Determinantes estructurales del plegamiento y la interacción entre fragmentos transmembrana

El análisis de secuencias genómicas completas de diversos organismos muestra que, aproximádamente, un 20-30% de las pautas de lectura abierta codifica para proteínas que atraviesan la membrana (Arkin, Brunger et al. 1997) (Wallin and von Heijne 1998). Sin embargo el medio en que estas proteínas adoptan su estructura nativa y desarrollan su función, la membrana, ha dificultado la aplicación directa de las técnicas de estudio habituales para proteínas solubles. Esta dificultad se refleja en el hecho de que en la era de la explosción de obtención de estructuras proteícas de alta resolución, se limita a unas docenas el número de proteínas de membrana resueltas. En este trabajo se ha utilizado la oligomerización del segmento transmembrana (TM) de una proteína modelo, Glicoforina A(GpA), para tratar de aportar nuevos datos que faciliten la comprensión de los factores que contribuyen al proceso de plegamiento de proteínas en el interior de la membrana

A fluorescent polarization-based assay for the identification of disruptors of the RCAN1/calcineurin A protein complex

5 pages, 4 figures, a table. 19891949 [PubMed]Calcineurin is a Ca(2+)/calmodulin-dependent serine/threonine protein phosphatase involved in many biological processes and developmental programs, including immune response. One of the most studied substrates of calcineurin is the transcription factor NFAT (nuclear factor of activated T cells) responsible for T-cell activation. Different anticalcineurin drugs, such as cyclosporine A and FK506, are the most commonly used immunosuppressants in transplantation therapies. Unfortunately, their mechanism of action, completely blocking the calcineurin phosphatase activity while also requiring continuous administration, bears severe side effects. During recent years, the family of regulators of calcineurin (RCAN) has been described and studied extensively as modulators of calcineurin signaling pathways. The RCAN1 region, spanning amino acids 198 to 218 and responsible for inhibiting the calcineurin-NFAT signaling pathway in vivo, has been identified. An RCAN1-derived peptide spanning this sequence interferes with the calcineurin-NFAT interaction without affecting the general calcineurin phosphatase activity. Here we report the development of an optimized in vitro high-throughput fluorescence polarization assay based on the disruption of the RCAN1(198-218)-CnA interaction for identifying molecules with immunosuppressant potential. This approach led us to identify dipyridamole as a disruptor of such interaction. Moreover, three small molecules with a potential immunosuppressive effect were also identifiedThis work was supported by grants from Fundació La Marató de TV3 (Ref. 030830), the Spanish Ministry of Education and Science (SAF2006-04815, BIO2004-00998, BIO2007-60066, CTQ2005-00995/BQU), the Fundación Mutua Madrileña 2007 and from the Generalitat de Catalunya (Ref. 2006 BE 00051)Peer reviewe

Influence of proline residues in transmembrane helix packing

Integral membrane proteins often contain proline residues in their alpha-helical transmembrane (TM) fragments, which may strongly influence their folding and association. Pro-scanning mutagenesis of the helical domain of glycophorin A (GpA) showed that replacement of the residues located at the center abrogates helix packing while substitution of the residues forming the ending helical turns allows dimer formation. Synthetic TM peptides revealed that a point mutation of one of the residues of the dimerization motif (L75P) located at the N-terminal helical turn of the GpA TM fragment, adopts a secondary structure and oligomeric state similar to the wild-type sequence in detergents. In addition, both glycosylation mapping in biological membranes and molecular dynamics showed that the presence of a proline residue at the lipid/water interface has as an effect the extension of the helical end. Thus, helix packing can be an important factor that determines appearance of proline in TM helices. Membrane proteins might accumulate proline residues at the two ends of their TM segments in order to modulate the exposition of key amino acid residues at the interface for molecular recognition events while allowing stable association and native folding

Influence of hydrophobic matching on association of model transmembrane fragments containing a minimised glycophorin A dimerisation motif

The principles that govern the folding and packing of membrane proteins are still not completely understood. In the present work, we have revisited the glycophorin A (GpA) dimer- isation motif that mediates transmembrane (TM) helix associa- tion, one of the best-suited models of membrane protein oligomerisation. By using artificial polyleucine TM segments we have demonstrated in this study that a pattern of only five amino acids (GVxxGVxxT) promotes specific dimerisation. Fur- ther, we have used this minimised GpA motif to assess the influ- ence of hydrophobic matching on the TM helix packing process in detergent micelles and found that this factor modulates helix-helix association and/or dissociation between TM fragments

Polar/Ionizable Residues in Transmembrane Segments: Effects on Helix-Helix Packing

The vast majority of membrane proteins are anchored to biological membranes through hydrophobic α-helices. Sequence analysis of high-resolution membrane protein structures show that ionizable amino acid residues are present in transmembrane (TM) helices, often with a functional and/or structural role. Here, using as scaffold the hydrophobic TM domain of the model membrane protein glycophorin A (GpA), we address the consequences of replacing specific residues by ionizable amino acids on TM helix insertion and packing, both in detergent micelles and in biological membranes. Our findings demonstrate that ionizable residues are stably inserted in hydrophobic environments, and tolerated in the dimerization process when oriented toward the lipid face, emphasizing the complexity of protein-lipid interactions in biological membranes

Real-Time In Vivo Detection of Cellular Senescence through the Controlled Release of the NIR Fluorescent Dye Nile Blue

This is the peer reviewed version of the following article: B. Lozano-Torres, J. F. Blandez, I. Galiana, A. García-Fernández, M. Alfonso, M. D. Marcos, M. Orzáez, F. Sancenón, R. Martínez-Máñez, Angew. Chem. Int. Ed. 2020, 59, 15152., which has been published in final form at https://doi.org/10.1002/anie.202004142. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.[EN] In vivo detection of cellular senescence is accomplished by using mesoporous silica nanoparticles loaded with the NIR-FDA approved Nile blue (NB) dye and capped with a galactohexasaccharide (S3). NB emission at 672 nm is highly quenched inside S3, yet a remarkable emission enhancement is observed upon cap hydrolysis in the presence of beta-galactosidase and dye release. The efficacy of the probe to detect cellular senescence is tested in vitro in melanoma SK-Mel-103 and breast cancer 4T1 cells and in vivo in palbociclib-treated BALB/cByJ mice bearing breast cancer tumor.R.M. thanks financial support from the Spanish Government (RTI2018-100910-B-C41 and RTI2018-101599-B-C22 (MCUI/AEI/FEDER, UE)) and the Generalitat Valenciana (PROMETEO 2018/024). M.O. thanks the financial support from SAF2017-84689-R project and MINECO/AEI/FEDER, UE and the Generalitat Valenciana (PROMETEO/2019/065). B.L.-T. is grateful to the Spanish Ministry of Economy for her PhD grant. I.G. thanks her contract from IDM. J.F.-B. and M.A. thank the UPV for their postdoctoral fellowship.Lozano-Torres, B.; Blandez, JF.; Galiana, I.; García-Fernández, A.; Alfonso-Navarro, M.; Marcos Martínez, MD.; Orzáez, M.... (2020). Real-Time In Vivo Detection of Cellular Senescence through the Controlled Release of the NIR Fluorescent Dye Nile Blue. 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Gene-Directed Enzyme Prodrug Therapy by Dendrimer-Like Mesoporous Silica Nanoparticles against Tumor Cells

[EN] We report herein a gene-directed enzyme prodrug therapy (GDEPT) system using gated mesoporous silica nanoparticles (MSNs) in an attempt to combine the reduction of side effects characteristic of GDEPT with improved pharmacokinetics promoted by gated MSNs. The system consists of the transfection of cancer cells with a plasmid controlled by the cytomegalovirus promoter, which promotes ß-galactosidase (ß-gal) expression from the bacterial gene lacZ (CMV-lacZ). Moreover, dendrimer-like mesoporous silica nanoparticles (DMSNs) are loaded with the prodrug doxorubicin modified with a galactose unit through a self-immolative group (DOXO-Gal) and modified with a disulfide-containing polyethyleneglycol gatekeeper. Once in tumor cells, the reducing environment induces disulfide bond rupture in the gatekeeper with the subsequent DOXO-Gal delivery, which is enzymatically converted by ß-gal into the cytotoxic doxorubicin drug, causing cell death. The combined treatment of the pair enzyme/DMSNs-prodrug are more effective in killing cells than the free prodrug DOXO-Gal alone in cells transfected with ß-gal.This research was funded by the Spanish Government (projects RTI2018-100910-B-C41, RTI2018-101599-B-C22 and SAF2017-84689-R (MCUI/FEDER, EU)) and the Generalitat Valenciana (projects PROMETEO 2018/024 and PROMETEO/2019/065). Vicente Candela-Noguera thanks the Spanish Government for his fellowship (FPU15/02753). Gema Vivo-Llorca thanks the Generalitat Valenciana for her fellowship (ACIF/2017/072).Candela-Noguera, V.; Vivo-Llorca, G.; Díaz De Greñu, B.; Alfonso, M.; Aznar, E.; Orzáez, M.; Marcos Martínez, MD.... (2021). Gene-Directed Enzyme Prodrug Therapy by Dendrimer-Like Mesoporous Silica Nanoparticles against Tumor Cells. Nanomaterials. 11(5):1-16. https://doi.org/10.3390/nano1105129811611

Bax transmembrane domain interacts with prosurvival Bcl-2 proteins in biological membranes

The Bcl-2 (B-cell lymphoma 2) protein Bax (Bcl-2 associated X, apoptosis regulator) can commit cells to apoptosis via outer mitochondrial membrane permeabilization. Bax activity is controlled in healthy cells by prosurvival Bcl-2 proteins. C-terminal Bax transmembrane domain interactions were implicated recently in Bax pore formation. Here, we show that the isolated transmembrane domains of Bax, Bcl-xL (B-cell lymphoma-extra large), and Bcl-2 can mediate interactions between Bax and prosurvival proteins inside the membrane in the absence of apoptotic stimuli. Bcl-2 protein transmembrane domains specifically homooligomerize and heterooligomerize in bacterial and mitochondrial membranes. Their interactions participate in the regulation of Bcl-2 proteins, thus modulating apoptotic activity. Our results suggest that interactions between the transmembrane domains of Bax and antiapoptotic Bcl-2 proteins represent a previously unappreciated level of apoptosis regulation

Analyse der Arzneimitteltherapie von Rheumapatienten im Erwachsenenalter : das Versorgungsgeschehen im Lichte der Leitlinienempfehlungen

The aim of this dissertation is to demonstrate the further developments in the provision of rheumatological health care with particular focus on evidence-based guideline recommendations. One of the issues to be examined is to identify gaps - defined as conditions of insufficient medical care between practical daily care and (theoretical) guideline recommendations differentiating various aspects of care. Are there still references about a lack of provison of medical care or is it possible to reveal further developments that can be assessed as positive respectively? Avenues for future action are to be pointed out against the background of these findings with regard to measures which have already been established as well as to aspired solutions in order to optimate the provision of rheumatological health care. A further glimpse of the future is disclosed by taking up the biosimilar-issue: As many bio-pharmaceuticals lose patent protection in coming years, biosimilar products are expected to play a key role in controlling pharmaceutical expenditure - while maintaining consistent quality of medical care. Assessing opportunities and challenges of these new medical products will round off this dissertation

2,4-dinitrophenyl ether-containing chemodosimeters for the selective and sensitive 'in vitro' and 'in vivo' detection of hydrogen sulfide

[EN] Four probes (i.e. D1¿D4) for the selective and sensitive fluorogenic detection of HS2 have been prepared and characterised. HEPES (10 mM, pH 7.4)¿DMSO 99:1 v/v solutions of D1¿D4 are essentially non-fluorescent. Changes in the emission using D1¿D4 in the presence of anions (F2, Cl2, Br2, I2, N2 3 , CN2, SCN2, AcO2, CO22 3 , PO22 4 , SO22 4 , HS2 and OH2), biothiols (GSH, Cys, Hcy, Me ¿Cys and lipoic acid), reducing agents (SO22 3 and S2O22 3 ) and oxidants (H2O2) demonstrated that only HS2 is able to induce the appearance of intense emission bands in the 400¿ 520 nm range in the four probes. The selectivity observed was ascribed to a unique hydrogen sulfide-induced hydrolysis of the 2,4-dinitrophenyl ether moiety that yielded the corresponding free highly fluorescent alcohols. The potential detection of intracellular HS2 was also studied.Financial support from the Spanish Government (Project MAT2012-38429-C04-01) and the Generalitat Valencia (Project PROMETEO/2009/016) is gratefully acknowledged. S.E. is grateful to the Generalitat Valenciana for his Santiago Grisolia fellow. L.E.S.F. also thanks the Carolina Foundation and UPNFM-Honduras for his doctoral grant.El Sayed Shehata Nasr, S.; De La Torre, C.; Santos Figueroa, LE.; Martínez-Máñez, R.; Sancenón Galarza, F.; Orzáez, M.; Costero, AM.... (2015). 2,4-dinitrophenyl ether-containing chemodosimeters for the selective and sensitive 'in vitro' and 'in vivo' detection of hydrogen sulfide. Supramolecular Chemistry. 27(4):244-254. https://doi.org/10.1080/10610278.2014.977286S24425427