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2. Conformational buffering underlies functional selection in intrinsically disordered protein regions

Author

González-Foutel, Nicolás S., Glavina, Juliana, Borcherds, Wade M., Safranchik, Matías, Barrera-Vilarmau, Susana, Sagar, Amin, Estaña, Alejandro, Barozet, Amelie, Garrone, Nicolás A., Fernandez-Ballester, Gregorio, Blanes-Mira, Clara, Sánchez, Ignacio E., de Prat-Gay, Gonzalo, Cortés, Juan, Bernadó, Pau, Pappu, Rohit V., Holehouse, Alex S., Daughdrill, Gary W., and Chemes, Lucía B.

Published
2022
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9. Molten globule driven and self-downmodulated phase separation of a viral factory scaffold

Author

Salgueiro, Mariano, primary, Camporeale, Gabriela, additional, Visentin, Araceli, additional, Aran, Martin, additional, Pellizza, Leonardo, additional, Esperante, Sebastián, additional, Corbat, Agustín, additional, Grecco, Hernán, additional, Sousa, Belén, additional, Esperón, Ramiro, additional, Borkosky, Silvia S., additional, and de Prat-Gay, Gonzalo, additional

Published
2023
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10. Assembly of the Tripartite and RNA Condensates of the Respiratory Syncytial Virus Factory Proteins In Vitro : Role of the Transcription Antiterminator M 2-1.

Author

Visentin, Araceli, Demitroff, Nicolás, Salgueiro, Mariano, Borkosky, Silvia Susana, Uversky, Vladimir N., Camporeale, Gabriela, and de Prat-Gay, Gonzalo

Subjects
RESPIRATORY syncytial virus, VIRAL proteins, RNA, IONIC strength, PHASE separation
Abstract

A wide variety of viruses replicate in liquid-like viral factories. Non-segmented negative stranded RNA viruses share a nucleoprotein (N) and a phosphoprotein (P) that together emerge as the main drivers of liquid–liquid phase separation. The respiratory syncytial virus includes the transcription antiterminator M2-1, which binds RNA and maximizes RNA transcriptase processivity. We recapitulate the assembly mechanism of condensates of the three proteins and the role played by RNA. M2-1 displays a strong propensity for condensation by itself and with RNA through the formation of electrostatically driven protein–RNA coacervates based on the amphiphilic behavior of M2-1 and finely tuned by stoichiometry. M2-1 incorporates into tripartite condensates with N and P, modulating their size through an interplay with P, where M2-1 is both client and modulator. RNA is incorporated into the tripartite condensates adopting a heterogeneous distribution, reminiscent of the M2-1-RNA IBAG granules within the viral factories. Ionic strength dependence indicates that M2-1 behaves differently in the protein phase as opposed to the protein–RNA phase, in line with the subcompartmentalization observed in viral factories. This work dissects the biochemical grounds for the formation and fate of the RSV condensates in vitro and provides clues to interrogate the mechanism under the highly complex infection context. [ABSTRACT FROM AUTHOR]

Published
2023
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11. Conformational buffering underlies functional selection in intrinsically disordered protein regions

Author

0000-0003-4868-6593, 0000-0002-9625-6275, 0000-0002-4660-0306, 0000-0003-2568-1378, 0000-0002-4155-5729, 0000-0001-5655-6559, 0000-0003-0192-9906, González-Foutel, Nicolás S., Glavina, Juliana, Borcherds, Wade M., Safranchik, Matías, Barrera-Vilarmau, Susana, Sagar, Amin, Estaña, Alejandro, Barozet, Amelie, Garrone, Nicolás A., Fernandez-Ballester, Gregorio, Blanes-Mira, Clara, Sánchez, Ignacio E., de Prat-Gay, Gonzalo, Cortés, Juan, Bernadó, Pau, Pappu, Rohit V., Holehouse, Alex S., Daughdrill, Gary W., Chemes, Lucía B., 0000-0003-4868-6593, 0000-0002-9625-6275, 0000-0002-4660-0306, 0000-0003-2568-1378, 0000-0002-4155-5729, 0000-0001-5655-6559, 0000-0003-0192-9906, González-Foutel, Nicolás S., Glavina, Juliana, Borcherds, Wade M., Safranchik, Matías, Barrera-Vilarmau, Susana, Sagar, Amin, Estaña, Alejandro, Barozet, Amelie, Garrone, Nicolás A., Fernandez-Ballester, Gregorio, Blanes-Mira, Clara, Sánchez, Ignacio E., de Prat-Gay, Gonzalo, Cortés, Juan, Bernadó, Pau, Pappu, Rohit V., Holehouse, Alex S., Daughdrill, Gary W., and Chemes, Lucía B.

Abstract

Many disordered proteins conserve essential functions in the face of extensive sequence variation, making it challenging to identify the mechanisms responsible for functional selection. Here we identify the molecular mechanism of functional selection for the disordered adenovirus early gene 1A (E1A) protein. E1A competes with host factors to bind the retinoblastoma (Rb) protein, subverting cell cycle regulation. We show that two binding motifs tethered by a hypervariable disordered linker drive picomolar affinity Rb binding and host factor displacement. Compensatory changes in amino acid sequence composition and sequence length lead to conservation of optimal tethering across a large family of E1A linkers. We refer to this compensatory mechanism as conformational buffering. We also detect coevolution of the motifs and linker, which can preserve or eliminate the tethering mechanism. Conformational buffering and motif-linker coevolution explain robust functional encoding within hypervariable disordered linkers and could underlie functional selection of many disordered protein regions.

Published
2022

16. Deconstructing virus condensation

Author

Lopez, Nora, primary, Camporeale, Gabriela, additional, Salgueiro, Mariano, additional, Borkosky, Silvia Susana, additional, Visentín, Araceli, additional, Peralta-Martinez, Ramon, additional, Loureiro, María Eugenia, additional, and de Prat-Gay, Gonzalo, additional

Published
2021
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17. Conformational buffering underlies functional selection in intrinsically disordered protein regions

Author

Gonzalez-Foutel, Nicolas S., Borcherds, Wade M., Glavina, Juliana, Barrera-Vilarmau, Susana, Sagar, Amin, Esta��a, Alejandro, Barozet, Amelie, Fernandez-Ballester, Gregorio, Blanes-Mira, Clara, S��nchez, Ignacio E, de Prat-Gay, Gonzalo, Cort��s, Juan, Bernad��, Pau, Pappu, Rohit V., Holehouse, Alex S., Daughdrill, Gary W., and Chemes, Luc��a B.

Abstract

doi:10.1101/2021.05.14.444182, Many disordered proteins conserve essential functions in the face of extensive sequence variation. This makes it challenging to identify the forces responsible for functional selection. Viruses are robust model systems to investigate functional selection and they take advantage of protein disorder to acquire novel traits. Here, we combine structural and computational biophysics with evolutionary analysis to determine the molecular basis for functional selection in the intrinsically disordered adenovirus early gene 1A (E1A) protein. E1A competes with host factors to bind the retinoblastoma (Rb) protein, triggering early S-phase entry and disrupting normal cellular proliferation. We show that the ability to outcompete host factors depends on the picomolar binding affinity of E1A for Rb, which is driven by two binding motifs tethered by a hypervariable disordered linker. Binding affinity is determined by the spatial dimensions of the linker, which constrain the relative position of the two binding motifs. Despite substantial sequence variation across evolution, the linker dimensions are finely optimized through compensatory changes in amino acid sequence and sequence length, leading to conserved linker dimensions and maximal affinity. We refer to the mechanism that conserves spatial dimensions despite large-scale variations in sequence as conformational buffering. Conformational buffering explains how variable disordered proteins encode functions and could be a general mechanism for functional selection within disordered protein regions.

Published
2021
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18. Conformational buffering underlies functional selection in intrinsically disordered protein regions

Author

Gonzalez-Foutel, Nicolas S., primary, Borcherds, Wade M., additional, Glavina, Juliana, additional, Barrera-Vilarmau, Susana, additional, Sagar, Amin, additional, Estaña, Alejandro, additional, Barozet, Amelie, additional, Fernandez-Ballester, Gregorio, additional, Blanes-Mira, Clara, additional, Sánchez, Ignacio E, additional, de Prat-Gay, Gonzalo, additional, Cortés, Juan, additional, Bernadó, Pau, additional, Pappu, Rohit V., additional, Holehouse, Alex S., additional, Daughdrill, Gary W., additional, and Chemes, Lucía B., additional

Published
2021
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19. Antibody recognition of a flexible epitope at the DNA binding site of the human papillomavirus transcriptional regulator E2

Author

Cerutti, Maria Laura, Ferreiro, Diego U., Sanguineti, Santiago, Goldbaum, Fernando A., and de Prat-Gay, Gonzalo

Subjects
DNA-ligand interactions -- Research, Papillomavirus infections -- Genetic aspects, Papillomavirus infections -- Research, Monoclonal antibodies -- Research, Biological sciences, Chemistry
Abstract

The interaction of the C-terminal DNA-binding and dimerization domain (E2C) with the DNA-binding inhibitory murine monoclonal antibodies (mAb) ED15 is characterized. The results have shown that the ED15-E2C pair is an excellent antibody-protein model to study the nature of the forces driving the humoral immune recognition of flexible and positively charged antigens, as are most nucleic acid regulatory proteins.

Published
2006

32. Estado actual del desarrollo de vacunas terapéuticas contra el HPV

Author

Camporeale, Gabriela, de Prat Gay, Gonzalo, Tatti, Silvio Alejandro, Fleider, Laura, Tinnirello, María de los Ángeles, and Caruso, Ricardo

Subjects
Ciencias Biológicas, VPH, VACUNA, ONCOPROTEINAS, PATOLOGIA, Virología, CIENCIAS NATURALES Y EXACTAS
Abstract

La infección persistente por genotipos de HPV de alto riesgo es un factor necesario p ara el desarrollo del cáncer cervical. Más de la mitad de los casos son causados por el genotipo de alto riesgo HPV-16, el cual junto con los tipos HPV-18, -31 y -45 representan cerca del 80 % de todos los cánceres cervicales (1). Las vacunas profilácticas existentes en el mercado han demostrado una alta eficacia en la generación de inmunidad humoral protectiva contra infecciones por los genotipos 16 y 18. Sin embargo, estas vacunas no poseen efecto terapéutico alguno en procesos neoplásicos pre-existentes y, por lo tanto, no presentarán un impacto inmediato sobre la incidencia del cáncer cervical (2, 3). Asimismo, no previenen la infección de cepas de HPV distintas a la que componen la vacuna. Fil: Camporeale, Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: de Prat Gay, Gonzalo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina

Published
2017

37. Degenerate cysteine patterns mediate two redox sensing mechanisms in the papillomavirus E7 oncoprotein

Author

Camporeale, Gabriela, Lorenzo Lopez, Juan Ramiro, Thomas, Maria M.G., Salvatierra, Edgardo, Borkosky, Silvia S.S., Risso, Marikena M.G., Sánchez, Ignacio I.E., de Prat Gay, Gonzalo, Alonso, Leonardo L.G., Camporeale, Gabriela, Lorenzo Lopez, Juan Ramiro, Thomas, Maria M.G., Salvatierra, Edgardo, Borkosky, Silvia S.S., Risso, Marikena M.G., Sánchez, Ignacio I.E., de Prat Gay, Gonzalo, and Alonso, Leonardo L.G.

Abstract

Infection with oncogenic human papillomavirus induces deregulation of cellular redox homeostasis. Virus replication and papillomavirus-induced cell transformation require persistent expression of viral oncoproteins E7 and E6 that must retain their functionality in a persistent oxidative environment. Here, we dissected the molecular mechanisms by which E7 oncoprotein can sense and manage the potentially harmful oxidative environment of the papillomavirus-infected cell. The carboxy terminal domain of E7 protein from most of the 79 papillomavirus viral types of alpha genus, which encloses all the tumorigenic viral types, is a cysteine rich domain that contains two classes of cysteines: strictly conserved low reactive Zn+2 binding and degenerate reactive cysteine residues that can sense reactive oxygen species (ROS). Based on experimental data obtained from E7 proteins from the prototypical viral types 16, 18 and 11, we identified a couple of low pKa nucleophilic cysteines that can form a disulfide bridge upon the exposure to ROS and regulate the cytoplasm to nucleus transport. From sequence analysis and phylogenetic reconstruction of redox sensing states we propose that reactive cysteine acquisition through evolution leads to three separate E7s protein families that differ in the ROS sensing mechanism: non ROS-sensitive E7s; ROS-sensitive E7s using only a single or multiple reactive cysteine sensing mechanisms and ROS-sensitive E7s using a reactive-resolutive cysteine couple sensing mechanism., SCOPUS: ar.j, info:eu-repo/semantics/published

Published
2017

38. Thermodynamics of cooperative DNA recognition at a replication origin and transcription regulatory site

Author

Dellarole, Mariano, Sanchez, Ignacio E., and de Prat Gay, Gonzalo

Subjects
DNA-ligand interactions -- Analysis, DNA testing -- Methods, Isothermal titration calorimetry -- Usage, Genetic transcription -- Analysis, Biological sciences, Chemistry
Abstract

The isothermal titration calorimetry analysis approach is employed to explain the thermodynamics involved in the cooperative DNA recognition taking place at the transcription regulatory site. The DNA binding helix of the three different domains is shown to be extremely similar in nature.

Published
2010

39. Development of a second generation prophylactic vaccine against human papillomavirus

Author

Alonso, Leonardo Gabriel, Cerutti, Maria Laura, Risso, Marikena Guadalupe, González, Mariángeles, Camporeale, Gabriela, and de Prat Gay, Gonzalo

Subjects
lcsh:Immunologic diseases. Allergy, CIENCIAS MÉDICAS Y DE LA SALUD, Biotecnología relacionada con la Salud, lcsh:R, lcsh:Medicine, Papillomavirus, Cáncer cervical, Vacuna profiláctica, Biotecnología de la Salud, lcsh:Infectious and parasitic diseases, Cervical cancer, Prophylactic vaccine, purl.org/becyt/ford/3 [https], lcsh:RC109-216, purl.org/becyt/ford/3.4 [https], lcsh:RC581-607, Papilomavirus
Abstract

Human papillomaviruses (HPV) are the etiologic agent for cervical cancer (CC), the second cause of cancer death in women worldwide. It is estimated that half a million new cases are diagnosed each year, mostly in developing countries due to the lack of massive programs for early detection of the virus. Recently, two prophylactic vaccines against the main oncogenic HPV types 16 and 18 (responsible for 80% of CC) have been introduced into market. Both of these vaccines, obtained as recombinants, have been shown to be safe and effective; however, their high cost works against its immediate impact in the incidence of HPV infection in developing and low-income countries. There is a need to have in hand second generation, low cost vaccines of massive use that will decrease CC cases in a large extent. With this in mind, we have developed a recombinant expression platform that allows us to obtain virus-like particles (VLPs) to formulate both effective and accessible vaccines against HPV infection. Human papillomaviruses (HPV) are the etiologic agent for cervical cancer (CC), the second cause of cancer death in women worldwide. It is estimated that half a million new cases are diagnosed each year, mostly in developing countries due to the lack of massive programs for early detection of the virus. Recently, two prophylactic vaccines against the main oncogenic HPV types 16 and 18 (responsible for 80% of CC) have been introduced into market. Both of these vaccines, obtained as recombinants, have been shown to be safe and effective; however, their high cost works against its immediate impact in the incidence of HPV infection in developing and low-income countries. There is a need to have in hand second generation, low cost vaccines of massive use that will decrease CC cases in a large extent. With this in mind, we have developed a recombinant expression platform that allows us to obtain virus-like particles (VLPs) to formulate both effective and accessible vaccines against HPV infection. Fil: Alonso, Leonardo Gabriel. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Xbio S.a; Argentina Fil: Cerutti, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina. Xbio S.a; Argentina Fil: Risso, Marikena Guadalupe. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Xbio S.a; Argentina Fil: González, Mariángeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina. Xbio S.a; Argentina Fil: Camporeale, Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina Fil: de Prat Gay, Gonzalo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina

Published
2011

40. High-risk HPV E6 oncoproteins assemble into large oligomers that allow localization of endogenous species in prototypic HPV-transformed cell lines

Author

Garcia-Alai, Maria M., Dantur, Karina I., Smal, Clara, Pietrasanta, Lia, and de Prat-Gay, Gonzalo

Subjects
Papillomavirus infections -- Physiological aspects, Oligomers -- Chemical properties, Oligomers -- Physiological aspects, Biological sciences, Chemistry
Abstract

A study was conducted to show that recombinant high-risk HPV 16 and HPV 18 E6 readily fold into very stable soluble oligomers (E6SOs) that exhibit secondary and tertiary structure, cooperatively lost upon denaturation. The results indicate that the cytoplasmic species contain conformational epitopes that might arise from yet undefined homo or hetero-oligomers, but its localization otherwise agrees with that of the other group of major E6 targets.

Published
2007

41. Structural and thermodynamics basis for the enhanced transcriptional control by the human papillomavirus strain-16 E2 protein

Author

Cicero, Daniel O., Nadra, Alejandro D., Eliseo, Tommaso, Dellarole, Mariano, Paci, Maurizio, and de Prat-Gay, Gonzalo

Subjects
Viral carcinogenesis -- Research, Papillomavirus infections -- Research, DNA binding proteins -- Structure, DNA binding proteins -- Research, Biological sciences, Chemistry
Abstract

The first structure for the DNA binding protein domain of human papillomavirus 16 (HPV16) E2 protein bound to DNA and to a natural cognate sequence is presented. The novel features have provided the structural and thermodynamic basis for the tight transcriptional control, the loss of which correlates with carcinogenesis.

Published
2006

42. Hidden Structural Codes in Protein Intrinsic Disorder

Author

Borkosky, Silvia S., primary, Camporeale, Gabriela, additional, Chemes, Lucía B., additional, Risso, Marikena, additional, Noval, María Gabriela, additional, Sánchez, Ignacio E., additional, Alonso, Leonardo G., additional, and de Prat Gay, Gonzalo, additional

Published
2017
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45. Specific antibody-DNA interaction: a novel strategy for tight DNA recognition

Author

Di Pietro, Santiago M., Centeno, Juan M., Cerutti, M. Laura, Lodeiro, M. Fernanda, Ferreiro, Diego U., Alonso, Leonardo G., Schwarz, Frederick P., Goldbaum, Fernando A., and de Prat-Gay, Gonzalo

Subjects
DNA antibodies -- Physiological aspects, DNA-ligand interactions -- Physiological aspects, Immune recognition -- Physiological aspects, Papillomaviruses -- Research, Structure-activity relationships (Biochemistry) -- Analysis, Biological sciences, Chemistry
Abstract

Results demonstrate that the anti-double-stranded DNA monoclonal antibodies against the DNA binding site of the transcriptional regulator E2 C-terminal domain from human papillomavirus 16 uses an indirect readout with a distortion of the DNA double helix. Data indicate an unusual molecular recognition mechanism of a specific DNA sequence by these antibodies, which facilitate DNA antigen recognition by the immune system.

Published
2003

46. Hidden Structural Codes in Protein Intrinsic Disorder

Author

Borkosky, Silvia S., Camporeale, Gabriela, Chemes, Lucía B., Risso, Marikena, Noval, María Gabriela, Sánchez, Ignacio E., Alonso, Leonardo G., and de Prat Gay, Gonzalo

Abstract

Intrinsic disorder is a major structural category in biology, accounting for more than 30% of coding regions across the domains of life, yet consists of conformational ensembles in equilibrium, a major challenge in protein chemistry. Anciently evolved papillomavirus genomes constitute an unparalleled case for sequence to structure–function correlation in cases in which there are no folded structures. E7, the major transforming oncoprotein of human papillomaviruses, is a paradigmatic example among the intrinsically disordered proteins. Analysis of a large number of sequences of the same viral protein allowed for the identification of a handful of residues with absolute conservation, scattered along the sequence of its N-terminal intrinsically disordered domain, which intriguingly are mostly leucine residues. Mutation of these led to a pronounced increase in both α-helix and β-sheet structural content, reflected by drastic effects on equilibrium propensities and oligomerization kinetics, and uncovers the existence of local structural elements that oppose canonical folding. These folding relays suggest the existence of yet undefined hidden structural codes behind intrinsic disorder in this model protein. Thus, evolution pinpoints conformational hot spots that could have not been identified by direct experimental methods for analyzing or perturbing the equilibrium of an intrinsically disordered protein ensemble.

Published
2024
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47. Argentophilic nucleolus organizer region as a proliferation marker in cervical intraepithelial neoplasia grade 1 of the uterine cervix

Author

Guerra, Fernando Andres, Rocher, Adriana Esther, Villacorta Hidalgo, José, Díaz, Lilí, Vighi, Susana, Cardinal, Lucía, Tatti, Silvio, Cúneo, Nicasio, de Prat Gay, Gonzalo, Camporeale, Gabriela, and Palaoro, Luis Alberto

Subjects
HPV, CIENCIAS MÉDICAS Y DE LA SALUD, proliferation marker, p16, Obstetricia y Ginecología, Medicina Clínica, Cervical Cancer
Abstract

AIM: p16INK4a and argentophilic nucleolus organizer region (AgNOR) can be used as markers for progression of cervical intraepithelial neoplasia grade 1 (CIN1) of the uterine cervix. Our objective was to study the predictive value of the AgNOR technique as a progression marker of CIN1 and its correlation with p16INK4A. MATERIAL AND METHODS: One uterine cervix biopsy from each of 75 patients with diagnosis of CIN1 was selected. All of these patients underwent a second biopsy, and these were also used for the study. RESULTS: The second biopsies showed: regression (20 patients), persistent CIN1 (38 patients), progression to CIN2 (10 patients) and progression to CIN3 (seven patients). p16INK4A showed reactivity in 67 of the 75 first CIN1 biopsies: 12 of the 20 cases that cleared the lesions and the 55 cases with persistent or progressive lesions were positive for p16INK4a (specificity: 40%; sensitivity: 100%; positive predictive value [PPV]: 82%; negative predictive value [NPV]: 100%). Samples with AgNOR areas less than 3.0 μ(2) returned in all cases, but patients whose lesions persisted or progressed to CIN2/CIN3, showed AgNOR areas greater than 3.0 μ(2) in 50/55 cases (specificity: 100%; sensitivity: 91%; PPV: 100%; NPV: 80%). CONCLUSIONS: p16INK4a is expressed in a high percentage of returning lesions. AgNOR might be a better marker of proliferation of CIN1 than p16INK4a (PPV = 100%), which means that a value greater than 3.0 μ(2) indicates the persistence or progression of the lesion. As its NPV is 80%, a value of AgNOR area less than 3.0 μ(2) in CIN1 leaves a margin of doubt about the future behavior of the lesion. Fil: Guerra, Fernando Andres. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina Fil: Rocher, Adriana Esther. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina Fil: Villacorta Hidalgo, José. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina Fil: Díaz, Lilí. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina Fil: Vighi, Susana. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina Fil: Cardinal, Lucía. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina Fil: Tatti, Silvio. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina Fil: Cúneo, Nicasio. Hospital de Oncología María Curie; Argentina Fil: de Prat Gay, Gonzalo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina Fil: Camporeale, Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina Fil: Palaoro, Luis Alberto. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina

Published
2014

48. Folding of a cyclin box: linking multitarget binding to marginal stability, oligomerization, and aggregation of the retinoblastoma tumor suppressor AB pocket domain

Author

Chemes, Lucia Beatriz, Noval, María Gabriela, Sánchez Miguel, Ignacio Enrique, and de Prat Gay, Gonzalo

Subjects
Ciencias Biológicas, purl.org/becyt/ford/1 [https], tumor suppressor, protein folding, Retinoblastoma, Bioquímica y Biología Molecular, protein misfolding, purl.org/becyt/ford/1.6 [https], CIENCIAS NATURALES Y EXACTAS
Abstract

The retinoblastoma tumor suppressor (Rb) controls the proliferation, differentiation, and survival of cells in most eukaryotes with a role in the fate of stem cells. Its inactivation by mutation or oncogenic viruses is required for cellular transformation and eventually carcinogenesis. The high conservation of the Rb cyclin fold prompted us to investigate the link between conformational stability and ligand binding properties of the RbAB pocket domain. RbAB unfolding presents a three-state transition involving cooperative secondary and tertiary structure changes and a partially folded intermediate that can oligomerize. The first transition corresponds to unfolding of the metastable B subdomain containing the binding site for the LXCXE motif present in cellular and viral targets, and the second transition corresponds to the stable A subdomain. The low thermodynamic stability of RbAB translates into a propensity to rapidly oligomerize and aggregate at 37 °C (T50 = 28 min) that is suppressed by human papillomavirus E7 and E2F peptide ligands, suggesting that Rb is likely stabilized in vivo through binding to target proteins. We propose that marginal stability and associated oligomerization may be conserved for function as a "hub" protein, allowing the formation of multiprotein complexes, which could constitute a robust mechanism to retain its cell cycle regulatory role throughout evolution. Decreased stability and oligomerization are shared with the p53 tumor suppressor, suggesting a link between folding and function in these two essential cell regulators that are inactivated in most cancers and operate within multitarget signaling pathways. Fil: Chemes, Lucia Beatriz. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: Noval, María Gabriela. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina Fil: Sánchez Miguel, Ignacio Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: de Prat Gay, Gonzalo. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina

Published
2013

49. Structure and stability of the <italic>Human respiratory syncytial virus</italic> M2–1 RNA‐binding core domain reveals a compact and cooperative folding unit.

Author

Molina, Ivana G., Josts, Inokentijs, Almeida Hernandez, Yasser, Esperante, Sebastian, Salgueiro, Mariano, Garcia Alai, Maria M., de Prat-Gay, Gonzalo, and Tidow, Henning

Subjects
RESPIRATORY syncytial virus, PEDIATRIC respiratory diseases, RNA polymerases
Abstract

Human syncytial respiratory virus is a nonsegmented negative‐strand RNA virus with serious implications for respiratory disease in infants, and has recently been reclassified into a new family, Pneumoviridae. One of the main reasons for this classification is the unique presence of a transcriptional antiterminator, called M2–1. The puzzling mechanism of action of M2–1, which is a rarity among antiterminators in viruses and is part of the RNA polymerase complex, relies on dissecting the structure and function of this multidomain tetramer. The RNA‐binding activity is located in a monomeric globular `core' domain, a high‐resolution crystal structure of which is now presented. The structure reveals a compact domain which is superimposable on the full‐length M2–1 tetramer, with additional electron density for the C‐terminal tail that was not observed in the previous models. Moreover, its folding stability was determined through chemical denaturation, which shows that the secondary and tertiary structure unfold concomitantly, which is indicative of a two‐state equilibrium. These results constitute a further step in the understanding of this unique RNA‐binding domain, for which there is no sequence or structural counterpart outside this virus family, in addition to its implications in transcription regulation and its likeliness as an antiviral target. [ABSTRACT FROM AUTHOR]

Published
2018
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50. Circular Dichroism Techniques for the identification of Intrinsically Disordered Proteins

Author

Chemes, Lucia Beatriz, Alonso, Leonardo Gabriel, Noval, María Gabriela, and de Prat Gay, Gonzalo

Subjects
Ciencias Biológicas, Desorden, Intrinsico, dicroismo, Bioquímica y Biología Molecular, Proteinas, CIENCIAS NATURALES Y EXACTAS
Abstract

Circular dichroism (CD) spectroscopy is a simple and powerful technique, which allows for the assessment of the conformational properties of a protein or protein domain. Intrinsically disordered proteins (IDPs), as discussed throughout this series, differ from random coil polypeptides in that different regions present specific conformational preferences, exhibiting dynamic secondary structure content [1]. These dynamic secondary structure elements can be stabilized or perturbed by different chemical (solvent, ionic strength, pH) or physical (temperature) agents, by posttranslational modifications, and by ligands. This information is important for defining ID nature. As IDPs present dynamic conformations, circular dichroism measurements (and other approaches as well) should be carried out not as single spectra performed in unique conditions, but instead changing the chemical conditions and observing the behavior, as part of the determination of the ID nature.In this chapter, we present the basic methodology for performing Far-UV CD measurements on a protein of interest and for identifying and characterizing intrinsically disordered regions, and several protocols for the analysis of residual secondary structure present in the protein under study. These techniques are straightforward to perform; they require minimal training and can be preliminary to more complex methodologies such as NMR. Fil: Chemes, Lucia Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Alonso, Leonardo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Noval, María Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: de Prat Gay, Gonzalo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina

Published
2012

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