Tetraspanins, Another Piece in the HIV-1 Replication Puzzle

Despite the great research effort placed during the last decades in HIV-1 study, still some aspects of its replication cycle remain unknown. All this powerful research has succeeded in developing different drugs for AIDS treatment, but none of them can completely remove the virus from infected patients, who require life-long medication. The classical approach was focused on the study of virus particles as the main target, but increasing evidence highlights the importance of host cell proteins in HIV-1 cycle. In this context, tetraspanins have emerged as critical players in different steps of the viral infection cycle. Through their association with other molecules, including membrane receptors, cytoskeletal proteins, and signaling molecules, tetraspanins organize specialized membrane microdomains called tetraspanin-enriched microdomains (TEMs). Within these microdomains, several tetraspanins have been described to regulate HIV-1 entry, assembly, and transfer between cells. Interestingly, the importance of tetraspanins CD81 and CD63 in the early steps of viral replication has been recently pointed out. Indeed, CD81 can control the turnover of the HIV-1 restriction factor SAMHD1. This deoxynucleoside triphosphate triphosphohydrolase counteracts HIV-1 reverse transcription (RT) in resting cells via its dual function as dNTPase, catalyzing deoxynucleotide triphosphates into deoxynucleosides and inorganic triphosphate, and as exonuclease able to degrade single-stranded RNAs. SAMHD1 has also been related with the detection of viral nucleic acids, regulating the innate immune response and would promote viral latency. New evidences demonstrating the ability of CD81 to control SAMHD1 expression, and as a consequence, HIV-1 RT activity, highlight the importance of TEMs for viral replication. Here, we will briefly review how tetraspanins modulate HIV-1 infection, focusing on the latest findings that link TEMs to viral replication.This work was supported by grants BFU2014-55478-R; BIO2017-86500-R; Fundacion Ramon Areces and RYC-2012-11025 to MY-M; and was co-funded by Fondo Europeo de Desarrollo Regional (FEDER). HS was supported by a FPI-UAM Fellowship.S

Unimolecular reactivity upon collision of uracil-Ca2+ complexes in the gas phase: Comparison with uracil-M+ (M = H, alkali metals) and uracil-M2+ (M = Cu, Pb) systems

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A COMBINED EXPERIMENTAL AND DFT INVESTIGATION OF ISOMERIC HEPARIN DISACCHARIDES METAL COMPLEXES

Communication par afficheHeparin (HP) glycosaminoglycans (GAGs) 1 , an anticoagulant drug, are recognized to be a biologically important polysaccharide, and have been involved in many biological processes such as blood coagulation, cell-cell and cell-matrix interaction inflammatory processes, cell growth, lipid transport and metabolism.  Why is it important to study the interaction between HP and metal cations? The effect of metal ions on protein-carbohydrate complexes is largely unknown. Heparin-biomolecule interaction can be influenced by the binding of metal ions to these complexes 2. For example, it has been reported that physiological Ca 2+ induces conformational changes in heparin that are necessary for the interaction between the anticoagulant Heparin and Annexin V, a protein proposed to play an important role in the inhibition of blood coagulation 3. It is therefore a Calcium-dependant interaction.  What is our strategy? Experimentally our aim was to study (Ca(II-H)) + and (Ca(II-A)) + complexes by tandem ESI/MS. Once generated in the gas phase, ions then undergo a fragmentation process by Collision Induced Dissociation (CID). The Ca 2+ cation induces different conformational changes in both isomers, resulting in completely different fragmentation pathways. Theoretically our aim is to explain this Metal-HP interaction by DFT calculations and delineate mechanisms of dissociation accounting for the experimental data.  Why II-A and II-H isomers? Without metal there is no difference in the MS/MS spectra of these two isomers. Only 0,2 A 2 fragmentation is observed. With Ca 2+ dissociation pattern changes drastically. Interaction between Acetyl/Ca 2+ must be important in the dissociation process. O O COOH HO OH O OH OH NH 2 OSO 3 O O COOH HO OH O OH OH NHAc OSO 3 0,2 A 2 * 0,2 X 1 * II-H II-A MS/MS results: Experiments were carried out on a LTQ Orbitrap XL mass spectrometer coupled to an ESI source. Nitrogen gas was used as collision gas. Computational results: The geometries were optimized using the density functional theory (DFT) with the B3LYP hybrid functional and 6-311G** basis set. Refined relative energies were obtained at the 6-311++G(3df,2p) level. Without Ca 2+ all the calculated 50 conformers, for each disaccharide, are very close in energy (50KJ/mol). High Binding Energy (BE) values (~1400 KJ/mol) are obtained. As deduced from the conformers calculation and the BE values, the metal complex stabilizes strongly one structure. It seems safe to deduce that both sugars lose partially their possibilities to change structurally. Biologically, this consideration could be critical in order to explain the strong interactions aforementioned. Analytically, when (Ca(HP)) + is formed, the molecule loses it flexibility due to the fixation structure effect and therefore it is noticed a decrease in the number of fragments. Leary et al. 4 delineates mechanisms of dissociation for isomeric HP without metal based upon CID experiments and H/D exchange. Another mechanism has been tested in this work but those pathways remain the most favorable ones.  0,2 A 2 Dissociation The acetyl group in II-A blocks the R1 (Rearrangement) step. Nevertheless, it is still possible to transfer the proton through the acetyl carbonyl group. The energy associated (PT2 barrier) is however bigger (163 KJ/mol) than for II-H (-55 KJ/mol).  0,2 X 1 Dissociation Starting from the initial structures, and using the dissociation mechanism given by Leary et al. shown below, the same methodology will be employed in order to unravel the 0,2 X 1 fragmentation pattern. , Ca 2

Paracrine effects of TLR4-polarised mesenchymal stromal cells are mediated by extracellular vesicles

Mesenchymal stromal cells (MSCs) are adult stem cells able to give rise to bone, cartilage and fat cells. In addition, they possess immunomodulatory and immunosuppressive properties that are mainly mediated through secretion of extracellular vesicles (EVs). In a previous issue of Journal of Translational Medicine, Ti and colleagues demonstrated that preconditioning of MSCs with bacterial lipopolysaccharides results in secretion of EVs that can polarise mac‑ rophages towards anti-inflammatory M2 phenotype. Moreover, the authors suggest that EVs of lipopolysaccharide (LPS)-treated MSCs are superior to EVs of untreated MSCs concerning their ability to support wound healing. Our commentary critically discusses parallel efforts of other laboratories to generate conditioned media from stem cells for therapeutic applications, and highlights impact and significance of the study of Ti et al. Finally, we summarise its limitations and spotlight areas that need to be addressed to better define the underlying molecular mechanisms

Remodeling of extracellular matrix by normal and tumor-associated fibroblasts promotes cervical cancer progression

Background: Comparison of tissue microarray results of 29 cervical cancer and 27 normal cervix tissue samples using immunohistochemistry revealed considerable reorganization of the fibrillar stroma of these tumors. Preliminary densitometry analysis of laminin-1, α -smooth muscle actin (SMA) and fibronectin immunostaining demonstrated 3.8-fold upregulation of laminin-1 and 5.2-fold increase of SMA in the interstitial stroma, indicating that these proteins and the activated fibroblasts play important role in the pathogenesis of cervical cancer. In the present work we investigated the role of normal and tumor-associated fibroblasts. Methods: In vitro models were used to throw light on the multifactorial process of tumor-stroma interaction, by means of studying the cooperation between tumor cells and fibroblasts. Fibroblasts from normal cervix and cervical cancers were grown either separately or in co-culture with CSCC7 cervical cancer cell line. Changes manifest in secreted glycoproteins, integrins and matrix metallo-proteases (MMPs) were explored. Results: While normal fibroblasts produced components of interstitial matrix and TGF- β 1 that promoted cell proliferation, cancer-associated fibroblasts (CAFs) synthesized ample amounts of laminin-1. The following results support the significance of laminin-1 in the invasion of CSCC7 cells: 1.) Tumor-associated fibroblasts produced more laminin-1 and less components of fibrillar ECM than normal cells; 2.) The production of laminin chains was further increased when CSCC7 cells were grown in co-culture with fibroblasts; 3.) CSCC7 cells were capable of increasing their laminin production; 4.) Tumor cells predominantly expressed integrin α 6 β 4 laminin receptors and migrated towards laminin. The integrin profile of both normal and tumor-associated fibroblasts was similar, expressing receptors for fibronectin, vitronectin and osteopontin. MMP-7 secreted by CSCC7 cells was upregulated by the presence of normal fibroblasts, whereas MMP-2 produced mainly by fibroblasts was activated in the presence of CSCC7 cells. Conclusions: Our results indicate that in addition to degradation of the basement membrane, invasion of cervical cancer is accomplished by the remodeling of the interstitial stroma, which process includes decrease and partial replacement of fibronectin and collagens by a laminin-rich matrix

Therapeutic prospects of extracellular vesicles in cancer treatment

Extracellular vesicles (EVs) are released by all cells within the tumor microenvironment, such as endothelial cells, tumor-associated fibroblasts, pericytes and immune system cells. The EVs carry the cargo of parental cells formed of proteins and nucleic acids, which can convey cell-to-cell communication influencing the maintenance and spread of the malignant neoplasm, for example promoting angiogenesis, tumor cell invasion and immune escape. However, EVs can also suppress tumor progression, either by the direct influence of the protein and nucleic acid cargo of the EVs or via antigen presentation to immune cells as tumor derived EVs carry on their surface some of the same antigens as the donor cells. Moreover, dendritic cell-derived EVs carry MHC class I and class II/peptide complexes and are able to prime other immune system cell types and activate an anti-tumor immune response. Given the relative longevity of vesicles within the circulation and their ability to cross blood-brain barriers, modification of these unique organelles offers the potential to create new biological-tools for cancer therapy. This review examines how modification of the EV cargo has the potential to target specific tumor mechanisms responsible for tumor formation and progression to develop new therapeutic strategies and to increase the efficacy of antitumor therapies

Functional molecules in mesothelial to mesenchymal transition revealed by transcriptome analyses

Peritoneal fibrosis is a common complication of abdominal and pelvic surgery, and can also be triggered by peritoneal dialysis, resulting in treatment failure. In these settings, fibrosis is driven by activated myofibroblasts that are considered to be partly derived by mesothelial‐to‐mesenchymal transition (MMT). We hypothesized that, if the molecular signature of MMT could be better defined, these insights could be exploited to block this pathological cellular transition. Rat peritoneal mesothelial cells were purified by the use of an antibody against HBME1, a protein present on mesothelial cell microvilli, and streptavidin nanobead technology. After exposure of sorted cells to a well‐known mediator of MMT, transforming growth factor (TGF)‐β1, RNA sequencing was undertaken to define the transcriptomes of mesothelial cells before and during early‐phase MMT. MMT was associated with dysregulation of transcripts encoding molecules involved in insulin‐like growth factor (IGF) and bone morphogenetic protein (BMP) signalling. The application of either recombinant BMP4 or IGF‐binding protein 4 (IGFBP4) ameliorated TGF‐β1‐induced MMT in culture, as judged from the retention of epithelial morphological and molecular phenotypes, and reduced migration. Furthermore, peritoneal tissue from peritoneal dialysis patients showed less prominent immunostaining than control tissue for IGFBP4 and BMP4 on the peritoneal surface. In a mouse model of TGF‐β1‐induced peritoneal thickening, BMP4 immunostaining on the peritoneal surface was attenuated as compared with healthy controls. Finally, genetic lineage tracing of mesothelial cells was used in mice with peritoneal injury. In this model, administration of BMP4 ameliorated the injury‐induced shape change and migration of mesothelial cells. Our findings demonstrate a distinctive MMT signature, and highlight the therapeutic potential for BMP4, and possibly IGFBP4, to reduce MMT

Regulation of MT1-MMP Activity through Its Association with ERMs

Membrane-bound proteases play a key role in biology by degrading matrix proteins or shedding adhesion receptors. MT1-MMP metalloproteinase is critical during cancer invasion, angiogenesis, and development. MT1-MMP activity is strictly regulated by internalization, recycling, autoprocessing but also through its incorporation into tetraspanin-enriched microdomains (TEMs), into invadopodia, or by its secretion on extracellular vesicles (EVs). We identified a juxtamembrane positively charged cluster responsible for the interaction of MT1-MMP with ERM (ezrin/radixin/moesin) cytoskeletal connectors in breast carcinoma cells. Linkage to ERMs regulates MT1-MMP subcellular distribution and internalization, but not its incorporation into extracellular vesicles. MT1-MMP association to ERMs and insertion into TEMs are independent phenomena, so that mutation of the ERM-binding motif in the cytoplasmic region of MT1-MMP does not preclude its association with the tetraspanin CD151, but impairs the accumulation and coalescence of CD151/MT1-MMP complexes at actin-rich structures. Conversely, gene deletion of CD151 does not impact on MT1-MMP colocalization with ERM molecules. At the plasma membrane MT1-MMP autoprocessing is severely dependent on ERM association and seems to be the dominant regulator of the enzyme collagenolytic activity. This newly characterized MT1-MMP/ERM association can thus be of relevance for tumor cell invasion.This work has been supported by grants BFU2014-55478-R, REDIEX. SAF2015-71231-REDT and BIO2017-86500-R from Ministerio Español de Economía y Competitividad (MINECO) and by a grant from Fundación Ramón Areces “Ayudas a la Investigación en Ciencias de la Vida y de la Materia, 2014” to M.Y.-M. H.S. was supported by a FPI-UAM fellowship. The CNIC is supported by the Ministry of Ciencia, Innovacion y Universidades and the Pro CNIC Foundation, is a Severo Ochoa Center of Excellence (SEV-2015-0505), also supported by European Regional Development Fund (FEDER) “Una manera de hacer Europa”.S

A transcriptomic analysis of Echinococcus granulosus larval stages:implications for parasite biology and host adaptation

The cestode Echinococcus granulosus--the agent of cystic echinococcosis, a zoonosis affecting humans and domestic animals worldwide--is an excellent model for the study of host-parasite cross-talk that interfaces with two mammalian hosts. To develop the molecular analysis of these interactions, we carried out an EST survey of E. granulosus larval stages. We report the salient features of this study with a focus on genes reflecting physiological adaptations of different parasite stages.We generated ~10,000 ESTs from two sets of full-length enriched libraries (derived from oligo-capped and trans-spliced cDNAs) prepared with three parasite materials: hydatid cyst wall, larval worms (protoscoleces), and pepsin/H(+)-activated protoscoleces. The ESTs were clustered into 2700 distinct gene products. In the context of the biology of E. granulosus, our analyses reveal: (i) a diverse group of abundant long non-protein coding transcripts showing homology to a middle repetitive element (EgBRep) that could either be active molecular species or represent precursors of small RNAs (like piRNAs); (ii) an up-regulation of fermentative pathways in the tissue of the cyst wall; (iii) highly expressed thiol- and selenol-dependent antioxidant enzyme targets of thioredoxin glutathione reductase, the functional hub of redox metabolism in parasitic flatworms; (iv) candidate apomucins for the external layer of the tissue-dwelling hydatid cyst, a mucin-rich structure that is critical for survival in the intermediate host; (v) a set of tetraspanins, a protein family that appears to have expanded in the cestode lineage; and (vi) a set of platyhelminth-specific gene products that may offer targets for novel pan-platyhelminth drug development.This survey has greatly increased the quality and the quantity of the molecular information on E. granulosus and constitutes a valuable resource for gene prediction on the parasite genome and for further genomic and proteomic analyses focused on cestodes and platyhelminths

Mechanisms of Epithelial-Mesenchymal Transition of Peritoneal Mesothelial Cells During Peritoneal Dialysis

A growing body of evidence indicates that epithelial-mesenchymal transition (EMT) of human peritoneal mesothelial cells (HPMC) may play an important role in the development and progression of peritoneal fibrosis during long-term peritoneal dialysis (PD) leading to failure of peritoneal membrane function. Here, we review our own observations and those of others on the mechanisms of EMT of HPMC and suggest potential therapeutic strategies to prevent EMT and peritoneal fibrosis during long-term PD. We found that high glucose and H2O2 as well as transforming growth factor-β1 (TGF-β1) induced EMT in HPMC and that high glucose-induced EMT was blocked not only by inhibition of TGF-β1 but also by antioxidants or inhibitors of mitogen-activated protein kinases (MAPK). Since MAPKs are downstream target molecules of reactive oxygen species (ROS), these data suggest that high glucose-induced generation of ROS and subsequent MAPK activation mediate high glucose-induced EMT in HPMC. We and others also observed that bone morphogenetic protein-7 (BMP-7) prevented EMT in HPMC. Glucose degradation products (GDP) were shown to play a role in inducing EMT. Involvement of a mammalian target of rapamycin (mTOR) in TGF-β1-induced EMT has also been proposed in cultured HPMC. A better understanding of the precise mechanisms involved in EMT of HPMC may provide new therapeutic strategies for inhibiting peritoneal fibrosis in long-term PD patients