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Jose Luis Rosa - One of the best experts on this subject based on the ideXlab platform.
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HERC Ubiquitin Ligases in Cancer.
Cancers, 2020Co-Authors: Joan Sala-gaston, Francesc Ventura, Arturo Martinez-martinez, Leonardo Pedrazza, L. Francisco Lorenzo-martín, Ruben Caloto, Xosé R. Bustelo, Jose Luis RosaAbstract:HERC proteins are ubiquitin E3 ligases of the HECT family. The HERC subfamily is composed of six members classified by size into large (HERC1 and HERC2) and small (HERC3–HERC6). HERC family ubiquitin ligases regulate important cellular processes, such as neurodevelopment, DNA damage response, cell proliferation, cell migration, and immune responses. Accumulating evidence also shows that this family plays critical roles in cancer. In this review, we provide an integrated view of the role of these ligases in cancer, highlighting their bivalent functions as either oncogenes or tumor suppressors, depending on the tumor type. We include a discussion of both the molecular mechanisms involved and the potential therapeutic strategies.
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Regulation of the MDM2-p53 pathway by the ubiquitin ligase HERC2.
Molecular oncology, 2019Co-Authors: Jesús García-cano, Ramon Bartrons, Francesc Ventura, Susana Sánchez-tena, Joan Sala-gaston, Agnès Figueras, Francesc Viñals, Jose Luis RosaAbstract:The p53 tumor suppressor protein is a transcription factor that plays a prominent role in protecting cells from malignant transformation. Protein levels of p53 and its transcriptional activity are tightly regulated by the ubiquitin E3 ligase MDM2, the gene expression of which is transcriptionally regulated by p53 in a negative feedback loop. The p53 protein is transcriptionally active as a tetramer, and this oligomerization state is modulated by a complex formed by NEURL4 and the ubiquitin E3 ligase HERC2. Here, we report that MDM2 forms a complex with oligomeric p53, HERC2, and NEURL4. HERC2 knockdown results in a decline in MDM2 protein levels without affecting its protein stability, as it reduces its mRNA expression by inhibition of its promoter activation. DNA damage induced by bleomycin dissociates MDM2 from the p53/HERC2/NEURL4 complex and increases the phosphorylation and acetylation of oligomeric p53 bound to HERC2 and NEURL4. Moreover, the MDM2 promoter, which contains p53-response elements, competes with HERC2 for binding of oligomeric, phosphorylated and acetylated p53. We integrate these findings in a model showing the pivotal role of HERC2 in p53-MDM2 loop regulation. Altogether, these new insights in p53 pathway regulation are of great interest in cancer and may provide new therapeutic targets.
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NEURL4 regulates the transcriptional activity of tumor suppressor protein p53 by modulating its oligomerization
Oncotarget, 2017Co-Authors: Monica Cubillos-rojas, Ramon Bartrons, Francesc Ventura, Taiane Schneider, Jose Luis RosaAbstract:// Monica Cubillos-Rojas 1 , Taiane Schneider 1 , Ramon Bartrons 1 , Francesc Ventura 1 and Jose Luis Rosa 1 1 Departament de Ciencies Fisiologiques, Campus de Bellvitge, Institut d’Investigacio Biomedica de Bellvitge (IDIBELL), Universitat de Barcelona, L’Hospitalet de Llobregat, Barcelona E-08907, Spain Correspondence to: Jose Luis Rosa, email: joseluisrosa@ub.edu Keywords: NEURL4, p53, oligomerization, tetramerization, HERC2 Received: December 28, 2016 Accepted: May 22, 2017 Published: June 27, 2017 ABSTRACT p53 is a transcription factor that regulates important cellular processes related to tumor suppression, including induction of senescence, apoptosis, and DNA repair as well as the inhibition of angiogenesis and cell migration. Therefore, it is critical to understand the molecular mechanism that regulates it. p53 tetramerization is a key step in its activation process and the regulation of this oligomerization, an important control point. The E3 ubiquitin ligase HERC2 controls the p53 transcriptional activity by regulation of its oligomerization state. HERC2-interacting proteins such as the adaptor-like protein with six neuralized domains NEURL4 are also candidates to regulate p53 activity. Here, we demonstrate the existence of an interaction network between NEURL4, HERC2 and p53 proteins. We report a functional interaction between NEURL4 and p53, involving the C-terminal region of p53 and the neuralized domains 3 and 4 of NEURL4. Through this interaction, NEURL4 regulates the transcriptional activity of p53. Thus, NEURL4 depletion reduced the transcriptional activity whereas NEURL4 overexpression increased it. In both cases, p53 stability was not affected. Although NEURL4 may interact with p53 independently of the E3 ubiquitin ligase HERC2, we observed that both proteins are needed to regulate the transcriptional activity of p53. Clonogenic assays confirmed the functional relevance of this interaction observing a decrease in cell growth by NEURL4 overexpression correlated to the increase of cellular cycle inhibitor p21 by p53 activation. Under these conditions, NEURL4 activated p53 oligomerization. All these findings identify NEURL4 as a novel regulator of the p53’s signaling.
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Functional and pathological relevance of HERC family proteins: a decade later
Cellular and Molecular Life Sciences, 2016Co-Authors: Susana Sánchez-tena, Monica Cubillos-rojas, Taiane Schneider, Jose Luis RosaAbstract:The HERC gene family encodes proteins with two characteristic domains in their sequence: the HECT domain and the RCC1-like domain (RLD). In humans, the HERC family comprises six members that can be divided into two groups based on their molecular mass and domain structure. Whereas large HERCs (HERC1 and HERC2) contain one HECT and more than one RLD, small HERCs (HERC3-6) possess single HECT and RLD domains. Accumulating evidence shows the HERC family proteins to be key components of a wide range of cellular functions, including neurodevelopment, DNA damage repair, cell growth and immune response. Considering the significant recent advances made regarding HERC functionality, an updated review summarizing the progress is greatly needed at 10 years since the last HERC review. We provide an integrated view of HERC function and go into detail about its implications for several human diseases such as cancer and neurological disorders.
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The E3 Ubiquitin Protein Ligase HERC2 Modulates the Activity of Tumor Protein p53 by Regulating Its Oligomerization
The Journal of biological chemistry, 2014Co-Authors: Monica Cubillos-rojas, Ramon Bartrons, Francesc Ventura, Fabiola Amair-pinedo, Roser Peiró-jordán, Jose Luis RosaAbstract:The tumor suppressor p53 is a transcription factor that coordinates the cellular response to several kinds of stress. p53 inactivation is an important step in tumor progression. Oligomerization of p53 is critical for its posttranslational modification and its ability to regulate the transcription of target genes necessary to inhibit tumor growth. Here we report that the HECT E3 ubiquitin ligase HERC2 interacts with p53. This interaction involves the CPH domain of HERC2 (a conserved domain within Cul7, PARC, and HERC2 proteins) and the last 43 amino acid residues of p53. Through this interaction, HERC2 regulates p53 activity. RNA interference experiments showed how HERC2 depletion reduces the transcriptional activity of p53 without affecting its stability. This regulation of p53 activity by HERC2 is independent of proteasome or MDM2 activity. Under these conditions, up-regulation of cell growth and increased focus formation were observed, showing the functional relevance of the HERC2-p53 interaction. This interaction was maintained after DNA damage caused by the chemotherapeutic drug bleomycin. In these stressed cells, p53 phosphorylation was not impaired by HERC2 knockdown. Interestingly, p53 mutations that affect its tetramerization domain disrupted the HERC2-p53 interaction, suggesting a role for HERC2 in p53 oligomerization. This regulatory role was shown using cross-linking assays. Thus, the inhibition of p53 activity after HERC2 depletion can be attributed to a reduction in p53 oligomerization. Ectopic expression of HERC2 (residues 2292–2923) confirmed these observations. Together, these results identify HERC2 as a novel regulator of p53 signaling.
Ji Liao - One of the best experts on this subject based on the ideXlab platform.
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HERC2 usp20 coordinates chk1 activation by modulating claspin stability
Nucleic Acids Research, 2014Co-Authors: Min Zhu, Hongchang Zhao, Ji LiaoAbstract:CLASPIN is an essential mediator in the DNA replication checkpoint, responsible for ATR (ataxia telangiectasia and Rad3-related protein)-dependent activation of CHK1 (checkpoint kinase 1). Here we found a dynamic signaling pathway that regulates CLASPIN turn over. Under unperturbed conditions, the E3 ubiquitin ligase HERC2 regulates the stability of the deubiquitinating enzyme USP20 by promoting ubiquitination-mediated proteasomal degradation. Under replication stress, ATR-mediated phosphorylation of USP20 results in the disassociation of HERC2 from USP20. USP20 in turn deubiquitinates K48-linked-polyubiquitinated CLASPIN, stabilizing CLASPIN and ultimately promoting CHK1 phosphorylation and CHK1-directed checkpoint activation. Inhibition of USP20 expression promotes chromosome instability and xenograft tumor growth. Taken together, our findings demonstrated a novel function of HERC2/USP20 in coordinating CHK1 activation by modulating CLASPIN stability, which ultimately promotes genome stability and suppresses tumor growth.
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HERC2/USP20 coordinates CHK1 activation by modulating CLASPIN stability
Nucleic acids research, 2014Co-Authors: Min Zhu, Hongchang Zhao, Ji LiaoAbstract:CLASPIN is an essential mediator in the DNA replication checkpoint, responsible for ATR (ataxia telangiectasia and Rad3-related protein)-dependent activation of CHK1 (checkpoint kinase 1). Here we found a dynamic signaling pathway that regulates CLASPIN turn over. Under unperturbed conditions, the E3 ubiquitin ligase HERC2 regulates the stability of the deubiquitinating enzyme USP20 by promoting ubiquitination-mediated proteasomal degradation. Under replication stress, ATR-mediated phosphorylation of USP20 results in the disassociation of HERC2 from USP20. USP20 in turn deubiquitinates K48-linked-polyubiquitinated CLASPIN, stabilizing CLASPIN and ultimately promoting CHK1 phosphorylation and CHK1-directed checkpoint activation. Inhibition of USP20 expression promotes chromosome instability and xenograft tumor growth. Taken together, our findings demonstrated a novel function of HERC2/USP20 in coordinating CHK1 activation by modulating CLASPIN stability, which ultimately promotes genome stability and suppresses tumor growth.
Monica Cubillos-rojas - One of the best experts on this subject based on the ideXlab platform.
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NEURL4 regulates the transcriptional activity of tumor suppressor protein p53 by modulating its oligomerization
Oncotarget, 2017Co-Authors: Monica Cubillos-rojas, Ramon Bartrons, Francesc Ventura, Taiane Schneider, Jose Luis RosaAbstract:// Monica Cubillos-Rojas 1 , Taiane Schneider 1 , Ramon Bartrons 1 , Francesc Ventura 1 and Jose Luis Rosa 1 1 Departament de Ciencies Fisiologiques, Campus de Bellvitge, Institut d’Investigacio Biomedica de Bellvitge (IDIBELL), Universitat de Barcelona, L’Hospitalet de Llobregat, Barcelona E-08907, Spain Correspondence to: Jose Luis Rosa, email: joseluisrosa@ub.edu Keywords: NEURL4, p53, oligomerization, tetramerization, HERC2 Received: December 28, 2016 Accepted: May 22, 2017 Published: June 27, 2017 ABSTRACT p53 is a transcription factor that regulates important cellular processes related to tumor suppression, including induction of senescence, apoptosis, and DNA repair as well as the inhibition of angiogenesis and cell migration. Therefore, it is critical to understand the molecular mechanism that regulates it. p53 tetramerization is a key step in its activation process and the regulation of this oligomerization, an important control point. The E3 ubiquitin ligase HERC2 controls the p53 transcriptional activity by regulation of its oligomerization state. HERC2-interacting proteins such as the adaptor-like protein with six neuralized domains NEURL4 are also candidates to regulate p53 activity. Here, we demonstrate the existence of an interaction network between NEURL4, HERC2 and p53 proteins. We report a functional interaction between NEURL4 and p53, involving the C-terminal region of p53 and the neuralized domains 3 and 4 of NEURL4. Through this interaction, NEURL4 regulates the transcriptional activity of p53. Thus, NEURL4 depletion reduced the transcriptional activity whereas NEURL4 overexpression increased it. In both cases, p53 stability was not affected. Although NEURL4 may interact with p53 independently of the E3 ubiquitin ligase HERC2, we observed that both proteins are needed to regulate the transcriptional activity of p53. Clonogenic assays confirmed the functional relevance of this interaction observing a decrease in cell growth by NEURL4 overexpression correlated to the increase of cellular cycle inhibitor p21 by p53 activation. Under these conditions, NEURL4 activated p53 oligomerization. All these findings identify NEURL4 as a novel regulator of the p53’s signaling.
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The HERC2 ubiquitin ligase is essential for embryonic development and regulates motor coordination.
Oncotarget, 2016Co-Authors: Monica Cubillos-rojas, Taiane Schneider, Leonardo Pedrazza, Ouadah Hadjebi, Jarbas Rodrigues De Oliveira, Francina Langa, Jean-louis Guénet, Joan Duran, Josep Maria De Anta, Soledad AlcántaraAbstract:// Monica Cubillos-Rojas 1 , Taiane Schneider 1 , Ouadah Hadjebi 1 , Leonardo Pedrazza 1,2 , Jarbas Rodrigues de Oliveira 2 , Francina Langa 3 , Jean-Louis Guenet 3 , Joan Duran 4 , Josep Maria de Anta 4 , Soledad Alcantara 4 , Rocio Ruiz 5,6 , Eva Maria Perez-Villegas 6 , Francisco J. Aguilar-Montilla 6 , Angel M. Carrion 6 , Jose Angel Armengol 6 , Emma Baple 7 , Andrew H. Crosby 7 , Ramon Bartrons 1 , Francesc Ventura 1 and Jose Luis Rosa 1 1 Departament de Ciencies Fisiologiques, IDIBELL, Campus de Bellvitge, Universitat de Barcelona, L’Hospitalet de Llobregat, Barcelona, Spain 2 Laboratorio de Pesquisa em Biofisica Celular e Inflamacao, Pontificia Universidade Catolica do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil 3 Departement de Biologie du Developpement, Institut Pasteur, Paris, France 4 Departament de Patologia i Terapeutica Experimental, Campus de Bellvitge, Universitat de Barcelona, L’Hospitalet de Llobregat, Barcelona, Spain 5 Departamento de Bioquimica y Biologia Molecular, Facultad de Farmacia, Universidad de Sevilla, Sevilla, Spain 6 Departamento de Fisiologia, Anatomia y Biologia Celular, Universidad Pablo de Olavide, Sevilla, Spain 7 Institute of Biomedical and Clinical Science, University of Exeter Medical School, RILD Wellcome Wolfson Centre, Exeter, UK Correspondence to: Jose Luis Rosa, email: // Keywords : ubiquitin, p53, Angelman syndrome, Purkinje cells, behavioural analysis, Pathology Section Received : June 07, 2016 Accepted : August 01, 2016 Published : August 12, 2016 Abstract A mutation in the HERC2 gene has been linked to a severe neurodevelopmental disorder with similarities to the Angelman syndrome. This gene codifies a protein with ubiquitin ligase activity that regulates the activity of tumor protein p53 and is involved in important cellular processes such as DNA repair, cell cycle, cancer, and iron metabolism. Despite the critical role of HERC2 in these physiological and pathological processes, little is known about its relevance in vivo . Here, we described a mouse with targeted inactivation of the HERC2 gene. Homozygous mice were not viable. Distinct from other ubiquitin ligases that interact with p53, such as MDM2 or MDM4, p53 depletion did not rescue the lethality of homozygous mice. The HERC2 protein levels were reduced by approximately one-half in heterozygous mice. Consequently, HERC2 activities, including ubiquitin ligase and stimulation of p53 activity, were lower in heterozygous mice. A decrease in HERC2 activities was also observed in human skin fibroblasts from individuals with an Angelman-like syndrome that express an unstable mutant protein of HERC2. Behavioural analysis of heterozygous mice identified an impaired motor synchronization with normal neuromuscular function. This effect was not observed in p53 knockout mice, indicating that a mechanism independent of p53 activity is involved. Morphological analysis showed the presence of HERC2 in Purkinje cells and a specific loss of these neurons in the cerebella of heterozygous mice. In these animals, an increase of autophagosomes and lysosomes was observed. Our findings establish a crucial role of HERC2 in embryonic development and motor coordination.
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Functional and pathological relevance of HERC family proteins: a decade later
Cellular and Molecular Life Sciences, 2016Co-Authors: Susana Sánchez-tena, Monica Cubillos-rojas, Taiane Schneider, Jose Luis RosaAbstract:The HERC gene family encodes proteins with two characteristic domains in their sequence: the HECT domain and the RCC1-like domain (RLD). In humans, the HERC family comprises six members that can be divided into two groups based on their molecular mass and domain structure. Whereas large HERCs (HERC1 and HERC2) contain one HECT and more than one RLD, small HERCs (HERC3-6) possess single HECT and RLD domains. Accumulating evidence shows the HERC family proteins to be key components of a wide range of cellular functions, including neurodevelopment, DNA damage repair, cell growth and immune response. Considering the significant recent advances made regarding HERC functionality, an updated review summarizing the progress is greatly needed at 10 years since the last HERC review. We provide an integrated view of HERC function and go into detail about its implications for several human diseases such as cancer and neurological disorders.
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The E3 Ubiquitin Protein Ligase HERC2 Modulates the Activity of Tumor Protein p53 by Regulating Its Oligomerization
The Journal of biological chemistry, 2014Co-Authors: Monica Cubillos-rojas, Ramon Bartrons, Francesc Ventura, Fabiola Amair-pinedo, Roser Peiró-jordán, Jose Luis RosaAbstract:The tumor suppressor p53 is a transcription factor that coordinates the cellular response to several kinds of stress. p53 inactivation is an important step in tumor progression. Oligomerization of p53 is critical for its posttranslational modification and its ability to regulate the transcription of target genes necessary to inhibit tumor growth. Here we report that the HECT E3 ubiquitin ligase HERC2 interacts with p53. This interaction involves the CPH domain of HERC2 (a conserved domain within Cul7, PARC, and HERC2 proteins) and the last 43 amino acid residues of p53. Through this interaction, HERC2 regulates p53 activity. RNA interference experiments showed how HERC2 depletion reduces the transcriptional activity of p53 without affecting its stability. This regulation of p53 activity by HERC2 is independent of proteasome or MDM2 activity. Under these conditions, up-regulation of cell growth and increased focus formation were observed, showing the functional relevance of the HERC2-p53 interaction. This interaction was maintained after DNA damage caused by the chemotherapeutic drug bleomycin. In these stressed cells, p53 phosphorylation was not impaired by HERC2 knockdown. Interestingly, p53 mutations that affect its tetramerization domain disrupted the HERC2-p53 interaction, suggesting a role for HERC2 in p53 oligomerization. This regulatory role was shown using cross-linking assays. Thus, the inhibition of p53 activity after HERC2 depletion can be attributed to a reduction in p53 oligomerization. Ectopic expression of HERC2 (residues 2292–2923) confirmed these observations. Together, these results identify HERC2 as a novel regulator of p53 signaling.
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Mutation of HERC2 causes developmental delay with Angelman-like features
Journal of medical genetics, 2012Co-Authors: Gaurav V. Harlalka, Monica Cubillos-rojas, Emma L. Baple, Harold E. Cross, Simone Kühnle, Konstantin Matentzoglu, Michael A. Patton, Karin Wagner, Roselyn Coblentz, Debra L FordAbstract:Background Deregulation of the activity of the ubiquitin ligase E6AP (UBE3A) is well recognised to contribute to the development of Angelman syndrome (AS). The ubiquitin ligase HERC2, encoded by the HERC2 gene in patients with Angelman/Prader-Willi syndrome, is thought to be a key regulator of E6AP. Methods and results Using a combination of autozygosity mapping and linkage analysis, we studied an autosomal-recessive neurodevelopmental disorder with some phenotypic similarities to AS, found among the Old Order Amish. Our molecular investigation identified a mutation in HERC2 associated with the disease phenotype. We establish that the encoded mutant HERC2 protein has a reduced half-life compared with its wild-type counterpart, which is associated with a significant reduction in HERC2 levels in affected individuals. Conclusions Our data implicate a model in which disruption of HERC2 function relates to a reduction in E6AP activity resulting in neurodevelopmental delay, suggesting a previously unrecognised role of HERC2 in the pathogenesis of AS.
Francesc Ventura - One of the best experts on this subject based on the ideXlab platform.
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HERC Ubiquitin Ligases in Cancer.
Cancers, 2020Co-Authors: Joan Sala-gaston, Francesc Ventura, Arturo Martinez-martinez, Leonardo Pedrazza, L. Francisco Lorenzo-martín, Ruben Caloto, Xosé R. Bustelo, Jose Luis RosaAbstract:HERC proteins are ubiquitin E3 ligases of the HECT family. The HERC subfamily is composed of six members classified by size into large (HERC1 and HERC2) and small (HERC3–HERC6). HERC family ubiquitin ligases regulate important cellular processes, such as neurodevelopment, DNA damage response, cell proliferation, cell migration, and immune responses. Accumulating evidence also shows that this family plays critical roles in cancer. In this review, we provide an integrated view of the role of these ligases in cancer, highlighting their bivalent functions as either oncogenes or tumor suppressors, depending on the tumor type. We include a discussion of both the molecular mechanisms involved and the potential therapeutic strategies.
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Regulation of the MDM2-p53 pathway by the ubiquitin ligase HERC2.
Molecular oncology, 2019Co-Authors: Jesús García-cano, Ramon Bartrons, Francesc Ventura, Susana Sánchez-tena, Joan Sala-gaston, Agnès Figueras, Francesc Viñals, Jose Luis RosaAbstract:The p53 tumor suppressor protein is a transcription factor that plays a prominent role in protecting cells from malignant transformation. Protein levels of p53 and its transcriptional activity are tightly regulated by the ubiquitin E3 ligase MDM2, the gene expression of which is transcriptionally regulated by p53 in a negative feedback loop. The p53 protein is transcriptionally active as a tetramer, and this oligomerization state is modulated by a complex formed by NEURL4 and the ubiquitin E3 ligase HERC2. Here, we report that MDM2 forms a complex with oligomeric p53, HERC2, and NEURL4. HERC2 knockdown results in a decline in MDM2 protein levels without affecting its protein stability, as it reduces its mRNA expression by inhibition of its promoter activation. DNA damage induced by bleomycin dissociates MDM2 from the p53/HERC2/NEURL4 complex and increases the phosphorylation and acetylation of oligomeric p53 bound to HERC2 and NEURL4. Moreover, the MDM2 promoter, which contains p53-response elements, competes with HERC2 for binding of oligomeric, phosphorylated and acetylated p53. We integrate these findings in a model showing the pivotal role of HERC2 in p53-MDM2 loop regulation. Altogether, these new insights in p53 pathway regulation are of great interest in cancer and may provide new therapeutic targets.
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NEURL4 regulates the transcriptional activity of tumor suppressor protein p53 by modulating its oligomerization
Oncotarget, 2017Co-Authors: Monica Cubillos-rojas, Ramon Bartrons, Francesc Ventura, Taiane Schneider, Jose Luis RosaAbstract:// Monica Cubillos-Rojas 1 , Taiane Schneider 1 , Ramon Bartrons 1 , Francesc Ventura 1 and Jose Luis Rosa 1 1 Departament de Ciencies Fisiologiques, Campus de Bellvitge, Institut d’Investigacio Biomedica de Bellvitge (IDIBELL), Universitat de Barcelona, L’Hospitalet de Llobregat, Barcelona E-08907, Spain Correspondence to: Jose Luis Rosa, email: joseluisrosa@ub.edu Keywords: NEURL4, p53, oligomerization, tetramerization, HERC2 Received: December 28, 2016 Accepted: May 22, 2017 Published: June 27, 2017 ABSTRACT p53 is a transcription factor that regulates important cellular processes related to tumor suppression, including induction of senescence, apoptosis, and DNA repair as well as the inhibition of angiogenesis and cell migration. Therefore, it is critical to understand the molecular mechanism that regulates it. p53 tetramerization is a key step in its activation process and the regulation of this oligomerization, an important control point. The E3 ubiquitin ligase HERC2 controls the p53 transcriptional activity by regulation of its oligomerization state. HERC2-interacting proteins such as the adaptor-like protein with six neuralized domains NEURL4 are also candidates to regulate p53 activity. Here, we demonstrate the existence of an interaction network between NEURL4, HERC2 and p53 proteins. We report a functional interaction between NEURL4 and p53, involving the C-terminal region of p53 and the neuralized domains 3 and 4 of NEURL4. Through this interaction, NEURL4 regulates the transcriptional activity of p53. Thus, NEURL4 depletion reduced the transcriptional activity whereas NEURL4 overexpression increased it. In both cases, p53 stability was not affected. Although NEURL4 may interact with p53 independently of the E3 ubiquitin ligase HERC2, we observed that both proteins are needed to regulate the transcriptional activity of p53. Clonogenic assays confirmed the functional relevance of this interaction observing a decrease in cell growth by NEURL4 overexpression correlated to the increase of cellular cycle inhibitor p21 by p53 activation. Under these conditions, NEURL4 activated p53 oligomerization. All these findings identify NEURL4 as a novel regulator of the p53’s signaling.
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The E3 Ubiquitin Protein Ligase HERC2 Modulates the Activity of Tumor Protein p53 by Regulating Its Oligomerization
The Journal of biological chemistry, 2014Co-Authors: Monica Cubillos-rojas, Ramon Bartrons, Francesc Ventura, Fabiola Amair-pinedo, Roser Peiró-jordán, Jose Luis RosaAbstract:The tumor suppressor p53 is a transcription factor that coordinates the cellular response to several kinds of stress. p53 inactivation is an important step in tumor progression. Oligomerization of p53 is critical for its posttranslational modification and its ability to regulate the transcription of target genes necessary to inhibit tumor growth. Here we report that the HECT E3 ubiquitin ligase HERC2 interacts with p53. This interaction involves the CPH domain of HERC2 (a conserved domain within Cul7, PARC, and HERC2 proteins) and the last 43 amino acid residues of p53. Through this interaction, HERC2 regulates p53 activity. RNA interference experiments showed how HERC2 depletion reduces the transcriptional activity of p53 without affecting its stability. This regulation of p53 activity by HERC2 is independent of proteasome or MDM2 activity. Under these conditions, up-regulation of cell growth and increased focus formation were observed, showing the functional relevance of the HERC2-p53 interaction. This interaction was maintained after DNA damage caused by the chemotherapeutic drug bleomycin. In these stressed cells, p53 phosphorylation was not impaired by HERC2 knockdown. Interestingly, p53 mutations that affect its tetramerization domain disrupted the HERC2-p53 interaction, suggesting a role for HERC2 in p53 oligomerization. This regulatory role was shown using cross-linking assays. Thus, the inhibition of p53 activity after HERC2 depletion can be attributed to a reduction in p53 oligomerization. Ectopic expression of HERC2 (residues 2292–2923) confirmed these observations. Together, these results identify HERC2 as a novel regulator of p53 signaling.
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Simultaneous electrophoretic analysis of proteins of very high and low molecular mass using Tris-acetate polyacrylamide gels.
Electrophoresis, 2010Co-Authors: Monica Cubillos-rojas, Ramon Bartrons, Francesc Ventura, Fabiola Amair-pinedo, Irantzu Tato, Jose Luis RosaAbstract:To separate and analyze giant and small proteins in the same electrophoresis gel, we have used a 3-15% polyacrylamide gradient gel containing 2.6% of the crosslinker bisacrylamide and 0.2 M of Tris-acetate buffer (pH 7.0). Samples were prepared in a sample buffer containing lithium dodecyl sulphate and were run in the gel described above using Tris-Tricine-SDS-sodium bisulfite buffer, pH 8.2, as electrophoresis buffer. Here, we show that this system can be successfully used for general applications of SDS-PAGE such as CBB staining and immunoblot. Thus, by using Tris-acetate 3-15% polyacrylamide gels, it is possible to simultaneously analyze proteins, in the mass range of 10-500 kDa, such as HERC1 (532 kDa), HERC2 (528 kDa), mTOR (289 kDa), Clathrin heavy chain (192 kDa), RSK (90 kDa), S6K (70 kDa), beta-actin (42 kDa), Ran (24 kDa) and LC3 (18 kDa). This system is highly sensitive since it allows detection from as low as 10 microg of total protein per lane. Moreover, it has a good resolution, low cost, high reproducibility and allows for analysis of proteins in a wide range of weights within a short period of time. All these features together with the use of a standard electrophoresis apparatus make the Tris-acetate-PAGE system a very helpful tool for protein analysis.
Min Zhu - One of the best experts on this subject based on the ideXlab platform.
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HERC2 usp20 coordinates chk1 activation by modulating claspin stability
Nucleic Acids Research, 2014Co-Authors: Min Zhu, Hongchang Zhao, Ji LiaoAbstract:CLASPIN is an essential mediator in the DNA replication checkpoint, responsible for ATR (ataxia telangiectasia and Rad3-related protein)-dependent activation of CHK1 (checkpoint kinase 1). Here we found a dynamic signaling pathway that regulates CLASPIN turn over. Under unperturbed conditions, the E3 ubiquitin ligase HERC2 regulates the stability of the deubiquitinating enzyme USP20 by promoting ubiquitination-mediated proteasomal degradation. Under replication stress, ATR-mediated phosphorylation of USP20 results in the disassociation of HERC2 from USP20. USP20 in turn deubiquitinates K48-linked-polyubiquitinated CLASPIN, stabilizing CLASPIN and ultimately promoting CHK1 phosphorylation and CHK1-directed checkpoint activation. Inhibition of USP20 expression promotes chromosome instability and xenograft tumor growth. Taken together, our findings demonstrated a novel function of HERC2/USP20 in coordinating CHK1 activation by modulating CLASPIN stability, which ultimately promotes genome stability and suppresses tumor growth.
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HERC2/USP20 coordinates CHK1 activation by modulating CLASPIN stability
Nucleic acids research, 2014Co-Authors: Min Zhu, Hongchang Zhao, Ji LiaoAbstract:CLASPIN is an essential mediator in the DNA replication checkpoint, responsible for ATR (ataxia telangiectasia and Rad3-related protein)-dependent activation of CHK1 (checkpoint kinase 1). Here we found a dynamic signaling pathway that regulates CLASPIN turn over. Under unperturbed conditions, the E3 ubiquitin ligase HERC2 regulates the stability of the deubiquitinating enzyme USP20 by promoting ubiquitination-mediated proteasomal degradation. Under replication stress, ATR-mediated phosphorylation of USP20 results in the disassociation of HERC2 from USP20. USP20 in turn deubiquitinates K48-linked-polyubiquitinated CLASPIN, stabilizing CLASPIN and ultimately promoting CHK1 phosphorylation and CHK1-directed checkpoint activation. Inhibition of USP20 expression promotes chromosome instability and xenograft tumor growth. Taken together, our findings demonstrated a novel function of HERC2/USP20 in coordinating CHK1 activation by modulating CLASPIN stability, which ultimately promotes genome stability and suppresses tumor growth.