RESUMO
Trypanosoma brucei are protozoan parasites that cause sleeping sickness in humans and nagana in cattle. Inside the mammalian host, a quorum sensing-like mechanism coordinates its differentiation from a slender replicative form into a quiescent stumpy form, limiting growth and activating metabolic pathways that are beneficial to the parasite in the insect host. The post-translational modification of proteins with the Small Ubiquitin-like MOdifier (SUMO) enables dynamic regulation of cellular metabolism. SUMO can be conjugated to its targets as a monomer but can also form oligomeric chains. Here, we have investigated the role of SUMO chains in T. brucei by abolishing the ability of SUMO to polymerize. We have found that parasites able to conjugate only SUMO monomers are primed for differentiation. This was demonstrated for monomorphic lines that are normally unable to produce stumpy forms in response to quorum sensing signaling in mice, and also for pleomorphic cell lines in which stumpy cells were observed at unusually low parasitemia levels. SUMO chain mutants showed a stumpy compatible transcriptional profile and better competence to differentiate into procyclics. Our study indicates that SUMO depolymerization may represent a coordinated signal triggered during stumpy activation program.
Assuntos
Trypanosoma brucei brucei , Animais , Trypanosoma brucei brucei/metabolismo , Camundongos , Tripanossomíase Africana/parasitologia , Diferenciação Celular , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Processamento de Proteína Pós-Traducional , Percepção de Quorum/fisiologia , Humanos , SumoilaçãoRESUMO
SUMOylation is a post-translational modification (PTM) whereby members of the Small Ubiquitin-like MOdifier (SUMO) family of proteins are conjugated to lysine residues in target proteins. SUMOylation has been implicated in a wide range of physiological and pathological processes, and much attention has been given to its role in neurodegenerative conditions. Due to its reported role in neuroprotection, pharmacological modulation of SUMOylation represents an attractive potential therapeutic strategy in a number of different brain disorders. However, very few compounds that target the SUMOylation pathway have been identified. Guanosine is an endogenous nucleoside with important neuromodulatory and neuroprotective effects. Experimental evidence has shown that guanosine can modulate different intracellular pathways, including PTMs. In the present study we examined whether guanosine alters global protein SUMOylation. Primary cortical neurons and astrocytes were treated with guanosine at 1, 10, 100, 300, or 500 µM at four time points, 1, 6, 24, or 48 h. We show that guanosine increases global SUMO2/3-ylation in neurons and astrocytes at 1 h at concentrations above 10 µM. The molecular mechanisms involved in this effect were evaluated in neurons. The guanosine-induced increase in global SUMO2/3-ylation was still observed in the presence of dipyridamole, which prevents guanosine internalization, demonstrating an extracellular guanosine-induced effect. Furthermore, the A1 adenosine receptor antagonist DPCPX abolished the guanosine-induced increase in SUMO2/3-ylation. The A2A adenosine receptor antagonist ZM241385 increased SUMOylation per se, but did not alter guanosine-induced SUMOylation, suggesting that guanosine may modulate SUMO2/3-ylation through an A1-A2A receptor interaction. Taken together, this is the first report to show guanosine as a SUMO2/3-ylation enhancer in astrocytes and neurons.
Assuntos
Astrócitos/efeitos dos fármacos , Guanosina/farmacologia , Neurônios/efeitos dos fármacos , Receptores Purinérgicos P1/metabolismo , Sumoilação/efeitos dos fármacos , Animais , Astrócitos/metabolismo , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Neurônios/metabolismo , Ratos , Ratos Wistar , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismoRESUMO
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/metabolismoRESUMO
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 , Proteína Ribossômica L3 , 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 TranscriçãoRESUMO
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
Humanos , Derivado da Hematoporfirina/farmacologia , Redes Reguladoras de Genes/genética , Adenocarcinoma de Pulmão/genética , Neoplasias Pulmonares/genética , Proteínas Ribossômicas/efeitos dos fármacos , Proteínas Ribossômicas/genética , Fatores de Transcrição , Análise por Conglomerados , Regulação Neoplásica da Expressão Gênica , Análise de Sequência de RNA , Proteínas de Choque Térmico HSP90/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/efeitos dos fármacos , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , MicroRNAs/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas Inibidoras de STAT Ativados/efeitos dos fármacos , Proteínas Inibidoras de STAT Ativados/genética , Citometria de Fluxo , 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 , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/radioterapiaRESUMO
CoREST family of transcriptional co-repressors regulates gene expression and cell fate determination during development. CoREST co-repressors recruit with different affinity the histone demethylase LSD1 (KDM1A) and the deacetylases HDAC1/2 to repress with variable strength the expression of target genes. CoREST protein levels are differentially regulated during cell fate determination and in mature tissues. However, regulatory mechanisms of CoREST co-repressors at the protein level have not been studied. Here, we report that CoREST (CoREST1, RCOR1) and its homologs CoREST2 (RCOR2) and CoREST3 (RCOR3) interact with PIASγ (protein inhibitor of activated STAT), a SUMO (small ubiquitin-like modifier)-E3-ligase. PIASγ increases the stability of CoREST proteins and facilitates their SUMOylation by SUMO-2. Interestingly, the SUMO-conjugating enzyme, Ubc9 also facilitates the SUMOylation of CoREST proteins. However, it does not change their protein levels. Specificity was shown using the null enzymatic form of PIASγ (PIASγ-C342A) and the SUMO protease SENP-1, which reversed SUMOylation and the increment of CoREST protein levels induced by PIASγ. The major SUMO acceptor lysines are different and are localized in nonconserved sequences among CoREST proteins. SUMOylation-deficient CoREST1 and CoREST3 mutants maintain a similar interaction profile with LSD1 and HDAC1/2, and consequently maintain similar repressor capacity compared with wild-type counterparts. In conclusion, CoREST co-repressors form protein complexes with PIASγ, which acts both as SUMO E3-ligase and as a protein stabilizer for CoREST proteins. This novel regulation of CoREST by PIASγ interaction and SUMOylation may serve to control cell fate determination during development.
Assuntos
Proteínas Correpressoras/química , Proteínas Correpressoras/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Transcrição Gênica , Animais , Proteínas Correpressoras/genética , Feminino , Células HEK293 , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/genética , Histona Desacetilase 2/metabolismo , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Humanos , Proteínas do Tecido Nervoso/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas Inibidoras de STAT Ativados/genética , Ratos , Ratos Sprague-Dawley , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
Ki-1/57 is a nuclear and cytoplasmic regulatory protein first identified in malignant cells from Hodgkin's lymphoma. It is involved in gene expression regulation on both transcriptional and mRNA metabolism levels. Ki-1/57 belongs to the family of intrinsically unstructured proteins and undergoes phosphorylation by PKC and methylation by PRMT1. Previous characterization of its protein interaction profile by yeast two-hybrid screening showed that Ki-1/57 interacts with proteins of the SUMOylation machinery, the SUMO E2 conjugating enzyme UBC9 and the SUMO E3 ligase PIAS3, which suggested that Ki-1/57 could be involved with this process. Here we identified seven potential SUMO target sites (lysine residues) on Ki-1/57 sequence and observed that Ki-1/57 is modified by SUMO proteins in vitro and in vivo. We showed that SUMOylation of Ki-1/57 occurred on lysines 213, 276, and 336. In transfected cells expressing FLAG-Ki-1/57 wild-type, its paralog FLAG-CGI-55 wild-type, or their non-SUMOylated triple mutants, the number of PML-nuclear bodies (PML-NBs) is reduced compared with the control cells not expressing the constructs. More interestingly, after treating cells with arsenic trioxide (As2O3), the number of PML-NBs is no longer reduced when the non-SUMOylated triple mutant Ki-1/57 is expressed, suggesting that the SUMOylation of Ki-1/57 has a role in the control of As2O3-induced PML-NB formation. A proteome-wide analysis of Ki-1/57 partners in the presence of either SUMO-1 or SUMO-2 suggests that the involvement of Ki-1/57 with the regulation of gene expression is independent of the presence of either SUMO-1 or SUMO-2; however, the presence of SUMO-1 strongly influences the interaction of Ki-1/57 with proteins associated with cellular metabolism, maintenance, and cell cycle.
Assuntos
Fatores de Regulação Miogênica/metabolismo , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Proteínas de Ligação a RNA/metabolismo , Proteína SUMO-1/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Trióxido de Arsênio , Arsenicais/farmacologia , Ciclo Celular/genética , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Células HEK293 , Células HeLa , Humanos , Lisina , Fatores de Regulação Miogênica/genética , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Óxidos/farmacologia , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteína SUMO-1/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Sumoilação , Transcrição GênicaRESUMO
The unicellular protozoa Giardia lamblia is a food- and waterborne parasite that causes giardiasis. This illness is manifested as acute and self-limited diarrhea and can evolve to long-term complications. Successful establishment of infection by Giardia trophozoites requires adhesion to host cells and colonization of the small intestine, where parasites multiply by mitotic division. The tight binding of trophozoites to host cells occurs by means of the ventral adhesive disc, a spiral array of microtubules and associated proteins such as giardins. In this work we show that knock down of the Small Ubiquitin-like MOdifier (SUMO) results in less adhesive trophzoites, decreased cell proliferation and deep morphological alterations, including at the ventral disc. Consistent with the reduced proliferation, SUMO knocked-down trophozoites were arrested in G1 and in S phases of the cell cycle. Mass spectrometry analysis of anti-SUMO immunoprecipitates was performed to identify SUMO substrates possibly involved in these events. Among the identified SUMOylation targets, α-tubulin was further validated by Western blot and confirmed to be a SUMO target in Giardia trophozoites.
Assuntos
Giardia lamblia/fisiologia , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Tubulina (Proteína)/metabolismo , Ciclo Celular , Técnicas de Silenciamento de Genes , Giardia lamblia/metabolismo , Espectrometria de Massas , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Trofozoítos/fisiologiaRESUMO
Danos em biomoléculas podem ocorrer a partir de uma interação direta entre as biomoléculas e espécies reativas de oxigênio e nitrogênio como também, pela reação de produtos secundários dessas espécies como eletrófilos gerados na peroxidação lipídica. Alguns desses produtos secundários possuem estabilidade química maior que as espécies reativas das quais foram derivadas e podem se ligar covalentemente as biomoléculas comprometendo o funcionamento normal das mesmas. Portanto, modificações em proteínas por aldeídos gerados na lipoperoxidação têm sido investigadas por suas implicações com desordens patológicas relacionadas à agregação proteica, e modificações em diversas vias de sinalização amplificando os efeitos deletérios em sistemas biológicos. Os objetivos desse trabalho foi contribuir na elucidação dos mecanismos moleculares associados ao desenvolvimento da esclerose lateral amiotrófica (ELA) através da identificação, caracterização e quantificação de modificações póstraducionais em proteínas pelos aldeídos 4-hidroxi-2-hexenal (HHE) e trans-4-hidroxi-2-nonenal (HNE) in vitro (citocromo c) e in vivo (modelo ELA) a partir de técnicas de Western blot, imunoprecipitação e espectrometria de massa com abordagem proteômica de "shotgun" em ratosSOD1G93A modelo de esclerose lateral amiotrófica (ELA). Estudos com citocromo c mostraram a ligação dos aldeídos ao citocromo c e mecanismos de reação foram propostos. Foram encontrados seis peptídeos modificados por HHE e um para o HNE, e o peptídeo TGPNLHGLFGR se mostrou modificado pelos dois aldeídos paralelamente. Foi demonstrado que a histidina 33 é um "hot spot" frente as adições pelos aldeídos. Nas análises por western blot das proteínas ligadas a aldeídos foi possível observar uma tendência de aumento na concentração de proteínas ligadas ao HNE nos animais ELA, mais acentuada nas amostras de 70 dias comparadas ao controle. Com relação aos resultados obtidos com HHE tanto os animais pré-sintomáticos quanto os sintomáticos não apresentaram diferenças de HHE-proteína, tantonos controles quanto nos animais ELA. Nas amostras dos animais sintomáticos não detectamos diferença significativa na concentração de aldeído-proteína entre os grupos. Já as análises proteômicas revelaram 24 proteínas diferencialmente expressas, com destaque para proteínas com os maiores valores de significância (p-value), como a ubiquitina no grupo dos pré- sintomáticos e a neurogranina, no grupo dos animais sintomáticos e várias proteínas de metabolismo energético, de neurofilamentos, proteínas de processos redox e proteínas ligadas o metabolismo de cálcio (fundamentais na fisiopatologia em ELA). Algumas proteínas importantes foram encontradas com exclusividades nos grupos pré-sintomáticos e sintomáticos pelo diagrama de Venn. Com relação a proteínas modificadas pelos aldeídos, foram encontradas algumas relevantes como a proteína 2 de interação com a polimerase delta que foi modificada por HNE via adição de Michael encontrada nos animais ELA pré-sintomáticos e sintomáticos, a catalase que foi encontrada modificada por HNE via base de Schiff apenas nos ELA pré- sintomáticos, e a tiol redutase induzível por interferon gama no grupo dos animais ELA sintomáticos. Com relação a proteínas modificadas por HHE, foram encontradas a Janus quinase e proteína 3 de interação com microtúbulo, modificadas tanto por adição de Michael quanto via base de Schiff nos animais ELA sintomáticos. É interessante ressaltar que algumas modificações encontradas em proteínas não caracterizadas podem indicar proteínas novas ainda não descritas como modificadas por esses aldeídos. Os resultados mostram que algumas das proteínas modificadas por HNE e HHE encontradas neste trabalho, estão relacionadas ao estresse redox, vias metabólicas energéticas, proteínas envolvidas na resposta a danos oxidativos, e processos inflamatórios. Tais modificações ocorrem não só no modelo de neurodegeneração, mas foram previamente descritas em outros processos patológicos, como doença cardiovascular, lesão hepática por uso crônico de álcool
Damage to biomolecules can occur from a direct interaction between biomolecules and reactive of oxygen and nitrogen species as well as from the reaction of secondary products of these species as electrophiles generated in lipid peroxidation. Some of these by-products have greater chemical stability than the derived reactive species and can bind to biomolecules compromising their normal function. Therefore, protein modifications by aldehydes generated during lipoperoxidation have been investigated for their implications with pathological disorders related to protein aggregation and modifications in signaling pathways amplifying the deleterious effects in biological systems. The aim of this work was to contribute to the elucidation of the molecular mechanisms associated with the development of amyotrophic lateral sclerosis (ALS) through the identification, characterization and quantification of posttranslational modifications in proteins by 4-hydroxy-2-hexenal (HHE) and trans-4-hydroxy-2- nonenal (HNE) in vitro, cytochrome c, and in vivo, rat model (SOD1G93A) of amyotrophic lateral sclerosis (ALS), throught Western blot techniques, and mass spectrometry with shotgun proteomics approach. The results showed the binding of aldehydes to cytochrome c. Six peptides were modified by HHE and one by HNE. The peptide TGPNLHGLFGR was modified by the two aldehydes. Histidine 33 has been shown to be a hot spot against aldehydes additions. By western blot analysis of the aldehyde-bound proteins, it was possible to observe a tendency of increase in the concentration of HNE-bound proteins in the ALS animals, more pronounced in the samples of 70 days compared to control samples. Regarding the results obtained with HHE, both pre-symptomatic and symptomatic animals did not show HHE-protein differences, both in controls and in ALS animals. We did not detect a significant difference in the aldehyde-protein concentration between the groups in the samples of the symptomatic animals. Proteomic analysis revealed 24 differentially expressed proteins, with emphasis on proteins with thehighest values of significance (p-value), such as the ubiquitin in the pre-symptomatic group and neurogranin in the group of the symptomatic animals and several proteins of the energetic metabolism pathways, neurofilaments, proteins of redox processes and proteins linked to calcium metabolism (fundamental in the pathophysiology of ALS). Some important proteins were found exclusivity in the pre-symptomatic and symptomatic groups by the Venn diagram. With regard to aldehyde-modified proteins, some relevant ones such as Delta-2 polymerase interaction protein, that was modified by HNE via the addition of Michael found in presymptomatic and symptomatic ELA animals, catalase that was found to be modified by HNE via Schiff's base only in pre-symptomatic ALS, and gamma interferon-inducible thiol reductase in the group of symptomatic ALS animals. Janus kinase and microtubule interaction protein 3, were found to be modified by Michael addition and Schiff base pathway respectively in symptomatic ALS animals. It is interesting to note that some modifications found in uncharacterized proteins may indicate new proteins not yet described as modified by these aldehydes. The results show that some of the proteins modified by HNE and HHE found in this work are related to redox stress, energetic metabolic pathways, proteins involved in the response to oxidative damage, and inflammatory processes. Such modifications occur not only in the neurodegeneration model, but were previously described in other pathological processes, such as cardiovascular disease, liver injury due to chronic alcohol use
Assuntos
Animais , Feminino , Ratos , Proteínas/análise , Esclerose Lateral Amiotrófica/fisiopatologia , Espectrometria de Massas/métodos , Biomarcadores/metabolismo , Western Blotting/métodos , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina , Proteômica/instrumentação , Citocromos c , Modificação Traducional de Proteínas , Aldeídos/análise , Cromatografia de Fase Reversa/métodos , Técnicas de Genotipagem/instrumentaçãoRESUMO
Isolating endogenous SUMOylated proteins is a challenging task due to the high reversibility of this posttranslational modification. We have shown that SUMO traps are useful tools for the enrichment and isolation of proteins modified by SUMO in vitro and in vivo. To characterize the affinity and specificity of different SUMO chains for these traps, that are based on SUMO-interacting motifs, we have used real-time surface plasmon resonance (SPR), which allows a label-free analysis of protein/protein interactions. Here, a protocol to determine the affinities of multivalent SUMO traps for polySUMO chains or mono-SUMO molecules by SPR is presented.
Assuntos
Processamento de Proteína Pós-Traducional , Proteína SUMO-1/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ressonância de Plasmônio de Superfície/métodos , Anticorpos/química , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Procedimentos Analíticos em Microchip , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteína SUMO-1/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , SumoilaçãoRESUMO
Parkinson's disease (PD) is the second most common neurodegenerative disorder characterized by cardinal motor signs such as rigidity, bradykinesia or rest tremor that arise from a significant death of dopaminergic neurons. Non-dopaminergic degeneration also occurs and it seems to induce the deficits in olfactory, emotional, and memory functions that precede the classical motor symptoms in PD. Despite the majority of PD cases being sporadic, several genes have previously been associated with the hereditary forms of the disease. The proteins encoded by some of these genes, including α-synuclein, DJ-1, and parkin, are modified by small ubiquitin-like modifier (SUMO), a post-translational modification that regulates a variety of cellular processes. Among the several pathogenic mechanisms proposed for PD is mitochondrial dysfunction. Recent studies suggest that SUMOylation can interfere with mitochondrial dynamics, which is essential for neuronal function, and may play a pivotal role in PD pathogenesis. Here, we present an overview of recent studies on mitochondrial disturbance in PD and the potential SUMO-modified proteins and pathways involved in this process. SUMOylation, a post-translational modification, interferes with mitochondrial dynamics, and may play a pivotal role in Parkinson's disease (PD). SUMOylation maintains α-synuclein (α-syn) in a soluble form and activates DJ-1, decreasing mitochondrial oxidative stress. SUMOylation may reduce the amount of parkin available for mitochondrial recruitment and decreases mitochondrial biogenesis through suppression of peroxisomal proliferator-activated receptor-γ co-activator 1 α (PGC-1α). Mitochondrial fission can be regulated by dynamin-related protein 1 SUMO-1- or SUMO-2/3-ylation. A fine balance for the SUMOylation/deSUMOylation of these proteins is required to ensure adequate mitochondrial function in PD.
Assuntos
Mitocôndrias/metabolismo , Doença de Parkinson/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação/fisiologia , Animais , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Humanos , Mitocôndrias/patologia , Estresse Oxidativo/fisiologia , Doença de Parkinson/patologiaRESUMO
SUMO (small ubiquitin-like modifier) conjugation is a critically important control process in all eukaryotic cells, because it acts as a biochemical switch and regulates the function of hundreds of proteins in many different pathways. Although the diverse functional consequences and molecular targets of SUMOylation remain largely unknown, SUMOylation is becoming increasingly implicated in the pathophysiology of Alzheimer's disease (AD). Apart from the central SUMO-modified disease-associated proteins, such as amyloid precursor protein, amyloid ß, and tau, SUMOylation also regulates several other processes underlying AD. These are involved in inflammation, mitochondrial dynamics, synaptic transmission and plasticity, as well as in protective responses to cell stress. Herein, we review current reports on the involvement of SUMOylation in AD, and present an overview of potential SUMO targets and pathways underlying AD pathogenesis.
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Doença de Alzheimer/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Envelhecimento/metabolismo , Doença de Alzheimer/tratamento farmacológico , Animais , Humanos , Terapia de Alvo Molecular , Transdução de Sinais , SumoilaçãoRESUMO
O fator CIITA é a proteína responsável por controlar a transcrição de genes do complexo principal de histocompatibilidade de classe II (MHC II) envolvidos na apresentação antigênica a linfócitos T CD4+. A expressão desta proteína é complexa e célula-específica, dependendo de mecanismos de regulação transcricionais e póstranscricionais. Com o intuito de investigar o potencial do fator CIITA como adjuvante molecular, no presente estudo desenvolvemos e validamos sistemas de transferência gênica capazes de promover a eficiente expressão de CIITA em vários tipos celulares. Além disso, investigamos a regulação pós-traducional deste fator em células não hematopoéticas. Desta forma, foram produzidos um vetor plasmidial e um vetor lentiviral, ambos carreando a sequência do fator CIITA humano desenhada in silico visando a eliminação de elementos cis-reguladores, e otimizada para eficiente expressão em células humanas. A transfecção/transdução de três linhagens de células humanas não hematopoéticas resultou na eficiente expressão de CIITA com localização nuclear apropriada. Células expressando CIITA apresentaram síntese de novo do MHC II, confirmando a funcionalidade da proteína e validando ambos os vetores para a análise futura da atividade adjuvante do CIITA em imunizações gênicas. Ensaios preliminares de inoculação de explantes de pele humana com o vetor lentiviral evidenciaram a eficiente transdução e expressão do CIITA exógeno em células primárias. Em seguida, células dendríticas (DCs) derivadas de monócitos de indivíduos saudáveis ou infectados com HIV-1 foram transduzidas com o vetor lentiviral para confirmar a expressão do CIITA em células primárias e avaliar a aplicação desse sistema adjuvante no aprimoramento da vacina de DCs anti-HIV. DCs de indivíduos saudáveis ou infectados foram transduzidas com sucesso pelo lentivírus, o qual induziu uma produção prolongada do mRNA codificando CIITA. Entretanto, os vetores lentivirais induziram um...
The CIITA factor is a protein responsible for controlling the transcription of major histocompatibility complex class II (MHC II) genes involved on antigen presentation to CD4+ T helper cells. The expression of this transcription factor is complex and differs in various cell types depending on transcriptional and post-transcriptional regulatory mechanisms. In order to investigate the CIITA factor potential as molecular adjuvant, here we developed and validated two gene delivery systems capable of promoting efficient CIITA expression in various human cell types. Additionally, we applied the delivery systems to investigate the post-translational regulation of this factor in nonimmune cells. A DNA plasmid and a lentiviral vector were produced, both carrying the human CIITA DNA sequence in silico designed to avoid cis-regulatory elements, and genetic optimized for expression efficacy in human cells. Transfection or transduction of three different non-immune human cell lines resulted in efficient CIITA expression with proper nuclear localization. The CIITA-expressing cells presented de novo MHC II molecules expression confirming the functionality of the exogenous protein, and validating both delivery systems for the future analysis of the CIITA adjuvant activity in genetic immunizations. Preliminary assays involving the inoculation of the lentiviral vector into human skin explants showed efficient transduction and expression of exogenous CIITA in primary cells. Next, monocyte-derived dendritic cells (DCs) from healthy individuals and HIV-1-infected patients were transduced with the lentiviral vector to confirm the exogenous CIITA expression in primary human cells and also evaluate the applicability of this adjuvant system to improve the DC-based vaccines against HIV. DCs from healthy and infected individuals were successfully transduced by the lentivirus, which induced a sustained CIITA mRNA production. However, the vector particles by themselves induced an...
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Proteínas Modificadoras Pequenas Relacionadas à UbiquitinaRESUMO
Gain of function mutation of Janus kinase 2 (JAK2V617F) has been identified in Philadelphia-negative myeloproliferative diseases; about half of essential thrombocythemia (ET) patients harbor this mutation. The activated JAK-STAT pathway promotes cell proliferation, differentiation and anti-apoptosis. We studied the role of negative regulators of the JAK-STAT pathway, PIAS, and SOCS in ET patients. Twenty ET patients and 20 healthy individuals were enrolled in the study. Thirteen of the ET patients harbored the JAK2V617F mutation based on mutation analysis. Quantitative-PCR was applied to assay the expression of SOCS1, SOCS3, PIAS1, PIAS3. The expression levels of PIAS1 and PIAS3 were significantly lower in ET groups than that in normal individuals. There was no significant difference between JAK2V617F (+) and JAK2V617F (-) patients. SOCS1 and SOCS3 expression did not differ between ET patients and normal individuals, or between JAK2V617F (+) and JAK2V617F (-) patients. We suggest that failed negative regulators of the JAK-STAT pathway take part in the pathomechanism of ET.
Assuntos
Chaperonas Moleculares/genética , Proteínas Inibidoras de STAT Ativados/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Trombocitemia Essencial/genética , Estudos de Casos e Controles , Feminino , Humanos , Janus Quinase 2/genética , Masculino , Pessoa de Meia-Idade , Chaperonas Moleculares/metabolismo , Mutação de Sentido Incorreto , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Proteína 1 Supressora da Sinalização de Citocina , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Trombocitemia Essencial/metabolismoRESUMO
Akt/PKB is a key signaling molecule in higher eukaryotes and a crucial protein kinase in human health and disease. Phosphorylation, acetylation, and ubiquitylation have been reported as important regulatory post-translational modifications of this kinase. We describe here that Akt is modified by SUMO conjugation, and show that lysine residues 276 and 301 are the major SUMO attachment sites within this protein. We found that phosphorylation and SUMOylation of Akt appear as independent events. However, decreasing Akt SUMOylation levels severely affects the role of this kinase as a regulator of fibronectin and Bcl-x alternative splicing. Moreover, we observed that the Akt mutant (Akt E17K) found in several human tumors displays increased levels of SUMOylation and also an enhanced capacity to regulate fibronectin splicing patterns. This splicing regulatory activity is completely abolished by decreasing Akt E17K SUMO conjugation levels. Additionally, we found that SUMOylation controls Akt regulatory function at G1/S transition during cell cycle progression. These findings reveal SUMO conjugation as a novel level of regulation for Akt activity, opening new areas of exploration related to the molecular mechanisms involved in the diverse cellular functions of this kinase.
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Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Processamento Alternativo , Fibronectinas/genética , Fibronectinas/metabolismo , Fase G1 , Células HEK293 , Células HeLa , Humanos , Mutagênese Sítio-Dirigida , Fosforilação , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fase S , Sumoilação , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
The Vacuole Membrane Protein 1 -VMP1- is a pancreatitis-associated transmembrane protein whose expression triggers autophagy in several human diseases. In the current study, we unveil the mechanism through which this protein induces autophagosome formation in mammalian cells. We show that VMP1 autophagy-related function requires its 20-aminoacid C-terminus hydrophilic domain (VMP1-AtgD). This is achieved through its direct binding to the BH3 motif of Beclin 1 leading to the formation of a complex with the Class III phosphatidylinositol-3 kinase (PI3K) hVps34, a key positive regulator of autophagy, at the site where autophagosomes are generated. This interaction also concomitantly promotes the dissociation of Bcl-2, an autophagy inhibitor, from Beclin 1. Moreover, we show that the VMP1-Beclin 1-hVps34 complex favors the association of Atg16L1 and LC3 with the autophagosomal membranes. Collectively, these findings reveal that VMP1 expression recruits and activates the Class III PI3K complex at the site of autophagosome formation during mammalian autophagy.
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Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/fisiologia , Proteínas de Membrana/metabolismo , Animais , Proteínas Reguladoras de Apoptose/química , Proteína Beclina-1 , Linhagem Celular , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Humanos , Proteínas de Membrana/química , Camundongos , Complexos Multiproteicos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismoRESUMO
Nurr1 (NR4A2) is a transcription factor that belongs to the orphan NR4A group of the nuclear receptor superfamily. Nurr1 plays key roles in the origin and maintenance of midbrain dopamine neurons, and peripheral inflammatory processes. PIASγ, a SUMO-E3 ligase, represses Nurr1 transcriptional activity. We report that Nurr1 is SUMOylated by SUMO-2 in the lysine 91 located in the transcriptional activation function 1 domain of Nurr1. Nurr1 SUMOylation by SUMO-2 is markedly facilitated by overexpressing wild type PIASγ, but not by a mutant form of PIASγ lacking its first LXXLL motif (PIASγmut1). This PIASγmut1 is also unable to interact with Nurr1 and to repress Nurr1 transcriptional activity. Interestingly, the mutant PIASγC342A that lacks SUMO ligase activity is still able to significantly repress Nurr1-dependent transcriptional activity, but not to enhance Nurr1 SUMOylation. A SUMOylation-deficient Nurr1 mutant displays higher transcriptional activity than the wild type Nurr1 only in promoters harboring more than one Nurr1 response element. Furthermore, lysine 91, the major target of Nurr1 SUMOylation is contained in a canonical synergy control motif, indicating that SUMO-2 posttranslational modification of Nurr1 regulates its transcriptional synergy in complex promoters. In conclusion, PIASγ can exert two types of negative regulations over Nurr1. On one hand, PIASγ limits Nurr1 transactivation in complex promoters by SUMOylating its lysine 91. On the other hand, PIASγ fully represses Nurr1 transactivation through a direct interaction, independently of its E3-ligase activity.
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Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Proteínas Inibidoras de STAT Ativados/fisiologia , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Transcrição Gênica , Western Blotting , Linhagem Celular , Humanos , Imunoprecipitação , Microscopia ConfocalRESUMO
Serine/arginine-rich (SR) proteins are among the most studied splicing regulators. They constitute a family of evolutionarily conserved proteins that, apart from their initially identified and deeply studied role in splicing regulation, have been implicated in genome stability, chromatin binding, transcription elongation, mRNA stability, mRNA export and mRNA translation. Remarkably, this list of SR protein activities seems far from complete, as unexpected functions keep being unraveled. An intriguing aspect that awaits further investigation is how the multiple tasks of SR proteins are concertedly regulated within mammalian cells. In this article, we first discuss recent findings regarding the regulation of SR protein expression, activity and accessibility. We dive into recent studies describing SR protein auto-regulatory feedback loops involving different molecular mechanisms such asunproductive splicing, microRNA-mediated regulation and translational repression. In addition, we take into account another step of regulation of SR proteins, presenting new findings about a variety of post-translational modifications by proteomics approaches and how some of these modifications can regulate SR protein sub-cellular localization or stability. Towards the end, we focus in two recently revealed functions of SR proteins beyond mRNA biogenesis and metabolism, the regulation of micro-RNA processing and the regulation of small ubiquitin-like modifier (SUMO) conjugation.
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Regulação da Expressão Gênica , Proteínas Nucleares/metabolismo , Processamento de Proteína Pós-Traducional , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Processamento Alternativo , Animais , Sequência Conservada , Retroalimentação Fisiológica , Humanos , MicroRNAs , Proteínas Nucleares/química , Proteínas Nucleares/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Fatores de Processamento de Serina-Arginina , Transdução de Sinais , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismoRESUMO
ARHGAP21 is a 217 kDa RhoGAP protein shown to modulate cell migration through the control of Cdc42 and FAK activities. In the present work a 250 kDa-ARHGAP21 was identified by mass spectrometry. This modified form is differentially expressed among cell lines and human primary cells. Co-immunoprecipitations and in vitro SUMOylation confirmed ARHGAP21 specific modification by SUMO2/3 and mapped the SUMOylation site to ARHGAP21 lysine K1443. Immunofluorescence staining revealed that ARHGAP21 co-localizes with SUMO2/3 in the cytoplasm and membrane compartments. Interestingly, our results suggest that ARHGAP21 SUMOylation may be related to cell proliferation. Therefore, SUMOylation of ARHGAP21 may represent a way of guiding its function.
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Proteínas Ativadoras de GTPase/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/genética , Células HEK293 , Humanos , Lisina/química , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , SumoilaçãoRESUMO
Sumoylation is a reversible post-translational modification that targets a variety of proteins mainly within the nucleus, but also in the plasma membrane and cytoplasm of the cell. It controls diverse cellular mechanisms such as subcellular localization, protein-protein interactions, or transcription factor activity. In recent years, the use of several developmental model systems has unraveled many critical functions for the sumoylation system in the early life of diverse species. In particular, detailed analyses of mutant organisms in both the components of the SUMO pathway and their targets have established the importance of the SUMO system in early developmental processes, such as cell division, cell lineage commitment, specification, and/or differentiation. In addition, an increasing number of developmental proteins, including transcription factors and epigenetic regulators, have been identified as sumoylation substrates. Sumoylation acts on these targets through various mechanisms. For example, this modification has been involved in converting a transcription factor from an activator to a repressor or in regulating the localization and/or stability of numerous transcription factors. This review will summarize current information on the function of sumoylation in embryonic development in different species from yeast to mammals.