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2. Biallelic mutations in neurofascin cause neurodevelopmental impairment and peripheral demyelination

Author

Efthymiou, S, Salpietro, V, Malintan, N, Poncelet, M, Kriouile, Y, Fortuna, S, De Zorzi, R, Payne, K, Henderson, LB, Cortese, A, Maddirevula, S, Alhashmi, N, Wiethoff, S, Ryten, M, Botia, JA, Provitera, V, Schuelke, M, Vandrovcova, J, SYNAPS Study Group, Walsh, L, Torti, E, Iodice, V, Najafi, M, Karimiani, EG, Maroofian, R, Siquier-Pernet, K, Boddaert, N, De Lonlay, P, Cantagrel, V, Aguennouz, M, El Khorassani, M, Schmidts, M, Alkuraya, FS, Edvardson, S, Nolano, M, Devaux, J, and Houlden, H

Abstract

Axon pathfinding and synapse formation are essential processes for nervous system development and function. The assembly of myelinated fibres and nodes of Ranvier is mediated by a number of cell adhesion molecules of the immunoglobulin superfamily including neurofascin, encoded by the NFASC gene, and its alternative isoforms Nfasc186 and Nfasc140 (located in the axonal membrane at the node of Ranvier) and Nfasc155 (a glial component of the paranodal axoglial junction). We identified 10 individuals from six unrelated families, exhibiting a neurodevelopmental disorder characterized with a spectrum of central (intellectual disability, developmental delay, motor impairment, speech difficulties) and peripheral (early onset demyelinating neuropathy) neurological involvement, who were found by exome or genome sequencing to carry one frameshift and four different homozygous non-synonymous variants in NFASC. Expression studies using immunostaining-based techniques identified absent expression of the Nfasc155 isoform as a consequence of the frameshift variant and a significant reduction of expression was also observed in association with two non-synonymous variants affecting the fibronectin type III domain. Cell aggregation studies revealed a severely impaired Nfasc155-CNTN1/CASPR1 complex interaction as a result of the identified variants. Immunofluorescence staining of myelinated fibres from two affected individuals showed a severe loss of myelinated fibres and abnormalities in the paranodal junction morphology. Our results establish that recessive variants affecting the Nfasc155 isoform can affect the formation of paranodal axoglial junctions at the nodes of Ranvier. The genetic disease caused by biallelic NFASC variants includes neurodevelopmental impairment and a spectrum of central and peripheral demyelination as part of its core clinical phenotype. Our findings support possible overlapping molecular mechanisms of paranodal damage at peripheral nerves in both the immune-mediated and the genetic disease, but the observation of prominent central neurological involvement in NFASC biallelic variant carriers highlights the importance of this gene in human brain development and function.

Published
2019

3. Biallelic mutations in neurofascin cause neurodevelopmental impairment and peripheral demyelination

Author

Efthymiou, S., Salpietro, V., Malintan,N., Poncelet, M., Kriouile, Y., Fortuna, S., De Zorzi, R., Payne, K., Henderson, L.B., Cortese, A., Maddirevula, S., Alhashmi, N., Wiethoff, S., Ryten, M., Botia, J.A., Provitera, V., Schuelke, M., Vandrovcova, J., SYNAPS Study Group, Walsh, L., Torti, E., Iodice, V., Najafi, M., Karimiani, E.G., Maroofian, R., Siquier-Pernet, K., Boddaert, N., De Lonlay, P., Cantagrel, V., Aguennouz, M., El Khorassani, M., Schmidts, M., Alkuraya, F.S., Edvardson, S., Nolano, M., Devaux, J., Houlden, H., and Cormand Rifà, Bru

Subjects
Neurobiologia del desenvolupament, Neurons, Malalties del sistema nerviós, Neurones, Nervous system Diseases, Developmental neurobiology, Axons
Abstract

Axon pathfinding and synapse formation are essential processes for nervous system development and function. The assembly of myelinated fibres and nodes of Ranvier is mediated by a number of cell adhesion molecules of the immunoglobulin superfamily including neurofascin, encoded by the NFASC gene, and its alternative isoforms Nfasc186 and Nfasc140 (located in the axonal membrane at the node of Ranvier) and Nfasc155 (a glial component of the paranodal axoglial junction). We identified 10 individuals from six unrelated families, exhibiting a neurodevelopmental disorder characterized with a spectrum of central (intellectual disability, developmental delay, motor impairment, speech difficulties) and peripheral (early onset demyelinating neuropathy) neurological involvement, who were found by exome or genome sequencing to carry one frameshift and four different homozygous non-synonymous variants in NFASC. Expression studies using immunostaining-based techniques identified absent expression of the Nfasc155 isoform as a consequence of the frameshift variant and a significant reduction of expression was also observed in association with two non-synonymous variants affecting the fibronectin type III domain. Cell aggregation studies revealed a severely impaired Nfasc155-CNTN1/CASPR1 complex interaction as a result of the identified variants. Immunofluorescence staining of myelinated fibres from two affected individuals showed a severe loss of myelinated fibres and abnormalities in the paranodal junction morphology. Our results establish that recessive variants affecting the Nfasc155 isoform can affect the formation of paranodal axoglial junctions at the nodes of Ranvier. The genetic disease caused by biallelic NFASC variants includes neurodevelopmental impairment and a spectrum of central and peripheral demyelination as part of its core clinical phenotype. Our findings support possible overlapping molecular mechanisms of paranodal damage at peripheral nerves in both the immune-mediated and the genetic disease, but the observation of prominent central neurological involvement in NFASC biallelic variant carriers highlights the importance of this gene in human brain development and function.

Published
2019

4. AMPA receptor GluA2 subunit defects are a cause of neurodevelopmental disorders

Author

Salpietro, V. Dixon, C.L. Guo, H. Bello, O.D. Vandrovcova, J. Efthymiou, S. Maroofian, R. Heimer, G. Burglen, L. Valence, S. Torti, E. Hacke, M. Rankin, J. Tariq, H. Colin, E. Procaccio, V. Striano, P. Mankad, K. Lieb, A. Chen, S. Pisani, L. Bettencourt, C. Männikkö, R. Manole, A. Brusco, A. Grosso, E. Ferrero, G.B. Armstrong-Moron, J. Gueden, S. Bar-Yosef, O. Tzadok, M. Monaghan, K.G. Santiago-Sim, T. Person, R.E. Cho, M.T. Willaert, R. Yoo, Y. Chae, J.-H. Quan, Y. Wu, H. Wang, T. Bernier, R.A. Xia, K. Blesson, A. Jain, M. Motazacker, M.M. Jaeger, B. Schneider, A.L. Boysen, K. Muir, A.M. Myers, C.T. Gavrilova, R.H. Gunderson, L. Schultz-Rogers, L. Klee, E.W. Dyment, D. Osmond, M. Parellada, M. Llorente, C. Gonzalez-Peñas, J. Carracedo, A. Van Haeringen, A. Ruivenkamp, C. Nava, C. Heron, D. Nardello, R. Iacomino, M. Minetti, C. Skabar, A. Fabretto, A. Hanna, M.G. Bugiardini, E. Hostettler, I. O’Callaghan, B. Khan, A. Cortese, A. O’Connor, E. Yau, W.Y. Bourinaris, T. Kaiyrzhanov, R. Chelban, V. Madej, M. Diana, M.C. Vari, M.S. Pedemonte, M. Bruno, C. Balagura, G. Scala, M. Fiorillo, C. Nobili, L. Malintan, N.T. Zanetti, M.N. Krishnakumar, S.S. Lignani, G. Jepson, J.E.C. Broda, P. Baldassari, S. Rossi, P. Fruscione, F. Madia, F. Traverso, M. De-Marco, P. Pérez-Dueñas, B. Munell, F. Kriouile, Y. El-Khorassani, M. Karashova, B. Avdjieva, D. Kathom, H. Tincheva, R. Van-Maldergem, L. Nachbauer, W. Boesch, S. Gagliano, A. Amadori, E. Goraya, J.S. Sultan, T. Kirmani, S. Ibrahim, S. Jan, F. Mine, J. Banu, S. Veggiotti, P. Zuccotti, G.V. Ferrari, M.D. Van Den Maagdenberg, A.M.J. Verrotti, A. Marseglia, G.L. Savasta, S. Soler, M.A. Scuderi, C. Borgione, E. Chimenz, R. Gitto, E. Dipasquale, V. Sallemi, A. Fusco, M. Cuppari, C. Cutrupi, M.C. Ruggieri, M. Cama, A. Capra, V. Mencacci, N.E. Boles, R. Gupta, N. Kabra, M. Papacostas, S. Zamba-Papanicolaou, E. Dardiotis, E. Maqbool, S. Rana, N. Atawneh, O. Lim, S.Y. Shaikh, F. Koutsis, G. Breza, M. Coviello, D.A. Dauvilliers, Y.A. AlKhawaja, I. AlKhawaja, M. Al-Mutairi, F. Stojkovic, T. Ferrucci, V. Zollo, M. Alkuraya, F.S. Kinali, M. Sherifa, H. Benrhouma, H. Turki, I.B.Y. Tazir, M. Obeid, M. Bakhtadze, S. Saadi, N.W. Zaki, M.S. Triki, C.C. Benfenati, F. Gustincich, S. Kara, M. Belcastro, V. Specchio, N. Capovilla, G. Karimiani, E.G. Salih, A.M. Okubadejo, N.U. Ojo, O.O. Oshinaike, O.O. Oguntunde, O. Wahab, K. Bello, A.H. Abubakar, S. Obiabo, Y. Nwazor, E. Ekenze, O. Williams, U. Iyagba, A. Taiwo, L. Komolafe, M. Senkevich, K. Shashkin, C. Zharkynbekova, N. Koneyev, K. Manizha, G. Isrofilov, M. Guliyeva, U. Salayev, K. Khachatryan, S. Rossi, S. Silvestri, G. Haridy, N. Ramenghi, L.A. Xiromerisiou, G. David, E. Aguennouz, M. Fidani, L. Spanaki, C. Tucci, A. Raspall-Chaure, M. Chez, M. Tsai, A. Fassi, E. Shinawi, M. Constantino, J.N. De Zorzi, R. Fortuna, S. Kok, F. Keren, B. Bonneau, D. Choi, M. Benzeev, B. Zara, F. Mefford, H.C. Scheffer, I.E. Clayton-Smith, J. Macaya, A. Rothman, J.E. Eichler, E.E. Kullmann, D.M. Houlden, H. SYNAPS Study Group

Abstract

AMPA receptors (AMPARs) are tetrameric ligand-gated channels made up of combinations of GluA1-4 subunits encoded by GRIA1-4 genes. GluA2 has an especially important role because, following post-transcriptional editing at the Q607 site, it renders heteromultimeric AMPARs Ca2+-impermeable, with a linear relationship between current and trans-membrane voltage. Here, we report heterozygous de novo GRIA2 mutations in 28 unrelated patients with intellectual disability (ID) and neurodevelopmental abnormalities including autism spectrum disorder (ASD), Rett syndrome-like features, and seizures or developmental epileptic encephalopathy (DEE). In functional expression studies, mutations lead to a decrease in agonist-evoked current mediated by mutant subunits compared to wild-type channels. When GluA2 subunits are co-expressed with GluA1, most GRIA2 mutations cause a decreased current amplitude and some also affect voltage rectification. Our results show that de-novo variants in GRIA2 can cause neurodevelopmental disorders, complementing evidence that other genetic causes of ID, ASD and DEE also disrupt glutamatergic synaptic transmission. © 2019, The Author(s).

Published
2019

5. Mutations in the Neuronal Vesicular SNARE VAMP2 Affect Synaptic Membrane Fusion and Impair Human Neurodevelopment

Author

Salpietro V, Malintan NT, Llano-Rivas I, Spaeth CG, Efthymiou S, Striano P, Vandrovcova J, Cutrupi MC, Chimenz R, David E, Di Rosa G, Marce-Grau A, Raspall-Chaure M, Martin-Hernandez E, Zara F, Minetti C, Deciphering Developmental Disorders Study, SYNAPS Study Group, Bello OD, De Zorzi R, Fortuna S, Dauber A, Alkhawaja M, Sultan T, Mankad K, Vitobello A, Thomas Q, Mau-Them FT, Faivre L, Martinez-Azorin F, Prada CE, Alfons Macaya, Kullmann DM, Rothman JE, Krishnakumar SS, and Houlden H

Subjects
SNARE, VAMP2, autism, epilepsy, movement disorders, neurodevelopmental disorders, neuronal exocytosis, synaptobrevin, synaptopathy, vesicle fusion, sense organs
Abstract

VAMP2 encodes the vesicular SNARE protein VAMP2 (also called synaptobrevin-2). Together with its partners syntaxin-1A and synaptosomal-associated protein 25 (SNAP25), VAMP2 mediates fusion of synaptic vesicles to release neurotransmitters. VAMP2 is essential for vesicular exocytosis and activity-dependent neurotransmitter release. Here, we report five heterozygous de novo mutations in VAMP2 in unrelated individuals presenting with a neurodevelopmental disorder characterized by axial hypotonia (which had been present since birth), intellectual disability, and autistic features. In total, we identified two single-amino-acid deletions and three non-synonymous variants affecting conserved residues within the C terminus of the VAMP2 SNARE motif. Affected individuals carrying de novo non-synonymous variants involving the C-terminal region presented a more severe phenotype with additional neurological features, including central visual impairment, hyperkinetic movement disorder, and epilepsy or electroencephalography abnormalities. Reconstituted fusion involving a lipid-mixing assay indicated impairment in vesicle fusion as one of the possible associated disease mechanisms. The genetic synaptopathy caused by VAMP2 de novo mutations highlights the key roles of this gene in human brain development and function.

Published
2019

6. AMPA receptor GluA2 subunit defects are a cause of neurodevelopmental disorders

Author

Salpietro, V, Dixon, CL, Guo, H, Bello, OD, Vandrovcova, J, Efthymiou, S, Maroofian, R, Heimer, G, Burglen, L, Valence, S, Torti, E, Hacke, M, Rankin, J, Tariq, H, Colin, E, Procaccio, V, Striano, P, Mankad, K, Lieb, A, Chen, S, Pisani, L, Bettencourt, C, Mannikko, R, Manole, A, Brusco, A, Grosso, E, Ferrero, GB, Armstrong-Moron, J, Gueden, S, Bar-Yosef, O, Tzadok, M, Monaghan, KG, Santiago-Sim, T, Person, RE, Cho, MT, Willaert, R, Yoo, Y, Chae, J-H, Quan, Y, Wu, H, Wang, T, Bernier, RA, Xia, K, Blesson, A, Jain, M, Motazacker, MM, Jaeger, B, Schneider, AL, Boysen, K, Muir, AM, Myers, CT, Gavrilova, RH, Gunderson, L, Schultz-Rogers, L, Klee, EW, Dyment, D, Osmond, M, Parellada, M, Llorente, C, Gonzalez-Penas, J, Carracedo, A, Van Haeringen, A, Ruivenkamp, C, Nava, C, Heron, D, Nardello, R, Iacomino, M, Minetti, C, Skabar, A, Fabretto, A, Chez, M, Tsai, A, Fassi, E, Shinawi, M, Constantino, JN, De Zorzi, R, Fortuna, S, Kok, F, Keren, B, Bonneau, D, Choi, M, Benzeev, B, Zara, F, Mefford, HC, Scheffer, IE, Clayton-Smith, J, Macaya, A, Rothman, JE, Eichler, EE, Kullmann, DM, Houlden, H, Raspall-Chaure, M, Hanna, MG, Bugiardini, E, Hostettler, I, O'Callaghan, B, Khan, A, Cortese, A, O'Connor, E, Yau, WY, Bourinaris, T, Kaiyrzhanov, R, Chelban, V, Madej, M, Diana, MC, Vari, MS, Pedemonte, M, Bruno, C, Balagura, G, Scala, M, Fiorillo, C, Nobili, L, Malintan, NT, Zanetti, MN, Krishnakumar, SS, Lignani, G, Jepson, JEC, Broda, P, Baldassari, S, Rossi, P, Fruscione, F, Madia, F, Traverso, M, De-Marco, P, Perez-Duenas, B, Munell, F, Kriouile, Y, El-Khorassani, M, Karashova, B, Avdjieva, D, Kathom, H, Tincheva, R, Van-Maldergem, L, Nachbauer, W, Boesch, S, Gagliano, A, Amadori, E, Goraya, JS, Sultan, T, Kirmani, S, Ibrahim, S, Jan, F, Mine, J, Banu, S, Veggiotti, P, Zuccotti, G, Ferrari, MD, Van Den Maagdenberg, AMJ, Verrotti, A, Marseglia, GL, Savasta, S, Soler, MA, Scuderi, C, Borgione, E, Chimenz, R, Gitto, E, Dipasquale, V, Sallemi, A, Fusco, M, Cuppari, C, Cutrupi, MC, Ruggieri, M, Cama, A, Capra, V, Mencacci, NE, Boles, R, Gupta, N, Kabra, M, Papacostas, S, Zamba-Papanicolaou, E, Dardiotis, E, Maqbool, S, Rana, N, Atawneh, O, Lim, SY, Shaikh, F, Koutsis, G, Breza, M, Coviello, DA, Dauvilliers, YA, AlKhawaja, I, AlKhawaja, M, Al-Mutairi, F, Stojkovic, T, Ferrucci, V, Zollo, M, Alkuraya, FS, Kinali, M, Sherifa, H, Benrhouma, H, Turki, IBY, Tazir, M, Obeid, M, Bakhtadze, S, Saadi, NW, Zaki, MS, Triki, CC, Benfenati, F, Gustincich, S, Kara, M, Belcastro, V, Specchio, N, Capovilla, G, Karimiani, EG, Salih, AM, Okubadejo, NU, Ojo, OO, Oshinaike, OO, Oguntunde, O, Wahab, K, Bello, AH, Abubakar, S, Obiabo, Y, Nwazor, E, Ekenze, O, Williams, U, Iyagba, A, Taiwo, L, Komolafe, M, Senkevich, K, Shashkin, C, Zharkynbekova, N, Koneyev, K, Manizha, G, Isrofilov, M, Guliyeva, U, Salayev, K, Khachatryan, S, Rossi, S, Silvestri, G, Haridy, N, Ramenghi, LA, Xiromerisiou, G, David, E, Aguennouz, M, Fidani, L, Spanaki, C, Tucci, A, Salpietro, V, Dixon, CL, Guo, H, Bello, OD, Vandrovcova, J, Efthymiou, S, Maroofian, R, Heimer, G, Burglen, L, Valence, S, Torti, E, Hacke, M, Rankin, J, Tariq, H, Colin, E, Procaccio, V, Striano, P, Mankad, K, Lieb, A, Chen, S, Pisani, L, Bettencourt, C, Mannikko, R, Manole, A, Brusco, A, Grosso, E, Ferrero, GB, Armstrong-Moron, J, Gueden, S, Bar-Yosef, O, Tzadok, M, Monaghan, KG, Santiago-Sim, T, Person, RE, Cho, MT, Willaert, R, Yoo, Y, Chae, J-H, Quan, Y, Wu, H, Wang, T, Bernier, RA, Xia, K, Blesson, A, Jain, M, Motazacker, MM, Jaeger, B, Schneider, AL, Boysen, K, Muir, AM, Myers, CT, Gavrilova, RH, Gunderson, L, Schultz-Rogers, L, Klee, EW, Dyment, D, Osmond, M, Parellada, M, Llorente, C, Gonzalez-Penas, J, Carracedo, A, Van Haeringen, A, Ruivenkamp, C, Nava, C, Heron, D, Nardello, R, Iacomino, M, Minetti, C, Skabar, A, Fabretto, A, Chez, M, Tsai, A, Fassi, E, Shinawi, M, Constantino, JN, De Zorzi, R, Fortuna, S, Kok, F, Keren, B, Bonneau, D, Choi, M, Benzeev, B, Zara, F, Mefford, HC, Scheffer, IE, Clayton-Smith, J, Macaya, A, Rothman, JE, Eichler, EE, Kullmann, DM, Houlden, H, Raspall-Chaure, M, Hanna, MG, Bugiardini, E, Hostettler, I, O'Callaghan, B, Khan, A, Cortese, A, O'Connor, E, Yau, WY, Bourinaris, T, Kaiyrzhanov, R, Chelban, V, Madej, M, Diana, MC, Vari, MS, Pedemonte, M, Bruno, C, Balagura, G, Scala, M, Fiorillo, C, Nobili, L, Malintan, NT, Zanetti, MN, Krishnakumar, SS, Lignani, G, Jepson, JEC, Broda, P, Baldassari, S, Rossi, P, Fruscione, F, Madia, F, Traverso, M, De-Marco, P, Perez-Duenas, B, Munell, F, Kriouile, Y, El-Khorassani, M, Karashova, B, Avdjieva, D, Kathom, H, Tincheva, R, Van-Maldergem, L, Nachbauer, W, Boesch, S, Gagliano, A, Amadori, E, Goraya, JS, Sultan, T, Kirmani, S, Ibrahim, S, Jan, F, Mine, J, Banu, S, Veggiotti, P, Zuccotti, G, Ferrari, MD, Van Den Maagdenberg, AMJ, Verrotti, A, Marseglia, GL, Savasta, S, Soler, MA, Scuderi, C, Borgione, E, Chimenz, R, Gitto, E, Dipasquale, V, Sallemi, A, Fusco, M, Cuppari, C, Cutrupi, MC, Ruggieri, M, Cama, A, Capra, V, Mencacci, NE, Boles, R, Gupta, N, Kabra, M, Papacostas, S, Zamba-Papanicolaou, E, Dardiotis, E, Maqbool, S, Rana, N, Atawneh, O, Lim, SY, Shaikh, F, Koutsis, G, Breza, M, Coviello, DA, Dauvilliers, YA, AlKhawaja, I, AlKhawaja, M, Al-Mutairi, F, Stojkovic, T, Ferrucci, V, Zollo, M, Alkuraya, FS, Kinali, M, Sherifa, H, Benrhouma, H, Turki, IBY, Tazir, M, Obeid, M, Bakhtadze, S, Saadi, NW, Zaki, MS, Triki, CC, Benfenati, F, Gustincich, S, Kara, M, Belcastro, V, Specchio, N, Capovilla, G, Karimiani, EG, Salih, AM, Okubadejo, NU, Ojo, OO, Oshinaike, OO, Oguntunde, O, Wahab, K, Bello, AH, Abubakar, S, Obiabo, Y, Nwazor, E, Ekenze, O, Williams, U, Iyagba, A, Taiwo, L, Komolafe, M, Senkevich, K, Shashkin, C, Zharkynbekova, N, Koneyev, K, Manizha, G, Isrofilov, M, Guliyeva, U, Salayev, K, Khachatryan, S, Rossi, S, Silvestri, G, Haridy, N, Ramenghi, LA, Xiromerisiou, G, David, E, Aguennouz, M, Fidani, L, Spanaki, C, and Tucci, A

Abstract

AMPA receptors (AMPARs) are tetrameric ligand-gated channels made up of combinations of GluA1-4 subunits encoded by GRIA1-4 genes. GluA2 has an especially important role because, following post-transcriptional editing at the Q607 site, it renders heteromultimeric AMPARs Ca2+-impermeable, with a linear relationship between current and trans-membrane voltage. Here, we report heterozygous de novo GRIA2 mutations in 28 unrelated patients with intellectual disability (ID) and neurodevelopmental abnormalities including autism spectrum disorder (ASD), Rett syndrome-like features, and seizures or developmental epileptic encephalopathy (DEE). In functional expression studies, mutations lead to a decrease in agonist-evoked current mediated by mutant subunits compared to wild-type channels. When GluA2 subunits are co-expressed with GluA1, most GRIA2 mutations cause a decreased current amplitude and some also affect voltage rectification. Our results show that de-novo variants in GRIA2 can cause neurodevelopmental disorders, complementing evidence that other genetic causes of ID, ASD and DEE also disrupt glutamatergic synaptic transmission.

Published
2019

17. Comparison of the structure of a potassium channel in both 2D and 3D crystals.

Author

De Zorzi, R., Bannwarth, L., Nicholson, W., and Venien-Bryan, C.

Subjects
*PHYSIOLOGICAL effects of potassium channels, *CELL transformation, *CRYSTALLOGRAPHY
Abstract

Inwardly-rectifying potassium (Kir) channels regulate membrane electrical excitability and K+ transport in many cell types where they control such diverse processes as heart rate, vascular tone, insulin secretion and salt/fluid balance. Their physiological importance is highlighted by the fact that genetically inherited defects in Kir channels are responsible for a wide-range of channelopathies. To elucidate how channel function becomes defective in the disease state requires a detailed understanding of channel structure in both the open and closed states. Here for the first time, we report the structure of a KirBac potassium channel with an open bundle crossing indicating a mechanism of channel gating determined by Xray crystallography at 3Å resolution. In this model, the rotational twist of the cytoplasmic domain is coupled to opening of the bundle-crossing gate via a network of inter- and intra-subunit interactions [1]. In addition, we have also used EM analysis of 2D crystals of the same Kir channel trapped in an open state and compared these results with the 3D structure. Intriguingly, the projection maps from the EM experiments suggest a larger opening of the pore in the 2D crystal form compared to that observed in the 3D crystal structure [2]. The organization of these two crystal forms is different and suggests that the 2D crystals may permit stabilisation of an open state structure that is not compatible with 3D crystallisation. These results not only have major implications for our understanding of the open state structure of the Kir channel, but more importantly they demonstrate the general utility and importance of methods such as electron microscopy and 2D crystallography for the study of membrane protein structure. [ABSTRACT FROM AUTHOR]

Published
2013

19. Hierarchical self-assembly and controlled disassembly of a cavitand-based host-guest supramolecular polymer

Author

Roberta Pinalli, Enrico Dalcanale, Cristiano Zuccaccia, Silvano Geremia, Monica Semeraro, Alceo Macchioni, Alberto Credi, Daniele Zuccaccia, Rita De Zorzi, Zuccaccia, D., Pinalli, R., De Zorzi, R., Semeraro, M., Credi, A., Zuccaccia, C., Macchioni, A., Geremia, S., Dalcanale, E., Zuccaccia D., Pinalli R., De Zorzi R., Semeraro M., Credi A., Zuccaccia C., MacChioni A., Geremia S., and Dalcanale E.

Subjects
Materials science, Polymers and Plastics, polymer, Supramolecular chemistry, Bioengineering, Biochemistry, supramolecular chemistry, chemistry.chemical_compound, Calixarene, host-guest, chemistry.chemical_classification, Isodesmic reaction, Organic Chemistry, cavitand, Cavitand, pyridinium, Supramolecular polymers, Crystallography, Monomer, chemistry, Polymerization, electrochemistry, calixarene, Self-assembly
Abstract

There is considerable interest in dynamic materials featuring modular components with nano-scale dimensions and controlled responsiveness to external stimuli. Supramolecular polymers are a class of materials that fulfil all these conditions well. Here, we present a family of host–guest supramolecular polymers that combine the outstanding complexing properties of tetraphosphonate cavitands toward N-methylpyridinium guests with molecular switching. The designed monomer is a cavitand featuring four inward facing PO groups at the upper rim and a single N-methylpyridinium unit at the lower rim, forming instantaneously a polymeric species in solution, thanks to the high complexation constants measured for these host–guest interactions. This system has been analyzed by NMR spectroscopy and electrochemical techniques. In order to interpret the results of diffusion-sensitive experiments, we took advantage of the X-ray crystal structure obtained for the polymeric species and developed an original treatment for the PGSE data by non-linear fitting. The analysis of the experimental data identified an isodesmic polymerization model at a monomer concentration below 20 mM, driven by intrachain host–guest interactions, and an additional level of tetrameric bundle aggregation above 20 mM, due to interchain dipolar and quadrupolar interactions. Two orthogonal disassembly procedures have been implemented: electrochemical reduction for the linear chains and solvent-driven dissolution for the bundles.

Published
2021

20. Heterochirality and Halogenation Control Phe-Phe Hierarchical Assembly

Author

Rita De Zorzi, Paola D'Andrea, Caterina Deganutti, Evelina Parisi, Ana M. Garcia, Silvano Geremia, Ottavia Bellotto, Sabrina Semeraro, Silvia Marchesan, Daniel Iglesias, Slavko Kralj, Attilio Vittorio Vargiu, Kralj, S., Bellotto, O., Parisi, E., Garcia, A. M., Iglesias, D., Semeraro, S., Deganutti, C., D'Andrea, P., Vargiu, A. V., Geremia, S., De Zorzi, R., and Marchesan, S.

Subjects
Biocompatibility, phenylalanine, Supramolecular chemistry, chirality, d -amino acid, General Physics and Astronomy, 02 engineering and technology, 010402 general chemistry, 01 natural sciences, Article, chemistry.chemical_compound, halogenation, Side chain, General Materials Science, Diphenylalanine, hydrogels, Halogen bond, Chemistry, General Engineering, d -amino acids, peptides, self-assembly, Halogenation, 021001 nanoscience & nanotechnology, Combinatorial chemistry, peptide, 0104 chemical sciences, Self-healing hydrogels, d-amino acids, Self-assembly, hydrogel, 0210 nano-technology
Abstract

Diphenylalanine is an amyloidogenic building block that can form a versatile array of supramolecular materials. Its shortcomings, however, include the uncontrolled hierarchical assembly into microtubes of heterogeneous size distribution and well-known cytotoxicity. This study rationalized heterochirality as a successful strategy to address both of these pitfalls and it provided an unprotected heterochiral dipeptide that self-organized into a homogeneous and optically clear hydrogel with excellent ability to sustain fibroblast cell proliferation and viability. Substitution of one l-amino acid with its d-enantiomer preserved the ability of the dipeptide to self-organize into nanotubes, as shown by single-crystal XRD analysis, whereby the pattern of electrostatic and hydrogen bonding interactions of the backbone was unaltered. The effect of heterochirality was manifested in subtle changes in the positioning of the aromatic side chains, which resulted in weaker intermolecular interactions between nanotubes. As a result, d-Phe-l-Phe self-organized into homogeneous nanofibrils with a diameter of 4 nm, corresponding to two layers of peptides around a water channel, and yielded a transparent hydrogel. In contrast with homochiral Phe-Phe stereoisomer, it formed stable hydrogels thermoreversibly. d-Phe-l-Phe displayed no amyloid toxicity in cell cultures with fibroblast cells proliferating in high numbers and viability on this biomaterial, marking it as a preferred substrate over tissue-culture plastic. Halogenation also enabled the tailoring of d-Phe-l-Phe self-organization. Fluorination allowed analogous supramolecular packing as confirmed by XRD, thus nanotube formation, and gave intermediate levels of bundling. In contrast, iodination was the most effective strategy to augment the stability of the resulting hydrogel, although at the expense of optical transparency and biocompatibility. Interestingly, iodine presence hindered the supramolecular packing into nanotubes, resulting instead into amphipathic layers of stacked peptides without the occurrence of halogen bonding. By unravelling fine details to control these materials at the meso- and macro-scale, this study significantly advanced our understanding of these systems.

Published
2020
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21. Neutralization of Reactive Oxygen Species at Dinuclear Cu(II)-Cores: Tuning the Antioxidant Manifold in Water by Ligand Design

Author

Marco Bortolus, Alice Santoro, Rita De Zorzi, Silvano Geremia, Marcella Bonchio, Marilena Di Valentin, Mauro Carraro, Andrea Squarcina, Neal Hickey, Squarcina, Andrea, Santoro, Alice, Hickey, JAMES NEIL, De Zorzi, R., Carraro, Mauro, Geremia, Silvano, Bortolus, Marco, Di Valentin, Marilena, and Bonchio, Marcella

Subjects
Antioxidant, medicine.medical_treatment, 010402 general chemistry, 01 natural sciences, Medicinal chemistry, Catalysis, Neutralization, antioxidant catalysis, Superoxide dismutase, chemistry.chemical_compound, parasitic diseases, medicine, Phenol, heterocyclic compounds, chemistry.chemical_classification, artificial enzyme, Reactive oxygen species, copper catalysis, biology, 010405 organic chemistry, Chemistry, Ligand, copper catalysi, organic chemicals, catalase, General Chemistry, superoxide dismutase, artificial enzymes, 0104 chemical sciences, artificial enzymes, copper catalysis, superoxide dismutase, catalase, Catalase, biology.protein
Abstract

Dinuclear Cu-2(II,II)-cores stabilized by the N3O donorset of HL1 = (2-{[[di(2-pyridyl)methyl](methyl)amino]methyl}phenol), HL2 = 2-({[di(2-pyridyl)methyl] amino}methyl)phenol), and HL3 = 2-({[di(2-pyridyl)methyl]amino}methyl)-4-nitrophenol display a unique superoxide dismutase (SOD) combined with catalase (CAT)-like activity in water, at neutral pH. The Cu2L21 < Cu2L22 < Cu2L23 structure-reactivity trend puts a spotlight on the electron-deficient core of Cu2L23 that exhibits the highest SOD (log k(cat) (O-2(center dot-)) = 7.55) and CAT-like (k(H2O2) = 0.66 M(-1)s(-1)) performance. Time-lapse ESI-MS and EPR experiments indicate that a dimeric core is essential for oxygenic turnover upon H2O2 decomposition.

Published
2020
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22. Self‐Assembly of Unprotected Dipeptides into Hydrogels: Water‐Channels Make the Difference

Author

Ottavia Bellotto, Paolo Pengo, Marjetka Podobnik, Matic Kisovec, Silvia Marchesan, Slavko Kralj, Rita De Zorzi, Michele Melchionna, Bellotto, O., Kralj, S., Melchionna, M., Pengo, P., Kisovec, M., Podobnik, M., De Zorzi, R., and Marchesan, S.

Subjects
D-amino acid, Circular dichroism, Supramolecular chemistry, chirality, Infrared spectroscopy, Biochemistry, Amphiphile, Molecular Biology, D-amino acids, hydrogels, peptides, self-assembly, Rheometry, Chemistry, Organic Chemistry, Water, Hydrogels, Stereoisomerism, Dipeptides, peptide, Chemical engineering, Attenuated total reflection, Self-healing hydrogels, Molecular Medicine, Self-assembly, hydrogel
Abstract

Unprotected dipeptides are attractive building blocks for environmentally friendly hydrogel biomaterials by virtue of their low-cost and ease of preparation. This work investigates the self-assembling behaviour of the distinct stereoisomers of Ile-Phe and Phe-Ile in phosphate buffered saline (PBS) to form hydrogels, using transmission electron microscopy (TEM), attenuated total reflectance infrared spectroscopy (ATR-IR), circular dichroism (CD), and oscillatory rheometry. Each peptide purity and identity was also confirmed by 1 H- and 13 C-NMR spectroscopy and HPLC-MS. Finally, single-crystal XRD data allowed the key interactions responsible for the supramolecular packing into amphipathic layers or water-channels to be revealed. The presence of the latter in the crystal structure is a distinctive feature of the only gelator of this work that self-organizes into stable hydrogels, with fast kinetics and the highest elastic modulus amongst its structural isomers and stereoisomers.

Published
2021
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23. Nanoscale Assembly of Functional Peptides with Divergent Programming Elements

Author

Ana M. Garcia, Paola D'Andrea, Slavko Kralj, Ottavia Bellotto, Daniel Iglesias, Rita De Zorzi, Evelina Parisi, Michele Melchionna, Sabrina Semeraro, Attilio Vittorio Vargiu, Silvia Marchesan, Garcia, A. M., Melchionna, M., Bellotto, O., Kralj, S., Semeraro, S., Parisi, E., Iglesias, D., D'Andrea, P., De Zorzi, R., Vargiu, A. V., and Marchesan, S.

Subjects
D-amino acid, Amyloid, Proline, General Physics and Astronomy, chirality, Sequence (biology), Peptide, 02 engineering and technology, Tripeptide, 010402 general chemistry, 01 natural sciences, Article, Chirality, D-amino acids, Hydrogels, Self-assembly, chemistry.chemical_compound, Nanotechnology, General Materials Science, Diphenylalanine, proline, chemistry.chemical_classification, Chemistry, General Engineering, self-assembly, 021001 nanoscience & nanotechnology, peptide, 0104 chemical sciences, Amino acid, Nanostructures, Hydrogel, Self-healing hydrogels, Biophysics, d-amino acids, 0210 nano-technology, Chirality (chemistry), Peptides
Abstract

Self-assembling peptides are being applied both in the biomedical area and as building blocks in nanotechnology. Their applications are closely linked to their modes of self-assembly, which determine the functional nanostructures that they form. This work brings together two structural elements that direct nanoscale self-association in divergent directions: proline as a β-breaker and the β-structure-associated diphenylalanine motif, into a single tripeptide sequence. Amino acid chirality was found to resolve the tension inherent to these conflicting self-assembly instructions. Stereoconfiguration determined the ability of each of the eight possible Pro-Phe-Phe stereoisomers to self-associate into diverse nanostructures, including nanoparticles, nanotapes, or fibrils, which yielded hydrogels with gel-to-sol transition at a physiologically relevant temperature. Three single-crystal structures and all-atom molecular dynamics simulations elucidated the ability of each peptide to establish key interactions to form long-range assemblies (i,e., stacks leading to gelling fibrils), medium-range assemblies (i.e., stacks yielding nanotapes), or short-range assemblies (i.e., dimers or trimers that further associated into nanoparticles). Importantly, diphenylalanine is known to serve as a binding site for pathological amyloids, potentially allowing these heterochiral systems to influence the fibrillization of other biologically relevant peptides. To probe this hypothesis, all eight Pro-Phe-Phe stereoisomers were tested in vitro on the Alzheimer's disease-associated Aβ(1-42) peptide. Indeed, one nonfibril-forming stereoisomer effectively inhibited Aβ fibrillization through multivalent binding between diphenylalanine motifs. This work thus defined heterochirality as a useful feature to strategically develop future therapeutics to interfere with pathological processes, with the additional value of resistance to protease-mediated degradation and biocompatibility.

Published
2021

24. Characterization of the multidrug efflux transporter styMdtMfrom Salmonella enterica serovar Typhi

Author

Thomas Walz, Fouzia Ismat, Aqsa Shaheen, Zaheer Ul-Haq, Mazhar Iqbal, Rita De Zorzi, Abdul Haque, Moazur Rahman, Osman Mirza, Shaheen, A., Ismat, F., Iqbal, M., Haque, A., Ul-Haq, Z., Mirza, O., De Zorzi, R., Walz, T., and Rahman, M.

Subjects
Models, Molecular, Protein Conformation, alpha-Helical, Salmonella, Mutant, Gene Expression, medicine.disease_cause, Salmonella typhi, Biochemistry, Salmonella Typhi, Substrate Specificity, E. coli MdfA, efflux pump, foodborne pathogen, MdtM, multidrug resistance, Structural Biology, 0303 health sciences, Chemistry, 030302 biochemistry & molecular biology, Recombinant Proteins, Anti-Bacterial Agents, Transmembrane domain, Thermodynamics, Efflux, Protein Binding, Monosaccharide Transport Proteins, Microbial Sensitivity Tests, Arginine, Article, Microbiology, 03 medical and health sciences, Bacterial Proteins, Drug Resistance, Bacterial, medicine, Humans, Protein Interaction Domains and Motifs, Typhoid Fever, Molecular Biology, 030304 developmental biology, Aspartic Acid, Binding Sites, Transporter, Biological Transport, Major facilitator superfamily, Multiple drug resistance, Kinetics, Chloramphenicol, Amino Acid Substitution, Mutation, Protein Conformation, beta-Strand
Abstract

Salmonellae are foodborne pathogens and the major cause of gastroenteritis in humans. Salmonellae express multidrug efflux transporters that play a key role in their drug resistance, which is becoming an increasing problem for therapeutic intervention. Despite their biomedical importance, the mechanisms underlying substrate transport by multidrug efflux transporters remain poorly understood. Here, we describe the first characterization of a multidrug transporter belonging to the major facilitator superfamily from the genus Salmonella. We show that several clinical Salmonella Typhi (S. Typhi) isolates constitutively express the styMdtM (STY4874) gene, which encodes a known multidrug-resistance (MDR) transporter. Guided by the structure of the Escherichia coli (E. coli) homolog, we studied two residues critical for substrate transport, Asp25 and Arg111. Mutation of Asp25 to glutamate did not affect the transport function of styMdtM, whereas mutation to alanine reduced its transport activity, suggesting that a negative charge at this position is critical for substrate translocation across the membrane. Substrate-affinity measurements by intrinsic fluorescence spectroscopy showed that the Asp25Ala mutant retained its capacity to bind substrate, albeit at a lower level. Mutation of Arg111 to alanine resulted in a decrease in secondary structure content of the transporter, and mutation to lysine completely destabilized the structure of the transporter. A homology model of styMdtM suggests that Arg111 is important for stabilizing the transmembrane domain by mediating necessary interactions between neighboring helices. Together, our studies provide new structural and mechanistic insights into the Salmonella MDR transporter styMdtM.

Published
2021
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25. Unexpected Gating Behaviour of an Engineered Potassium Channel Kir

Author

Saïd Bendahhou, Dania Zuniga, David Perahia, Charline Fagnen, Iman Oubella, Catherine Vénien-Bryan, Samuel Guilbault, Rita De Zorzi, Ludovic Bannwarth, Rosa Scala, Eric Forest, Fagnen, C, Bannwarth, L, Zuniga, D, Oubella, I, De Zorzi, R, Forest, E, Scala, R, Guilbault, S, Bendahhou, S, Perahia, D, Venien-Bryan, C, Institut de minéralogie, de physique des matériaux et de cosmochimie (IMPMC), Muséum national d'Histoire naturelle (MNHN)-Institut de recherche pour le développement [IRD] : UR206-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de biologie et pharmacologie appliquée (LBPA), Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Ecole Normale Supérieure Paris-Saclay (ENS Paris Saclay), Università degli studi di Trieste = University of Trieste, Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Laboratoire de PhysioMédecine Moléculaire (LP2M), Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), University of Trieste, and Université Nice Sophia Antipolis (... - 2019) (UNS)

Subjects
0301 basic medicine, molecular dynamics and normal modes, HDX-mass spectrometry, single channel recording, potassium channel KirBac3, 1, mutation effect, QH301-705.5, Mutant, Gating, 01 natural sciences, Biochemistry, Genetics and Molecular Biology (miscellaneous), Biochemistry, 03 medical and health sciences, Molecular dynamics, Normal mode, 0103 physical sciences, Molecular Biosciences, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Biology (General), Molecular Biology, Original Research, Physics, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 010304 chemical physics, Potassium channel, potassium channel KirBac3.1, 030104 developmental biology, Helix, Mutation (genetic algorithm), Biophysics, molecular dynamics and normal mode, Communication channel
Abstract

In this study, we investigated the dynamics and functional characteristics of the KirBac3.1 S129R, a mutated bacterial potassium channel for which the inner pore-lining helix (TM2) was engineered so that the bundle crossing is trapped in an open conformation. The structure of this channel has been previously determined at high atomic resolution. We explored the dynamical characteristics of this open state channel using an in silico method MDeNM that combines molecular dynamics simulations and normal modes. We captured the global and local motions at the mutation level and compared these data with HDX-MS experiments. MDeNM provided also an estimation of the probability of the different opening states that are in agreement with our electrophysiological experiments. In the S129R mutant, the Arg129 mutation releases the two constriction points in the channel that existed in the wild type but interestingly creates another restriction point.

Published
2021
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26. (R)-10-Hydroxystearic Acid: Crystals vs. Organogel

Author

Carla Boga, Patrizia Nitti, Sabrina Semeraro, Lara Gigli, Fioretta Asaro, Rita De Zorzi, Silvano Geremia, Asaro, F., Boga, C., De Zorzi, R., Geremia, S., Gigli, L., Nitti, P., Semeraro, S., Asaro F., Boga C., Zorzi R., Geremia S., Gigli L., Nitti P., and Semeraro S.

Subjects
crystal structure, Infrared spectroscopy, 02 engineering and technology, Crystal structure, 010402 general chemistry, 01 natural sciences, Catalysis, polymorphism, Inorganic Chemistry, lcsh:Chemistry, chemistry.chemical_compound, (R)-hydroxystearic acid, IR spectroscopy, organogel, X-ray diffraction, Physical and Theoretical Chemistry, Molecular Biology, lcsh:QH301-705.5, Spectroscopy, Chemistry, Organic Chemistry, Hydroxystearic Acid, General Medicine, 021001 nanoscience & nanotechnology, 0104 chemical sciences, Computer Science Applications, body regions, Crystallography, Polymorphism (materials science), lcsh:Biology (General), lcsh:QD1-999, X-ray crystallography, embryonic structures, Stearic acid, 0210 nano-technology, Single crystal, Powder diffraction
Abstract

The chiral (R)-10-hydroxystearic acid ((R)-10-HSA) is a positional homologue of both (R)-12-HSA and (R)-9-HSA with the OH group in an intermediate position. While (R)-12-HSA is one of the best-known low-molecular-weight organogelators, (R)-9-HSA is not, but it forms crystals in several solvents. With the aim to gain information on the structural role of hydrogen-bonding interactions of the carbinol OH groups, we investigated the behavior of (R)-10-HSA in various solvents. This isomer displays an intermediate behavior between (R)-9 and (R)-12-HSA, producing a stable gel exclusively in paraffin oil, while it crystallizes in other organic solvents. Here, we report the X-ray structure of a single crystal of (R)-10-HSA as well as some structural information on its polymorphism, obtained through X-ray Powder Diffraction (XRPD) and Infrared Spectroscopy (IR). This case study provides new elements to elucidate the structural determinants of the microscopic architectures that lead to the formation of organogels of stearic acid derivatives.

Published
2020

27. New Structural insights into Kir channel gating from molecular simulations, HDX-MS and functional studies

Author

Iman Oubella, David Perahia, Catherine Vénien-Bryan, Yasmina Mhoumadi, Aline de Araujo, Saïd Bendahhou, Charline Fagnen, Rita De Zorzi, Ludovic Bannwarth, Eric Forest, Institut de minéralogie, de physique des matériaux et de cosmochimie (IMPMC), Muséum national d'Histoire naturelle (MNHN)-Institut de recherche pour le développement [IRD] : UR206-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de biologie et pharmacologie appliquée (LBPA), Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Ecole Normale Supérieure Paris-Saclay (ENS Paris Saclay), Université Grenoble Alpes (UGA), University of Trieste, Laboratoire de PhysioMédecine Moléculaire (LP2M), Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), Fagnen, C., Bannwarth, L., Oubella, I., Forest, E., De Zorzi, R., de Araujo, A., Mhoumadi, Y., Bendahhou, S., Perahia, D., Venien-Bryan, C., Università degli studi di Trieste = University of Trieste, and Université Nice Sophia Antipolis (1965 - 2019) (UNS)

Subjects
0301 basic medicine, Guanine Nucleotide Binding Protein, Protein Conformation, In silico, Lipid Bilayers, lcsh:Medicine, Inwardly Rectifying Potassium Channels, Kir2.1 Channel, Hydrogen Deuterium Exchange-Mass Spectrometry, Gating, Molecular Dynamics Simulation, Article, 03 medical and health sciences, Molecular dynamics, 0302 clinical medicine, Protein structure, Computer Simulation, Potassium Channels, Inwardly Rectifying, lcsh:Science, Physics, Inwardly Rectifying Potassium Channel, Multidisciplinary, Mass spectrometry, Inward-rectifier potassium ion channel, lcsh:R, [PHYS.PHYS.PHYS-ATM-PH]Physics [physics]/Physics [physics]/Atomic and Molecular Clusters [physics.atm-clus], Potassium channel, Electrophysiology, Transmembrane domain, 030104 developmental biology, lcsh:Q, Molecular modelling, Ion Channel Gating, Neuroscience, 030217 neurology & neurosurgery
Abstract

Inward rectifier potassium (Kir) channels play diverse and important roles in shaping action potentials in biological membranes. An increasing number of diseases are now known to be directly associated with abnormal Kir function. However, the gating of Kir still remains unknown. To increase our understanding of its gating mechanism, a dynamical view of the entire channel is essential. Here the gating activation was studied using a recent developped in silico method, MDeNM, which combines normal mode analysis and molecular dynamics simulations that showed for the very first time the importance of interrelated collective and localized conformational movements. In particular, we highlighted the role played by concerted movements of the different regions throughout the entire protein, such as the cytoplasmic and transmembrane domains and the slide helices. In addition, the HDX-MS analysis achieved in these studies provided a comprehensive and detailed view of the dynamics associated with open/closed transition of the Kir channel in coherence with the theoretical results. MDeNM gives access to the probability of the different opening states that are in agreement with our electrophysiological experiments. The investigations presented in this article are important to remedy dysfunctional channels and are of interest for designing new pharmacological compounds.

Published
2020
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28. Bioadhesive supramolecular hydrogel from unprotected, short d,l-peptides with Phe-Phe and Leu-Asp-Val motifs

Author

Evelina Parisi, Rita De Zorzi, Michele Melchionna, Chiara D Romano, Maria C. Cringoli, Silvia Marchesan, Sabrina Semeraro, Lynne J. Waddington, Mikaela Grönholm, Cringoli, M. C., Romano, C., Parisi, E., Waddington, L. J., Melchionna, M., Semeraro, S., De Zorzi, R., Gronholm, M., and Marchesan, S.

Subjects
Stereochemistry, Cell Survival, Macromolecular Substances, Surface Properties, Bioadhesive, Surface Propertie, Cell Adhesion, Fibroblasts, Humans, Hydrogels, Molecular Conformation, Particle Size, Peptides, Optical Imaging, Supramolecular chemistry, Peptide, macromolecular substances, 02 engineering and technology, Tripeptide, 010402 general chemistry, 01 natural sciences, Catalysis, Materials Chemistry, Macromolecular Substance, Cell adhesion, chemistry.chemical_classification, biology, Chemistry, technology, industry, and agriculture, Metals and Alloys, General Chemistry, Adhesion, 021001 nanoscience & nanotechnology, 3. Good health, 0104 chemical sciences, Surfaces, Coatings and Films, Electronic, Optical and Magnetic Materials, Fibronectin, Hydrogel, Self-healing hydrogels, Ceramics and Composites, biology.protein, Fibroblast, 0210 nano-technology, Human
Abstract

The uncapped tripeptide DPhe-Phe-Leu acts as self-assembly template to yield supramolecular hydrogel biomaterials. As an example, self-assembling DPhe-Phe-Leu-Asp-Val contains the LDV bioadhesive motif for β1 integrin activation. Hydrogels made of the two peptides successfully mimic fibronectin of the extracellular matrix and lead to high cell viability, adhesion, and spreading.

Published
2020

29. Self-Assembling l-d-l -Tripeptides Dance the Twist

Author

Maria C. Cringoli, Attilio Vittorio Vargiu, Ottavia Bellotto, Silvia Marchesan, Rita De Zorzi, Cringoli, M. C., Bellotto, O., De Zorzi, R., Vargiu, A. V., and Marchesan, S.

Subjects
chirality, d -amino acids, hydrogels, peptides, self-assembly, chemistry.chemical_classification, 010405 organic chemistry, Stereochemistry, Chemistry, Organic Chemistry, Supramolecular chemistry, d -amino acid, Tripeptide, 010402 general chemistry, 01 natural sciences, peptide, 0104 chemical sciences, Amino acid, Turn (biochemistry), Molecular dynamics, Self-assembly, Twist, hydrogel, Chirality (chemistry)
Abstract

Minimalistic peptides composed of d- and l-amino acids are attractive building blocks for functional supramolecular materials, including catalysts. d-Amino acids have long been known to promote turn conformations in peptides, yet unexpected twists continue to emerge on their effects on self-assembly. The combination of single-crystal X-ray diffraction and full-atom molecular dynamics have finally unraveled fine details of how l-d-l-tripeptides visit different conformations in solution and establish key interactions in supramolecular structures.

Published
2020

30. Supramolecular hydrogels from unprotected dipeptides: a comparative study on stereoisomers and structural isomers

Author

Slavko Kralj, Silvano Geremia, Ottavia Bellotto, Rita De Zorzi, Silvia Marchesan, Bellotto, O., Kralj, S., De Zorzi, R., Geremia, S., and Marchesan, S.

Subjects
Phe, D-amino acid, Stereochemistry, phenylalanine, Supramolecular chemistry, chirality, hydrogels: gels, macromolecular substances, Tripeptide, 010402 general chemistry, 01 natural sciences, chemistry.chemical_compound, Structural isomer, dipeptides, Diphenylalanine, chemistry.chemical_classification, D-amino acids, peptides, leucine, Leu, soft matter, self-assembly, supramolecular, Dipeptide, 010405 organic chemistry, Chemistry, gel [hydrogels], technology, industry, and agriculture, Hydrogels, Stereoisomerism, General Chemistry, Condensed Matter Physics, peptide, 0104 chemical sciences, Amino acid, Supramolecular hydrogels, Self-healing hydrogels, Hydrophobic and Hydrophilic Interactions, dipeptide
Abstract

Amino acid stereoconfiguration has been shown to play a key role in the self-assembly of unprotected tripeptides into hydrogels under physiological conditions. Dramatic changes were noted for hydrophobic sequences based on the diphenylalanine motif from the formation of amorphous aggregates in the case of homochiral peptides to nanostructured and stable hydrogels in the case of heterochiral stereoisomers. Herein, we report that by further shortening the sequence to a dipeptide, the overall differences between isomers are less marked, with both homo- and hetero-chiral dipeptides forming gels, although with different stability over time. The soft materials are studied by a number of spectroscopic and microcopic techniques, and single-crystal X-ray diffraction to unveil the supramolecular interactions of these hydrogel building blocks.

Published
2020

31. Mutations in the Neuronal Vesicular SNARE VAMP2 Affect Synaptic Membrane Fusion and Impair Human Neurodevelopment

Author

Salpietro, Vincenzo, Malintan, Nancy T, Llano-Rivas, Isabel, Spaeth, Christine G, Efthymiou, Stephanie, Striano, Pasquale, Vandrovcova, Jana, Cutrupi, Maria C, Chimenz, Roberto, David, Emanuele, Di Rosa, Gabriella, Marce-Grau, Anna, Raspall-Chaure, Miquel, Martin-Hernandez, Elena, Zara, Federico, Minetti, Carlo, Bello, Oscar D, De Zorzi, Rita, Fortuna, Sara, Dauber, Andrew, Alkhawaja, Mariam, Sultan, Tipu, Mankad, Kshitij, Vitobello, Antonio, Thomas, Quentin, Mau-Them, Frederic Tran, Faivre, Laurence, Martinez-Azorin, Francisco, Prada, Carlos E, Macaya, Alfons, Kullmann, Dimitri M, Rothman, James E, Krishnakumar, Shyam S, Houlden, Henry, Kriouile, Yamna, El Khorassani, Mohamed, Aguennouz, Mhammed, Karashova, Blagovesta, Avdjieva, Daniela, Kathom, Hadil, Tincheva, Radka, Van Maldergem, Lionel, Nachbauer, Wolfgang, Boesch, Sylvia, Arning, Larissa, Timmann, Dagmar, Cormand, Bru, Pérez-Dueñas, Belen, Pironti, Erica, Goraya, Jatinder S, Kirmani, Salman, Ibrahim, Shahnaz, Jan, Farida, Mine, Jun, Banu, Selina, Veggiotti, Pierangelo, Ferrari, Michel D, Verrotti, Alberto, Marseglia, Gian Luigi, Savasta, Salvatore, Garavaglia, Barbara, Scuderi, Carmela, Borgione, Eugenia, Dipasquale, Valeria, Cutrupi, Maria Concetta, Portaro, Simona, Sanchez, Benigno Monteagudo, Pineda-Marfa’, Mercedes, Munell, Francina, Boles, Richard, Heimer, Gali, Papacostas, Savvas, Manole, Andreea, Malintan, Nancy, Zanetti, Maria Natalia, Hanna, Michael G, Houlden, Henry., Salpietro, V., Malintan, N. T., Llano-Rivas, I., Spaeth, C. G., Efthymiou, S., Striano, P., Vandrovcova, J., Cutrupi, M. C., Chimenz, R., David, E., Di Rosa, G., Marce-Grau, A., Raspall-Chaure, M., Martin-Hernandez, E., Zara, F., Minetti, C., Kriouile, Y., El Khorassani, M., Aguennouz, M., Karashova, B., Avdjieva, D., Kathom, H., Tincheva, R., Van Maldergem, L., Nachbauer, W., Boesch, S., Arning, L., Timmann, D., Cormand, B., Perez-Duenas, B., Pironti, E., Goraya, J. S., Sultan, T., Kirmani, S., Ibrahim, S., Jan, F., Mine, J., Banu, S., Veggiotti, P., Ferrari, M. D., Verrotti, A., Marseglia, G. L., Savasta, S., Garavaglia, B., Scuderi, C., Borgione, E., Dipasquale, V., Portaro, S., Sanchez, B. M., Pineda-Marfa, M., Munell, F., Macaya, A., Boles, R., Heimer, G., Papacostas, S., Manole, A., Malintan, N., Zanetti, M. N., Hanna, M. G., Rothman, J. E., Kullmann, D. M., Houlden, H., Bello, O. D., De Zorzi, R., Fortuna, S., Dauber, A., Alkhawaja, M., Mankad, K., Vitobello, A., Thomas, Q., Mau-Them, F. T., Faivre, L., Martinez-Azorin, F., Prada, C. E., and Krishnakumar, S. S.

Subjects
Male, Heterozygote, Adolescent, Vesicle-Associated Membrane Protein 2, neuronal exocytosi, synaptopathy, autism, synaptobrevin, Membrane Fusion, Exocytosis, R-SNARE Proteins, Protein Domains, Report, Intellectual Disability, Genetics, Humans, Autistic Disorder, Child, Genetics (clinical), Neurons, Neurotransmitter Agents, neurodevelopmental disorders, vesicle fusion, Brain, epilepsy, movement disorders, neuronal exocytosis, SNARE, VAMP2, Lipids, Magnetic Resonance Imaging, neurodevelopmental disorder, autism, epilepsy, movement disorders, neurodevelopmental disorders, neuronal exocytosis, SNARE, synaptobrevin, synaptopathy, VAMP2, vesicle fusion, Genetics, Genetics (clinical), Phenotype, Child, Preschool, Mutation, Synapses, Muscle Hypotonia, Female, sense organs, movement disorder
Abstract

VAMP2 encodes the vesicular SNARE protein VAMP2 (also called synaptobrevin-2). Together with its partners syntaxin-1A and synaptosomal-associated protein 25 (SNAP25), VAMP2 mediates fusion of synaptic vesicles to release neurotransmitters. VAMP2 is essential for vesicular exocytosis and activity-dependent neurotransmitter release. Here, we report five heterozygous de novo mutations in VAMP2 in unrelated individuals presenting with a neurodevelopmental disorder characterized by axial hypotonia (which had been present since birth), intellectual disability, and autistic features. In total, we identified two single-amino-acid deletions and three non-synonymous variants affecting conserved residues within the C terminus of the VAMP2 SNARE motif. Affected individuals carrying de novo non-synonymous variants involving the C-terminal region presented a more severe phenotype with additional neurological features, including central visual impairment, hyperkinetic movement disorder, and epilepsy or electroencephalography abnormalities. Reconstituted fusion involving a lipid-mixing assay indicated impairment in vesicle fusion as one of the possible associated disease mechanisms. The genetic synaptopathy caused by VAMP2 de novo mutations highlights the key roles of this gene in human brain development and function.

Published
2019

32. AMPA receptor GluA2 subunit defects are a cause of neurodevelopmental disorders

Author

Vincenzo Salpietro1, 2 3, 140, Christine L. Dixon4, Hui Guo5, 6 140, Oscar D. Bello Stephanie Efthymiou 1, 4, Reza Maroofian1, Gali Heimer7, Lydie Burglen 8, Stephanie Valence 9, Erin Torti 10, Moritz Hacke11, Julia Rankin12, Huma Tariq1, Estelle Colin13, Vincent Procaccio13, Pasquale Striano2, 3, Kshitij Mankad15, Andreas Lieb 4, Sharon Chen16, Laura Pisani16, Conceicao Bettencourt 17, Roope Männikkö 1, Andreea Manole1, Alfredo Brusco 18, Enrico Grosso18, Giovanni Battista Ferrero19, Judith Armstrong-Moron20, Sophie Gueden21, Omer Bar-Yosef7, Michal Tzadok7, Kristin G. Monaghan10, Teresa Santiago-Sim10, Richard E. Person10, Megan T. Cho10, Rebecca Willaert10, Yongjin Yoo22, Jong-Hee Chae23, Yingting Quan6, Huidan Wu6, Tianyun Wang5, 6, Raphael A. Bernier24, Kun Xia6, Alyssa Blesson25, Mahim Jain25, Mohammad M. Motazacker26, Bregje Jaeger27, Amy L. Schneider 28, Katja Boysen28, Alison M. Muir 29, Candace T. Myers30, Ralitza H. Gavrilova31, Lauren Gunderson31, Laura Schultz-Rogers 31, Eric W. Klee31, David Dyment32, Matthew Osmond32, 33 34, Mara Parellada35, Cloe Llorente36, Javier Gonzalez-Peñas37, Angel Carracedo38, Arie Van Haeringen40, Claudia Ruivenkamp40, Caroline Nava41, Delphine Heron41, Rosaria Nardello42, Michele Iacomino43, Carlo Minetti2, Aldo Skabar44, Antonella Fabretto44, SYNAPS Study GroupMiquel Raspall-Chaure45, Michael Chez46, Anne Tsai47, Emily Fassi48, Marwan Shinawi48, John N. Constantino49, Rita De Zorzi50, Sara Fortuna 50, Fernando Kok51, Boris Keren41, Dominique Bonneau13, Murim Choi 22, Bruria Benzeev7, Federico Zara43, Heather C. Mefford29, Ingrid E. Scheffer28, Jill Clayton-Smith53, Alfons Macaya45, James E. Rothman4, Evan E. Eichler 5, Dimitri M. Kullmann 4, Henry Houlden 1, SYNAPS Study Group Michael G. Hanna1, Enrico Bugiardini1, Isabel Hostettler1, Benjamin O’Callaghan1, Alaa Khan1, Andrea Cortese1, Emer O’Connor1, Wai Y. Yau1, Thomas Bourinaris1, Rauan Kaiyrzhanov1, Viorica Chelban1, Monika Madej1, Maria C. Diana2, Maria S. Vari2, Marina Pedemonte2, Claudio Bruno2, Ganna Balagura3, Marcello Scala3, Chiara Fiorillo3, Lino Nobili3, Nancy T. Malintan4, Maria N. Zanetti4, Shyam S. Krishnakumar4, Gabriele Lignani4, James E. C. Jepson4, Paolo Broda43, Simona Baldassari43, Pia Rossi43, Floriana Fruscione43, Francesca Madia43, Monica Traverso43, Patrizia De-Marco43, Belen Pérez-Dueñas45, Francina Munell45, Yamna Kriouile57, Mohamed El-Khorassani57, Blagovesta Karashova58, Daniela Avdjieva58, Hadil Kathom58, Radka Tincheva58, Lionel Van-Maldergem59, Wolfgang Nachbauer60, Sylvia Boesch60, Antonella Gagliano61, Elisabetta Amadori62, Jatinder S. Goraya63, Tipu Sultan64, Salman Kirmani65, Shahnaz Ibrahim66, Farida Jan66, Jun Mine67, Selina Banu68, Pierangelo Veggiotti69, Gian V. Zuccotti69, Michel D. Ferrari70, Arn M. J. Van Den Maagdenberg70, Alberto Verrotti71, Gian L. Marseglia72, Salvatore Savasta72, Miguel A. Soler73, Carmela Scuderi74, Eugenia Borgione74, Roberto Chimenz75, Eloisa Gitto75, Valeria Dipasquale75, Alessia Sallemi75, Monica Fusco75, Caterina Cuppari75, Maria C. Cutrupi75, Martino Ruggieri76, Armando Cama77, Valeria Capra77, Niccolò E. Mencacci78, Richard Boles79, Neerja Gupta80, Madhulika Kabra80, Savvas Papacostas81, Eleni Zamba-Papanicolaou81, Efthymios Dardiotis82, Shazia Maqbool83, Nuzhat Rana84, Osama Atawneh85, Shen Y. Lim86, Farooq Shaikh87, George Koutsis88, Marianthi Breza88, Domenico A. Coviello89, Yves A. Dauvilliers90, Issam AlKhawaja91, Mariam AlKhawaja92, Fuad Al-Mutairi93, Tanya Stojkovic94, Veronica Ferrucci, Massimo Zollo, Fowzan S. Alkuraya96, Maria Kinali97, Hamed Sherifa98, Hanene Benrhouma99, Ilhem B. Y. Turki99, Meriem Tazir100, Makram Obeid101, Sophia Bakhtadze102, Nebal W. Saadi103, Maha S. Zaki104, Chahnez C. Triki105, Fabio Benfenati106, Stefano Gustincich106, Majdi Kara107, Vincenzo Belcastro108, Nicola Specchio109, Giuseppe Capovilla110, Ehsan G. Karimiani111, Ahmed M. Salih112, Njideka U. Okubadejo113, Oluwadamilola O. Ojo113, Olajumoke O. Oshinaike113, Olapeju Oguntunde113, Kolawole Wahab114, Abiodun H. Bello114, Sanni Abubakar115, Yahaya Obiabo116, Ernest Nwazor117, Oluchi Ekenze118, Uduak Williams119, Alagoma Iyagba120, Lolade Taiwo121, Morenikeji Komolafe122, Konstantin Senkevich123, Chingiz Shashkin124, Nazira Zharkynbekova125, Kairgali Koneyev126, Ganieva Manizha127, Maksud Isrofilov127, Ulviyya Guliyeva128, Kamran Salayev129, Samson Khachatryan130, Salvatore Rossi131, Gabriella Silvestri131, Nourelhoda Haridy132, Luca A. Ramenghi133, Georgia Xiromerisiou134, Emanuele David135, Mhammed Aguennouz136, Liana Fidani137, Cleanthe Spanaki138, Arianna Tucci139, University College of London [London] (UCL), Instituto Giannina Gaslini, Genoa, University of Genoa (UNIGE), University of Washington [Seattle], Institute of Neurology, Queen Square, London, King‘s College London, UCL Institute of Neurology, National Hospital for Neurology and Neurosurgery, Queen Square, London, Molecular and Clinical Sciences Institute - St George’s [London, UK] (Genetics Research Centre), University of London [London], Tel Aviv University Sackler School of Medicine [Tel Aviv, Israël], Service de génétique et embryologie médicales [CHU Trousseau], CHU Trousseau [APHP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Service de Neuropédiatrie [CHU Trousseau], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-CHU Trousseau [APHP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Sorbonne Université (SU), GeneDx [Gaithersburg, MD, USA], Heidelberg University Hospital [Heidelberg], Royal Devon and Exeter NHS Foundation Trust [UK], Biologie Neurovasculaire et Mitochondriale Intégrée (BNMI), Université d'Angers (UA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre Hospitalier Universitaire d'Angers (CHU Angers), PRES Université Nantes Angers Le Mans (UNAM), Universita degli studi di Genova, Great Ormond Street Hospital for Children [London] (GOSH), The University of Sydney, Hofstra University [Hempstead], Università degli studi di Torino (UNITO), Hospital Sant Joan de Déu [Barcelona], Safra Children's Hospital, Seoul National University Hospital, Central South University [Changsha], Kennedy Krieger Institute [Baltimore], University of Amsterdam [Amsterdam] (UvA), University of Melbourne, Mayo Clinic [Rochester], Department of Health Sciences Research [Mayo Clinic] (HSR), Mayo Clinic, University of Ottawa [Ottawa], University of British Columbia (UBC), Universidad Complutense de Madrid = Complutense University of Madrid [Madrid] (UCM), Universidade de Santiago de Compostela [Spain] (USC ), Universiteit Leiden [Leiden], Institut du Cerveau et de la Moëlle Epinière = Brain and Spine Institute (ICM), Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Università degli studi di Palermo - University of Palermo, University of Trieste, Universitat Autònoma de Barcelona (UAB), Department of Neurology and Center for Neuroscience, University of California at Davis, Sacramento, University of California [Davis] (UC Davis), University of California-University of California, Children’s Hospital Colorado, University of Colorado Anschutz [Aurora], Washington University in Saint Louis (WUSTL), Department of Molecular and Human Genetics, Baylor College of Medicine (BCM), Baylor University-Baylor University, Department of Psychiatry, Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, University of Oxford [Oxford], University of São Paulo (USP), Service de Génétique Cytogénétique et Embryologie [CHU Pitié-Salpêtrière], Service de Pédiatrie, CHUR Poitiers, Seoul National University [Seoul] (SNU), Pediatric Neurology and Neuromuscular Diseases Unit, University of Manchester [Manchester], Yale University School of Medicine, Centre Hospitalier Régional Universitaire [Montpellier] (CHRU Montpellier), Salvy-Córdoba, Nathalie, Università degli studi di Genova = University of Genoa (UniGe), Tel Aviv University (TAU), Università degli studi di Torino = University of Turin (UNITO), Institut du Cerveau = Paris Brain Institute (ICM), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Sorbonne Université (SU)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Università degli studi di Trieste = University of Trieste, University of California (UC)-University of California (UC), University of Oxford, Universidade de São Paulo = University of São Paulo (USP), Yale School of Medicine [New Haven, Connecticut] (YSM), Salpietro V, Dixon CL, Guo H, Bello OD, Vandrovcova J, Efthymiou S, Maroofian R, Heimer G, Burglen L, Valence S, Torti E, Hacke M, Rankin J, Tariq H, Colin E, Procaccio V, Striano P, Mankad K, Lieb A, Chen S, Pisani L, Bettencourt C, Männikkö R, Manole A, Brusco A, Grosso E, Ferrero GB, Armstrong-Moron J, Gueden S, Bar-Yosef O, Tzadok M, Monaghan KG, Santiago-Sim T, Person RE, Cho MT, Willaert R, Yoo Y, Chae JH, Quan Y, Wu H, Wang T, Bernier RA, Xia K, Blesson A, Jain M, Motazacker MM, Jaeger B, Schneider AL, Boysen K, Muir AM, Myers CT, Gavrilova RH, Gunderson L, Schultz-Rogers L, Klee EW, Dyment D, Osmond M, Parellada M, Llorente C, Gonzalez-Peñas J, Carracedo A, Van Haeringen A, Ruivenkamp C, Nava C, Heron D, Nardello R, Iacomino M, Minetti C, Skabar A, Fabretto A, SYNAPS Study Group, Raspall-Chaure M, Chez M, Tsai A, Fassi E, Shinawi M, Constantino JN, De Zorzi R, Fortuna S, Kok F, Keren B, Bonneau D, Choi M, Benzeev B, Zara F, Mefford HC, Scheffer IE, Clayton-Smith J, Macaya A, Rothman JE, Eichler EE, Kullmann DM, Houlden H, Salpietro, Vincenzo, Dixon, Christine L, Guo, Hui, Bello, Oscar D, Vandrovcova, Jana, Efthymiou, Stephanie, Maroofian, Reza, Heimer, Gali, Burglen, Lydie, Valence, Stephanie, Torti, Erin, Hacke, Moritz, Rankin, Julia, Tariq, Huma, Colin, Estelle, Procaccio, Vincent, Striano, Pasquale, Mankad, Kshitij, Lieb, Andrea, Chen, Sharon, Pisani, Laura, Bettencourt, Conceicao, Männikkö, Roope, Manole, Andreea, Brusco, Alfredo, Grosso, Enrico, Ferrero, Giovanni Battista, Armstrong-Moron, Judith, Gueden, Sophie, Bar-Yosef, Omer, Tzadok, Michal, Monaghan, Kristin G, Santiago-Sim, Teresa, Person, Richard E, Cho, Megan T, Willaert, Rebecca, Yoo, Yongjin, Chae, Jong-Hee, Quan, Yingting, Wu, Huidan, Wang, Tianyun, Bernier, Raphael A, Xia, Kun, Blesson, Alyssa, Jain, Mahim, Motazacker, Mohammad M, Jaeger, Bregje, Schneider, Amy L, Boysen, Katja, Muir, Alison M, Myers, Candace T, Gavrilova, Ralitza H, Gunderson, Lauren, Schultz-Rogers, Laura, Klee, Eric W, Dyment, David, Osmond, Matthew, Parellada, Mara, Llorente, Cloe, Gonzalez-Peñas, Javier, Carracedo, Angel, Van Haeringen, Arie, Ruivenkamp, Claudia, Nava, Caroline, Heron, Delphine, Nardello, Rosaria, Iacomino, Michele, Minetti, Carlo, Skabar, Aldo, Fabretto, Antonella, Raspall-Chaure, Miquel, Chez, Michael, Tsai, Anne, Fassi, Emily, Shinawi, Marwan, Constantino, John N, De Zorzi, Rita, Fortuna, Sara, Kok, Fernando, Keren, Bori, Bonneau, Dominique, Choi, Murim, Benzeev, Bruria, Zara, Federico, Mefford, Heather C, Scheffer, Ingrid E, Clayton-Smith, Jill, Macaya, Alfon, Rothman, James E, Eichler, Evan E, Kullmann, Dimitri M, Houlden, Henry, Salpietro1, Vincenzo, 3, 2, Dixon4, Christine L., Guo5, Hui, 140, 6, Bello Stephanie Efthymiou 1, Oscar D., Maroofian1, Reza, Heimer7, Gali, 8, Lydie Burglen, 9, Stephanie Valence, Torti 10, Erin, Hacke11, Moritz, Rankin12, Julia, Tariq1, Huma, Colin13, Estelle, Procaccio13, Vincent, Striano2, Pasquale, Mankad15, Kshitij, 4, Andreas Lieb, Chen16, Sharon, Pisani16, Laura, Bettencourt 17, Conceicao, 1, Roope Männikkö, Manole1, Andreea, Brusco 18, Alfredo, Grosso18, Enrico, Battista Ferrero19, Giovanni, Armstrong-Moron20, Judith, Gueden21, Sophie, Bar-Yosef7, Omer, Tzadok7, Michal, Monaghan10, Kristin G., Santiago-Sim10, Teresa, Person10, Richard E., Cho10, Megan T., Willaert10, Rebecca, Yoo22, Yongjin, Chae23, Jong-Hee, Quan6, Yingting, Wu6, Huidan, Wang5, Tianyun, Bernier24, Raphael A., Xia6, Kun, Blesson25, Alyssa, Jain25, Mahim, Motazacker26, Mohammad M., Jaeger27, Bregje, Schneider 28, Amy L., Boysen28, Katja, Muir 29, Alison M., Myers30, Candace T., Gavrilova31, Ralitza H., Gunderson31, Lauren, Schultz-Rogers 31, Laura, Klee31, Eric W., Dyment32, David, Osmond32, Matthew, 34, 33, Parellada35, Mara, Llorente36, Cloe, Gonzalez-Peñas37, Javier, Carracedo38, Angel, Van Haeringen40, Arie, Ruivenkamp40, Claudia, Nava41, Caroline, Heron41, Delphine, Nardello42, Rosaria, Iacomino43, Michele, Minetti2, Carlo, Skabar44, Aldo, Fabretto44, Antonella, Study GroupMiquel Raspall-Chaure45, Synap, Chez46, Michael, Tsai47, Anne, Fassi48, Emily, Shinawi48, Marwan, Constantino49, John N., De Zorzi50, Rita, Fortuna 50, Sara, Kok51, Fernando, Keren41, Bori, Bonneau13, Dominique, Choi 22, Murim, Benzeev7, Bruria, Zara43, Federico, Mefford29, Heather C., Scheffer28, Ingrid E., Clayton-Smith53, Jill, Macaya45, Alfon, Rothman4, James E., Eichler 5, Evan E., Kullmann 4 &amp, Dimitri M., 1, Henry Houlden, Hanna1, SYNAPS Study Group Michael G., Bugiardini1, Enrico, Hostettler1, Isabel, O’Callaghan1, Benjamin, Khan1, Alaa, Cortese1, Andrea, O’Connor1, Emer, Yau1, Wai Y., Bourinaris1, Thoma, Kaiyrzhanov1, Rauan, Chelban1, Viorica, Madej1, Monika, Diana2, Maria C., Vari2, Maria S., Pedemonte2, Marina, Bruno2, Claudio, Balagura3, Ganna, Scala3, Marcello, Fiorillo3, Chiara, Nobili3, Lino, Malintan4, Nancy T., Zanetti4, Maria N., Krishnakumar4, Shyam S., Lignani4, Gabriele, Jepson4, James E. C., Broda43, Paolo, Baldassari43, Simona, Rossi43, Pia, Fruscione43, Floriana, Madia43, Francesca, Traverso43, Monica, De-Marco43, Patrizia, Pérez-Dueñas45, Belen, Munell45, Francina, Kriouile57, Yamna, El-Khorassani57, Mohamed, Karashova58, Blagovesta, Avdjieva58, Daniela, Kathom58, Hadil, Tincheva58, Radka, Van-Maldergem59, Lionel, Nachbauer60, Wolfgang, Boesch60, Sylvia, Gagliano61, Antonella, Amadori62, Elisabetta, Goraya63, Jatinder S., Sultan64, Tipu, Kirmani65, Salman, Ibrahim66, Shahnaz, Jan66, Farida, Mine67, Jun, Banu68, Selina, Veggiotti69, Pierangelo, Zuccotti69, Gian V., Ferrari70, Michel D., Van Den Maagdenberg70, Arn M. J., Verrotti71, Alberto, Marseglia72, Gian L., Savasta72, Salvatore, Soler73, Miguel A., Scuderi74, Carmela, Borgione74, Eugenia, Chimenz75, Roberto, Gitto75, Eloisa, Dipasquale75, Valeria, Sallemi75, Alessia, Fusco75, Monica, Cuppari75, Caterina, Cutrupi75, Maria C., Ruggieri76, Martino, Cama77, Armando, Capra77, Valeria, Mencacci78, Niccolò E., Boles79, Richard, Gupta80, Neerja, Kabra80, Madhulika, Papacostas81, Savva, Zamba-Papanicolaou81, Eleni, Dardiotis82, Efthymio, Maqbool83, Shazia, Rana84, Nuzhat, Atawneh85, Osama, Lim86, Shen Y., Shaikh87, Farooq, Koutsis88, George, Breza88, Marianthi, Coviello89, Domenico A., Dauvilliers90, Yves A., Alkhawaja91, Issam, Alkhawaja92, Mariam, Al-Mutairi93, Fuad, Stojkovic94, Tanya, Ferrucci, Veronica, Zollo, Massimo, Alkuraya96, Fowzan S., Kinali97, Maria, Sherifa98, Hamed, Benrhouma99, Hanene, Turki99, Ilhem B. Y., Tazir100, Meriem, Obeid101, Makram, Bakhtadze102, Sophia, Saadi103, Nebal W., Zaki104, Maha S., Triki105, Chahnez C., Benfenati106, Fabio, Gustincich106, Stefano, Kara107, Majdi, Belcastro108, Vincenzo, Specchio109, Nicola, Capovilla110, Giuseppe, Karimiani111, Ehsan G., Salih112, Ahmed M., Okubadejo113, Njideka U., Ojo113, Oluwadamilola O., Oshinaike113, Olajumoke O., Oguntunde113, Olapeju, Wahab114, Kolawole, Bello114, Abiodun H., Abubakar115, Sanni, Obiabo116, Yahaya, Nwazor117, Ernest, Ekenze118, Oluchi, Williams119, Uduak, Iyagba120, Alagoma, Taiwo121, Lolade, Komolafe122, Morenikeji, Senkevich123, Konstantin, Shashkin124, Chingiz, Zharkynbekova125, Nazira, Koneyev126, Kairgali, Manizha127, Ganieva, Isrofilov127, Maksud, Guliyeva128, Ulviyya, Salayev129, Kamran, Khachatryan130, Samson, Rossi131, Salvatore, Silvestri131, Gabriella, Haridy132, Nourelhoda, Ramenghi133, Luca A., Xiromerisiou134, Georgia, David135, Emanuele, Aguennouz136, Mhammed, Fidani137, Liana, Spanaki138 &amp, Cleanthe, and Tucci139, Arianna

Subjects
Male, [SDV.GEN] Life Sciences [q-bio]/Genetics, Ion channels in the nervous system, Cohort Studies, fluids and secretions, Loss of Function Mutation, Receptors, AMPA, AMPA receptor, lcsh:Science, Child, reproductive and urinary physiology, AMPA receptor, GluA2, neurodevelopmental disorders, autism spectrum disorder, glutamatergic synaptic transmission, GRIA2, neurodevelopmental disorders, Developmental disorders, Neurodevelopmental disorders, Brain, Magnetic Resonance Imaging, Settore MED/26 - NEUROLOGIA, GluA2, Child, Preschool, Female, Adult, Heterozygote, Adolescent, Science, autism spectrum disorder, Article, Young Adult, [SDV.MHEP.PED] Life Sciences [q-bio]/Human health and pathology/Pediatrics, MESH: Intellectual Disability/genetics, Neurodevelopmental Disorders/genetics, Receptors AMPA/genetics, Intellectual Disability, mental disorders, Humans, Infant, Neurodevelopmental Disorders, Receptors, AMPA, GRIA2, Preschool, Ion channel in the nervous system, Developmental disorders, Synaptic development, NG sequencing, [SDV.GEN]Life Sciences [q-bio]/Genetics, [SDV.MHEP.PED]Life Sciences [q-bio]/Human health and pathology/Pediatrics, glutamatergic synaptic transmission, [SCCO.NEUR]Cognitive science/Neuroscience, [SCCO.NEUR] Cognitive science/Neuroscience, NG sequencing, Synaptic development, Ion channel in the nervous system, Next-generation sequencing, lcsh:Q
Abstract

AMPA receptors (AMPARs) are tetrameric ligand-gated channels made up of combinations of GluA1-4 subunits encoded by GRIA1-4 genes. GluA2 has an especially important role because, following post-transcriptional editing at the Q607 site, it renders heteromultimeric AMPARs Ca2+-impermeable, with a linear relationship between current and trans-membrane voltage. Here, we report heterozygous de novo GRIA2 mutations in 28 unrelated patients with intellectual disability (ID) and neurodevelopmental abnormalities including autism spectrum disorder (ASD), Rett syndrome-like features, and seizures or developmental epileptic encephalopathy (DEE). In functional expression studies, mutations lead to a decrease in agonist-evoked current mediated by mutant subunits compared to wild-type channels. When GluA2 subunits are co-expressed with GluA1, most GRIA2 mutations cause a decreased current amplitude and some also affect voltage rectification. Our results show that de-novo variants in GRIA2 can cause neurodevelopmental disorders, complementing evidence that other genetic causes of ID, ASD and DEE also disrupt glutamatergic synaptic transmission., Genetic variants in ionotropic glutamate receptors have been implicated in neurodevelopmental disorders. Here, the authors report heterozygous de novo mutations in the GRIA2 gene in 28 individuals with intellectual disability and neurodevelopmental abnormalities associated with reduced Ca2+ transport and AMPAR currents.”

Published
2019
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33. Enantiospecific recognition of 2-butanol by an inherently chiral cavitand in the solid state

Author

Enrico Dalcanale, R. De Zorzi, Jean-Pierre Dutasta, Tahnie Barboza, Roberta Pinalli, Carlo Nicosia, Silvano Geremia, Nicola Demitri, Laure Guy, Giovanna Brancatelli, Brancatelli, G., Nicosia, C., Barboza, T., Guy, L., Dutasta, J. -. P., De Zorzi, R., Demitri, N., Dalcanale, E., Geremia, S., Pinalli, R., Laboratoire de Chimie - UMR5182 (LC), Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-École normale supérieure - Lyon (ENS Lyon)-Institut de Chimie du CNRS (INC), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Stereochemistry, PROTEINS, RESORCINARENES, 010402 general chemistry, CALIXARENES, 01 natural sciences, Desymmetrization, General Materials Science, SELECTIVE DETECTION, PHOSPHONATE, ALCOHOLS, AIR, CONFIGURATION, SUBSTITUTION, RECEPTORS, ComputingMilieux_MISCELLANEOUS, 010405 organic chemistry, Chemistry, Enantioselective synthesis, Cavitand, General Chemistry, Condensed Matter Physics, 0104 chemical sciences, Chiral column chromatography, [CHIM.THEO]Chemical Sciences/Theoretical and/or physical chemistry, Enantiopure drug, Racemic mixture, Enantiomer, Chirality (chemistry)
Abstract

A new inherently chiral cavitand is described, in which the desymmetrization of the rigid concave cavity is achieved by introducing three different bridging groups at the upper rim, namely two inward P=O groups, one inward P=S moiety and one methylene bridge in the AABC mode. The racemic mixture of cR and cS enantiomers is resolved by semi-preparative chiral HPLC and the enantiopurity confirmed through circular dichroism. The properties of the enantiopure cR-cav in the enantioselective recognition of racemic 2-butanol is studied by co-crystallization experiments. The exclusive formation of the complex R-BuOH@cR-cav demonstrates that cR-cav is able to discriminate between the two enantiomers of the alcohol in the solid state. The enantiospecific recognition exhibited by the cR-cav towards racemic 2-butanol is particularly relevant because of the low degree of chirality of the alcohol (1.9 calculated by CCM algorithm).

Published
2017
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34. Synthesis, Photophysical, Electrochemical, and Electrochemiluminescent Properties of 5,15-bis(9-anthracenyl)porphyrin Derivatives

Author

Alessandra Magistrato, Carlo Bruno, Andrea Listorti, Davide Bonifazi, Chloe Sooambar, Francesco Paolucci, Abdelhalim Belbakra, Silvano Geremia, Massimo Marcaccio, Vincent Troiani, Rita De Zorzi, Nicola Armaroli, Sooambar C., Troiani V., Bruno C., Marcaccio M., Paolucci F., Listorti A., Belbakra A., Armaroli N., Magistrato A., De Zorzi R., Geremia S., Bonifazi D., Sooambar, CHLOE MARIE-NELLY, Troiani, V., Bruno, C., Marcaccio, M., Paolucci, F., Listorti, A., Belbakra, A., Armaroli, N., Magistrato, A., DE ZORZI, Rita, Geremia, Silvano, and Bonifazi, Davide

Subjects
Models, Molecular, Luminescence, Porphyrins, Absorption spectroscopy, CONJUGATED PORPHYRIN OLIGOMERS, Intervalence charge transfer, Crystallography, X-Ray, Photochemistry, Biochemistry, CARBON NANOTUBES, HIGHLY EFFICIENT, chemistry.chemical_compound, Electron transfer, Electrochemistry, Moiety, EXCITATION-ENERGY TRANSFER, Computer Simulation, DONOR-ACCEPTOR DIAD, ELECTROGENERATED CHEMILUMINESCENCE, ELECTRONIC-STRUCTURE, SPECTRAL CHARACTERIZATION, SUPRAMOLECULAR CHEMISTRY, MOLECULAR-DYNAMICS, ORGANIC CHEMISTRY, Singlet state, Physical and Theoretical Chemistry, Settore CHIM/03 - Chimica Generale e Inorganica, Anthracenes, Molecular Structure, Organic Chemistry, Chromophore, Photochemical Processes, Porphyrin, chemistry, Spectrophotometry, Quantum Theory, Phosphorescence
Abstract

Novel 5,15-bis(9-anthracenyl)porphyrin derivatives (1a, 1b) were synthesized by stepwise Suzuki-type coupling reactions using anthracenyl-boronates bearing various electronically active moieties. Absorption spectra of these porphyrin conjugates reveal some degree of delocalisation with the directly linked chromophores, particularly in the case of anthracenyl-porphyrin bearing dimethylanilino moieties at the two extremities. Fluorescence and 77 K phosphorescence properties indicate that the excitation energy is invariably funnelled to the lowest singlet and triplet states of the porphyrin chromophore. The latter levels have been probed also by transient absorption spectroscopy, showing the typical triplet features detected in meso-substituted porphyrins. Extensive electrochemical studies have been performed to unravel the electronic properties of the newly synthesized porphyrins. Low-temperature cyclic voltammetry investigations showed that the anthracenyl-porphyrins are capable of undergoing as many as four electron transfer processes. In particular, by means of UV-Vis-NIR spectroelectrochemical measurements, a NIR-centred intramolecular photoinduced intervalence charge transfer (IV-CT) from a neutral N,N-dimethylanilino moiety to the N,N-dimethylanilino radical cation has been observed for the doubly-oxidised porphyrin 1b2+. The molecules also showed unexpected electrogenerated chemiluminescence properties, which revealed to be largely controlled by the electronic characteristics of the peripheral anthracenyl substituents. The structural and the electronic properties of these complexes have been also characterised by DFT calculations, as well as by X-ray crystallographic analyses. © The Royal Society of Chemistry 2009.

Published
2009

35. Noncovalent synthesis in aqueous solution and spectroscopic characterization of multi-porphyrin complexes

Author

Silvano Geremia, Rita De Zorzi, Roberto Purrello, Rosaria Lauceri, Fabio Giuseppe Gulino, Limor Frish, Yoram Cohen, Lucio Randaccio, Tamar Evan-Salem, Luigi Di Costanzo, Domenico Sciotto, Gulino, F. G., Lauceri, R., Frish, L., EVAN SALEM, T., Cohen, Y., DE ZORZI, Rita, Geremia, Silvano, DI COSTANZO, L., Randaccio, Lucio, Sciotto, D., Purrello, R., Evan-Salem, T., De Zorzi, R., Geremia, S., DI COSTANZO, Luigi, Randaccio, L., and Purrello, Francesco

Subjects
Magnetic Resonance Spectroscopy, Porphyrins, Stereochemistry, chemistry.chemical_element, Noncovalent synthesi, Zinc, Catalysis, WATER CHEMISTRY, Porphyrin, chemistry.chemical_compound, CALIXARENES, SUPRAMOLECULAR CHEMISTRY, Calixarene, Deuterium Oxide, Porphyrin Complex, Noncovalent interaction, Aqueous solution, biology, Molecular Structure, Chemistry, Organic Chemistry, Cationic polymerization, Water, Self-assembly, General Chemistry, biology.organism_classification, Template synthesi, Crystallography, Spectrometry, Fluorescence, Solubility, Tetra, Spectrophotometry, Ultraviolet, Calixarenes, Porphyrinoid, Stoichiometry
Abstract

The interactions of the tetra- cationic meso-tetrakis(N-methyl-4-pyri- dyl)porphyrin (H2TMPyP) and its met- allo derivatives (MTMPyP) (where M = copper(ii), zinc(ii), and gold(iii) with the octa-anionic form (at neutral pH) of 5,11,17,23-tetrasulfonato- 25,26,27,28-tetrakis(hydroxycarbonyl- methoxy)calix(4)arene (C4TsTc) lead to a series of complex species whose stoichiometry and porphyrin sequence can be easily tuned. Crystallographic, spectroscopic, and diffusion NMR stud- ies converge towards a common picture in which a central 1:4 porphyrin/calix- arene unit serves as a template for the formation of more complex species. These species arise by successive, step- wise addition of single porphyrin mole- cules above and below the plane of the 1:4 central core to ultimately give a 7:4 complex. Noticeably, the stoichiometry of the various complex species corre- sponds to the actual concentration ratio of porphyrins and calixarenes in solution allowing the stoichiometry of these species to be easily tuned. This behavior and the remarkable stability of these species allow homo-porphyrin and hetero-(metallo)porphyrin species to be formed with control of not only the stoichiometry but also the sequence of the porphyrin array. The flexibility and ease of this approach permit, in principle, the design and synthesis of porphyrin arrays for predetermined purposes. For example, we have shown that it is very easy to design and obtain mixed porphyrin species in which a foreseen photoinduced electron-trans- fer is indeed observed.

Published
2006

36. A water playground for peptide re-assembly from fibrils to plates.

Author

Adorinni S, Kurbasic M, Garcia AM, Kralj S, Bellotto O, Scarel E, Pengo P, De Zorzi R, Melchionna M, Vargiu AV, and Marchesan S

Abstract

Short-peptide amyloid assembly and disassembly play crucial roles in various research fields, which range from addressing pathologies that lack therapeutic solutions to the development of innovative soft (bio)materials. Hydrogels from short peptides typically show thermo-reversible gel-to-sol transition, whereby fibrils disassemble upon heating, and re-assemble upon cooling down to room temperature (rt). Despite ongoing intense research studies in this area, the majority focus on peptide-peptide interaction and neglect the structuring role of water in peptide supramolecular behavior. This study describes an unprotected tetrapeptide gelator that forms highly stable fibrils which, upon heating, re-organize into plates that persist upon cooling to rt. All-atom molecular dynamics (MD) simulations and experimental methods reveal water as a key player in the thermodynamics that accompany this irreversible morphological transition, and advance our understanding of supramolecular structures.

Published
2024
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37. Interactions of biofilm polysaccharides produced by human infective bacteria with molecules of the quorum sensing system. A microscopy and NMR study.

Author

Bellich B, Cacioppo M, De Zorzi R, Rizzo R, Brady JW, and Cescutti P

Subjects
Humans, Rhamnose metabolism, Rhamnose chemistry, Bacteria metabolism, Quorum Sensing, Biofilms growth & development, Polysaccharides, Bacterial chemistry, Polysaccharides, Bacterial metabolism, Magnetic Resonance Spectroscopy methods
Abstract

Biofilms are the most common lifestyle adopted by bacterial communities where cells live embedded in a self-produced hydrated matrix. Although polysaccharides are considered essential for matrix architecture, their possible functional roles are still rather unexplored. The primary structure of polysaccharides produced by Klebsiella pneumoniae and species of the Burkholderia cepacia Complex revealed a composition rich in rhamnose. The methyl group on carbon 6 of rhamnose units lowers the polymer hydrophilicity and can form low polarity regions on the polysaccharide chains. These regions promote chain-chain interactions that contribute to the biofilm matrix stability, but may also act as binding sites for low-polarity molecules, aiding their mobility through the hydrated matrix. In particular, quorum sensing system components crucial for the biofilm life cycle often display poor solubility in water. Therefore, cis-11-methyl-2-dodecenoic acid and L-homoserine-lactones were investigated by NMR spectroscopy for their possible interaction with polysaccharides. In addition, the macromolecular morphology of the polysaccharides was assessed using atomic force and electron microscopies to define the role of Rha residues on the three-dimensional conformation of the polymer. NMR data revealed that quorum sensing components interact with Rhamnose-rich polysaccharides, and the extent of interaction depends on the specific primary structure of each polysaccharide., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Published
2024
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38. On the Cholesterol Raising Effect of Coffee Diterpenes Cafestol and 16- O -Methylcafestol: Interaction with Farnesoid X Receptor.

Author

Guercia E, Berti F, De Zorzi R, Navarini L, Geremia S, Medagli B, De Conto M, Cassetta A, and Forzato C

Subjects
Humans, Molecular Docking Simulation, Protein Binding, Molecular Dynamics Simulation, Circular Dichroism, Diterpenes pharmacology, Diterpenes chemistry, Receptors, Cytoplasmic and Nuclear metabolism, Receptors, Cytoplasmic and Nuclear agonists, Cholesterol metabolism, Coffee chemistry
Abstract

The diterpene cafestol represents the most potent cholesterol-elevating compound known in the human diet, being responsible for more than 80% of the effect of coffee on serum lipids, with a mechanism still not fully clarified. In the present study, the interaction of cafestol and 16- O -methylcafestol with the stabilized ligand-binding domain (LBD) of the Farnesoid X Receptor was evaluated by fluorescence and circular dichroism. Fluorescence quenching was observed with both cafestol and 16- O -methylcafestol due to an interaction occurring in the close environment of the tryptophan W454 residue of the protein, as confirmed by docking and molecular dynamics. A conformational change of the protein was also observed by circular dichroism, particularly for cafestol. These results provide evidence at the molecular level of the interactions of FXR with the coffee diterpenes, confirming that cafestol can act as an agonist of FXR, causing an enhancement of the cholesterol level in blood serum.

Published
2024
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39. Peptide Stereochemistry Effects from p K a -Shift to Gold Nanoparticle Templating in a Supramolecular Hydrogel.

Author

Adorinni S, Gentile S, Bellotto O, Kralj S, Parisi E, Cringoli MC, Deganutti C, Malloci G, Piccirilli F, Pengo P, Vaccari L, Geremia S, Vargiu AV, De Zorzi R, and Marchesan S

Subjects
Gold chemistry, Peptides chemistry, Hydrogels chemistry, Metal Nanoparticles chemistry
Abstract

The divergent supramolecular behavior of a series of tripeptide stereoisomers was elucidated through spectroscopic, microscopic, crystallographic, and computational techniques. Only two epimers were able to effectively self-organize into amphipathic structures, leading to supramolecular hydrogels or crystals, respectively. Despite the similarity between the two peptides' turn conformations, stereoconfiguration led to different abilities to engage in intramolecular hydrogen bonding. Self-assembly further shifted the p K a value of the C-terminal side chain. As a result, across the pH range 4-6, only one epimer predominated sufficiently as a zwitterion to reach the critical molar fraction, allowing gelation. By contrast, the differing p K a values and higher dipole moment of the other epimer favored crystallization. The four stereoisomers were further tested for gold nanoparticle (AuNP) formation, with the supramolecular hydrogel being the key to control and stabilize AuNPs, yielding a nanocomposite that catalyzed the photodegradation of a dye. Importantly, the AuNP formation occurred without the use of reductants other than the peptide, and the redox chemistry was investigated by LC-MS, NMR, and infrared scattering-type near field optical microscopy (IR s-SNOM). This study provides important insights for the rational design of simple peptides as minimalistic and green building blocks for functional nanocomposites.

Published
2024
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40. Identification of additional mechanistically important residues in the multidrug transporter styMdtM of Salmonella Typhi.

Author

Shaheen A, Tariq A, Ismat F, Naveed H, De Zorzi R, Iqbal M, Storici P, Mirza O, Walz T, and Rahman M

Subjects
Membrane Transport Proteins metabolism, Membrane Transport Proteins chemistry, Membrane Transport Proteins genetics, Mutation, Crystallography, X-Ray, Protein Conformation, Drug Resistance, Multiple, Bacterial genetics, Salmonella typhi metabolism, Salmonella typhi genetics, Bacterial Proteins chemistry, Bacterial Proteins metabolism, Bacterial Proteins genetics, Models, Molecular
Abstract

Multidrug efflux is a well-established mechanism of drug resistance in bacterial pathogens like Salmonella Typhi. styMdtM (locus name; STY4874) is a multidrug efflux transporter of the major facilitator superfamily expressed in S . Typhi. Functional assays identified several residues important for its transport activity. Here, we used an AlphaFold model to identify additional residues for analysis by mutagenesis. Mutation of peripheral residue Cys185 had no effect on the structure or function of the transporter. However, substitution of channel-lining residues Tyr29 and Tyr231 completely abolished transport function. Finally, mutation of Gln294, which faces peripheral helices of the transporter, resulted in the loss of transport of some substrates. Crystallization studies yielded diffraction data for the wild-type protein at 4.5 Å resolution and allowed the unit cell parameters to be established as a  =  b  = 64.3 Å, c  = 245.4 Å, α = β = γ = 90°, in space group P4. Our studies represent a further stepping stone towards a mechanistic understanding of the clinically important multidrug transporter styMdtM.Communicated by Ramaswamy H. Sarma.

Published
2024
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41. Microscopy and modelling investigations on the morphology of the biofilm exopolysaccharide produced by Burkholderia multivorans strain C1576.

Author

Cacioppo M, De Zorzi R, Syrgiannis Z, Bellich B, Bertoncin P, Jou IA, Brady JW, Rizzo R, and Cescutti P

Subjects
Humans, Biofilms, Microscopy, Atomic Force, Polysaccharides, Bacterial chemistry, Burkholderia metabolism
Abstract

Bacteria form very often biofilms where they embed in a self-synthesized matrix exhibiting a gel-like appearance. Matrices offer several advantages, including defence against external threats and the easiness of intercellular communication. In infections, biofilm formation enhances bacteria resistance against antimicrobials, causing serious clinical problems for patients' treatments. Biofilm matrices are composed of proteins, extracellular DNA, and polysaccharides, the latter being the major responsible for matrix architecture. The repeating unit of the biofilm polysaccharide synthesized by Burkholderia multivorans strain C1576 contains two mannoses and two sequentially linked rhamnoses, one of them 50 % methylated on C-3. Rhamnose, a 6-deoxysugar, has lower polarity than other common monosaccharides and its methylation further reduces polarity. This suggests a possible role of this polysaccharide in the biofilm matrix; in fact, computer modelling and atomic force microscopy studies evidenced intra- and inter-molecular non-polar interactions both within polysaccharides and with aliphatic molecules. In this paper, the polysaccharide three-dimensional morphology was investigated using atomic force microscopy in both solid and solution states. Independent evidence of the polymer conformation was obtained by transmission electron microscopy which confirmed the formation of globular compact structures. Finally, data from computer dynamic simulations were used to model the three-dimensional structure., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Published
2023
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42. Self-assembling tripeptide forming water-bound channels and hydrogels.

Author

Parisi E, Adorinni S, Garcia AM, Kralj S, De Zorzi R, and Marchesan S

Subjects
Microscopy, Electron, Transmission, Crystallography, X-Ray, Circular Dichroism, Water, Hydrogels chemistry, Peptides chemistry
Abstract

D-Ser(tBu)-L-Phe-L-Trp is described as a self-assembling tripeptide that yields nanofibrillar hydrogels at physiological conditions (phosphate buffer at pH 7.4). The peptide is characterized by several spectroscopic methods, such as circular dichroism and fluorescence, oscillatory rheometry, and transmission electron microscopy. Single-crystal X-ray diffraction reveals supramolecular packing into water-bound channels and allows the visualization of the intermolecular interactions holding together peptide stacks., (© 2023 The Authors. Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.)

Published
2023
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43. Self-assembly of benzophenone-diphenylalanine conjugate into a nanostructured photocatalyst.

Author

Adorinni S, Goti G, Rizzo L, Grassi F, Kralj S, Matroodi F, Natali M, De Zorzi R, Marchesan S, and Dell'Amico L

Subjects
Phenylalanine, Benzophenones, Dipeptides, Nanostructures
Abstract

The conjugation of photoactive benzophenone with diphenylalanine yielded a self-assembling photocatalyst that was probed in the E → Z photoisomerisation of stilbene derivatives.

Published
2023
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44. Molecular Dynamics and Structural Studies of Zinc Chloroquine Complexes.

Author

Paulikat M, Vitone D, Schackert FK, Schuth N, Barbanente A, Piccini G, Ippoliti E, Rossetti G, Clark AH, Nachtegaal M, Haumann M, Dau H, Carloni P, Geremia S, De Zorzi R, Quintanar L, and Arnesano F

Subjects
Humans, Chloroquine pharmacology, Chloroquine chemistry, Molecular Dynamics Simulation, Zinc chemistry, Chlorides, COVID-19 Drug Treatment, SARS-CoV-2, Metals, COVID-19, Coordination Complexes
Abstract

Chloroquine (CQ) is a first-choice drug against malaria and autoimmune diseases. It has been co-administered with zinc against SARS-CoV-2 and soon dismissed because of safety issues. The structural features of Zn-CQ complexes and the effect of CQ on zinc distribution in cells are poorly known. In this study, state-of-the-art computations combined with experiments were leveraged to solve the structural determinants of zinc-CQ interactions in solution and the solid state. NMR, ESI-MS, and X-ray absorption and diffraction methods were combined with ab initio molecular dynamics calculations to address the kinetic lability of this complex. Within the physiological pH range, CQ binds Zn 2+ through the quinoline ring nitrogen, forming [Zn(CQH)Cl x (H 2 O) 3- x ] (3+)- x ( x = 0, 1, 2, and 3) tetrahedral complexes. The Zn(CQH)Cl 3 species is stable at neutral pH and at high chloride concentrations typical of the extracellular medium, but metal coordination is lost at a moderately low pH as in the lysosomal lumen. The pentacoordinate complex [Zn(CQH)(H 2 O) 4 ] 3+ may exist in the absence of chloride. This in vitro / in silico approach can be extended to other metal-targeting drugs and bioinorganic systems.

Published
2023
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45. Antibody Affinity Maturation Using Computational Methods: From an Initial Hit to Small-Scale Expression of Optimized Binders.

Author

Medagli B, Soler MA, De Zorzi R, and Fortuna S

Subjects
Antibody Affinity, Epitopes, Recombinant Proteins genetics, Single-Domain Antibodies chemistry
Abstract

Nanobodies (VHHs) are engineered fragments of the camelid single-chain immunoglobulins. The VHH domain contains the highly variable segments responsible for antigen recognition. VHHs can be easily produced as recombinant proteins. Their small size is a good advantage for in silico approaches. Computer methods represent a valuable strategy for the optimization and improvement of their binding affinity. They also allow for epitope selection offering the possibility to design new VHHs for regions of a target protein that are not naturally immunogenic. Here we present an in silico mutagenic protocol developed to improve the binding affinity of nanobodies together with the first step of their in vitro production. The method, already proven successful in improving the low Kd of a nanobody hit obtained by panning, can be employed for the ex novo design of antibody fragments against selected protein target epitopes., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

Published
2023
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46. Single-atom substitution enables supramolecular diversity from dipeptide building blocks.

Author

Scarel E, Bellotto O, Rozhin P, Kralj S, Tortora M, Vargiu AV, De Zorzi R, Rossi B, and Marchesan S

Subjects
Circular Dichroism, Microscopy, Electron, Transmission, Phenylalanine chemistry, Dipeptides chemistry, Hydrogels chemistry
Abstract

Dipeptides are popular building blocks for supramolecular gels that do not persist in the environment and may find various applications. In this work, we show that a simple substitution on the aromatic side-chain of phenylalanine with either fluorine or iodine enables supramolecular diversity upon self-assembly at neutral pH, leading to hydrogels or crystals. Each building block is characterized by 1 H- and 13 C-NMR spectroscopy, LC-MS, circular dichroism, and molecular models. The supramolecular behaviour is monitored with a variety of techniques, including circular dichroism, oscillatory rheology, transmission electron microscopy, attenuated total reflectance Fourier-transformed infrared spectroscopy, visible Raman spectroscopy, synchrotron-radiation single-crystal X-ray diffraction and UV Resonance Raman spectroscopy, allowing key differences to be pinpointed amongst the halogenated analogues.

Published
2022
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47. Self-Assembly of Unprotected Dipeptides into Hydrogels: Water-Channels Make the Difference.

Author

Bellotto O, Kralj S, Melchionna M, Pengo P, Kisovec M, Podobnik M, De Zorzi R, and Marchesan S

Subjects
Stereoisomerism, Dipeptides chemistry, Hydrogels chemistry, Water chemistry
Abstract

Unprotected dipeptides are attractive building blocks for environmentally friendly hydrogel biomaterials by virtue of their low-cost and ease of preparation. This work investigates the self-assembling behaviour of the distinct stereoisomers of Ile-Phe and Phe-Ile in phosphate buffered saline (PBS) to form hydrogels, using transmission electron microscopy (TEM), attenuated total reflectance infrared spectroscopy (ATR-IR), circular dichroism (CD), and oscillatory rheometry. Each peptide purity and identity was also confirmed by 1 H- and 13 C-NMR spectroscopy and HPLC-MS. Finally, single-crystal XRD data allowed the key interactions responsible for the supramolecular packing into amphipathic layers or water-channels to be revealed. The presence of the latter in the crystal structure is a distinctive feature of the only gelator of this work that self-organizes into stable hydrogels, with fast kinetics and the highest elastic modulus amongst its structural isomers and stereoisomers., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Published
2022
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48. Integrative Study of the Structural and Dynamical Properties of a KirBac3.1 Mutant: Functional Implication of a Highly Conserved Tryptophan in the Transmembrane Domain.

Author

Fagnen C, Bannwarth L, Oubella I, Zuniga D, Haouz A, Forest E, Scala R, Bendahhou S, De Zorzi R, Perahia D, and Vénien-Bryan C

Subjects
Amino Acid Sequence, Crystallography, X-Ray, Hydrogen Deuterium Exchange-Mass Spectrometry, Ion Channel Gating, Mutant Proteins chemistry, Mutant Proteins metabolism, Protein Domains, Protein Interaction Maps, Protein Structure, Secondary, Conserved Sequence, Mutation genetics, Potassium Channels, Inwardly Rectifying chemistry, Potassium Channels, Inwardly Rectifying genetics, Tryptophan chemistry
Abstract

ATP-sensitive potassium (K-ATP) channels are ubiquitously expressed on the plasma membrane of cells in several organs, including the heart, pancreas, and brain, and they govern a wide range of physiological processes. In pancreatic β-cells, K-ATP channels composed of Kir6.2 and SUR1 play a key role in coupling blood glucose and insulin secretion. A tryptophan residue located at the cytosolic end of the transmembrane helix is highly conserved in eukaryote and prokaryote Kir channels. Any mutation on this amino acid causes a gain of function and neonatal diabetes mellitus. In this study, we have investigated the effect of mutation on this highly conserved residue on a KirBac channel (prokaryotic homolog of mammalian Kir6.2). We provide the crystal structure of the mutant KirBac3.1 W46R (equivalent to W68R in Kir6.2) and its conformational flexibility properties using HDX-MS. In addition, the detailed dynamical view of the mutant during the gating was investigated using the in silico method. Finally, functional assays have been performed. A comparison of important structural determinants for the gating mechanism between the wild type KirBac and the mutant W46R suggests interesting structural and dynamical clues and a mechanism of action of the mutation that leads to the gain of function.

Published
2021
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49. Characterization of the multidrug efflux transporter styMdtM from Salmonella enterica serovar Typhi.

Author

Shaheen A, Ismat F, Iqbal M, Haque A, Ul-Haq Z, Mirza O, De Zorzi R, Walz T, and Rahman M

Subjects
Amino Acid Substitution, Anti-Bacterial Agents pharmacology, Arginine chemistry, Arginine metabolism, Aspartic Acid chemistry, Aspartic Acid metabolism, Bacterial Proteins genetics, Bacterial Proteins metabolism, Binding Sites, Biological Transport, Chloramphenicol pharmacology, Gene Expression, Humans, Kinetics, Microbial Sensitivity Tests, Models, Molecular, Monosaccharide Transport Proteins genetics, Monosaccharide Transport Proteins metabolism, Protein Binding, Protein Conformation, alpha-Helical, Protein Conformation, beta-Strand, Protein Interaction Domains and Motifs, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Salmonella typhi drug effects, Salmonella typhi genetics, Salmonella typhi isolation & purification, Salmonella typhi metabolism, Substrate Specificity, Thermodynamics, Typhoid Fever microbiology, Anti-Bacterial Agents chemistry, Bacterial Proteins chemistry, Chloramphenicol chemistry, Drug Resistance, Bacterial genetics, Monosaccharide Transport Proteins chemistry, Mutation
Abstract

Salmonellae are foodborne pathogens and the major cause of gastroenteritis in humans. Salmonellae express multidrug efflux transporters that play a key role in their drug resistance, which is becoming an increasing problem for therapeutic intervention. Despite their biomedical importance, the mechanisms underlying substrate transport by multidrug efflux transporters remain poorly understood. Here, we describe the first characterization of a multidrug transporter belonging to the major facilitator superfamily from the genus Salmonella. We show that several clinical Salmonella Typhi (S. Typhi) isolates constitutively express the styMdtM (STY4874) gene, which encodes a known multidrug-resistance (MDR) transporter. Guided by the structure of the Escherichia coli (E. coli) homolog, we studied two residues critical for substrate transport, Asp25 and Arg111. Mutation of Asp25 to glutamate did not affect the transport function of styMdtM, whereas mutation to alanine reduced its transport activity, suggesting that a negative charge at this position is critical for substrate translocation across the membrane. Substrate-affinity measurements by intrinsic fluorescence spectroscopy showed that the Asp25Ala mutant retained its capacity to bind substrate, albeit at a lower level. Mutation of Arg111 to alanine resulted in a decrease in secondary structure content of the transporter, and mutation to lysine completely destabilized the structure of the transporter. A homology model of styMdtM suggests that Arg111 is important for stabilizing the transmembrane domain by mediating necessary interactions between neighboring helices. Together, our studies provide new structural and mechanistic insights into the Salmonella MDR transporter styMdtM., (© 2021 Wiley Periodicals LLC.)

Published
2021
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50. Unexpected Gating Behaviour of an Engineered Potassium Channel Kir.

Author

Fagnen C, Bannwarth L, Zuniga D, Oubella I, De Zorzi R, Forest E, Scala R, Guilbault S, Bendahhou S, Perahia D, and Vénien-Bryan C

Abstract

In this study, we investigated the dynamics and functional characteristics of the KirBac3.1 S129R, a mutated bacterial potassium channel for which the inner pore-lining helix (TM2) was engineered so that the bundle crossing is trapped in an open conformation. The structure of this channel has been previously determined at high atomic resolution. We explored the dynamical characteristics of this open state channel using an in silico method MDeNM that combines molecular dynamics simulations and normal modes. We captured the global and local motions at the mutation level and compared these data with HDX-MS experiments. MDeNM provided also an estimation of the probability of the different opening states that are in agreement with our electrophysiological experiments. In the S129R mutant, the Arg129 mutation releases the two constriction points in the channel that existed in the wild type but interestingly creates another restriction point., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Fagnen, Bannwarth, Zuniga, Oubella, De Zorzi, Forest, Scala, Guilbault, Bendahhou, Perahia and Vénien-Bryan.)

Published
2021
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