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

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

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

Interaction of pregnancy-specific glycoprotein 1 with integrin Α5β1 is a modulator of extravillous trophoblast functions

Human pregnancy-specific glycoproteins (PSGs) serve immunomodulatory and pro-angiogenic functions during pregnancy and are mainly expressed by syncytiotrophoblast cells. While PSG mRNA expression in extravillous trophoblasts (EVTs) was reported, the proteins were not previously detected. By immunohistochemistry and immunoblotting, we show that PSGs are expressed by invasive EVTs and co-localize with integrin α5. In addition, we determined that native and recombinant PSG1, the most highly expressed member of the family, binds to α5β1 and induces the formation of focal adhesion structures resulting in adhesion of primary EVTs and EVT-like cell lines under 21% oxygen and 1% oxygen conditions. Furthermore, we found that PSG1 can simultaneously bind to heparan sulfate in the extracellular matrix and to α5β1 on the cell membrane. Wound healing assays and single-cell movement tracking showed that immobilized PSG1 enhances EVT migration. Although PSG1 did not affect EVT invasion in the in vitro assays employed, we found that the serum PSG1 concentration is lower in African-American women diagnosed with early-onset and late-onset preeclampsia, a pregnancy pathology characterized by shallow trophoblast invasion, than in their respective healthy controls only when the fetus was a male; therefore, the reduced expression of this molecule should be considered in the context of preeclampsia as a potential therapy

Inhibition of tetraspanin functions impairs human papillomavirus and cytomegalovirus infections

Tetraspanins are suggested to regulate the composition of cell membrane components and control intracellular transport, which leaves them vulnerable to utilization by pathogens such as human papillomaviruses (HPV) and cytomegaloviruses (HCMV) to facilitate host cell entry and subsequent infection. In this study, by means of cellular depletion, the cluster of differentiation (CD) tetraspanins CD9, CD63, and CD151 were found to reduce HPV16 infection in HeLa cells by 50 to 80%. Moreover, we tested recombinant proteins or peptides of specific tetraspanin domains on their effect on the most oncogenic HPV type, HPV16, and HCMV. We found that the C-terminal tails of CD63 and CD151 significantly inhibited infections of both HPV16 and HCMV. Although CD9 was newly identified as a key cellular factor for HPV16 infection, the recombinant CD9 C-terminal peptide had no effect on infection. Based on the determined half-maximal inhibitory concentration (IC50), we classified CD63 and CD151 C-terminal peptides as moderate to potent inhibitors of HPV16 infection in HeLa and HaCaT cells, and in EA.hy926, HFF (human foreskin fibroblast) cells, and HEC-LTT (human endothelial cell-large T antigen and telomerase) cells for HCMV, respectively. These results indicate that HPV16 and HCMV share similar cellular requirements for their entry into host cells and reveal the necessity of the cytoplasmic CD151 and CD63 C-termini in virus infections. Furthermore, this highlights the suitability of these peptides for functional investigation of tetraspanin domains and as inhibitors of pathogen infections

BIOKID: Randomized controlled trial comparing bicarbonate and lactate buffer in biocompatible peritoneal dialysis solutions in children [ISRCTN81137991]

BACKGROUND: Peritoneal dialysis (PD) is the preferred dialysis modality in children. Its major drawback is the limited technique survival due to infections and progressive ultrafiltration failure. Conventional PD solutions exert marked acute and chronic toxicity to local tissues. Prolonged exposure is associated with severe histopathological alterations including vasculopathy, neoangiogenesis, submesothelial fibrosis and a gradual loss of the mesothelial cell layer. Recently, more biocompatible PD solutions containing reduced amounts of toxic glucose degradation products (GDPs) and buffered at neutral pH have been introduced into clinical practice. These solutions contain lactate, bicarbonate or a combination of both as buffer substance. Increasing evidence from clinical trials in adults and children suggests that the new PD fluids may allow for better long-term preservation of peritoneal morphology and function. However, the relative importance of the buffer in neutral-pH, low-GDP fluids is still unclear. In vitro, lactate is cytotoxic and vasoactive at the concentrations used in PD fluids. The BIOKID trial is designed to clarify the clinical significance of the buffer choice in biocompatible PD fluids. METHODS/DESIGN: The objective of the study is to test the hypothesis that bicarbonate based PD solutions may allow for a better preservation of peritoneal transport characteristics in children than solutions containing lactate buffer. Secondary objectives are to assess any impact of the buffer system on acid-base status, peritoneal tissue integrity and the incidence and severity of peritonitis. After a run-in period of 2 months during which a targeted cohort of 60 patients is treated with a conventional, lactate buffered, acidic, GDP containing PD fluid, patients will be stratified according to residual renal function and type of phosphate binding medication and randomized to receive either the lactate-containing Balance solution or the bicarbonate-buffered Bicavera(® )solution for a period of 10 months. Patients will be monitored by monthly physical and laboratory examinations. Peritoneal equilibration tests, 24-h dialysate and urine collections will be performed 4 times. Peritoneal biopsies will be obtained on occasion of intraabdominal surgery. Changes in small solute transport rates, markers of peritoneal tissue turnover in the effluent, acid-base status and peritonitis rates and severity will be analyzed

Mesothelial cell differentiation into osteoblast- and adipocyte-like cells

Serosal pathologies including malignant mesothelioma (MM) can show features of osseous and/or cartilaginous differentiation although the mechanism for its formation is unknown. Mesothelial cells have the capacity to differentiate into cells with myofibroblast, smooth muscle and endothelial cell characteristics. Whether they can differentiate into other cell types is unclear. This study tests the hypothesis that mesothelial cells can differentiate into cell lineages of the embryonic mesoderm including osteoblasts and adipocytes. To examine this, a functional assay of bone formation and an adipogenic assay were performed in vitro with primary rat and human mesothelial cells maintained in osteogenic or adipogenic medium (AM) for 0–26 days. Mesothelial cells expressed increasing levels of alkaline phosphatase, an early marker of the osteoblast phenotype, and formed mineralized bone-like nodules. Mesothelial cells also accumulated lipid indicative of a mature adipocyte phenotype when cultured in AM. All cells expressed several key osteoblast and adipocyte markers, including osteoblast-specific runt-related transcription factor 2, and demonstrated changes in mRNA expression consistent with epithelial-to-mesenchymal transition. In conclusion, these studies confirm that mesothelial cells have the capacity to differentiate into osteoblast- and adipocyte-like cells, providing definitive evidence of their multipotential nature. These data strongly support mesothelial cell differentiation as the potential source of different tissue types in MM tumours and other serosal pathologies, and add support for the use of mesothelial cells in regenerative therapies

Different states of integrin LFA-1 aggregation are controlled through its association with tetraspanin CD9

This is the author’s version of a work that was accepted for publication in Biochimica et Biophysica Acta - Mollecular Cell Research. A definitive version was subsequently published in Biochimica et Biophysica Acta - Mollecular Cell Research, 1853.10 (2015): 2464-2480 DOI: 10.1016/j.bbamcr.2015.05.018The tetraspanin CD9 has been shown to interact with different members of the β1 and β3 subfamilies of integrins, regulating through these interactions cell adhesion, migration and signaling. Based on confocal microscopy co-localization and on coimmunoprecipitation results, we report here that CD9 associates with the β2 integrin LFA-1 in different types of leukocytes including T, B and monocytic cells. This association is resistant to stringent solubilisation conditions which, together with data from chemical crosslinking, in situ Proximity Ligation Assays and pull-down experiments, suggests a primary/direct type of interaction mediated by the Large Extracellular Loop of the tetraspanin. CD9 exerts inhibitory effects on the adhesive function of LFA-1 and on LFA-1-dependent leukocyte cytotoxic activity. The mechanism responsible for this negative regulation exerted by CD9 on LFA-1 adhesion does not involve changes in the affinity state of this integrin but seems to be related to alterations in its state of aggregationThis work was supported by grant SAF2012-34561 from the Spanish «Ministerio de Economía y Competitividad-MINECO», (to C.C.). R.R.M. salary is supported by a «Profesor Ayudante» position from Departamento de Biología, Facutad de Ciencias, Universidad Autónoma de Madri