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1.
Adv Exp Med Biol ; 1233: 263-277, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32274761

RESUMO

SUMO is a ubiquitin-like protein that covalently binds to lysine residues of target proteins and regulates many biological processes such as protein subcellular localization or stability, transcription, DNA repair, innate immunity, or antiviral defense. SUMO has a critical role in the signaling pathway governing type I interferon (IFN) production, and among the SUMOylation substrates are many IFN-induced proteins. The overall effect of IFN is increasing global SUMOylation, pointing to SUMO as part of the antiviral stress response. Viral agents have developed different mechanisms to counteract the antiviral activities exerted by SUMO, and some viruses have evolved to exploit the host SUMOylation machinery to modify their own proteins. The exploitation of SUMO has been mainly linked to nuclear replicating viruses due to the predominant nuclear localization of SUMO proteins and enzymes involved in SUMOylation. However, SUMOylation of numerous viral proteins encoded by RNA viruses replicating at the cytoplasm has been lately described. Whether nuclear localization of these viral proteins is required for their SUMOylation is unclear. Here, we summarize the studies on exploitation of SUMOylation by cytoplasmic RNA viruses and discuss about the requirement for nuclear localization of their proteins.


Assuntos
Citoplasma/virologia , Vírus de RNA/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Proteínas Virais/metabolismo , Núcleo Celular/metabolismo , Humanos , Sumoilação
2.
Nat Commun ; 11(1): 834, 2020 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-32047143

RESUMO

The protein inhibitor of activated STAT1 (PIAS1) is an E3 SUMO ligase that plays important roles in various cellular pathways. Increasing evidence shows that PIAS1 is overexpressed in various human malignancies, including prostate and lung cancers. Here we used quantitative SUMO proteomics to identify potential substrates of PIAS1 in a system-wide manner. We identified 983 SUMO sites on 544 proteins, of which 62 proteins were assigned as putative PIAS1 substrates. In particular, vimentin (VIM), a type III intermediate filament protein involved in cytoskeleton organization and cell motility, was SUMOylated by PIAS1 at Lys-439 and Lys-445 residues. VIM SUMOylation was necessary for its dynamic disassembly and cells expressing a non-SUMOylatable VIM mutant showed a reduced level of migration. Our approach not only enables the identification of E3 SUMO ligase substrates but also yields valuable biological insights into the unsuspected role of PIAS1 and VIM SUMOylation on cell motility.


Assuntos
Movimento Celular/fisiologia , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteômica , Proteína SUMO-1/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Proliferação de Células , Proteínas do Citoesqueleto/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Células HeLa , Humanos , Proteínas Inibidoras de STAT Ativados/genética , Mapas de Interação de Proteínas , Proteína SUMO-1/genética , Análise de Sequência de Proteína , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Sumoilação , Ubiquitina-Proteína Ligases/metabolismo , Vimentina/metabolismo
3.
Mol Cell ; 77(3): 556-570.e6, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-31901446

RESUMO

Regulation of transcription is the main mechanism responsible for precise control of gene expression. Whereas the majority of transcriptional regulation is mediated by DNA-binding transcription factors that bind to regulatory gene regions, an elegant alternative strategy employs small RNA guides, Piwi-interacting RNAs (piRNAs) to identify targets of transcriptional repression. Here, we show that in Drosophila the small ubiquitin-like protein SUMO and the SUMO E3 ligase Su(var)2-10 are required for piRNA-guided deposition of repressive chromatin marks and transcriptional silencing of piRNA targets. Su(var)2-10 links the piRNA-guided target recognition complex to the silencing effector by binding the piRNA/Piwi complex and inducing SUMO-dependent recruitment of the SetDB1/Wde histone methyltransferase effector. We propose that in Drosophila, the nuclear piRNA pathway has co-opted a conserved mechanism of SUMO-dependent recruitment of the SetDB1/Wde chromatin modifier to confer repression of genomic parasites.


Assuntos
Proteínas de Drosophila/metabolismo , Proteínas Inibidoras de STAT Ativados/metabolismo , RNA Interferente Pequeno/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Animais , Proteínas Argonauta/metabolismo , Núcleo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Elementos de DNA Transponíveis , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica/genética , Inativação Gênica/fisiologia , Ligação Proteica , Proteínas Inibidoras de STAT Ativados/genética , RNA Interferente Pequeno/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Transcrição Genética/genética
4.
Mol Cell ; 77(3): 571-585.e4, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-31901448

RESUMO

Сhromatin is critical for genome compaction and gene expression. On a coarse scale, the genome is divided into euchromatin, which harbors the majority of genes and is enriched in active chromatin marks, and heterochromatin, which is gene-poor but repeat-rich. The conserved molecular hallmark of heterochromatin is the H3K9me3 modification, which is associated with gene silencing. We found that in Drosophila, deposition of most of the H3K9me3 mark depends on SUMO and the SUMO ligase Su(var)2-10, which recruits the histone methyltransferase complex SetDB1/Wde. In addition to repressing repeats, H3K9me3 influences expression of both hetero- and euchromatic host genes. High H3K9me3 levels in heterochromatin are required to suppress spurious transcription and ensure proper gene expression. In euchromatin, a set of conserved genes is repressed by Su(var)2-10/SetDB1-induced H3K9 trimethylation, ensuring tissue-specific gene expression. Several components of heterochromatin are themselves repressed by this pathway, providing a negative feedback mechanism to ensure chromatin homeostasis.


Assuntos
Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica/genética , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Animais , Proteínas Cromossômicas não Histona/metabolismo , Metilação de DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Eucromatina/metabolismo , Retroalimentação Fisiológica , Expressão Gênica/genética , Inativação Gênica/fisiologia , Heterocromatina/genética , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Histonas/metabolismo , Ligases/genética , Metiltransferases/genética , Proteínas Inibidoras de STAT Ativados/genética , Proteínas Repressoras/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética
5.
Nat Commun ; 11(1): 240, 2020 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-31932588

RESUMO

Farnesoid X receptor (FXR) is a promising target for nonalcoholic steatohepatitis (NASH) and fibrosis. Although various FXR agonists have shown anti-fibrotic effects in diverse preclinical animal models, the response rate and efficacies in clinical trials were not optimum. Here we report that prophylactic but not therapeutic administration of obeticholic acid (OCA) prevents hepatic stellate cell (HSC) activation and fibrogenesis. Activated HSCs show limited response to OCA and other FXR agonists due to enhanced FXR SUMOylation. SUMOylation inhibitors rescue FXR signaling and thereby increasing the efficacy of OCA against HSC activation and fibrosis. FXR upregulates Perilipin-1, a direct target gene of FXR, to stabilize lipid droplets and thereby prevent HSC activation. Therapeutic coadministration of OCA and SUMOylation inhibitors drastically impedes liver fibrosis induced by CCl4, bile duct ligation, and more importantly NASH. In conclusion, we propose a promising therapeutic approach by combining SUMOylation inhibitors and FXR agonists for liver fibrosis.


Assuntos
Cirrose Hepática/tratamento farmacológico , Receptores Citoplasmáticos e Nucleares/agonistas , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/antagonistas & inibidores , Sumoilação , Animais , Células Cultivadas , Ácido Quenodesoxicólico/administração & dosagem , Ácido Quenodesoxicólico/análogos & derivados , Ácido Quenodesoxicólico/farmacologia , Modelos Animais de Doenças , Quimioterapia Combinada , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/patologia , Humanos , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/patologia , Cirrose Hepática/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/patologia , Perilipina-1/genética , Perilipina-1/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ativação Transcricional/efeitos dos fármacos , Resultado do Tratamento
6.
Biochem Soc Trans ; 48(1): 123-135, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31872228

RESUMO

The small ubiquitin-like modifier (SUMO) is a post-translational modifier that can regulate the function of hundreds of proteins inside the cell. SUMO belongs to the ubiquitin-like family of proteins that can be attached to target proteins by a dedicated enzymatic cascade pathway formed by E1, E2 and E3 enzymes. SUMOylation is involved in many cellular pathways, having in most instances essential roles for their correct function. In this review, we want to highlight the latest research on the molecular mechanisms that lead to the formation of the isopeptidic bond between the lysine substrate and the C-terminus of SUMO. In particular, we will focus on the recent discoveries on the catalytic function of the SUMO E3 ligases revealed by structural and biochemical approaches. Also, we will discuss important questions regarding specificity in SUMO conjugation, which it still remains as a major issue due to the small number of SUMO E3 ligases discovered so far, in contrast with the large number of SUMO conjugated proteins in the cell.


Assuntos
Processamento de Proteína Pós-Traducional , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Domínio Catalítico , Humanos , Modelos Moleculares , Especificidade por Substrato , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
7.
PLoS Pathog ; 15(12): e1008174, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31830143

RESUMO

Primary effusion lymphoma (PEL) is an aggressive B-cell malignancy without effective treatment, and caused by the infection of Kaposi's sarcoma-associated herpesvirus (KSHV), predominantly in its latent form. Previously we showed that the SUMO2-interacting motif within the viral latency-associated nuclear antigen (LANASIM) is essential for establishment and maintenance of KSHV latency. Here, we developed a luciferase based live-cell reporter system to screen inhibitors selectively targeting the interaction between LANASIM and SUMO2. Cambogin, a bioactive natural product isolated from the Garcinia genus (a traditional herbal medicine used for cancer treatment), was obtained from the reporter system screening to efficiently inhibit the association of SUMO2 with LANASIM, in turn reducing the viral episome DNA copy number for establishment and maintenance of KSHV latent infection at a low concentration (nM). Importantly, Cambogin treatments not only specifically inhibited proliferation of KSHV-latently infected cells in vitro, but also induced regression of PEL tumors in a xenograft mouse model. This study has identified Cambogin as a novel therapeutic agent for treating PEL as well as eliminating persistent infection of oncogenic herpesvirus.


Assuntos
Antineoplásicos/farmacologia , Linfoma de Efusão Primária/virologia , Terpenos/farmacologia , Latência Viral/efeitos dos fármacos , Animais , Antígenos Virais/efeitos dos fármacos , Antígenos Virais/metabolismo , Células HEK293 , Infecções por Herpesviridae/metabolismo , Herpesvirus Humano 8 , Humanos , Camundongos , Proteínas Nucleares/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Extratos Vegetais/farmacologia , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/efeitos dos fármacos , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Nat Commun ; 10(1): 4452, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31575873

RESUMO

The covalent attachment of the cytokine-inducible ubiquitin-like modifier HLA-F adjacent transcript 10 (FAT10) to hundreds of substrate proteins leads to their rapid degradation by the 26 S proteasome independently of ubiquitylation. Here, we identify another function of FAT10, showing that it interferes with the activation of SUMO1/2/3 in vitro and down-regulates SUMO conjugation and the SUMO-dependent formation of promyelocytic leukemia protein (PML) bodies in cells. Mechanistically, we show that FAT10 directly binds to and impedes the activity of the heterodimeric SUMO E1 activating enzyme AOS1/UBA2 by competing very efficiently with SUMO for activation and thioester formation. Nevertheless, activation of FAT10 by AOS1/UBA2 does not lead to covalent conjugation of FAT10 with substrate proteins which relies on its cognate E1 enzyme UBA6. Hence, we report that one ubiquitin-like modifier (FAT10) inhibits the conjugation and function of another ubiquitin-like modifier (SUMO) by impairing its activation.


Assuntos
Proteína da Leucemia Promielocítica/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Proteína SUMO-1/metabolismo , Ubiquitinas/metabolismo , Regulação para Baixo , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Recombinantes , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Fatores de Transcrição/metabolismo , Enzimas Ativadoras de Ubiquitina/genética , Enzimas Ativadoras de Ubiquitina/metabolismo , Ubiquitinação , Ubiquitinas/genética
9.
Nat Commun ; 10(1): 3987, 2019 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-31485003

RESUMO

In contrast to our extensive knowledge on ubiquitin polymer signaling, we are severely limited in our understanding of poly-SUMO signaling. We set out to identify substrates conjugated to SUMO polymers, using knockdown of the poly-SUMO2/3 protease SENP6. We identify over 180 SENP6 regulated proteins that represent highly interconnected functional groups of proteins including the constitutive centromere-associated network (CCAN), the CENP-A loading factors Mis18BP1 and Mis18A and DNA damage response factors. Our results indicate a striking protein group de-modification by SENP6. SENP6 deficient cells are severely compromised for proliferation, accumulate in G2/M and frequently form micronuclei. Accumulation of CENP-T, CENP-W and CENP-A to centromeres is impaired in the absence of SENP6. Surprisingly, the increase of SUMO chains does not lead to ubiquitin-dependent proteasomal degradation of the CCAN subunits. Our results indicate that SUMO polymers can act in a proteolysis-independent manner and consequently, have a more diverse signaling function than previously expected.


Assuntos
Centrômero/metabolismo , Cisteína Endopeptidases/metabolismo , Mapas de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Linhagem Celular Tumoral , Proliferação de Células/genética , Proteína Centromérica A/genética , Proteína Centromérica A/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Cisteína Endopeptidases/genética , Células HEK293 , Células HeLa , Humanos , Interferência de RNA , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Ubiquitinas/genética , Ubiquitinas/metabolismo
10.
J Chem Theory Comput ; 15(11): 6403-6410, 2019 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-31525924

RESUMO

Interaction of the small ubiquitin-related modifier (SUMO) and peptides containing a SUMO-interacting motif (SIM) has attracted a lot of interest in recent years, yet their structural properties and relationships between the composition of the peptide and binding free energy are not completely understood. We perform molecular dynamics simulations of the complex formed by SUMO and a peptide containing the tight-binding SIM of the protein inhibitor of activated STAT. The calculated standard binding free energy of -5.06 kcal/mol is in reasonable agreement with the experimental value of -6.54 kcal/mol. Experimental results for complexes formed by SUMO and SIM dimers indicate the existence of a parallel and an antiparallel binding mode for similar SIM peptides. We find that the parallel binding mode is highly favored in the present case. Furthermore, the simulations show that residues neighboring the SIM core motif contribute strongly to the binding energy. Structurally, the complex shows differences from the picture in which the SIM core motif lies deep in the SUMO binding groove. This also supports the idea that neighboring residues play an important role in binding.


Assuntos
Peptídeos/química , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Motivos de Aminoácidos , Dimerização , Simulação de Dinâmica Molecular , Peptídeos/metabolismo , Ligação Proteica , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Termodinâmica
11.
Nat Commun ; 10(1): 3678, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31417085

RESUMO

Modification with SUMO regulates many eukaryotic proteins. Down-regulation of sumoylated forms of proteins involves either their desumoylation, and hence recycling of the unmodified form, or their proteolytic targeting by ubiquitin ligases that recognize their SUMO modification (termed STUbL or ULS). STUbL enzymes such as Uls1 and Slx5-Slx8 in budding yeast or RNF4 and Arkadia/RNF111 in humans bear multiple SUMO interaction motifs to recognize substrates carrying poly-SUMO chains. Using yeast as experimental system and isothermal titration calorimetry, we here show that Arkadia specifically selects substrates carrying SUMO1-capped SUMO2/3 hybrid conjugates and targets them for proteasomal degradation. Our data suggest that a SUMO1-specific binding site in Arkadia with sequence similarity to a SUMO1-binding site in DPP9 is required for targeting endogenous hybrid SUMO conjugates and PML nuclear bodies in human cells. We thus characterize Arkadia as a STUbL with a preference for substrate proteins marked with distinct hybrid SUMO chains.


Assuntos
Proteínas Nucleares/metabolismo , Sumoilação , Ubiquitina-Proteína Ligases/metabolismo , Escherichia coli , Células HeLa , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proteína SUMO-1/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ubiquitinação , Ubiquitinas/metabolismo
12.
Methods Mol Biol ; 1934: 223-233, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31256382

RESUMO

Protein regulation by reversible attachment of SUMO (small ubiquitin-related modifier) plays an important role in several cellular processes such as transcriptional regulation, nucleo-cytoplasmic transport, cell-cycle progression, meiosis, and DNA repair. However, most sumoylated proteins are of marginal abundance at steady state levels, which is due to strict regulation and/or rapid turnover of modification and de-modification. Consequently, analysis of protein sumoylation in vivo is very challenging. Nonetheless, a novel method was established that allows detection of sumoylated proteins at endogenous levels from vertebrate cells and tissues. This approach involves the enrichment of sumoylated proteins by immunoprecipitation followed by peptide elution. After endogenous substrate sumoylation is verified, addressing its functional consequences is the next logical step. This requires SUMO site mapping that benefits from larger quantities of modified protein. Here, we shortly describe strategies to achieve efficient in vitro sumoylation of many substrates.


Assuntos
Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Cromatografia de Afinidade , Humanos , Processamento de Proteína Pós-Traducional , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/isolamento & purificação , Especificidade por Substrato , Sumoilação
13.
Plant Physiol Biochem ; 141: 206-214, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31176880

RESUMO

SUMOylation is an important protein modification that regulates the properties of substrate proteins in a variety of cellular processes. SUMOylation is catalyzed via a cascade of enzymes and is usually stimulated by SUMO E3 ligases. However, the molecular functions and regulatory mechanisms of SUMOylation in forage crops are unknown. Here, we isolated and functionally characterized DiMMS21, a homolog of the Arabidopsis thaliana SUMO ligase AtMMS21, from the forage legume Desmodium intortum. DiMMS21 is expressed ubiquitously in various D. intortum organs and its encoded protein is found in the cytoplasm and nucleus. Bioinformatics analysis indicated that DiMMS21 contains a conserved SP-RING domain that is required for its activity. Biochemical evidence supports the notion that this protein is a functional SUMO ligase. When expressed in an Arabidopsis mms21 mutant, DiMMS21 completely rescued the defects in root, leaf, and silique development. The results from cotyledon greening and marker gene expression suggested that DiMMS21 can only partially complements the role of AtMMS21 in abscisic acid (ABA) responses. In summary, we characterized the molecular features of DiMMS21 and uncovered potential roles of this SUMO ligase in development and ABA responses, increasing our understanding on the function of SUMOylation in forage crops.


Assuntos
Fabaceae/enzimologia , Regulação da Expressão Gênica de Plantas , Ligases/metabolismo , Proteínas de Plantas/metabolismo , Ácido Abscísico/metabolismo , Arabidopsis/enzimologia , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Biologia Computacional , Citoplasma/metabolismo , DNA Complementar/metabolismo , Perfilação da Expressão Gênica , Mutação , Filogenia , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Sementes/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo
14.
Plant Cell Physiol ; 60(9): 2026-2039, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31076779

RESUMO

The tRNA modification at the wobble position of Lys, Glu and Gln (wobbleU* modification) is responsible for the fine-tuning of protein translation efficiency and translation rate. This modification influences organism function in accordance with growth and environmental changes. However, the effects of wobbleU* modification at the cellular, tissue, or individual level have not yet been elucidated. In this study, we show that sulfur modification of wobbleU* of the tRNAs affects leaf development in Arabidopsis thaliana. The sulfur modification was impaired in the two wobbleU*-modification mutants: the URM1-like protein-defective mutant and the Elongator complex-defective mutants. Analyses of the mutant phenotypes revealed that the deficiency in the wobbleU* modification increased the airspaces in the leaves and the leaf size without affecting the number and the area of palisade mesophyll cells. On the other hand, both mutants exhibited increased number of leaf epidermal pavement cells but with reduced cell size. The deficiency in the wobbleU* modification also delayed the initiation of the endoreduplication processes of mesophyll cells. The phenotype of ASYMMETRIC LEAVES2-defective mutant was enhanced in the Elongator-defective mutants, while it was unchanged in the URM1-like protein-defective mutant. Collectively, the findings of this study suggest that the tRNA wobbleU* modification plays an important role in leaf morphogenesis by balancing the development between epidermal and mesophyll tissues.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Processamento Pós-Transcricional do RNA , RNA de Transferência/metabolismo , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , Vias Biossintéticas , Células do Mesofilo/metabolismo , Mutação , Fenótipo , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , RNA de Plantas/genética , RNA de Plantas/metabolismo , RNA de Transferência/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Enxofre/metabolismo
15.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 5): 332-339, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31045562

RESUMO

SUMOylation is a post-translational modification in which a small ubiquitin-like molecule (SUMO) is appended to substrate proteins and is known to influence myriads of biological processes. A delicate interplay between several families of SUMOylation proteins and their substrates ensures the proper level of SUMOylation required for normal cell function. Among the SUMO proteins, SUMO2 is known to form mono-SUMOylated proteins and engage in poly-SUMO chain formation, while sentrin-specific protease 1 (SENP1) is a key enzyme in regulating both events. Determination of the SENP1-SUMO2 interaction is therefore necessary to better understand SUMOylation. In this regard, the current paper reports the noncovalent structure of SENP1 in complex with SUMO2, which was refined to a resolution of 2.62 Šwith R and Rfree values of 22.92% and 27.66%, respectively. The structure shows that SENP1-SUMO2 complex formation is driven largely by polar interactions and limited hydrophobic contacts. The essential C-terminal motif (QQTGG) of SUMO2 is stabilized by a number of specific bonding interactions that enable it to protrude into the catalytic triad of SENP1 and provide the arrangement necessary for maturation of SUMO and deSUMOylation activity. Overall, the structure shows a number of structural details that pinpoint the basis of SENP1-SUMO2 complex formation.


Assuntos
Cisteína Endopeptidases/química , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Homologia Estrutural de Proteína , Termodinâmica
16.
Virology ; 531: 269-279, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30974383

RESUMO

The study evaluated the effects of nucleoprotein viral and the infectious virus in SHK-1 cells. The results show a strong respiratory burst activation and the induction of p47phox, SOD, GLURED, and apoptotic genes. Additionally, the cells alter the profile of SUMOylated proteins by the effect of transfection and infection experiments. In silico analyses show a set of structural motifs in NP susceptible of post-translational modification by the SUMO protein. Interestingly, the inhibition of the NADPH oxidase complex blocked the production of reactive oxygen species and the high level of cellular ROS due to the nucleoprotein and the ISAv. At the same time, the blocking of the p38MAPK signaling pathway and the use of Aristotelia chilensis, decreased viral progeny production. These results suggest that the NP triggers a strong production of ROS and modifying the post-translational profile mediated by SUMO-2/3, a phenomenon that favors the production of new virions.


Assuntos
Doenças dos Peixes/metabolismo , Proteínas de Peixes/metabolismo , Isavirus/metabolismo , NADPH Oxidases/metabolismo , Nucleoproteínas/metabolismo , Infecções por Orthomyxoviridae/veterinária , Estresse Oxidativo , Proteínas Virais/metabolismo , Animais , Doenças dos Peixes/genética , Doenças dos Peixes/virologia , Proteínas de Peixes/genética , Interações Hospedeiro-Patógeno , Isavirus/genética , NADPH Oxidases/genética , Nucleoproteínas/genética , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/virologia , Espécies Reativas de Oxigênio/metabolismo , Explosão Respiratória , Salmão , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Proteínas Virais/genética , Vírion/genética , Vírion/metabolismo
17.
Life Sci Alliance ; 2(2)2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30979826

RESUMO

We have shown previously that the process of replication machinery (replisome) disassembly at the termination of DNA replication forks in the S-phase is driven through polyubiquitylation of one of the replicative helicase subunits (Mcm7) by Cul2LRR1 ubiquitin ligase. Interestingly, upon inhibition of this pathway in Caenorhabditis elegans embryos, the replisomes retained on chromatin were unloaded in the subsequent mitosis. Here, we show that this mitotic replisome disassembly pathway exists in Xenopus laevis egg extract and we determine the first elements of its regulation. The mitotic disassembly pathway depends on the formation of K6- and K63-linked ubiquitin chains on Mcm7 by TRAIP ubiquitin ligase and the activity of p97/VCP protein segregase. Unlike in lower eukaryotes, however, it does not require SUMO modifications. Importantly, we also show that this process can remove all replisomes from mitotic chromatin, including stalled ones, which indicates a wide application for this pathway over being just a "backup" for terminated replisomes. Finally, we characterise the composition of the replisome retained on chromatin until mitosis.


Assuntos
Replicação do DNA , Mitose/genética , Ubiquitina-Proteína Ligases/metabolismo , Xenopus laevis/genética , Animais , Afidicolina/farmacologia , Cafeína/farmacologia , Cromatina/metabolismo , Ciclinas/metabolismo , Ciclopentanos/farmacologia , Feminino , Masculino , Componente 7 do Complexo de Manutenção de Minicromossomo/metabolismo , Óvulo/efeitos dos fármacos , Pirimidinas/farmacologia , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Espermatozoides/efeitos dos fármacos , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação/fisiologia , Proteína com Valosina/metabolismo , Proteínas de Xenopus/metabolismo
18.
Biochim Biophys Acta Mol Cell Res ; 1866(8): 1282-1297, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30935967

RESUMO

Fas-associated factor 1 (FAF1) was originally isolated as a Fas-associated factor and was subsequently found to interact with numerous other proteins that are involved in various cellular events including Fas-mediated apoptosis, nuclear factor (NF)-κB, Wnt/ß-catenin, and transforming growth factor (TGF)-ß signaling pathways, mineralocorticoid receptor (MR)-mediated transactivation, and ubiquitin-dependent processes. Herein, we defined two small ubiquitin-like modifier (SUMO)-interacting motifs (SIMs) within FAF1 and demonstrated to be crucial for transcriptional modulation of the MR. Our study demonstrated that the SIMs of FAF1 do not play a significant role in regulating its subcellular localization, Fas-mediated apoptosis, or NF-κB or Wnt/ß-catenin pathways. Remarkably, FAF1 interacts with the sumoylated MR and represses aldosterone-activated MR transactivation in a SIM-dependent manner. Moreover, silencing of endogenous FAF1 in cells resulted in an increase in the induction of MR target genes by aldosterone, indicating that FAF1 functions as an MR co-repressor. We further provide evidence to suggest that the mechanisms of FAF1/SIM-mediated MR transrepression involve inhibition of MR N/C interactions and promotion of MR polyubiquitination and degradation. Sumoylation has been linked to impacting of repressive properties on several transcription factors and cofactors. Our findings therefore provide mechanistic insights underlying SUMO-dependent transcriptional repression of the MR.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Receptores de Mineralocorticoides/metabolismo , Transcrição Genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Aldosterona/farmacologia , Motivos de Aminoácidos , Animais , Células COS , Células HEK293 , Células HeLa , Humanos , Transporte Proteico , Receptores de Mineralocorticoides/agonistas , Receptores de Mineralocorticoides/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação/efeitos dos fármacos , Sumoilação/genética
19.
Int J Mol Sci ; 20(8)2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30991648

RESUMO

Protein SUMOylation is a dynamic post-translational modification which is involved in a diverse set of physiologic processes throughout the cell. Of note, SUMOylation also plays a role in the pathobiology of a myriad of cancers, one of which is glioblastoma (GBM). Accordingly, herein, we review core aspects of SUMOylation as it relates to GBM and in so doing highlight putative methods/modalities capable of therapeutically engaging the pathway for treatment of this deadly neoplasm.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Sumoilação/efeitos dos fármacos , Animais , Antineoplásicos/uso terapêutico , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Humanos , Terapia de Alvo Molecular/métodos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo
20.
Biomolecules ; 9(4)2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30999631

RESUMO

Death Associated Protein Kinase 1 (DAPK1) is an important signaling kinase mediating the biological effect of multiple natural biomolecules such as IFN-γ, TNF-α, curcumin, etc. DAPK1 is degraded through both ubiquitin-proteasomal and lysosomal degradation pathways. To investigate the crosstalk between these two DAPK1 degradation pathways, we carried out a screen using a set of ubiquitin E2 siRNAs at the presence of Tuberous Sclerous 2 (TSC2) and identified that the small ubiquitin-like molecule (SUMO) pathway is able to regulate the protein levels of DAPK1. Inhibition of the SUMO pathway enhanced DAPK1 protein levels and the minimum domain of DAPK1 protein required for this regulation is the kinase domain, suggesting that the SUMO pathway regulates DAPK1 protein levels independent of TSC2. Suppression of the SUMO pathway did not enhance DAPK1 protein stability. In addition, mutation of the potential SUMO conjugation sites on DAPK1 kinase domain did not alter its protein stability or response to SUMO pathway inhibition. These data suggested that the SUMO pathway does not regulate DAPK1 protein degradation. The exact molecular mechanism underlying this regulation is yet to be discovered.


Assuntos
Proteínas Quinases Associadas com Morte Celular/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sítios de Ligação , Proteínas Quinases Associadas com Morte Celular/química , Proteínas Quinases Associadas com Morte Celular/metabolismo , Células HCT116 , Células HEK293 , Humanos , Ligação Proteica , Proteólise , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Sumoilação , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo
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