Abstract:

Protein ubiquitination leading to degradation by the proteasome is an important mechanism in regulating key cellular functions. Protein ubiquitination is carried out by a three step process involving ubiquitin (Ub) activation by a E1 enzyme, the transfer of Ub to a protein E2, finally an ubiquitin ligase E3 catalyzes the transfer of the Ub peptide to an acceptor protein. The E3 component is responsible for the specific recognition of the target, making the unveiling of E3 components essential to understand the mechanisms regulating fundamental cell processes through the protein degradation pathways. The Arabidopsis thaliana seven in absentia-like 7 (AtSINAL7) gene encodes for a protein with characteristics from a C3HC4-type E3 ubiquitin ligase. We demonstrate here that AtSINAL7 protein is indeed an E3 protein ligase based on the self-ubiquitination in vitro assay. This activity is dependent of the presence of a Lys residue in position 124. We also found that higher AtSINAL7 transcript levels are present in tissues undergoing active cell division during floral development. An interesting observation is the circadian expression pattern of AtSINAL7 mRNA in floral buds. Furthermore, UV–B irradiation induces the expression of this transcript indicating that AtSINAL7 may be involved in a wide range of different cell processes.

Author affiliation: Peralta, Diego Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosinteticos y Bioquimicos. Universidad Nacional de Rosario. Facultad de Cs.bioquímicas y Farmaceuticas. Centro de Estudios Fotosinteticos y Bioquimicos; Argentina

Author affiliation: Araya, Alejandro. Centre National de la Recherche Scientifique; Francia. Institut National de la Recherche Agronomique; Francia

Author affiliation: Nardi, Cristina Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina

Author affiliation: Busi, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosinteticos y Bioquimicos. Universidad Nacional de Rosario. Facultad de Cs.bioquímicas y Farmaceuticas. Centro de Estudios Fotosinteticos y Bioquimicos; Argentina

Author affiliation: Gomez Casati, Diego Fabian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosinteticos y Bioquimicos. Universidad Nacional de Rosario. Facultad de Cs.bioquímicas y Farmaceuticas. Centro de Estudios Fotosinteticos y Bioquimicos; Argentina

Abstract:

The process of genome release or uncoating after viral entry is one of the least-studied steps in the flavivirus life cycle. Flaviviruses are mainly arthropod-borne viruses, including emerging and reemerging pathogens such as dengue, Zika, and West Nile viruses. Currently, dengue virus is one of the most significant human viral pathogens transmitted by mosquitoes and is responsible for about 390 million infections every year around the world. Here, we examined for the first time molecular aspects of dengue virus genome uncoating. We followed the fate of the capsid protein and RNA genome early during infection and found that capsid is degraded after viral internalization by the host ubiquitin-proteasome system. However, proteasome activity and capsid degradation were not necessary to free the genome for initial viral translation. Unexpectedly, genome uncoating was blocked by inhibiting ubiquitination. Using different assays to bypass entry and evaluate the first rounds of viral translation, a narrow window of time during infection that requires ubiquitination but not proteasome activity was identified. In this regard, ubiquitin E1-activating enzyme inhibition was sufficient to stabilize the incoming viral genome in the cytoplasm of infected cells, causing its retention in either endosomes or nucleocapsids. Our data support a model in which dengue virus genome uncoating requires a nondegradative ubiquitination step, providing new insights into this crucial but understudied viral process. IMPORTANCE: Dengue is the most significant arthropod-borne viral infection in humans. Although the number of cases increases every year, there are no approved therapeutics available for the treatment of dengue infection, and many basic aspects of the viral biology remain elusive. After entry, the viral membrane must fuse with the endosomal membrane to deliver the viral genome into the cytoplasm for translation and replication. A great deal of information has been obtained in the last decade regarding molecular aspects of the fusion step, but little is known about the events that follow this process, which leads to viral RNA release from the nucleocapsid. Here, we investigated the fate of nucleocapsid components (capsid protein and viral genome) during the infection process and found that capsid is degraded by the ubiquitin-proteasome system. However, in contrast to that observed for other RNA and DNA viruses, dengue virus capsid degradation was not responsible for genome uncoating. Interestingly, we found that dengue virus genome release requires a nondegradative ubiquitination step. These results provide the first insights into dengue virus uncoating and present new opportunities for antiviral intervention.

Author affiliation: Byk, Laura Andrea. 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

Author affiliation: Iglesias, Nestor 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

Author affiliation: de Maio, Federico Andres. 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

Author affiliation: Gebhard, Leopoldo German. 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

Author affiliation: Rossi, Mario. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina

Author affiliation: Gamarnik, Andrea Vanesa. 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

Abstract:

Author affiliation: Byk, Laura A. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina

Author affiliation: Byk, Laura A. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir; Argentina

Author affiliation: Iglesias, Nestor G. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina

Author affiliation: Iglesias, Nestor G. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina

Author affiliation: de Maio, Federico A. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina

Author affiliation: de Maio, Federico A. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina

Author affiliation: Gebhard, Leopoldo G. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina

Author affiliation: Gebhard, Leopoldo G. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina

Author affiliation: Rossi, Mario. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina

Author affiliation: Rossi, Mario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina

Author affiliation: Gamarnik, Andrea V. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina

Author affiliation: Gamarnik, Andrea V. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina

The process of genome release or uncoating after viral entry is one of the least-studied steps in the flavivirus life cycle. Flaviviruses are mainly arthropod-borne viruses, including emerging and reemerging pathogens such as dengue, Zika, and West Nile viruses. Currently, dengue virus is one of the most significant human viral pathogens transmitted by mosquitoes and is responsible for about 390 million infections every year around the world. Here, we examined for the first time molecular aspects of dengue virus genome uncoating. We followed the fate of the capsid protein and RNA genome early during infection and found that capsid is degraded after viral internalization by the host ubiquitin-proteasome system. However, proteasome activity and capsid degradation were not necessary to free the genome for initial viral translation. Unexpectedly, genome uncoating was blocked by inhibiting ubiquitination. Using different assays to bypass entry and evaluate the first rounds of viral translation, a narrow window of time during infection that requires ubiquitination but not proteasome activity was identified. In this regard, ubiquitin E1-activating enzyme inhibition was sufficient to stabilize the incoming viral genome in the cytoplasm of infected cells, causing its retention in either endosomes or nucleocapsids. Our data support a model in which dengue virus genome uncoating requires a nondegradative ubiquitination step, providing new insights into this crucial but understudied viral process. IMPORTANCE: Dengue is the most significant arthropod-borne viral infection in humans. Although the number of cases increases every year, there are no approved therapeutics available for the treatment of dengue infection, and many basic aspects of the viral biology remain elusive. After entry, the viral membrane must fuse with the endosomal membrane to deliver the viral genome into the cytoplasm for translation and replication. A great deal of information has been obtained in the last decade regarding molecular aspects of the fusion step, but little is known about the events that follow this process, which leads to viral RNA release from the nucleocapsid. Here, we investigated the fate of nucleocapsid components (capsid protein and viral genome) during the infection process and found that capsid is degraded by the ubiquitin-proteasome system. However, in contrast to that observed for other RNA and DNA viruses, dengue virus capsid degradation was not responsible for genome uncoating. Interestingly, we found that dengue virus genome release requires a nondegradative ubiquitination step. These results provide the first insights into dengue virus uncoating and present new opportunities for antiviral intervention.

Abstract:

Perinatal asphyxia (PA) is a medical condition associated with a high short-term morbimortality and different long-term neurological diseases. In previous work we have observed at 6 months post-synaptic densities (PSDs) alterations compatible with neurodegeneration highly correlated with the increment in the ubiquitination. Although alterations in the synaptic organization and function have been related with neuronal death after hypoxia, little is known about the synaptic changes in young animals exposed to PA. The main aim of this work is to study the PSDs changes in striatum of 30-day-old rats subjected to PA. Using two-dimensional electron microscopic analyses of synapses staining with ethanolic phosphotungstic acid we observed an increment of PSD thickness in severe hypoxic rats. These data are consistent with the western blot analysis that showed an increment in ubiquitination levels in the synapses of severe hypoxic rat. We did observe any alterations neither in synaptic structure nor in ubiquitinization in mild asphyctic rats. These data suggest that hypoxia might cause early misfolding and aggregation of synaptic proteins in severe anoxic animas that could induce long-term neurodegeneration.

Author affiliation: Grimaldi, M.. Universidad Argentina "John F. Kennedy"; Argentina

Author affiliation: Romer, I.. Universidad Argentina "John F. Kennedy"; Argentina

Author affiliation: González de Apodaca, M. T.. Universidad Argentina "John F. Kennedy"; Argentina

Author affiliation: Iturbe, L.. Universidad Argentina "John F. Kennedy"; Argentina

Author affiliation: Catania, I. D.. Universidad Argentina "John F. Kennedy"; Argentina

Author affiliation: González, J.. Pontificia Universidad Javeriana; Colombia

Author affiliation: Kolliker Fres, R.. Universidad Argentina "John F. Kennedy"; Argentina

Author affiliation: Barreto, G.. Pontificia Universidad Javeriana; Colombia

Author affiliation: Capani, Francisco. Universidad Argentina "John F. Kennedy"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Cardiológicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Cardiológicas; Argentina

Abstract:

El sistema ubiquitina-proteasoma es una vía reguladora fundamental para controlar la estabilidad de las proteínas dentro de varios procesos celulares, como la señalización y el ciclo celular. En respuesta a señales particulares, proteínas reguladoras específicas son marcadas con una cadena de moléculas de ubiquitina a través de la acción de una cascada enzimática compuesta por una enzima activadora de la ubi- quitina (E1), una enzima conjugadora de la ubiquitina (E2), y una ubiquitina ligasa (E3). Las E3 son responsables de unir los sustratos y acercarlos a la E2 cargada con ubiquitina, la cual luego transfiere la ubiquitina al sustrato y a la siguiente ubiqui- tina de la cadena en una reacción secuencial. Una de las ubiquitina ligasa E3 res- ponsables en el control directo de proteínas involucradas en el ciclo celular es el complejo SCF (SKP1-CUL1-F-box). En esta tesis se estudió cuál es la función de las proteínas que forman parte del complejo SCF en la regulación del ciclo celular de Trypanosoma brucei. El análisis de los genomas de Tripanosomátidos mostró que las cuatro proteínas que forman parte del complejo se encuentran conservadas, a excepción de las proteínas F-box. Utilizando la técnica de ARN de interferencia en los dos estadios del ciclo de vida de Trypanosoma brucei, se determinó en que fase del ciclo celular estas proteínas tienen una función preponderante. Los resultados obtenidos muestran que las subunidades del complejo actúan en diferentes momen- tos del ciclo celular. No solo se demostró que la actividad del complejo SCF es im- portante para el crecimiento de los parásitos in vitro, sino que la enzima conjugadora de ubiquitina es necesaria para que los parásitos establezcan una infección en un hospedador mamífero. A su vez, se utilizó la técnica de Electroforesis bidimensional diferencial en gel (2D-DIGE) con el objetivo de identificar posibles sustratos del complejo de ubiquitinación. Este trabajo describe por primera vez la actividad de las subunidades homólogas del complejo SCF en el parásito Trypanosoma brucei.

The ubiquitin–proteasome system (UPS) is a fundamental regulatory pathway for controlling protein stability that underlies many cellular processes, such as signaling and cell cycle progression. In response to particular signals, specific regulatory pro- teins are tagged with a chain of ubiquitin molecules through the action of an enzyma- tic cascade composed of an E1 ubiquitin activating enzyme, an E2 ubiquitin conjuga- ting enzyme (Ubc), and an E3 ubiquitin ligase. E3s are responsible for binding subs- trates and for bringing substrates into the proximity of a ubiquitin-charged E2, which then transfers ubiquitin to the substrate and to the ensuing ubiquitin chain in a pro- cessive reaction. One of the E3 responsible for the ubiquitination of cell cycle regula- tors is the SCF complex (SKP1-CUL1-F-box). In this thesis, we have studied the function of the proteins that form the core of the SCF complex in the Trypanosoma brucei cell cycle. The analysis of the Trypanosome genomes showed us that the four core subunit homologs are conserved, with the exception of the F-box proteins. Using RNA interference in both life cycle stages of Trypanosoma brucei, we were able to determine the cell cycle stages in which these proteins have important roles. The obtained results show that the subunits of the complex act at diferent points in the cell cycle. Not only we demonstrated that the activity of the complex is important for the parasites growth in vitro, but we showed that the ubiquitin conjugating enzyme is indispensable for establishing an infection in a mammalian host. At the same time, we utilized the Two-dimensional gel electrophoresis (2D-PAGE) technique for the purpose of identifiying possible substrates of the ubiquitination complex. This work analizes for the first time the activity of the subunits of the SCF complex in a lower eukaryote organism.

Author affiliation: Rojas, Federico. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Abstract:

A pesar de los formidables avances logrados en el tratamiento, prevención y detección temprana de un gran número de cánceres, el desarrollo de procesos metastásicos en pacientes afectados con esta enfermedad continúa representando una reducción significativa en su supervivencia y calidad de vida restante. De hecho, la diseminación metastásica de un tumor primario a un segundo sitio es la causa principal de muertes por tumores sólidos. El desarrollo de metástasis requiere que las células tumorales completen una cascada de acontecimientos constituida por etapas bien definidas, que implican interacciones muy diversas entre las células tumorales y el organismo hospedador. El potencial invasivo de las células malignas depende de tres fenómenos: alteraciones en la adhesión celular, degradación proteolítica localizada de la matriz extracelular y movilidad de las células tumorales. Dicho potencial puede verse modificado por alteraciones genéticas en las células tumorales que podrían constituir posibles puntos de intervención terapéutica. La ubiquitinación representa una de las modificaciones post-traduccionales cuyo estudio ha despertado un creciente interés en el ámbito de la oncología molecular, debido a su gran potencial a nivel terapéutico en el tratamiento del cáncer. Prueba de esto último es el hecho que, en muchos casos, alteraciones en distintos componentes de la cascada de ubiquitinación están asociadas a la trasformación maligna de células tumorales y al desarrollo de procesos metastásicos. La cascada de ubiquitinación consiste en una red compleja de enzimas que median la unión covalente de múltiples unidades de ubiquitina a las proteínas, y representa una de las vías metabólicas más importantes que participa en el control de la proteostasis a nivel celular. Por lo tanto, el estudio de los mecanismos moleculares que rigen esta cascada enzimática, junto con el desarrollo de compuestos que inhiben o activen componentes específicos, posee un gran potencial a nivel terapéutico. En el contexto de lo señalado en los párrafos anteriores, el objetivo de esta tesis doctoral es identificar y caracterizar componentes de la cascada de ubiquitinación que representen potenciales nuevos blancos moleculares que puedan ser utilizados en futuros desarrollos y mejoras de las terapias antitumorales actuales. A tal fin se realizó un screen genético utilizando tecnología de ARN de interferencia (shARN) para encontrar nuevos genes relacionados con el proceso de ubiquitinación que regularan positivamente el potencial migratorio de células tumorales. En particular se focalizó la atención en células derivadas de Cáncer de Mama Triple Negativo (CMTN), una neoplasia maligna que posee un mal pronóstico, presenta alto riesgo de recurrencia metastásica y para la cual no se posee en la actualidad una terapia eficaz y específica. Mediante este procedimiento se identificó USP19, una enzima deubiquitinasa (DUB), como un nuevo regulador de los procesos de migración e invasión celular. Asimismo se validó, tanto in vitro, como así también en modelos animales in vivo, el rol que este gen cumple en el control de procesos de tumorigénesis resaltando su alto potencial como posible futuro punto de intervención terapéutico en el tratamiento del cáncer.

Despite formidable advance in the prevention, early detection and treatment of a great number of cancers, the development of metastasis foci in patients suffering from this disease still represents a significant reduction in their survival and life quality. In fact, metastatic dissemination from a primary tumor to a secondary site is the principal cause of death from solid tumors. The invasive potential of malignant cells depends on three phenomena: alterations of cellular adhesion, localized proteolytic degradation of the extracellular matrix and tumor cell motility. The invasive ability is accomplished by a series of molecular and genetic alterations in tumor cells, which could represent putative therapeutic points of intervention. Due to its great potential as a new therapeutic option for the treatment of cancer, ubiquitination has attracted the attentions of the scientific community working on the molecular oncology field. This is based on the observations that several alterations in the levels of different ubiquitin-pathway components are associated with malignant transformation of tumor cells and the generation of metastatic processes. The Ubiquitination cascade consists of a complex network of enzymes that link ubiquitin units to proteins, and represents one of the most important pathways that regulate cellular proteostasis. Therefore, the study of the molecular mechanisms that regulate this enzymatic pathway, together with the development of new compounds that inhibit or activate specific components, may prove extremely important for pharmaceutical intervention in therapy. In relation to the hypothesis outlined in the previous paragraph, the focus of my PhD Thesis is to contribute to the development and improvement of anti-tumoral therapeutic strategies in cancer, by identifying and characterizing components of the Ubiquitination cascade that regulate tumor cell migration and invasion. To this end I performed a genetic screen using the shRNA technology using cells derived from Triple Negative Breast Cancers (TNBC), a malignant disease with poor prognosis, a high metastatic risk and for which there is no specific or effective treatment available. Using this strategy we identified USP19, a deubiquitinase enzyme (DUB), as a new regulator of cellular migration and invasion. Moreover, its role in tumorigenesis was validated both in vitro as well as in animal models in vivo, highlighting its potential as a possible future therapeutic intervention point for the treatment of cancer.

Author affiliation: Rossi, Fabiana Alejandra. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Abstract:

Post-translational arginylation of proteins is an important regulator of many physiological pathways in cells. This modification was originally noted in protein degradation during neurodegenerative processes, with an apparently different physiological relevance between central and peripheral nervous system. Subsequent studies have identified a steadily increasing number of proteins and proteolysis-derived polypeptides as arginyltransferase (ATE1) substrates, including β-amyloid, α-synuclein, and TDP43 proteolytic fragments. Arginylation is involved in signaling processes of proteins and polypeptides that are further ubiquitinated and degraded by the proteasome. In addition, it is also implicated in autophagy/lysosomal degradation pathway. Recent studies using mutant mouse strains deficient in ATE1 indicate additional roles of this modification in neuronal physiology. As ATE1 is capable of modifying proteins either at the N-terminus or middle-chain acidic residues, determining which proteins function are modulated by arginylation represents a big challenge. Here, we review studies addressing various roles of ATE1 activity in nervous system function, and suggest future research directions that will clarify the role of post-translational protein arginylation in brain development and various neurological disorders. (Figure presented.) Arginyltransferase (ATE1), the enzyme responsible for post-translational arginylation, modulates the functions of a wide variety of proteins and polypeptides, and is also involved in the main degradation pathways of intracellular proteins. Regulatory roles of ATE1 have been well defined for certain organs. However, its roles in nervous system development and neurodegenerative processes remain largely unknown, and present exciting opportunities for future research, as discussed in this review.

Author affiliation: Galiano, Mauricio Raul. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina

Author affiliation: Goitea, Victor Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina

Author affiliation: Hallak, Marta Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina

Abstract:

Somatic mutations or loss of von Hippel-Lindau (pVHL) happen in the majority of VHL disease tumors, which present a constitutively active Hypoxia Inducible Factor (HIF), essential for tumor growth. Recently described mechanisms for pVHL modulation shed light on the open question of the HIF/pVHL pathway regulation. The aim of the present study was to determine the molecular mechanism by which RSUME stabilizes HIFs, by studying RSUME effect on pVHL function and to determine the role of RSUME on pVHL-related tumor progression. We determined that RSUME sumoylates and physically interacts with pVHL and negatively regulates the assembly of the complex between pVHL, Elongins and Cullins (ECV), inhibiting HIF-1 and 2alpha ubiquitination and degradation. We found that RSUME is expressed in human VHL tumors (renal clear-cell carcinoma (RCC), pheochromocytoma and hemangioblastoma) and by overexpressing or silencing RSUME in a pVHL-HIF-oxygen-dependent degradation stability reporter assay, we determined that RSUME is necessary for the loss of function of type 2 pVHL mutants. The functional RSUME/pVHL interaction in VHL-related tumor progression was further confirmed using a xenograft assay in nude mice. RCC clones, in which RSUME was knocked down and express either pVHL wt or type 2 mutation, have an impaired tumor growth, as well as HIF-2alpha, vascular endothelial growth factor A and tumor vascularization diminution. This work shows a novel mechanism for VHL tumor progression and presents a new mechanism and factor for targeting tumor-related pathologies with pVHL/HIF altered function.

Author affiliation: Gerez, Juan Atilio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina

Author affiliation: Tedesco, Lucas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina

Author affiliation: Bonfiglio, Juan José. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina

Author affiliation: Fuertes, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina

Author affiliation: Barontini, Marta Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; Argentina

Author affiliation: Silberstein Cuña, Susana Iris. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina

Author affiliation: Wu, Y.. Max Planck Institute of Psychiatry; Alemania

Author affiliation: Renner, U.. Max Planck Institute of Psychiatry; Alemania

Author affiliation: Paez Pereda, M.. Max Planck Institute of Psychiatry; Alemania

Author affiliation: Holsboer, F.. Max Planck Institute of Psychiatry; Alemania

Author affiliation: Stalla, G. K.. Max Planck Institute of Psychiatry; Alemania

Author affiliation: Arzt, Eduardo Simon. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina

Abstract:

Los tumores de hipófisis son adenomas benignos de incidencia frecuente que se originan a partir de los distintos tipos celulares hipofisarios por expansión clonal. En todos los tipos de adenomas hipofisarios se ha visto una sobreexpresión de pituitary tumor transforming gene (PTTG). PTTG facilita la progresión del ciclo celular ya que actúa como securina, regulando la correcta separación de las cromátidas hermanas en metafase, entre otras funciones. Por tratarse de un regulador del ciclo celular, el balance de los niveles de expresión de PTTG en la célula es importante, y la desregulación de estos niveles por defecto o exceso conduce a inestabilidad genómica, aneuploidía, apoptosis, daño al ADN, y conlleva al desarrollo de tumores. Se desconoce la causa que provoca el desbalance de PTTG al momento inicial del desarrollo de tumores de hipófisis. En este trabajo identificamos y describimos por primera vez un mecanismo de desregulación de los niveles de expresión de la proteína PTTG, que desencadena su acción como oncogen, permitiendo el avance de la tumorigenicidad hipofisaria. El mismo involucra la sobreexpresión de RWD-containing sumoylation enhancer (RSUME) al momento inicial del proceso tumoral y, como consecuencia, la inducción de la modificación de PTTG por SUMO, reduciendo su degradación por ubiquitinación y aumentando su estabilidad proteica. Este efecto de RSUME conduce a un aumento de la actividad PTTG y conlleva a hiperactividad del ciclo celular, aneuploidía, anormalidades cromosómicas, células multinucleadas y otros efectos tumorigénicos mediados por PTTG. Además, demostramos que PTTG y RSUME colocalizan a nivel celular y su expresión correlaciona positivamente en adenomas hipofisarios humanos.

Pituitary tumors are benign adenomas of frequent incidence originating from different pituitary cell types by clonal expansion. In all types of pituitary adenomas has been an overexpression of pituitary tumor transforming gene (PTTG). PTTG facilitates cell cycle progression by acting as securin, regulating the correct separation of sister chromatids in metaphase. Being a cell cycle regulator, the balance of PTTG expression levels in the cell is important, and deregulation of these default levels or excess leads to genetic instability, aneuploidy, apoptosis, DNA damage, among others, and leads to the development of tumors. The underlying cause of the imbalance of PTTG the initial stage of development of pituitary tumors is unknown. In this paper we identify and describe for the first time a mechanism for deregulation of the expression levels of PTTG protein, which triggers its action as oncogene, allowing the advance of pituitary tumorigenicity. It involves overexpression of RWD-containing sumoylation enhancer (RSUME) at the initial moment of the tumor process and, as a result, induction of PTTG by SUMO modification, reducing degradation by ubiquitination and increasing protein stability. This effect of RSUME leads to an increase of PTTG activity and leads to hyperactivity of the cell cycle, aneuploidy, chromosomal abnormalities, multinucleated cells and other tumorigenic effects mediated by PTTG. Furthermore, we demonstrate that PTTG and RSUME colocalize at the cellular level and its expression correlates positively in human pituitary adenomas.

Author affiliation: Fuertes, Mariana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Abstract:

Tumour-necrosis factor (TNF) receptor-associated factor 2 (TRAF2) is a key component in NF-κB signalling triggered by TNF-α1, 2. Genetic evidence indicates that TRAF2 is necessary for the polyubiquitination of receptor interacting protein 1 (RIP1)3 that then serves as a platform for recruitment and stimulation of IκB kinase, leading to activation of the transcription factor NF-κB. Although TRAF2 is a RING domain ubiquitin ligase, direct evidence that TRAF2 catalyses the ubiquitination of RIP1 is lacking. TRAF2 binds to sphingosine kinase 1 (SphK1)4, one of the isoenzymes that generates the pro-survival lipid mediator sphingosine-1-phosphate (S1P) inside cells. Here we show that SphK1 and the production of S1P is necessary for lysine-63-linked polyubiquitination of RIP1, phosphorylation of IκB kinase and IκBα, and IκBα degradation, leading to NF-κB activation. These responses were mediated by intracellular S1P independently of its cell surface G-protein-coupled receptors. S1P specifically binds to TRAF2 at the amino-terminal RING domain and stimulates its E3 ligase activity. S1P, but not dihydro-S1P, markedly increased recombinant TRAF2-catalysed lysine-63-linked, but not lysine-48-linked, polyubiquitination of RIP1 in vitro in the presence of the ubiquitin conjugating enzymes (E2) UbcH13 or UbcH5a. Our data show that TRAF2 is a novel intracellular target of S1P, and that S1P is the missing cofactor for TRAF2 E3 ubiquitin ligase activity, indicating a new paradigm for the regulation of lysine-63-linked polyubiquitination. These results also highlight the key role of SphK1 and its product S1P in TNF-α signalling and the canonical NF-κB activation pathway important in inflammatory, antiapoptotic and immune processes.

Author affiliation: Alvarez, Sergio Eduardo. Virginia Commonwealth University. School of Medicine; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; Argentina

Author affiliation: Harikumar, Kuzhuvelil B.. Virginia Commonwealth University. School of Medicine; Estados Unidos

Author affiliation: Hait, Nitai C.. Virginia Commonwealth University. School of Medicine; Estados Unidos

Author affiliation: Allegood, Jeremy. Virginia Commonwealth University. School of Medicine; Estados Unidos

Author affiliation: Strub, Graham M.. Virginia Commonwealth University. School of Medicine; Estados Unidos

Author affiliation: Kim, Eugene Y.. Virginia Commonwealth University. School of Medicine; Estados Unidos

Author affiliation: Maceycka, Michael. Virginia Commonwealth University. School of Medicine; Estados Unidos

Author affiliation: Jiang, Hualiang. Chinese Academy of Sciences. Shangai Institute of Materia Medica. State Key Laboratory of Drug Research; China

Author affiliation: Lu, Cheng. Chinese Academy of Sciences. Shangai Institute of Materia Medica. State Key Laboratory of Drug Research; China

Author affiliation: Kordula, Tomasz. Virginia Commonwealth University. School of Medicine; Estados Unidos

Author affiliation: Milstien, Sheldon. Virginia Commonwealth University. School of Medicine; Estados Unidos

Author affiliation: Spiegel, Sarah. Virginia Commonwealth University. School of Medicine; Estados Unidos