BBA - General Subjects (v.1861, #5PA)

Evidence of proteolipid domain formation in an inner mitochondrial membrane mimicking model by Mouhedine Cheniour; Jonathan Brewer; Luis Bagatolli; Olivier Marcillat; Thierry Granjon (969-976).
Mitochondrial creatine kinase (mtCK) is highly abundant in mitochondria; its quantity is equimolecular to the Adenylic Nucleotide Translocator and represents 1% of the mitochondrial proteins. It is a multitask protein localized in the mitochondria intermembrane space where it binds to the specific cardiolipin (CL) phospholipid. If mtCK was initially thought to be exclusively implicated in energy transfer between mitochondria and cytosol through a mechanism referred to as the phosphocreatine shuttle, several recent studies suggested an additional role in maintaining mitochondria membrane structure.To further characterized mtCK binding process we used multiphoton excitation fluorescence microscopy coupled with Giant Unilamellar Vesicles (GUV) and laurdan as fluorescence probe.We gathered structural and dynamical information on the molecular events occurring during the binding of mtCK to the mitochondria inner membrane.We present the first visualization of mtCK-induced CL segregation on a bilayer model forming micrometer-size proteolipid domains at the surface of the GUV. Those microdomains, which only occurred when CL is included in the lipid mixture, were accompanied by the formation of protein multimolecular assembly, vesicle clamping, and changes in both vesicle curvature and membrane fluidityThose results highlighted the importance of the highly abundant mtCK in the lateral organization of the mitochondrial inner membrane.Microdomains were induced in mitochondria-mimicking membranes composed of natural phospholipids without cholesterol and/or sphingolipids differing from the proposed cytoplasmic membrane rafts. Those findings as well as membrane curvature modification were discussed in relation with protein-membrane interaction and protein cluster involvement in membrane morphology.Display Omitted
Keywords: GUVs; Fluorescence microscopy; Cardiolipin; Mitochondrial creatine kinase; Protein clusters; Membrane curvature;

Structure and function of Vibrio cholerae accessory cholera enterotoxin in presence of gold nanoparticles: Dependence on morphology by Tanaya Chatterjee; Barun K. Chatterjee; Tultul Saha; Kazi Mirajul Hoque; Pinak Chakrabarti (977-986).
Accessory cholera enterotoxin (Ace) is a classical enterotoxin produced by Vibrio cholerae, the causative agent for cholera. Considering the crucial role of Ace in pathogenesis of cholera, we explored the modulation of structure/function of Ace using gold nanoparticles (AuNPs) of different size and shape – spherical (AuNS10 and AuNS100, the number indicating the diameter in nm) and rod (AuNR10).Biophysical techniques have been used to find out structural modulation of Ace by AuNPs. Effect of AuNP on Ace conformation was monitored by far-UV CD; urea-induced unfolding and binding of Ace to various AuNPs were studied by tryptophan fluorescence. In vivo experiments using mouse ileal loop and Ussing chamber were carried out to corroborate biophysical data.Biophysical data revealed degradation of Ace by AuNR10 and AuNS100, not by AuNS10. The feature of AuNR10 having high aspect ratio, but with the same transverse diameter as that of AuNS10 enabled us to explore the importance of morphology on modulation of protein structure/function. The equilibration time for adsorption shows dependence on the radius of curvature, being largest for AuNR10. In vivo experiments revealed the efficacy of AuNR10 and AuNS100 for reduced fluid accumulation, indicative of the loss of activity of Ace.We show how biophysical studies and in vivo experiments go hand-in-hand in establishing the efficacy and role of size/shape of AuNPs on a toxin structure.The effect of AuNP on toxin depends on its morphology. The targeted modulation of Ace could be of therapeutic benefit for gastrointestinal disorders.Display Omitted
Keywords: Vibrio cholerae; Accessory cholera enterotoxin; Gold nanoparticles; Nanoparticle morphology; Structure-function modulation;

Global stability of an α-ketoglutarate-dependent dioxygenase (TauD) and its related complexes by Kate L. Henderson; Mingjie Li; Salette Martinez; Edwin A. Lewis; Robert P. Hausinger; Joseph P. Emerson (987-994).
TauD is a nonheme iron(II) and α-ketoglutarate (αKG) dependent dioxygenase, and a member of a broader family of enzymes that oxidatively decarboxylate αKG to succinate and carbon dioxide thereby activating O2 to perform a range of oxidation reactions. However before O2 activation can occur, these enzymes bind both substrate and cofactor in an effective manner. Here the thermodynamics associated with substrate and cofactor binding to FeTauD are explored.Thermal denaturation of TauD and its enzyme-taurine, enzyme-αKG, and enzyme-taurine-αKG complexes are explored using circular dichroism (CD) spectroscopy and differential scanning calorimetry (DSC).Taurine binding is endothermic (+ 26 kcal/mol) and entropically driven that includes burial of hydrophobic surfaces to close the lid domain. Binding of αKG is enthalpically favorable and shows cooperativity with taurine binding, where the change in enthalpy associated with αKG binding (δΔH cal) increases from − 30.1 kcal/mol when binding to FeTauD to − 65.2 kcal/mol when binding to the FeTauD-taurine complex.The intermolecular interactions that govern taurine and αKG binding impact the global stability of TauD and its complexes, with clear and dramatic cooperativity between substrate and cofactor.Thermal denaturation of TauD and its enzyme-taurine, enzyme-αKG, and enzyme-taurine-αKG complexes each exhibited increased temperature stability over the free enzyme. Through deconvolution of the energetic profiles for all species studied, a thermodynamic cycle was generated that shows significant cooperativity between substrate and cofactor binding which continues to clarity the events leading up O2 activation.Display Omitted
Keywords: Differential scanning calorimetry; Circular dichroism; Nonheme iron(II); Alpha-ketoglutarate; Taurine; And thermal stability;

We recently demonstrated that a novel storage and transport mechanism for nitric oxide (NO) mediated by glutathione-S-transferase P1 (GSTP1) and multidrug resistance protein 1 (MRP1/ABCC1), protects M1-macrophage (M1-MØ) models from large quantities of endogenous NO. This system stores and transports NO as dinitrosyl-dithiol-iron complexes (DNICs) composed of iron, NO and glutathione (GSH). Hence, this gas with contrasting anti- and pro-tumor effects, which has been assumed to be freely diffusible, is a tightly-regulated species in M1-MØs. These control systems prevent NO cytotoxicity and may be responsible for delivering cytotoxic NO as DNICs via MRP1 from M1-MØs, to tumor cell targets.
Keywords: Nitric oxide; Macrophages; MRP1;

A new light on Alkaptonuria: A Fourier-transform infrared microscopy (FTIRM) and low energy X-ray fluorescence (LEXRF) microscopy correlative study on a rare disease by Elisa Mitri; Lia Millucci; Lucia Merolle; Giulia Bernardini; Lisa Vaccari; Alessandra Gianoncelli; Annalisa Santucci (1000-1008).
Alkaptonuria (AKU) is an ultra-rare disease associated to the lack of an enzyme involved in tyrosine catabolism. This deficiency results in the accumulation of homogentisic acid (HGA) in the form of ochronotic pigment in joint cartilage, leading to a severe arthropathy. Secondary amyloidosis has been also unequivocally assessed as a comorbidity of AKU arthropathy. Composition of ochronotic pigment and how it is structurally related to amyloid is still unknown.We exploited Synchrotron Radiation Infrared and X-Ray Fluorescence microscopies in combination with conventional bio-assays and analytical tools to characterize chemical composition and morphology of AKU cartilage.We evinced that AKU cartilage is characterized by proteoglycans depletion, increased Sodium levels, accumulation of lipids in the peri-lacunar regions and amyloid formation. We also highlighted an increase of aromatic compounds and oxygen-containing species, depletion in overall Magnesium content (although localized in the peri-lacunar region) and the presence of calcium carbonate fragments in proximity of cartilage lacunae.We highlighted common features between AKU and arthropathy, but also specific signatures of the disease, like presence of amyloids and peculiar calcifications. Our analyses provide a unified picture of AKU cartilage, shedding a new light on the disease and opening new perspectives.Ochronotic pigment is a hallmark of AKU and responsible of tissue degeneration. Conventional bio-assays have not yet clarified its composition and its structural relationship with amyloids. The present work proposes new strategies for filling the aforementioned gap that encompass the integration of new analytical approaches with standardized analyses.Display Omitted
Keywords: Alkaptonuria; Arthropathy; Amyloids; Ochronosis; FTIR Microscopy; X-ray fluorescence microscopy;

Functions of Rho family of small GTPases and Rho-associated coiled-coil kinases in bone cells during differentiation and mineralization by Agnieszka Strzelecka-Kiliszek; Saida Mebarek; Monika Roszkowska; René Buchet; David Magne; Slawomir Pikula (1009-1023).
Members of Rho-associated coiled-coil kinases (ROCKs) are effectors of Rho family of small GTPases. ROCKs have multiple functions that include regulation of cellular contraction and polarity, adhesion, motility, proliferation, apoptosis, differentiation, maturation and remodeling of the extracellular matrix (ECM).Here, we focus on the action of RhoA and RhoA effectors, ROCK1 and ROCK2, in cells related to tissue mineralization: mesenchymal stem cells, chondrocytes, preosteoblasts, osteoblasts, osteocytes, lining cells and osteoclasts.The activation of the RhoA/ROCK pathway promotes stress fiber formation and reduces chondrocyte and osteogenic differentiations, in contrast to that in mesenchymal stem cells which stimulated the osteogenic and the chondrogenic differentiation. The effects of Rac1 and Cdc42 in promoting chondrocyte hypertrophy and of Rac1, Rac2 and Cdc42 in osteoclast are discussed. In addition, members of the Rho family of GTPases such Rac1, Rac2, Rac3 and Cdc42, acting upstream of ROCK and/or other protein effectors, may compensate the actions of RhoA, affecting directly or indirectly the actions of ROCKs as well as other protein effectors.ROCK activity can trigger cartilage degradation and affect bone formation, therefore these kinases may represent a possible therapeutic target to treat osteoarthritis and osseous diseases. Inhibition of Rho/ROCK activity in chondrocytes prevents cartilage degradation, stimulate mineralization of osteoblasts and facilitate bone formation around implanted metals. Treatment with osteoprotegerin results in a significant decrease in the expression of Rho GTPases, ROCK1 and ROCK2, reducing bone resorption. Inhibition of ROCK signaling increases osteoblast differentiation in a topography-dependent manner.Display Omitted
Keywords: Bone cells; Differentiation; Mineralization; ROCK; Signal transduction; Vesicles;

Stress granules assembly affects detection of mRNA in living cells by the NanoFlares; an important aspect of the technology by Magdalena Wolczyk; Paulina Podszywalow-Bartnicka; Lukasz Bugajski; Katarzyna Piwocka (1024-1035).
The recently announced new methodologies to detect mRNA molecules in single cells offer opportunities for research, medicine and molecular diagnostics. The NanoFlare RNA Detection Probes are tools for characterizing RNA content (not localization) using fluorescence-based approaches in living cells. Combined with flow cytometry, NanoFlares have expanded the available possibilities of quantitative analysis of mRNA level in a single cell. Herein we present that in some cases, the specific NanoFlare probes (SmartFlares) detect different amounts of mRNA compared to qPCR. Using the previously published model, in which we studied influence of BCR-ABL oncogene on BRCA1 mRNA translation, we found that the NanoFlare-mediated measurement of mRNA was affected by the assembly of stress granules, structures which store mRNA in complexes with RNA binding proteins. With the usage of chemical compounds we confirmed that under conditions supporting assembly of stress granules, the detection of mRNAs by these probes was decreased, whereas disassembly resulted in the increased mRNAs detection. Altogether, we showed that assembly of stress granules could interfere with mRNA accessibility to the NanoFlare RNA Detection Probes, indicating that the SmartFlares could recognize only the translationally active pool of mRNA, contrary to qPCR. This can significantly influence the quality of obtained data and should be taken into consideration while planning the analysis of mRNA markers using NanoFlares.
Keywords: NanoFlares; Stress granules; mRNA detection; mRNA biomarkers; Cancer; Diagnostic;

Alteration of N-glycan expression profile and glycan pattern of glycoproteins in human hepatoma cells after HCV infection by Tian Xiang; Ganglong Yang; Xiaoyu Liu; Yidan Zhou; Zhongxiao Fu; Fangfang Lu; Jianguo Gu; Naoyuki Taniguchi; Zengqi Tan; Xi Chen; Yan Xie; Feng Guan; Xiao-Lian Zhang (1036-1045).
Hepatitis C virus (HCV) infection causes chronic liver diseases, liver fibrosis and even hepatocellular carcinoma (HCC). However little is known about any information of N-glycan pattern in human liver cell after HCV infection.The altered profiles of N-glycans in HCV-infected Huh7.5.1 cell were analyzed by using mass spectrometry. Then, lectin microarray, lectin pull-down assay, reverse transcription-quantitative real time PCR (RT-qPCR) and western-blotting were used to identify the altered N-glycosylated proteins and glycosyltransferases.Compared to uninfected cells, significantly elevated levels of fucosylated, sialylated and complex N-glycans were found in HCV infected cells. Furthermore, Lens culinaris agglutinin (LCA)-binding glycoconjugates were increased most. Then, the LCA-agarose was used to precipitate the specific glycosylated proteins and identify that fucosylated modified annexin A2 (ANXA2) and heat shock protein 90 beta family member 1 (HSP90B1) was greatly increased in HCV-infected cells. However, the total ANXA2 and HSP90B1 protein levels remained unchanged. Additionally, we screened the mRNA expressions of 47 types of different glycosyltransferases and found that α1,6-fucosyltransferase 8 (FUT8) was the most up-regulated and contributed to strengthen the LCA binding capability to fucosylated modified ANXA2 and HSP90B1 after HCV infection.HCV infection caused the altered N-glycans profiles, increased expressions of FUT8, fucosylated ANXA2 and HSP90B1 as well as enhanced LCA binding to Huh7.5.1.Our results may lay the foundation for clarifying the role of N-glycans and facilitate the development of novel diagnostic biomarkers and therapeutic targets based on the increased FUT8, fucosylated ANXA2 and HSP90B1 after HCV infection.
Keywords: Glycosyltransferase expression analysis; Hepatitis C virus (HCV); Lectin microarray; Mass spectrometry; N-glycan; α1,6-fucosyltransferase 8 (FUT8);

miR-634 is a Pol III-dependent intronic microRNA regulating alternative-polyadenylated isoforms of its host gene PRKCA by Elvezia Maria Paraboschi; Giulia Cardamone; Valeria Rimoldi; Stefano Duga; Giulia Soldà; Rosanna Asselta (1046-1056).
The protein kinase C alpha (PRKCA) gene, coding for a Th17-cell-selective kinase, shows a complex splicing pattern, with at least 2 stable alternative transcripts characterized by an alternative upstream polyadenylation site. Polymorphisms in this gene were associated with several conditions, including multiple sclerosis, asthma, schizophrenia, and cancer. The presence of a microRNA (miRNA), i.e. miR-634, within intron 15 of the PRKCA gene, suggests the intriguing possibility that this miRNA might play a role in the susceptibility to these pathologies.Here, we characterized miR-634 expression profile and searched for its putative targets using a combination of RT-PCR and gene reporter assays.The quantitative analysis of PRKCA and miR-634 transcripts in a panel of human tissues and cell lines revealed discordant expression profiles, suggesting the presence of an independent miR-634 promoter and/or a possible direct role of miR-634 in modulating PRKCA expression. Functional studies demonstrated the existence of a miRNA-specific promoter, which was shown to be Pol-III-dependent. Furthermore, transfection experiments showed that miR-634 is able to target its host gene by specifically down-regulating the shorter alternative-polyadenylated isoforms.MiR-634 is a Pol III-dependent intronic miRNA, which could target its host gene through a “first-order” negative feedback.MiR-634 is one of the few characterized examples of Pol-III-dependent intronic miRNAs. Its independent transcription from the host gene suggests caution in using expression profiles of host genes as proxies for the expression of the corresponding intronic miRNAs.
Keywords: miR-634; PRKCA; Promoter; Target gene; Multiple sclerosis;

Multifaceted functions of the forkhead box transcription factors FoxO1 and FoxO3 in skin by Dimitrios Tsitsipatis; Lars-Oliver Klotz; Holger Steinbrenner (1057-1064).
The ubiquitously expressed forkhead box, class O (FoxO) transcription factors act as signaling integrators in extensive transcriptional networks, ensuring maintenance of cell and tissue homeostasis over time and in response to environmental challenges. Proteins whose biosynthesis is controlled through FoxOs fulfil key functions in antioxidant defense, metabolism, cell cycle regulation and apoptosis.All four mammalian FoxO isoforms (FoxO1, FoxO3, FoxO4 and FoxO6) are expressed in skin but functions have been specified only for FoxO1 and FoxO3. This review provides an overview on the roles of FoxO1 and FoxO3 in the major types of skin cells: fibroblasts, keratinocytes and melanocytes.As expected because of their target genes, FoxOs are involved in counter-acting oxidative stress and in decisions on cell fate regarding apoptosis or senescence. However, their role in skin surpasses these rather obvious tasks: FoxO1 is part of signaling axes related to the control of epidermal morphogenesis and the pathogenesis of acne. FoxO3 dampens the biosynthesis of melanin in melanocytes; on the other hand, FoxO3 suppression in melanoma is associated with impaired apoptosis and increased metastatic potential of melanoma cells. Upon skin injury, a well-balanced and -timed up-regulation of FoxOs appears to support the healing process through affecting proliferation, migration and apoptosis of keratinocytes, fibroblasts and other cells accumulating at the wounded site.FoxO1 and FoxO3 are discussed as homeostatic factors that influence morphogenesis, maintenance and repair processes in skin as well as the pathogenesis of disorders such as acne and skin cancer.
Keywords: Epidermis; p63; IGF-1; Melanogenesis; Wound healing; UV;

Glucose-derived AGEs promote migration and invasion of colorectal cancer by up-regulating Sp1 expression by Ruyuan Deng; Huo Wu; Hui Ran; Xiang Kong; Lei Hu; Xiao Wang; Qing Su (1065-1074).
It is well established that the risk of colorectal cancer (CRC) is significantly increased in diabetic patients. As one of main forms of the advanced glycation end products (AGEs) that accumulate in vivo, glucose-derived AGEs play an important role in the pathogenesis of diabetic complications and may contribute to CRC progression. However, to date, both the contribution of glucose-derived AGEs to the course of CRC and the underlying mechanism are unclear. In the present study, the concentration of glucose-derived AGEs in the serum and tumor tissue of patients with CRC increased. A clinical data analysis demonstrated that the expression of the receptor for AGEs (RAGE), Specificity Protein 1 (Sp1), and matrix metallopeptidase − 2 (MMP2) was significantly higher in cancerous tissues compared with non-tumor tissue in Chinese Han patients with CRC and that RAGE expression was closely associated with lymph node metastasis and TNM stage. Furthermore, in vivo and in vitro experiments showed that AGEs promoted invasion and migration of colorectal cancer, and the AGEs treatment increased the expression of RAGE, Sp1, and MMP2 in a dose-dependent manner. A RAGE blocking antibody and an Sp1-specific siRNA attenuated the AGE-induced effects. Moreover, the AGEs treatment increased the phosphorylation of ERK, and reducing the phosphorylation level of ERK by MEK1/2 inhibitor decreased the expression of Sp1. In conclusion, glucose-derived AGEs promote the invasion and metastasis of CRC partially through the RAGE/ERK/SP1/MMP2 cascade. These findings may provide an explanation for the poor prognoses of colorectal cancer in diabetic patients.
Keywords: AGEs; RAGE; Sp1; Colorectal cancer; Migration; Invasion;

ATP-dependent activity and mitochondrial localization of drug efflux pumps in doxorubicin-resistant breast cancer cells by Julie Dartier; Elsa Lemaitre; Igor Chourpa; Caroline Goupille; Stéphane Servais; Stéphan Chevalier; Karine Mahéo; Jean-François Dumas (1075-1084).
We hypothesized that, among the mechanisms of drug-resistance acquired by doxorubicin (DOX)-resistant breast cancer cells to maintain cell survival, ATP-dependent drug efflux pumps could be expressed in their mitochondrial membranes and this might limit the accumulation of DOX in this subcellular compartment in relation to mitochondrial ATP production.Mitochondrial DOX accumulation: the presence and the activity of mitochondrial efflux pumps and their relationship with mitochondrial ATP synthesis were analyzed in DOX-resistant (MCF-7doxR) and -sensitive (MCF-7S) breast cancer cells. Mitochondrial accumulation of DOX (autofluorescence) was decreased when ATP was produced, but only in MCF-7doxR. In these DOX-resistant cells, breast cancer resistance protein (BCRP) and multidrug resistance-associated protein (MRP1) were expressed and localized in mitochondria (confocal microscopy and confocal spectral imaging studies). In addition, mitochondrial accumulation of DOX was increased by BCRP and MRP1 inhibitors and, to a lower extent, by the mitochondrial ATP synthase inhibitor, oligomycin, in MCF-7doxR.Both BCRP and MRP1 were localized in mitochondria and participated to the reduction of mitochondrial accumulation of DOX in MCF-7doxR. This process was partly dependent of mitochondrial ATP synthesis. General Significance. The present study provides novel insights in the involvement of mitochondria in the underlying mechanisms of DOX-resistance in breast cancer cells.
Keywords: Breast cancer; Breast cancer resistance protein; Chemoresistance; Mitochondrial ATP synthesis; Multidrug resistance-associated protein;

Iron-sulfur (Fe-S) clusters are protein-bound cofactors associated with cellular electron transport and redox sensing, with multiple specific functions in oxygen-evolving photosynthetic cyanobacteria. The aim here was to elucidate protein-level effects of the transcriptional repressor SufR involved in the regulation of Fe-S cluster biogenesis in the cyanobacterium Synechocystis sp. PCC 6803.The approach was to quantitate 94 pre-selected target proteins associated with various metabolic functions using SRM in Synechocystis. The evaluation was conducted in response to sufR deletion under different iron conditions, and complemented with EPR analysis on the functionality of the photosystems I and II as well as with RT-qPCR to verify the effects of SufR also on transcript level.The results on both protein and transcript levels show that SufR acts not only as a repressor of the suf operon when iron is available but also has other direct and indirect functions in the cell, including maintenance of the expression of pyruvate:ferredoxin oxidoreductase NifJ and other Fe-S cluster proteins under iron sufficient conditions. Furthermore, the results imply that in the absence of iron the suf operon is repressed by some additional regulatory mechanism independent of SufR.The study demonstrates that Fe-S cluster metabolism in Synechocystis is stringently regulated, and has complex interactions with multiple primary functions in the cell, including photosynthesis and central carbon metabolism.The study provides new insight into the regulation of Fe-S cluster biogenesis via suf operon, and the associated wide-ranging protein-level changes in photosynthetic cyanobacteria.Display Omitted
Keywords: Synechocystis sp. PCC 6803; Iron-sulfur clusters; Gene regulation; SRM proteomics; EPR spectroscopy; RT-qPCR;

Effect of therapeutic concentration of lithium on live HEK293 cells; increase of Na+/K+-ATPase, change of overall protein composition and alteration of surface layer of plasma membrane by Miroslava Vosahlikova; Hana Ujcikova; Oleksandr Chernyavskiy; Jana Brejchova; Lenka Roubalova; Martin Alda; Petr Svoboda (1099-1112).
The effect of long-term exposure of live cells to lithium cations (Li) was studied in HEK293 cells cultivated in the presence of 1 mM LiCl for 7 or 21 days. The alteration of Na+/K+-ATPase level, protein composition and biophysical state of plasma membrane was determined with the aim to characterize the physiological state of Li-treated cells.Na+/K+-ATPase level was determined by [3H]ouabain binding and immunoblot assays. Overall protein composition was determined by 2D electrophoresis followed by proteomic analysis by MALDI-TOF MS/MS and LFQ. Li interaction with plasma membrane was characterized by fluorescent probes DPH, TMA-DPH and Laurdan.Na+/K+-ATPase was increased in plasma membranes isolated from cells exposed to Li. Identification of Li-altered proteins by 2D electrophoresis, MALDI-TOF MS/MS and LFQ suggests a change of energy metabolism in mitochondria and cytosol and alteration of cell homeostasis of calcium. Measurement of Laurdan generalized polarization indicated a significant alteration of surface layer of isolated plasma membranes prepared from both types of Li-treated cells.Prolonged exposure of HEK293 cells to 1 mM LiCl results in up-regulation of Na+/K+-ATPase expression, reorganization of overall cellular metabolism and alteration of the surface layer/polar head-group region of isolated plasma membranes.Our findings demonstrate adaptation of live HEK293 cell metabolism to prolonged exposure to therapeutic concentration of Li manifested as up-regulation of Na+/K+-ATPase expression, alteration of protein composition and change of the surface layer of plasma membrane.
Keywords: Lithium; HEK293 cells; Na+/K+-ATPase; Membrane biophysics; Proteomic analysis;

Purification, characterization and cytotoxicity assessment of Ageritin: The first ribotoxin from the basidiomycete mushroom Agrocybe aegerita by Nicola Landi; Severina Pacifico; Sara Ragucci; Rosario Iglesias; Simona Piccolella; Adolfo Amici; Antonella M.A. Di Giuseppe; Antimo Di Maro (1113-1121).
Several species belonging to Ascomycota phylum produce extracellular ribonucleases, known as ribotoxins, which exhibit RNase activity through the cleavage of a single phosphodiester bond, located at the universally conserved sarcin/ricin loop of the large rRNA leading to inhibition of protein biosynthesis. Clarifying the structure-function relationship in ribotoxins is interesting for their use in human tumour therapy and in construction of pest resistant transgenic plants.The ribotoxin Ageritin has been isolated for the first time from the Basidiomycetes class. The enzyme, characterized by means of its amino acid composition, N-terminal sequence and a circular dichroism, structurally differs from Ascomycota ribotoxin prototype, although it was able, as α-sarcin, to release a specific α-fragment. However, it does not display aspecific ribonucleolytic activity. Ageritin exerts cytotoxicity and cell death promoting effects towards CNS model cell lines (SK-N-BE(2)-C, U-251 and C6), as vinblastine, a plant alkaloid used in cancer therapy. Moreover, our results indicate that Ageritin initially activates caspase-8, whereas caspase-9 cleavage was not detected, demonstrating the involvement of an extrinsic apoptotic pathway.Our findings show that Ageritin is the earliest diverging member of the Ascomycota ribotoxin family, suggesting that ribotoxins are more widely distributed among fungi than previously believed.Ageritin, structurally different from the widely known Ascomycota ribotoxins, with promising anti-cancer properties vs. aggressive brain tumours, has been found from the basidiomycete fungus Agrocybe aegerita. Finally, this finding highlights that the ribotoxin family has divergent members in Basidiomycota phylum, whose structural and functional characterization can give new information on ribotoxin or ribonuclease superfamilies.Display Omitted
Keywords: Agrocybe aegerita; Ageritin; Ribotoxins; Ribonuclease activity; Cytotoxicity; Apoptosis;

Endothelial migration is a critical physiological process during vascular angiogenesis, growth and development, as well as in various disease conditions, such as cancer and cardiovascular diseases. Neutrophil migration, known as the important characteristic of immune responses, is also recognized as a contributor to the diseases involving endothelial migration. Herein, the mutually dependent relationship between neutrophil recruitment and endothelial migration was studied on a microfluidic platform for the first time.An in vivo-like microenvironment is created inside microfluidic devices by embedding a gel scaffold into the micro-chambers. This approach, with controllable stable chemical gradients and the ability to quantitate interaction characteristics, overcomes the limitations of the current in vivo and in vitro assays for cell migration studies.The number of neutrophils migrating through the endothelial cell layer is heavily influenced by the concentration of vascular endothelial growth factor (VEGF) that induces endothelial cell migration in the gel scaffold, and is not as correlated to the concentration of chemokine solution used for initiating neutrophil migration. More importantly, neutrophil migration diminishes the effects of the drug that inhibits endothelial migration and this process is regulated by the concentration of chemokine molecules instead of VEGF concentration.The results presented herein demonstrate the complicated cellular interactions between endothelial cells and neutrophils: endothelial migration delicately regulates neutrophil migration while the presence of neutrophils stabilizes the structures of endothelial migration.This study provides deeper understanding of the dynamic cellular interactions between neutrophils and endothelial cells as well as the pathogenesis of relevant diseases.
Keywords: Angiogenesis; Neutrophil; Microfluidic; Chemokine; Migration; Receptor expression;

Nitroarachidonic acid (NO2AA) inhibits protein disulfide isomerase (PDI) through reversible covalent adduct formation with critical cysteines by Lucía González-Perilli; Mauricio Mastrogiovanni; Denise de Castro Fernandes; Homero Rubbo; Francisco Laurindo; Andrés Trostchansky (1131-1139).
Nitroarachidonic acid (NO2AA) exhibits pleiotropic anti-inflammatory actions in a variety of cell types. We have recently shown that NO2AA inhibits phagocytic NADPH oxidase 2 (NOX2) by preventing the formation of the active complex. Recent work indicates the participation of protein disulfide isomerase (PDI) activity in NOX2 activation. Cysteine (Cys) residues at PDI active sites could be targets for NO2AA- nitroalkylation regulating PDI activity which could explain our previous observation.PDI reductase and chaperone activities were assessed using the insulin and GFP renaturation methods in the presence or absence of NO2AA. To determine the covalent reaction with PDI as well as the site of reaction, the PEG-switch assay and LC–MS/MS studies were performed.We determined that both activities of PDI were inhibited by NO2AA in a dose- and time- dependent manner and independent from release of nitric oxide. Since nitroalkenes are potent electrophiles and PDI has critical Cys residues for its activity, then formation of a covalent adduct between NO2AA and PDI is feasible. To this end we demonstrated the reversible covalent modification of PDI by NO2AA. Trypsinization of modified PDI confirmed that the Cys residues present in the active site a′ of PDI were key targets accounting for nitroalkene modification.PDI may contribute to NOX2 activation. As such, inhibition of PDI by NO2AA might be involved in preventing NOX2 activation. Future work will be directed to determine if the covalent modifications observed play a role in the reported NO2AA inhibition of NOX2 activity.
Keywords: Nitroarachidonic acid; Protein disulfide isomerase; Nitroalkene; Mass spectrometry;

Redox regulation of methionine in calmodulin affects the activity levels of senescence-related transcription factors in litchi by Guoxiang Jiang; Lu Xiao; Huiling Yan; Dandan Zhang; Fuwang Wu; Xuncheng Liu; Xinguo Su; Xinhong Dong; Jiasheng Wang; Xuewu Duan; Yueming Jiang (1140-1151).
Reactive oxygen species (ROS) play a role in aging and senescence in organisms. The oxidation of methionine (Met) residues in proteins to Met sulfoxide by ROS can cause conformational alteration and functional impairments. Met oxidation is reversed by Met sulfoxide reductase (Msr) A and B. Currently, the repair of oxidized proteins by Msr and Msr-mediated physiological functions are not well understood, especially in higher plants. The down-regulated expression of LcMsrA1/B1 may be involved in the senescence of litchi (Litchi chinensis) fruit. We verified that LcCaM1 is a substrate of LcMsrA1 and LcMsrB1 in vitro and in vivo, and oxidized LcCaM1 could be repaired by LcMsrA1 in combination with LcMsrB1. Moreover, LcMsrA1 and LcMsrB1 play important roles in repairing oxidized Met110 and Met125 residues, respectively, in LcCaM1. Furthermore, the Met oxidation in LcCaM1 did not affect its physical interactions with two LcCaM1-binding senescence-related transcription factors LcNAC13 and LcWRKY1, but enhanced their DNA-binding activities. Therefore, we hypothesized that the down-regulated expression of LcMsrA1/B1 results in the accelerated oxidation of LcCaM1, which enhanced the DNA-binding activities of LcNAC13 and LcWRKY1, thereby activating or repressing the expression of senescence-related genes.Display Omitted
Keywords: Methionine sulfoxide reductase; Calmodulin; Transcriptional regulation; Reactive oxygen species; Senescence; Litchi;

Effects of statins on the immunoglobulin G glycome by Toma Keser; Frano Vučković; Clara Barrios; Jonas Zierer; Annika Wahl; Akintunde O Akinkuolie; Jerko Štambuk; Natali Nakić; Tamara Pavić; Josipa Periša; Samia Mora; Christian Gieger; Cristina Menni; Tim D Spector; Olga Gornik; Gordan Lauc (1152-1158).
Statins are among the most widely prescribed medications worldwide and usually many individuals involved in clinical and population studies are on statin therapy. Immunoglobulin G (IgG) glycosylation has been associated with numerous cardiometabolic risk factors.The aim of this study was to investigate the possible association of statin use with N-glycosylation of IgG. The association was analyzed in two large population cohorts (TwinsUK and KORA) using hydrophilic interaction liquid chromatography (HILIC-UPLC) in the TwinsUK cohort and reverse phase liquid chromatography coupled with electrospray mass spectrometry (LC-ESI-MS) in the KORA cohort. Afterwards we investigated the same association for only one statin (rosuvastatin) in a subset of individuals from the randomized double-blind placebo-controlled JUPITER study using LC-ESI-MS for IgG glycome and HILIC-UPLC for total plasma N-glycome.In the TwinsUK population, the use of statins was associated with higher levels of core-fucosylated biantennary glycan structure with bisecting N-acetylglucosamine (FA2B) and lower levels of core-fucosylated biantennary digalactosylated monosialylated glycan structure (FA2G2S1). The association between statin use and FA2B was replicated in the KORA cohort. In the JUPITER trial we found no statistically significant differences between the randomly allocated placebo and rosuvastatin groups.In the TwinsUK and KORA cohorts, statin use was associated with a small increase of pro-inflammatory IgG glycan, although this finding was not confirmed in a subset of participants from the JUPITER trial.Even if the association between IgG N-glycome and statins exists, it is not large enough to pose a problem for glycomic studies.
Keywords: N-glycosylation; Immunoglobulin G; Total plasma N-glycome; Statin; Rosuvastatin;

Yeast cells as an assay system for in vivo O-GlcNAc modification by Hideki Nakanishi; Feng Li; Baoxian Han; Seisuke Arai; Xiao-Dong Gao (1159-1167).
O-GlcNAcylation is a reversible protein post-translational modification, where O-GlcNAc moiety is attached to nucleocytoplasmic protein by O-GlcNAc transferase (OGT) and removed by O-GlcNAcase (OGA). Although O-GlcNAc modification widely occurs in eukaryotic cells, the budding yeast Saccharomyces cerevisiae notably lacks this protein modification and the genes for the GlcNAc transferase and hydrolase.Human OGT isoforms and OGA were ectopically expressed in S. cerevisiae, and the effects of their expressions on yeast growth and O-GlcNAc modification levels were assessed.Expression of sOGT, in S. cerevisiae catalyzes the O-GlcNAc modification of proteins in vivo; conversely, the expression of OGA mediates the hydrolysis of these sugars. sOGT expression causes a severe growth defect in yeast cells, which is remediated by the co-expression of OGA. The direct analysis of yeast proteins demonstrates protein O-GlcNAcylation is dependent on sOGT expression; conversely, the hydrolysis of these sugar modifications is induced by co-expression of OGA. Protein O-GlcNAcylation and the growth defects of yeast cells are caused by the O-GlcNAc transferase activity because catalytically inactive sOGT does not exhibit toxicity in yeast cells. Expression of another OGT isoform, ncOGT, also results in a growth defect in yeast cells. However, its toxicity is largely attributed to the TPR domain rather than the O-GlcNAc transferase activity. O-GlcNAc cycling can occur in yeast cells, and OGT and OGA activities can be monitored via yeast growth.Yeast cells may be used to assess OGT and OGA.
Keywords: Saccharomyces cerevisiae; O-GlcNAcylation; O-linked N-acetylglucosamine transferase; O-GlcNAcase; Glycosylation; Post-translational modification;

Synthetic toxic Aβ1–42 oligomers can assemble in different morphologies by Claude Bobo; Stéphane Chaignepain; Sarah Henry; Hélène Vignaud; Alfred Améadan; Christelle Marchal; Enora Prado; James Doutch; Jean-Marie Schmitter; Corinne Nardin; Sophie Lecomte; Christophe Cullin (1168-1176).
Alzheimer's disease is the most common neurodegenerative disease associated with aggregation of Aβ peptides. Aβ toxicity is mostly related to the capacity of intermediate oligomers to disrupt membrane integrity. We previously expressed Aβ1–42 in a eukaryotic cellular system and selected synthetic variants on their sole toxicity. The most toxic mutant G37C forms stable oligomers.Different biophysical methods (Fluorescence spectroscopy, cross-linking, mass spectrometry (MS), Small Angle X-ray Scattering (SAXS), Atomic Force Microscopy (AFM), Transmission Electron Microscopy (TEM), calcein leakage) were used.The oligomers are mostly populated by a 14 mers resulting from the packing of homodimers. These homodimers come from the formation of a disulfide bridge between two monomers. This link stabilizes the multimers and prevents the assembly into amyloid fibrils. These oligomers affect the membrane integrity. The reduction of disulfide bonds leads to a rearrangement and redirects assembly of Aβ amyloid fibrils.The toxic synthetic AβG37C mutant can assemble into an amyloid of unusual morphology through the formation of anti-parallel β-sheets. This pathway involves the formation of oligomers resulting from the arrangement of Aβ dimers linked by covalent di-sulfide link, being these oligomers harmful for the membranes.The capacity to produce large amount of stable oligomers without additional detergents or extrinsic cross-linkers allow further structural and biophysical studies to understand their capacity to assemble and disrupt the membranes, a key event in Alzheimer's disease.
Keywords: Amyloid; Aβ1–42; Membrane; Oligomers;

Rutin protects against H2O2-triggered impaired relaxation of placental arterioles and induces Nrf2-mediated adaptation in Human Umbilical Vein Endothelial Cells exposed to oxidative stress by Mireille M.J.P.E. Sthijns; Paul M. Schiffers; Ger M. Janssen; Kristien J.A. Lemmens; Bart Ides; Philippe Vangrieken; Freek G. Bouwman; Edwin C. Mariman; Irina Pader; Elias S.J. Arnér; Katarina Johansson; Aalt Bast; Guido R.M.M. Haenen (1177-1189).
Rutin intake is associated with a reduced risk of cardiovascular disease (CVD). The exact mechanism by which rutin can protect against CVD development is still enigmatic. Since, rutin is a compound with a relatively short half-life, the direct antioxidant effect of rutin cannot explain the long-lasting effect on human health. We hypothesized that rutin next to its direct antioxidant effect that improves endothelial function, may also induce an adaptive response in endogenous antioxidant systems.In Human Umbilical Vein Endothelial Cells (HUVECs), the direct antioxidant effect was confirmed. During scavenging of Reactive Oxygen Species (ROS), rutin is oxidized into a quinone derivative. HUVECs pretreated with rutin quinone became better protected against a second challenge with oxidative stress 3 h later. LC-MS/MS analysis indicated that rutin quinone targets cysteine 151 of Keap1. Moreover, we found that the quinone is an inhibitor of the selenoprotein thioredoxin reductase 1. These properties correlated with an activation of Nrf2 and upregulation of Glutamate Cysteine Ligase, the rate-limiting enzyme of glutathione synthesis, while NF-κB and HIF activation became blunted by rutin treatment. Furthermore, rutin was found to prevent hydrogen peroxide from impairing relaxation of human chorionic plate placental vessels, which may help to protect endothelial function.Rutin functions as an antioxidant and is oxidized into a quinone that upregulates the Nrf2-mediated endogenous antioxidant response. This mechanism suggests that rutin selectively exerts its protective effects in regions with increased oxidative stress, and explains how rutin reduces the risk of developing CVD.The newly found mechanism behind the long-term protection of rutin against cardiovascular disease, the selective upregulation of endogenous antioxidant systems, contributes to the further understanding why rutin can reduce the risk on developing cardiovascular disease.Display Omitted
Keywords: Rutin; Flavonoids; Nrf2; Adaptation; Endothelial function;

Proteomics-based network analysis characterizes biological processes and pathways activated by preconditioned mesenchymal stem cells in cardiac repair mechanisms by Dario Di Silvestre; Francesca Brambilla; Giovanni Scardoni; Pietro Brunetti; Sara Motta; Marco Matteucci; Carlo Laudanna; Fabio A. Recchia; Vincenzo Lionetti; Pierluigi Mauri (1190-1199).
We have demonstrated that intramyocardial delivery of human mesenchymal stem cells preconditioned with a hyaluronan mixed ester of butyric and retinoic acid (MSCp+) is more effective in preventing the decay of regional myocardial contractility in a swine model of myocardial infarction (MI). However, the understanding of the role of MSCp+ in proteomic remodeling of cardiac infarcted tissue is not complete. We therefore sought to perform a comprehensive analysis of the proteome of infarct remote (RZ) and border zone (BZ) of pigs treated with MSCp+ or unconditioned stem cells.Heart tissues were analyzed by MudPIT and differentially expressed proteins were selected by a label-free approach based on spectral counting. Protein profiles were evaluated by using PPI networks and their topological analysis.The proteomic remodeling was largely prevented in MSCp+ group. Extracellular proteins involved in fibrosis were down-regulated, while energetic pathways were globally up-regulated. Cardioprotectant pathways involved in the production of keto acid metabolites were also activated. Additionally, we found that new hub proteins support the cardioprotective phenotype characterizing the left ventricular BZ treated with MSCp+. In fact, the up-regulation of angiogenic proteins NCL and RAC1 can be explained by the increase of capillary density induced by MSCp+.Our results show that angiogenic pathways appear to be uniquely positioned to integrate signaling with energetic pathways involving cardiac repair.Our findings prompt the use of proteomics-based network analysis to optimize new approaches preventing the post-ischemic proteomic remodeling that may underlie the limited self-repair ability of adult heart.
Keywords: Stem cells; Myocardial infarction; Sus scrofa; MudPIT; PPI network; Proteomics;