamer is a 15 nt deoxyribonucleic acid screened by SELEX, it can specifically bind to thrombin and inhibit blood coagulation. Since it is also endowed with excellent antitumor activity, the intrinsic anticoagulation advantage converted to a main potential side effect for its further application in antiproliferative therapy.Site-specific alkylation was conducted through nucleophilic reaction of phosphorothioated TBAs using bromide reagents. Circular dichroism (CD) spectroscopy and surface plasmon resonance (SPR) measurements were used to evaluate anticoagulation activity, and a CCK-8 assay was used to determine cell proliferation activity.The CD spectra of the modified TBAs were weakened, and their affinity for thrombin was dramatically reduced, as reflected by the K D values. On the other hand, their inhibition of A549 cells was retained.Incorporation of different alkyls apparently disrupted the binding of TBA to thrombin while maintaining the antitumor activity.A new modification strategy was established for the use of TBA as a more selective antitumor agent.Display Omitted
Keywords: Thrombin binding aptamer; Alkylation; Antitumor; Selectivity;

HmuS from Yersinia pseudotuberculosis is a non-canonical heme-degrading enzyme to acquire iron from heme by Masato Onzuka; Yukari Sekine; Takeshi Uchida; Koichiro Ishimori; Shin-ichi Ozaki (1870-1878).
Some Gram-negative pathogens import host heme into the cytoplasm and utilize it as an iron source for their survival. We report here that HmuS, encoded by the heme utilizing system (hmu) locus, cleaves the protoporphyrin ring to release iron from heme. A liquid chromatography/mass spectrometry analysis revealed that the degradation products of this reaction are two biliverdin isomers that result from transformation of a verdoheme intermediate. This oxidative heme degradation by HmuS required molecular oxygen and electrons supplied by either ascorbate or NADPH. Electrons could not be directly transferred from NADPH to heme; instead, ferredoxin-NADP+ reductase (FNR) functioned as a mediator. Although HmuS does not share amino acid sequence homology with heme oxygenase (HO), a well-known heme-degrading enzyme, absorption and resonance Raman spectral analyses suggest that the heme iron is coordinated with an axial histidine residue and a water molecule in both enzymes. The substitution of axial His196 or distal Arg102 with an alanine residue in HmuS almost completely eliminated heme-degradation activity, suggesting that Fe-His coordination and interaction of a distal residue with water molecules in the heme pocket are important for this activity.Display Omitted
Keywords: Heme; Biliverdin; Iron; Ferredoxin; Ferredoxin-NADP+ reductase; Yersinia pseudotuberculosis;

Loss of DUSP3 activity radiosensitizes human tumor cell lines via attenuation of DNA repair pathways by Thompson E.P. Torres; Lilian C. Russo; Alexsandro Santos; Gabriela R. Marques; Yuli T. Magalhaes; Sartaj Tabassum; Fabio L. Forti (1879-1894).
Radiotherapy causes the regression of many human tumors by increasing DNA damage, and the novel molecular mechanisms underlying the genomic instability leading to cancer progression and metastasis must be elucidated. Atypical dual-specificity phosphatase 3 (DUSP3) has been shown to down-regulate mitogen-activated protein kinases (MAPKs) to control the proliferation and apoptosis of human cancer cells. We have recently identified novel molecular targets of DUSP3 that function in DNA damage response and repair; however, whether DUSP3 affects these processes remains unknown.Tumor cell lines in which DUSP3 activity was suppressed by pharmacological inhibitors or a targeted siRNA were exposed to gamma radiation, and proliferation, survival, DNA strand breaks and recombination repair pathways were sequentially analyzed.The combination of reduced DUSP3 activity and gamma irradiation resulted in decreased cellular proliferation and survival and increased cellular senescence compared with the effects of radiation exposure alone. Gamma radiation-induced DNA damage was increased by the loss of DUSP3 activity and correlated with increased levels of phospho-H2AX protein and numbers of ionizing radiation-induced γ-H2AX foci, which were reflected in diminished efficiencies of homologous recombination (HR) and non-homologous end-joining (NHEJ) repair. Similar results were obtained in ATM-deficient cells, in which reduced DUSP3 activity increased radiosensitivity, independent of increased MAPK phosphorylation.The loss of DUSP3 activity markedly increases gamma radiation-induced DNA strand breaks, suggesting a potential novel role for DUSP3 in DNA repair.The radioresistance of tumor cells is effectively reduced by a combination of approaches through the inhibition of DUSPs.Display Omitted
Keywords: Dual-specificity tyrosine phosphatases; DUSP3/VHR; Gamma radiation; Recombination DNA repair mechanisms; DNA damage response; HeLa and MeWo tumor cells;