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1.
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
2.
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
3.
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
4.
BMC Plant Biol ; 19(1): 593, 2019 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-31884953

RESUMO

BACKGROUND: Posttranslational modification of proteins by small ubiquitin like modifier (SUMO) proteins play an important role during the developmental process and in response to abiotic stresses in plants. However, little is known about SUMOylation in peanut (Arachis hypogaea L.), one of the world's major food legume crops. In this study, we characterized the SUMOylation system from the diploid progenitor genomes of peanut, Arachis duranensis (AA) and Arachis ipaensis (BB). RESULTS: Genome-wide analysis revealed the presence of 40 SUMO system genes in A. duranensis and A. ipaensis. Our results showed that peanut also encodes a novel class II isotype of the SCE1, which was previously reported to be uniquely present in cereals. RNA-seq data showed that the core components of the SUMOylation cascade SUMO1/2 and SCE1 genes exhibited pod-specific expression patterns, implying coordinated regulation during pod development. Furthermore, both transcripts and conjugate profiles revealed that SUMOylation has significant roles during the pod development. Moreover, dynamic changes in the SUMO conjugates were observed in response to abiotic stresses. CONCLUSIONS: The identification and organization of peanut SUMO system revealed SUMOylation has important roles during stress defense and pod development. The present study will serve as a resource for providing new strategies to enhance agronomic yield and reveal the mechanism of peanut pod development.


Assuntos
Proteínas de Plantas/fisiologia , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/fisiologia , Arachis/crescimento & desenvolvimento , Flores/crescimento & desenvolvimento , Desenvolvimento Vegetal/genética , Desenvolvimento Vegetal/fisiologia , Proteínas de Plantas/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Estresse Fisiológico , Sumoilação , Transcrição Genética
5.
PLoS Genet ; 15(11): e1008426, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31765372

RESUMO

DNA polymerase epsilon (Pol ε) is critical for genome duplication, but little is known about how post-translational modification regulates its function. Here we report that the Pol ε catalytic subunit Pol2 in yeast is sumoylated at a single lysine within a catalytic domain insertion uniquely possessed by Pol2 family members. We found that Pol2 sumoylation occurs specifically in S phase and is increased under conditions of replication fork blockade. Analyses of the genetic requirements of this modification indicate that Pol2 sumoylation is associated with replication fork progression and dependent on the Smc5/6 SUMO ligase known to promote DNA synthesis. Consistently, the pol2 sumoylation mutant phenotype suggests impaired replication progression and increased levels of gross chromosomal rearrangements. Our findings thus indicate a direct role for SUMO in Pol2-mediated DNA synthesis and a molecular basis for Smc5/6-mediated regulation of genome stability.


Assuntos
Proteínas de Ciclo Celular/genética , DNA Polimerase II/genética , DNA/biossíntese , Proteínas de Saccharomyces cerevisiae/genética , Sumoilação/genética , Domínio Catalítico/genética , DNA/genética , Replicação do DNA/genética , Instabilidade Genômica/genética , Lisina/genética , Complexos Multiproteicos/genética , Mutação/genética , Fase S/genética , Saccharomyces cerevisiae/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Telômero/genética
6.
PLoS Genet ; 15(11): e1008427, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31765407

RESUMO

Replication fork stalling and accumulation of single-stranded DNA trigger the S phase checkpoint, a signalling cascade that, in budding yeast, leads to the activation of the Rad53 kinase. Rad53 is essential in maintaining cell viability, but its targets of regulation are still partially unknown. Here we show that Rad53 drives the hyper-SUMOylation of Pol2, the catalytic subunit of DNA polymerase ε, principally following replication forks stalling induced by nucleotide depletion. Pol2 is the main target of SUMOylation within the replisome and its modification requires the SUMO-ligase Mms21, a subunit of the Smc5/6 complex. Moreover, the Smc5/6 complex co-purifies with Pol ε, independently of other replisome components. Finally, we map Pol2 SUMOylation to a single site within the N-terminal catalytic domain and identify a SUMO-interacting motif at the C-terminus of Pol2. These data suggest that the S phase checkpoint regulate Pol ε during replication stress through Pol2 SUMOylation and SUMO-binding ability.


Assuntos
Proteínas de Ciclo Celular/genética , Quinase do Ponto de Checagem 2/genética , DNA Polimerase II/genética , DNA/biossíntese , Proteína SUMO-1/genética , Proteínas de Saccharomyces cerevisiae/genética , Sumoilação/genética , Domínio Catalítico/genética , DNA/genética , Replicação do DNA/genética , Complexos Multiproteicos/genética , Ligação Proteica , Fase S/genética , Saccharomyces cerevisiae/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética
7.
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
8.
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
9.
J Immunoassay Immunochem ; 40(4): 396-406, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31084243

RESUMO

Introduction: PIAS1 and PIAS2 (protein inhibitor of activated STAT 1,2) play key roles in the pathogenesis of autoimmune and inflammatory diseases. This study aims to evaluate the gene expression of these factors in multiple sclerosis (MS) patients compared to healthy individuals and correlate them with the severity of MS. Materials and methods: Sixty participants, including 30 patients with MS and 30 healthy controls were studied. The expression of PIAS1 and PIAS2 genes in peripheral blood samples of all participants was measured by real-time PCR. The severity of MS was evaluated using the Expanded Disability Status Scale (EDSS). Finally, we evaluated the correlation between the expression of PIAS1 and PIAS2 genes with disease severity. Results: The expression of PIAS1 gene was increased in patients with MS compared to healthy subjects (P value<.001). Also, there was a significant correlation between the expression of PIAS1 and PIAS2 genes with disease severity according to EDSS. Conclusion: Our study suggests the expression of PIAS1 and PIAS2 genes as a prognostic and diagnostic marker in MS disease.


Assuntos
Esclerose Múltipla/genética , Proteínas Inibidoras de STAT Ativados/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Adulto , Estudos de Casos e Controles , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/sangue , Proteínas Inibidoras de STAT Ativados/sangue , RNA/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/sangue , Adulto Jovem
10.
Neoplasia ; 21(7): 676-688, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31125786

RESUMO

Csk, a non-receptor tyrosine kinase, serves as an indispensable negative regulator of the Src family kinases (SFKs). However, little is known about regulation of Csk expression so far. SUMOylation, a reversible post-translational modification, has been shown to regulate many biological processes especially in tumor progression. Here we report that Csk is covalently modified by SUMO1 at lysine 53 (K53) both in vitro and in vivo. Treatment with hydrogen peroxide inhibited this modification to a certain extent, but PIAS3, identified as the main specific SUMO E3 ligase for Csk, could significantly enhance SUMO1-Csk level. In addition, phosphorylation at Ser364, the active site in Csk, had no effect on this modification. Ectopic expression of SUMO-defective mutant, Csk K53R, inhibited tumor cell growth more potentially than Csk wild-type. Consistent with the biological phenotype, the SUMO modification of Csk impaired its activity to interact with Cbp (Csk binding protein) leading to decreased c-Src phosphorylation at Y527. Our results suggest that SUMOylation of Csk mainly at lysine 53 negatively modulates its tumor suppressor function by reducing its binding with Cbp and consequently, inducing c-Src activation.


Assuntos
/genética , Neoplasias/genética , Proteína SUMO-1/genética , Sumoilação/genética , Humanos , Peróxido de Hidrogênio/farmacologia , Lisina/genética , Chaperonas Moleculares/química , Neoplasias/patologia , Fosforilação , Ligação Proteica/genética , Proteínas Inibidoras de STAT Ativados/genética , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Ubiquitina-Proteína Ligases/genética , Quinases da Família src/genética
11.
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
12.
PLoS One ; 14(4): e0213331, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30973885

RESUMO

Interruption of blood supply to the heart is a leading cause of death and disability. However, the molecular events that occur during heart ischemia, and how these changes prime consequent cell death upon reperfusion, are poorly understood. Protein SUMOylation is a post-translational modification that has been strongly implicated in the protection of cells against a variety of stressors, including ischemia-reperfusion. In particular, the SUMO2/3-specific protease SENP3 has emerged as an important determinant of cell survival after ischemic infarct. Here, we used the Langendorff perfusion model to examine changes in the levels and localisation of SUMOylated target proteins and SENP3 in whole heart. We observed a 50% loss of SENP3 from the cytosolic fraction of hearts after preconditioning, a 90% loss after ischemia and an 80% loss after ischemia-reperfusion. To examine these effects further, we performed ischemia and ischemia-reperfusion experiments in the cardiomyocyte H9C2 cell line. Similar to whole hearts, ischemia induced a decrease in cytosolic SENP3. Furthermore, shRNA-mediated knockdown of SENP3 led to an increase in the rate of cell death upon reperfusion. Together, our results indicate that cardiac ischemia dramatically alter levels of SENP3 and suggest that this may a mechanism to promote cell survival after ischemia-reperfusion in heart.


Assuntos
Endopeptidases/genética , Isquemia Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/genética , Sumoilação/genética , Animais , Morte Celular/genética , Sobrevivência Celular , Técnicas de Inativação de Genes , Humanos , Precondicionamento Isquêmico Miocárdico/métodos , Isquemia Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Estresse Oxidativo/genética , RNA Interferente Pequeno/genética , Ratos , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética
13.
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
14.
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
15.
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
16.
Biol Res ; 52(1): 4, 2019 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-30717818

RESUMO

BACKGROUND: Hematoporphyrin derivative (HPD) has a sensibilization effect in lung adenocarcinoma. This study was conducted to identify the target genes of HPD in lung adenocarcinoma. METHODS: RNA sequencing was performed using the lung adenocarcinoma cell line A549 after no treatment or treatment with X-ray or X-ray + HPD. The differentially expressed genes (DEGs) were screened using Mfuzz package by noise-robust soft clustering analysis. Enrichment analysis was carried out using "BioCloud" online tool. Protein-protein interaction (PPI) network and module analyses were performed using Cytoscape software. Using WebGestalt tool and integrated transcription factor platform (ITFP), microRNA target and transcription factor (TF) target pairs were separately predicted. An integrated regulatory network was visualized with Cytoscape software. RESULTS: A total of 815 DEGs in the gene set G1 (continuously dysregulated genes along with changes in processing conditions [untreated-treated with X-ray-X-ray + treated with HPD]) and 464 DEGs in the gene set G2 (significantly dysregulated between X-ray + HPD-treated group and untreated/X-ray-treated group) were screened. The significant module identified from the PPI network for gene set G1 showed that ribosomal protein L3 (RPL3) gene could interact with heat shock protein 90 kDa alpha, class A member 1 (HSP90AA1). TFs AAA domain containing 2 (ATAD2) and protein inhibitor of activated STAT 1 (PIAS1) were separately predicted for the genes in gene set G1 and G2, respectively. In the integrated network for gene set G2, ubiquitin-specific peptidase 25 (USP25) was targeted by miR-200b, miR-200c, and miR-429. CONCLUSION: RPL3, HSP90AA1, ATAD2, and PIAS1 as well as USP25, which is targeted by miR-200b, miR-200c, and miR-429, may be the potential targets of HPD in lung adenocarcinoma.


Assuntos
Adenocarcinoma de Pulmão/genética , Redes Reguladoras de Genes/genética , Derivado da Hematoporfirina/farmacologia , Neoplasias Pulmonares/genética , ATPases Associadas a Diversas Atividades Celulares/efeitos dos fármacos , ATPases Associadas a Diversas Atividades Celulares/genética , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/radioterapia , Linhagem Celular Tumoral , Análise por Conglomerados , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico HSP90/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/genética , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/radioterapia , MicroRNAs/metabolismo , Proteínas Inibidoras de STAT Ativados/efeitos dos fármacos , Proteínas Inibidoras de STAT Ativados/genética , Proteínas Ribossômicas/efeitos dos fármacos , Proteínas Ribossômicas/genética , Análise de Sequência de RNA , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/efeitos dos fármacos , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Fatores de Transcrição
17.
BMC Biotechnol ; 19(1): 13, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30770741

RESUMO

BACKGROUND: Despite the growing demand for antimicrobial peptides (AMPs) for clinical use as an alternative approach against antibiotic-resistant bacteria, the manufacture of AMPs relies on expensive, small-scale chemical methods. The small ubiquitin-related modifier (SUMO) tag is industrially practical for increasing the yield of recombinant proteins by increasing solubility and preventing degradation in expression systems. RESULTS: A new vector system, pKSEC1, was designed to produce AMPs, which can work in prokaryotic systems such as Escherichia coli and plant chloroplasts. 6xHis was tagged to SUMO for purification of SUMO-fused AMPs. Abaecin, a 34-aa-long antimicrobial peptide from honeybees, was expressed in a fusion form to 6xHis-SUMO in a new vector system to evaluate the prokaryotic expression platform of the antimicrobial peptides. The fusion sequences were codon-optimized in three different combinations and expressed in E. coli. The combination of the native SUMO sequence with codon-optimized abaecin showed the highest expression level among the three combinations, and most of the expressed fusion proteins were detected in soluble fractions. Cleavage of the SUMO tag by sumoase produced a 29-aa-long abaecin derivative with a C-terminal deletion. However, this abaecin derivative still retained the binding sequence for its target protein, DnaK. Antibacterial activity of the 29-aa long abaecin was tested against Bacillus subtilis alone or in combination with cecropin B. The combined treatment of the abaecin derivative and cecropin B showed bacteriolytic activity 2 to 3 times greater than that of abaecin alone. CONCLUSIONS: Using a SUMO-tag with an appropriate codon-optimization strategy could be an approach for the production of antimicrobial peptides in E.coli without affecting the viability of the host cell.


Assuntos
Anti-Infecciosos/síntese química , Peptídeos Catiônicos Antimicrobianos/síntese química , Escherichia coli/genética , Expressão Gênica , Vetores Genéticos/genética , Proteínas de Insetos/síntese química , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Anti-Infecciosos/administração & dosagem , Bacillus subtilis , Códon/genética , Proteínas Recombinantes de Fusão/genética
18.
BMB Rep ; 52(2): 113-118, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30638182

RESUMO

The small ubiquitin-related modification molecule (SUMO), one of the post-translational modification molecules, is involved in a variety of cellular functions where it regulates protein activity and stability, transcription, and cell cycling. Modulation of protein SUMOylation or deSUMOylation modification has been associated with regulation of carcinogenesis in breast cancer. In the dynamic processes of SUMOylation and deSUMOylation in a variety of cancers, SUMO proteases (SENPs), reverse SUMOylation by isopeptidase activity and SENPs are mostly elevated, and are related to poor patient prognosis. Although underlying mechanisms have been suggested for how SENPs participate in breast cancer tumorigenesis, such as through regulation of target protein transactivation, cancer cell survival, cell cycle, or other post-translational modification-related machinery recruitment, the effect of SENP isoform-specific inhibitors on the progression of breast cancer have not been well evaluated. This review will introduce the functions of SENP1 and SENP2 and the underlying signaling pathways in breast cancer for use in discovery of new biomarkers for diagnosis or therapeutic targets for treatment. [BMB Reports 2019; 52(2): 113-118].


Assuntos
Neoplasias da Mama/genética , Sumoilação/fisiologia , Neoplasias da Mama/metabolismo , Cisteína Endopeptidases/metabolismo , Feminino , Humanos , Peptídeo Hidrolases/metabolismo , Processamento de Proteína Pós-Traducional/genética , Processamento de Proteína Pós-Traducional/fisiologia , Transdução de Sinais , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo
19.
J Clin Invest ; 129(2): 786-801, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30480549

RESUMO

Tumor cure with conventional fractionated radiotherapy is 65%, dependent on tumor cell-autonomous gradual buildup of DNA double-strand break (DSB) misrepair. Here we report that single-dose radiotherapy (SDRT), a disruptive technique that ablates more than 90% of human cancers, operates a distinct dual-target mechanism, linking acid sphingomyelinase-mediated (ASMase-mediated) microvascular perfusion defects to DNA unrepair in tumor cells to confer tumor cell lethality. ASMase-mediated microcirculatory vasoconstriction after SDRT conferred an ischemic stress response within parenchymal tumor cells, with ROS triggering the evolutionarily conserved SUMO stress response, specifically depleting chromatin-associated free SUMO3. Whereas SUMO3, but not SUMO2, was indispensable for homology-directed repair (HDR) of DSBs, HDR loss of function after SDRT yielded DSB unrepair, chromosomal aberrations, and tumor clonogen demise. Vasoconstriction blockade with the endothelin-1 inhibitor BQ-123, or ROS scavenging after SDRT using peroxiredoxin-6 overexpression or the SOD mimetic tempol, prevented chromatin SUMO3 depletion, HDR loss of function, and SDRT tumor ablation. We also provide evidence of mouse-to-human translation of this biology in a randomized clinical trial, showing that 24 Gy SDRT, but not 3×9 Gy fractionation, coupled early tumor ischemia/reperfusion to human cancer ablation. The SDRT biology provides opportunities for mechanism-based selective tumor radiosensitization via accessing of SDRT/ASMase signaling, as current studies indicate that this pathway is tractable to pharmacologic intervention.


Assuntos
Recombinação Homóloga , Neoplasias , Traumatismo por Reperfusão , Transdução de Sinais , Animais , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Humanos , Camundongos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias/radioterapia , Transdução de Sinais/genética , Transdução de Sinais/efeitos da radiação , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismo
20.
Enzyme Microb Technol ; 120: 98-109, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30396406

RESUMO

The exploitation of SUMO (small ubiquitin-related modifier) fusion technology at a large scale for the production of therapeutic proteins with an authentic N-terminus is majorly limited due to the higher cost of ScUlp1 protease. Therefore, the cost-effective production of Saccharomyces cerevisiae Ulp1 protease catalytic domain (402-621 aa) was targeted via its cloning under strong T7 promoter with and without histidine tag. The optimization of cultivation conditions at shake flask resulted in ScUlp1 expression of 195 mg/L in TB medium with a specific product yield of 98 mg/g DCW. The leaky expression of the ScUlp1 protease was controlled using the chemically defined minimal medium. The Ni-NTA affinity purification of ScUlp1 was done near homogeneity using different additives (0.1% Triton X-100, 0.01 mM DTT, 0.02 mM EDTA and 1% glycerol) where a product purity of ∼95% with a recovery yield of 80% was obtained. The specific activity of purified ScUlp1 was found to be 3.986 × 105 U/mg. The ScUlp1 protease successfully cleaved the SUMO tag even at 1:10,000 enzyme to substrate ratio with high efficacy and also showed a comparable catalytic efficiency as of commercial control. Moreover, the in vivo cleavage of SUMO tag via co-expression strategy also resulted in more than 80% cleavage of SUMO fusion protein. The optimization of high cell density cultivation strategies and maintenance of higher plasmid stability at bioreactor level resulted in the ScUlp1 production of 3.25 g/L with a specific product yield of 45.41 mg/g DCW when cells were induced at an OD600 of 132 (63.66 g/L DCW).


Assuntos
Técnicas de Cultura Celular por Lotes/métodos , Cisteína Endopeptidases/metabolismo , Escherichia coli/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Reatores Biológicos , Domínio Catalítico , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/isolamento & purificação , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Ubiquitinas/metabolismo
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