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1.
Physiol Plant ; 171(1): 77-85, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32880960

RESUMO

Post-translational modifications (PTMs) play a critical role in regulating plant growth and development through the modulation of protein functionality and its interaction with its partners. Analysis of the functional implication of PTMs on plant cellular signalling presents grand challenges in understanding their significance. Proteins decorated or modified with another chemical group or polypeptide play a significant role in regulating physiological processes as compared with non-decorated or non-modified proteins. In the past decade, SUMOylation has been emerging as a potent PTM influencing the adaptability of plants to growth, in response to various environmental cues. Deciphering the SUMO-mediated regulation of plant stress responses and its consequences is required to understand the mechanism underneath. Here, we will discuss the recent advances in the role and significance of SUMOylation in plant growth, development and stress response.


Assuntos
Desenvolvimento Vegetal , Sumoilação , Plantas , Processamento de Proteína Pós-Traducional
2.
Nucleic Acids Res ; 48(21): 12151-12168, 2020 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-33231641

RESUMO

Histones are substrates of the SUMO (small ubiquitin-like modifier) conjugation pathway. Several reports suggest histone sumoylation affects transcription negatively, but paradoxically, our genome-wide analysis shows the modification concentrated at many active genes. We find that trans-tail regulation of histone-H2B ubiquitylation and H3K4 di-methylation potentiates subsequent histone sumoylation. Consistent with the known control of the Set3 histone deacetylase complex (HDAC) by H3K4 di-methylation, histone sumoylation directly recruits the Set3 complex to both protein-coding and noncoding RNA (ncRNA) genes via a SUMO-interacting motif in the HDAC Cpr1 subunit. The altered gene expression profile caused by reducing histone sumoylation matches well to the profile in cells lacking Set3. Histone H2B sumoylation and the Set3 HDAC coordinately suppress cryptic ncRNA transcription initiation internal to mRNA genes. Our results reveal an elaborate co-transcriptional histone crosstalk pathway involving the consecutive ubiquitylation, methylation, sumoylation and deacetylation of histones, which maintains transcriptional fidelity by suppressing spurious transcription.


Assuntos
Histona Desacetilases/genética , Histonas/genética , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transcrição Genética , Acetilação , Ciclofilina A/genética , Ciclofilina A/metabolismo , Regulação Fúngica da Expressão Gênica , Histona Desacetilases/metabolismo , Histonas/metabolismo , Metilação , RNA/genética , RNA/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sumoilação , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitinação
3.
Am J Pathol ; 190(12): 2464-2477, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33222991

RESUMO

Heat shock proteins (HSPs) are emerging as valuable potential molecular targets in breast cancer therapy owing to their diverse functions in cancer cells. This study investigated the potential role of heat shock protein 27 (HSP27, also known as HSPB1) in breast cancer through heat shock protein B8 (HSPB8). The correlation between HSP27 and HSPB8 was identified by using co-immunoprecipitation, immunoprecipitation, and SUMOylation assays. Through gain- and loss-of-function approaches in MCF-7 cells, the effect of HSP27 on HSPB8 expression, SUMOylation level, and protein stability of HSPB8, as well as on cell proliferation, migration, and stemness, was elucidated. A mouse xenograft model of breast cancer cells was established to verify the function of HSP27 in vivo. Results indicate that HSP27 and HSPB8 were highly expressed in breast cancer tissues and MCF-7 cells. HSP27 was also found to induce the SUMOylation of HSPB8 at the 106 locus and subsequently increased its protein stability, which resulted in accelerated proliferation, migration, and stemness of breast cancer cells in vitro along with increased tumor metastasis of breast cancer in vivo. However, these results could be reversed by the knockdown of HSPB8. Overall, HSP27 induces SUMOylation of HSPB8 to promote HSPB8 expression, thereby endorsing proliferation and metastasis of breast cancer cells. This study may provide insight for the development of new targets for breast cancer.


Assuntos
Neoplasias da Mama/patologia , Progressão da Doença , Proteínas de Choque Térmico HSP27/metabolismo , Metástase Neoplásica/patologia , Animais , Neoplasias da Mama/metabolismo , Proliferação de Células/fisiologia , Feminino , Proteínas de Choque Térmico/genética , Humanos , Camundongos , Proteínas Serina-Treonina Quinases/metabolismo , Sumoilação/fisiologia
4.
Nat Commun ; 11(1): 5746, 2020 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-33184279

RESUMO

The Mus81-Mms4 nuclease is activated in G2/M via Mms4 phosphorylation to allow resolution of persistent recombination structures. However, the fate of the activated phosphorylated Mms4 remains unknown. Here we find that Mms4 is engaged by (poly)SUMOylation and ubiquitylation and targeted for proteasome degradation, a process linked to the previously described Mms4 phosphorylation cycle. Mms4 is a mitotic substrate for the SUMO-Targeted Ubiquitin ligase Slx5/8, the SUMO-like domain-containing protein Esc2, and the Mms1-Cul8 ubiquitin ligase. In the absence of these activities, phosphorylated Mms4 accumulates on chromatin in an active state in the next G1, subsequently causing abnormal processing of replication-associated recombination intermediates and delaying the activation of the DNA damage checkpoint. Mus81-Mms4 mutants that stabilize phosphorylated Mms4 have similar detrimental effects on genome integrity. Overall, our findings highlight a replication protection function for Esc2-STUbL-Cul8 and emphasize the importance for genome stability of resetting phosphorylated Mms4 from one cycle to another.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Endonucleases Flap/metabolismo , Mitose/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Proteínas Culina/metabolismo , Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Endonucleases Flap/genética , Regulação Fúngica da Expressão Gênica , Instabilidade Genômica , Mitose/genética , Processamento de Proteína Pós-Traducional/genética , Processamento de Proteína Pós-Traducional/fisiologia , Reparo de DNA por Recombinação , Proteínas de Saccharomyces cerevisiae/genética , Sumoilação , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
5.
Mol Cell ; 80(5): 876-891.e6, 2020 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-33217318

RESUMO

Stress granules (SGs) are cytoplasmic assemblies of proteins and non-translating mRNAs. Whereas much has been learned about SG formation, a major gap remains in understanding the compositional changes SGs undergo during normal disassembly and under disease conditions. Here, we address this gap by proteomic dissection of the SG temporal disassembly sequence using multi-bait APEX proximity proteomics. We discover 109 novel SG proteins and characterize distinct SG substructures. We reveal dozens of disassembly-engaged proteins (DEPs), some of which play functional roles in SG disassembly, including small ubiquitin-like modifier (SUMO) conjugating enzymes. We further demonstrate that SUMOylation regulates SG disassembly and SG formation. Parallel proteomics with amyotrophic lateral sclerosis (ALS)-associated C9ORF72 dipeptides uncovered attenuated DEP recruitment during SG disassembly and impaired SUMOylation. Accordingly, SUMO activity ameliorated C9ORF72-ALS-related neurodegeneration in Drosophila. By dissecting the SG spatiotemporal proteomic landscape, we provide an in-depth resource for future work on SG function and reveal basic and disease-relevant mechanisms of SG disassembly.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Proteína C9orf72/metabolismo , Grânulos Citoplasmáticos/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/patologia , Animais , Proteína C9orf72/genética , Linhagem Celular Tumoral , Grânulos Citoplasmáticos/genética , Grânulos Citoplasmáticos/patologia , Dipeptídeos/genética , Dipeptídeos/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster , Humanos , Camundongos , Proteômica , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética
6.
Nucleic Acids Res ; 48(21): 11890-11912, 2020 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-33068409

RESUMO

Eukaryotic cells compartmentalize their internal milieu in order to achieve specific reactions in time and space. This organization in distinct compartments is essential to allow subcellular processing of regulatory signals and generate specific cellular responses. In the nucleus, genetic information is packaged in the form of chromatin, an organized and repeated nucleoprotein structure that is a source of epigenetic information. In addition, cells organize the distribution of macromolecules via various membrane-less nuclear organelles, which have gathered considerable attention in the last few years. The macromolecular multiprotein complexes known as Promyelocytic Leukemia Nuclear Bodies (PML NBs) are an archetype for nuclear membrane-less organelles. Chromatin interactions with nuclear bodies are important to regulate genome function. In this review, we will focus on the dynamic interplay between PML NBs and chromatin. We report how the structure and formation of PML NBs, which may involve phase separation mechanisms, might impact their functions in the regulation of chromatin dynamics. In particular, we will discuss how PML NBs participate in the chromatinization of viral genomes, as well as in the control of specific cellular chromatin assembly pathways which govern physiological mechanisms such as senescence or telomere maintenance.


Assuntos
Núcleo Celular/metabolismo , Cromatina/metabolismo , Genoma Viral , Corpos de Inclusão Intranuclear/metabolismo , Proteína da Leucemia Promielocítica/genética , Processamento de Proteína Pós-Traducional , Núcleo Celular/genética , Núcleo Celular/ultraestrutura , Núcleo Celular/virologia , Senescência Celular , Cromatina/química , Cromatina/ultraestrutura , Montagem e Desmontagem da Cromatina , Genoma Humano , Histonas/genética , Histonas/metabolismo , Interações Hospedeiro-Patógeno/genética , Humanos , Corpos de Inclusão Intranuclear/química , Corpos de Inclusão Intranuclear/ultraestrutura , Proteína da Leucemia Promielocítica/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Homeostase do Telômero , Vírus/genética , Vírus/metabolismo
7.
Nat Commun ; 11(1): 4980, 2020 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-33020477

RESUMO

The functions of the proto-oncoprotein c-Myc and the tumor suppressor p53 in controlling cell survival and proliferation are inextricably linked as "Yin and Yang" partners in normal cells to maintain tissue homeostasis: c-Myc induces the expression of ARF tumor suppressor (p14ARF in human and p19ARF in mouse) that binds to and inhibits mouse double minute 2 homolog (MDM2) leading to p53 activation, whereas p53 suppresses c-Myc through a combination of mechanisms involving transcriptional inactivation and microRNA-mediated repression. Nonetheless, the regulatory interactions between c-Myc and p53 are not retained by cancer cells as is evident from the often-imbalanced expression of c-Myc over wildtype p53. Although p53 repression in cancer cells is frequently associated with the loss of ARF, we disclose here an alternate mechanism whereby c-Myc inactivates p53 through the actions of the c-Myc-Inducible Long noncoding RNA Inactivating P53 (MILIP). MILIP functions to promote p53 polyubiquitination and turnover by reducing p53 SUMOylation through suppressing tripartite-motif family-like 2 (TRIML2). MILIP upregulation is observed amongst diverse cancer types and is shown to support cell survival, division and tumourigenicity. Thus our results uncover an inhibitory axis targeting p53 through a pan-cancer expressed RNA accomplice that links c-Myc to suppression of p53.


Assuntos
Neoplasias/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Longo não Codificante/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Carcinogênese , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/genética , RNA Longo não Codificante/genética , Sumoilação , Proteína Supressora de Tumor p53/genética , Ubiquitinação
8.
Sci Rep ; 10(1): 15513, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32968203

RESUMO

The insulin/IGF signalling pathway impacts lifespan across distant taxa, by controlling the activity of nodal transcription factors. In the nematode Caenorhabditis elegans, the transcription regulators DAF-16/FOXO and SKN-1/Nrf function to promote longevity under conditions of low insulin/IGF signalling and stress. The activity and subcellular localization of both DAF-16 and SKN-1 is further modulated by specific posttranslational modifications, such as phosphorylation and ubiquitination. Here, we show that ageing elicits a marked increase of SUMO levels in C. elegans. In turn, SUMO fine-tunes DAF-16 and SKN-1 activity in specific C. elegans somatic tissues, to enhance stress resistance. SUMOylation of DAF-16 modulates mitochondrial homeostasis by interfering with mitochondrial dynamics and mitophagy. Our findings reveal that SUMO is an important determinant of lifespan, and provide novel insight, relevant to the complexity of the signalling mechanisms that influence gene expression to govern organismal survival in metazoans.


Assuntos
Envelhecimento/fisiologia , Caenorhabditis elegans/fisiologia , Mitocôndrias/metabolismo , Proteína SUMO-1/fisiologia , Envelhecimento/metabolismo , Animais , Western Blotting , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/fisiologia , Clonagem Molecular , DNA Mitocondrial/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição Forkhead/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Homeostase/fisiologia , Longevidade/fisiologia , Mitocôndrias/fisiologia , Consumo de Oxigênio , Proteína SUMO-1/metabolismo , Sumoilação , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia
9.
Oncogene ; 39(43): 6692-6703, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32948837

RESUMO

Sumoylation is an essential posttranslational modification in eukaryotes that has emerged as an important pathway in oncogenic processes. Most human cancers display hyperactivated sumoylation and many cancer cells are remarkably sensitive to its inhibition, thus supporting application of chemical sumoylation inhibitors in cancer treatment. Here we show, first, that transformed embryonic fibroblasts derived from mice haploinsufficient for Ubc9, the essential and unique gene encoding the SUMO E2 conjugating enzyme, exhibit enhanced proliferation and transformed phenotypes in vitro and as xenografts ex vivo. To then evaluate the possible impact of loss of one Ubc9 allele in vivo, we used a mouse model of intestinal tumorigenesis. We crossed Ubc9+/- mice with mice harboring a conditional ablation of Apc either all along the crypt-villus axis or only in Lgr5+ crypt-based columnar (CBC) cells, the cell compartment that includes the intestinal stem cells proposed as cells-of-origin of intestinal cancer. While Ubc9+/- mice display no overt phenotypes and no globally visible hyposumoylation in cells of the small intestine, we found, strikingly, that, upon loss of Apc in both models, Ubc9+/- mice develop more (>2-fold) intestinal adenomas and show significantly shortened survival. This is accompanied by reduced global sumoylation levels in the polyps, indicating that Ubc9 levels become critical upon oncogenic stress. Moreover, we found that, in normal conditions, Ubc9+/- mice show a moderate but robust (15%) increase in the number of Lgr5+ CBC cells when compared to their wild-type littermates, and further, that these cells display higher degree of stemness and cancer-related and inflammatory gene expression signatures that, altogether, may contribute to enhanced intestinal tumorigenesis. The phenotypes of Ubc9 haploinsufficiency discovered here indicate an unanticipated tumor-suppressive role of sumoylation, one that may have important implications for optimal use of sumoylation inhibitors in the clinic.


Assuntos
Proteína da Polipose Adenomatosa do Colo/metabolismo , Transformação Celular Neoplásica/genética , Neoplasias Intestinais/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Proteína da Polipose Adenomatosa do Colo/genética , Animais , Células Cultivadas , Modelos Animais de Doenças , Embrião de Mamíferos , Fibroblastos , Haploinsuficiência , Humanos , Mucosa Intestinal/patologia , Neoplasias Intestinais/patologia , Camundongos , Camundongos Transgênicos , Cultura Primária de Células , Transdução de Sinais/genética , Sumoilação/genética , Enzimas de Conjugação de Ubiquitina/metabolismo
10.
Am J Physiol Cell Physiol ; 319(4): C657-C666, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32783654

RESUMO

Human flap endonuclease 1 (FEN1) is a structure-specific, multifunctional endonuclease essential for DNA replication and repair. Our previous study showed that in response to DNA damage, FEN1 interacts with the PCNA-like Rad9-Rad1-Hus1 complex instead of PCNA to engage in DNA repair activities, such as stalled DNA replication fork repair, and undergoes SUMOylation by SUMO-1. Here, we report that succinylation of FEN1 was stimulated in response to DNA replication fork-stalling agents, such as ultraviolet (UV) irradiation, hydroxyurea, camptothecin, and mitomycin C. K200 is a key succinylation site of FEN1 that is essential for gap endonuclease activity and could be suppressed by methylation and stimulated by phosphorylation to promote SUMO-1 modification. Succinylation at K200 of FEN1 promoted the interaction of FEN1 with the Rad9-Rad1-Hus1 complex to rescue stalled replication forks. Impairment of FEN1 succinylation led to the accumulation of DNA damage and heightened sensitivity to fork-stalling agents. Altogether, our findings suggest an important role of FEN1 succinylation in regulating its roles in DNA replication and repair, thus maintaining genome stability.


Assuntos
Endonucleases Flap/genética , Instabilidade Genômica/genética , Antígeno Nuclear de Célula em Proliferação/genética , Proteína SUMO-1/genética , Ácido Succínico/metabolismo , Camptotecina/farmacologia , Proteínas de Ciclo Celular/genética , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/efeitos da radiação , Exonucleases/genética , Genoma Humano/genética , Humanos , Hidroxiureia/farmacologia , Mitomicina/farmacologia , Complexos Multiproteicos/genética , Processamento de Proteína Pós-Traducional/genética , Sumoilação/genética , Raios Ultravioleta
11.
Proc Natl Acad Sci U S A ; 117(36): 22128-22134, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32848053

RESUMO

Dendritic spines are tiny membranous protrusions on the dendrites of neurons. Dendritic spines change shape in response to input signals, thereby strengthening the connections between neurons. The growth and stabilization of dendritic spines is thought to be essential for maintaining long-term memory. Actin cytoskeleton remodeling in spines is a key element of their formation and growth. More speculatively, the aggregation of CPEB3, a functional prion that binds RNA, has been reported to be involved in the maintenance of long-term memory. Here we study the interaction between actin and CPEB3 and propose a molecular model for the complex structure of CPEB3 and an actin filament (F-actin). The results of our computational modeling, including both energetic and structural analyses, are compared with novel data from peptide array experiments. Our model of the CPEB3/F-actin interaction suggests that F-actin potentially triggers the aggregation-prone structural transition of a short CPEB3 sequence by zipping it into a beta-hairpin form. We also propose that the CPEB3/F-actin interaction might be regulated by the SUMOylation of CPEB3, based on bioinformatic searches for potential SUMOylation sites as well as SUMO interacting motifs in CPEB3. On the basis of these results and the existing literature, we put forward a possible molecular mechanism underlying long-term memory that involves CPEB3's binding to actin, its aggregation, and its regulation by SUMOylation.


Assuntos
Actinas/química , Proteínas de Ligação a RNA/química , Actinas/metabolismo , Motivos de Aminoácidos , Simulação por Computador , Humanos , Memória de Longo Prazo , Modelos Moleculares , Neurônios/química , Neurônios/fisiologia , Conformação Proteica , Proteínas de Ligação a RNA/metabolismo , Sumoilação
12.
Neuron ; 107(6): 1141-1159.e7, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32735781

RESUMO

Diabetic peripheral neuropathy (DPN) is a highly frequent and debilitating clinical complication of diabetes that lacks therapies. Cellular oxidative stress regulates post-translational modifications, including SUMOylation. Here, using unbiased screens, we identified key enzymes in metabolic pathways and ion channels as novel molecular targets of SUMOylation that critically regulated their activity. Sensory neurons of diabetic patients and diabetic mice demonstrated changes in the SUMOylation status of metabolic enzymes and ion channels. In support of this, profound metabolic dysfunction, accelerated neuropathology, and sensory loss were observed in diabetic gene-targeted mice selectively lacking the ability to SUMOylate proteins in peripheral sensory neurons. TRPV1 function was impaired by diabetes-induced de-SUMOylation as well as by metabolic imbalance elicited by de-SUMOylation of metabolic enzymes, facilitating diabetic sensory loss. Our results unexpectedly uncover an endogenous post-translational mechanism regulating diabetic neuropathy in patients and mouse models that protects against metabolic dysfunction, nerve damage, and altered sensory perception.


Assuntos
Neuropatias Diabéticas/metabolismo , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Nociceptividade , Células Receptoras Sensoriais/metabolismo , Sumoilação , Canais de Cátion TRPV/metabolismo , Animais , Células Cultivadas , Ciclo do Ácido Cítrico , Neuropatias Diabéticas/fisiopatologia , Feminino , Gânglios Espinais/citologia , Glicólise , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL
13.
Curr Protoc Protein Sci ; 101(1): e111, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32633885

RESUMO

The small ubiquitin-like modifier (SUMO) is an important post-translational modifier that regulates various cellular processes. Extensive investigations have been made to comprehend the enzymatic process and consequence of SUMOylation. In vitro SUMOylation assays are invaluable for understanding the fundamental mechanisms of SUMOylation. A majority of these assays monitor changes in the size of the substrate upon SUMO conjugation. Current methods typically detect the size difference through SDS-PAGE and western blots, which makes these methods cumbersome, error-prone, and time-consuming. Here, we describe a fluorescence-based assay for real-time detection of SUMOylation. In the method, a fluorophore-tagged substrate is used in the SUMOylation reaction. Upon SUMOylation, the size and correlation time (τc ) of the substrate increases, and so does its anisotropy. The rate of change in anisotropy with time reflects the efficiency of the SUMOylation machinery. The real-time SUMOylation assay protocol is elegant, time-saving, and less prone to errors. © 2020 Wiley Periodicals LLC. Basic Protocol: Fluorescent anisotropy-based in vitro SUMOylation assay.


Assuntos
Processamento de Proteína Pós-Traducional , Proteína SUMO-1/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Espectrometria de Fluorescência/métodos , Enzimas de Conjugação de Ubiquitina/metabolismo , Fluoresceína-5-Isotiocianato/química , Fluoresceínas/química , Polarização de Fluorescência , Corantes Fluorescentes/química , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína SUMO-1/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Ácidos Sulfônicos/química , Sumoilação , Enzimas de Conjugação de Ubiquitina/genética
14.
Nat Microbiol ; 5(10): 1247-1261, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32690953

RESUMO

To avoid innate sensing and immune control, human immunodeficiency virus type 1 (HIV-1) has to prevent the accumulation of viral complementary DNA species. Here, we show that the late HIV-1 accessory protein Vpu hijacks DNA repair mechanisms to promote degradation of nuclear viral cDNA in cells that are already productively infected. Vpu achieves this by interacting with RanBP2-RanGAP1*SUMO1-Ubc9 SUMO E3-ligase complexes at the nuclear pore to reprogramme promyelocytic leukaemia protein nuclear bodies and reduce SUMOylation of Bloom syndrome protein, unleashing end degradation of viral cDNA. Concomitantly, Vpu inhibits RAD52-mediated homologous repair of viral cDNA, preventing the generation of dead-end circular forms of single copies of the long terminal repeat and permitting sustained nucleolytic attack. Our results identify Vpu as a key modulator of the DNA repair machinery. We show that Bloom syndrome protein eliminates nuclear HIV-1 cDNA and thereby suppresses immune sensing and proviral hyper-integration. Therapeutic targeting of DNA repair may facilitate the induction of antiviral immunity and suppress proviral integration replenishing latent HIV reservoirs.


Assuntos
Reparo do DNA , Infecções por HIV/virologia , HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Imunidade Inata , Proteínas Virais Reguladoras e Acessórias/metabolismo , Integração Viral , Regulação Viral da Expressão Gênica , Infecções por HIV/genética , Infecções por HIV/imunologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Modelos Biológicos , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Reparo de DNA por Recombinação , Sumoilação
15.
J Virol ; 94(19)2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32699085

RESUMO

Zika virus (ZIKV) is cytopathic to neurons and persistently infects brain microvascular endothelial cells (hBMECs), which normally restrict viral access to neurons. Despite replicating in the cytoplasm, ZIKV and Dengue virus (DENV) polymerases, NS5 proteins, are predominantly trafficked to the nucleus. We found that a SUMO interaction motif in ZIKV and DENV NS5 proteins directs nuclear localization. However, ZIKV NS5 formed discrete punctate nuclear bodies (NBs), while DENV NS5 was uniformly dispersed in the nucleoplasm. Yet, mutating one DENV NS5 SUMO site (K546R) localized the NS5 mutant to discrete NBs, and NBs formed by the ZIKV NS5 SUMO mutant (K252R) were restructured into discrete protein complexes. In hBMECs, NBs formed by STAT2 and promyelocytic leukemia (PML) protein are present constitutively and enhance innate immunity. During ZIKV infection or NS5 expression, we found that ZIKV NS5 evicts PML from STAT2 NBs, forming NS5/STAT2 NBs that dramatically reduce PML expression in hBMECs and inhibit the transcription of interferon-stimulated genes (ISG). Expressing the ZIKV NS5 SUMO site mutant (K252R) resulted in NS5/STAT2/PML NBs that failed to degrade PML, reduce STAT2 expression, or inhibit ISG induction. Additionally, the K252 SUMOylation site and NS5 nuclear localization were required for ZIKV NS5 to regulate hBMEC cell cycle transcriptional responses. Our data reveal NS5 SUMO motifs as novel NB coordinating factors that distinguish flavivirus NS5 proteins. These findings establish SUMOylation of ZIKV NS5 as critical in the regulation of antiviral ISG and cell cycle responses that permit ZIKV to persistently infect hBMECs.IMPORTANCE ZIKV is a unique neurovirulent flavivirus that persistently infects human brain microvascular endothelial cells (hBMECs), the primary barrier that restricts viral access to neuronal compartments. Here, we demonstrate that flavivirus-specific SIM and SUMO sites determine the assembly of NS5 proteins into discrete nuclear bodies (NBs). We found that NS5 SIM sites are required for NS5 nuclear localization and that SUMO sites regulate NS5 NB complex constituents, assembly, and function. We reveal that ZIKV NS5 SUMO sites direct NS5 binding to STAT2, disrupt the formation of antiviral PML-STAT2 NBs, and direct PML degradation. ZIKV NS5 SUMO sites also transcriptionally regulate cell cycle and ISG responses that permit ZIKV to persistently infect hBMECs. Our findings demonstrate the function of SUMO sites in ZIKV NS5 NB formation and their importance in regulating nuclear responses that permit ZIKV to persistently infect hBMECs and thereby gain access to neurons.


Assuntos
Encéfalo/metabolismo , Núcleo Celular/metabolismo , Células Endoteliais/metabolismo , Sumoilação/fisiologia , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Zika virus/genética , Zika virus/metabolismo , Células A549 , Antivirais/farmacologia , Sítios de Ligação , Encéfalo/virologia , Ciclo Celular , Células Endoteliais/virologia , Exorribonucleases/metabolismo , Regulação Viral da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Imunidade Inata/efeitos dos fármacos , Modelos Moleculares , Mutação , Proteína da Leucemia Promielocítica/metabolismo , Fator de Transcrição STAT2/metabolismo , Alinhamento de Sequência , Sumoilação/efeitos dos fármacos , Zika virus/imunologia , Infecção por Zika virus/imunologia , Infecção por Zika virus/metabolismo
16.
PLoS Pathog ; 16(7): e1008683, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32658923

RESUMO

Human herpesvirus 6B (HHV-6B) is a betaherpesvirus capable of integrating its genome into the telomeres of host chromosomes. Until now, the cellular and/or viral proteins facilitating HHV-6B integration have remained elusive. Here we show that a cellular protein, the promyelocytic leukemia protein (PML) that forms nuclear bodies (PML-NBs), associates with the HHV-6B immediate early 1 (IE1) protein at telomeres. We report enhanced levels of SUMOylated IE1 in the presence of PML and have identified a putative SUMO Interacting Motif (SIM) within IE1, essential for its nuclear distribution, overall SUMOylation and association with PML to nuclear bodies. Furthermore, using PML knockout cell lines we made the original observation that PML is required for efficient HHV-6B integration into host chromosomes. Taken together, we could demonstrate that PML-NBs are important for IE1 multiSUMOylation and that PML plays an important role in HHV-6B integration into chromosomes, a strategy developed by this virus to maintain its genome in its host over long periods of time.


Assuntos
Herpesvirus Humano 6/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Fosfoproteínas/metabolismo , Proteína da Leucemia Promielocítica/metabolismo , Infecções por Roseolovirus/metabolismo , Telômero/virologia , Linhagem Celular , Herpesvirus Humano 6/genética , Humanos , Infecções por Roseolovirus/genética , Sumoilação , Latência Viral/genética
17.
J Virol ; 94(18)2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32641476

RESUMO

Promyelocytic leukemia nuclear bodies (PML-NBs) possess an important intrinsic antiviral activity against alphaherpesvirus infection. PML is the structural backbone of NBs, comprising different isoforms. However, the contribution of each isoform to alphaherpesvirus restriction is not well understood. Here, we report the role of PML-NBs and swine PML (sPML) isoforms in pseudorabies virus (PRV) infection in its natural host swine cells. We found that sPML-NBs exhibit an anti-PRV activity in the context of increasing the expression level of endogenous sPML. Of four sPML isoforms cloned and examined, only isoforms sPML-II and -IIa, not sPML-I and -IVa, expressed in a sPML knockout cells inhibit PRV infection. Both the unique 7b region of sPML-II and the sumoylation-dependent normal formation of PML-NBs are required. 7b possesses a transcriptional repression activity and suppresses viral gene transcription during PRV infection with the cysteine residues 589 and 599 being critically involved. We conclude that sPML-NBs inhibit PRV infection partly by repressing viral gene transcription through the 7b region of sPML-II.IMPORTANCE PML-NBs are nuclear sites that mediate the antiviral restriction of alphaherpesvirus gene expression and replication. However, the contribution of each PML isoform to this activity of PML-NBs is not well characterized. Using PRV and its natural host swine cells as a system, we have discovered that the unique C terminus of sPML isoform II is required for PML-NBs to inhibit PRV infection by directly engaging in repression of viral gene transcription. Our study not only confirms in swine cells that PML-NBs have an antiviral function but also presents a mechanism to suggest that PML-NBs inhibit viral infection in an isoform specific manner.


Assuntos
Herpesvirus Suídeo 1/genética , Corpos de Inclusão Intranuclear/genética , Proteína da Leucemia Promielocítica/genética , Transcrição Genética , Proteínas Virais/genética , Animais , Linhagem Celular , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Regulação da Expressão Gênica , Células HEK293 , Herpesvirus Suídeo 1/metabolismo , Herpesvirus Suídeo 1/patogenicidade , Interações Hospedeiro-Patógeno/genética , Humanos , Corpos de Inclusão Intranuclear/metabolismo , Corpos de Inclusão Intranuclear/virologia , Macrófagos/metabolismo , Macrófagos/virologia , Proteína da Leucemia Promielocítica/metabolismo , Domínios Proteicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade , Sumoilação , Suínos , Proteínas Virais/metabolismo
18.
J Virol ; 94(19)2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32669341

RESUMO

Adeno-associated virus (AAV) has proven to be a promising candidate for gene therapy due to its nonpathogenic nature, ease of production, and broad tissue tropism. However, its transduction capabilities are not optimal due to the interaction with various host factors within the cell. In a previous study, we identified members of the small ubiquitin-like modifier (SUMO) pathway as significant restriction factors in AAV gene transduction. In the present study, we explored the scope of this restriction by focusing on the AAV capsid and host cell proteins as targets. We show that during vector production, the capsid protein VP2 becomes SUMOylated, as indicated by deletion and point mutations of VP2 or the obstruction of its N terminus via the addition of a tag. We observed that SUMOylated AAV capsids display higher stability than non-SUMOylated capsids. Prevention of capsid SUMOylation by VP2 mutations did not abolish transduction restriction by SUMOylation; however, it reduced activation of gene transduction by shutdown of the cellular SUMOylation pathway. This indicates a link between capsid SUMOylation and SUMOylation of cellular proteins in restricting gene transduction. Infection with AAV triggers general SUMOylation of cellular proteins. In particular, the DAXX protein, a putative host cell restriction factor that can become SUMOylated, is able to restrict AAV gene transduction by reducing the intracellular accumulation of AAV vectors. We also observe that the coexpression of a SUMOylation inhibitor with an AAV2 reporter gene vector increased gene transduction significantly.IMPORTANCE Host factors within the cell are the major mode of restriction of adeno-associated virus (AAV) and keep it from fulfilling its maximum potential as a gene therapy vector. A better understanding of the intricacies of restriction would enable the engineering of better vectors. Via a genome-wide short interfering RNA screen, we identified that proteins of the small ubiquitin-like modifier (SUMO) pathway play an important role in AAV restriction. In this study, we investigate whether this restriction is targeted to the AAV directly or indirectly through host cell factors. The results indicate that both targets act in concert to restrict AAV.


Assuntos
Capsídeo/metabolismo , Dependovirus/genética , Dependovirus/fisiologia , Sumoilação/fisiologia , Transdução Genética , Células A549 , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Terapia Genética , Vetores Genéticos/genética , Células HEK293 , Células HeLa , Humanos , Sumoilação/genética
19.
Life Sci ; 255: 117859, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32474020

RESUMO

Excessive fibrosis and extracellular matrix deposition resulting from upregulation of target genes expression mediated by transforming growth factor-beta (TGF-ß)/SMAD and hypoxia inducible factor-1 (HIF-1) signaling pathways are the main mechanisms that drive keloid formation. Sumoylation is a protein posttranslational modification that regulates the function of proteins in many biological processes. In the present study, we aimed to investigate the mechanism underlying the effects of sumoylation on the TGF-ß/SMAD and HIF-1 signaling pathways in keloids. We used 2-D08 to block sumoylation and silenced the expression of sentrin sumo-specific protease 1 (SENP1) to enhance sumoylation in human foreskin fibroblasts (HFFs) and human keloid fibroblasts (HKFs). We also reduced and increased intracellular SUMO1 levels by silencing SUMO1 and transfecting cells with a SUMO1 overexpression lentivirus, respectively. Sumoylation has the ability to amplify TGF-ß/SMAD and HIF-1 signals in keloids, while SUMO1, especially the SUMO1-RanGAP1 complex, is the key molecule affecting the TGF-ß/SMAD and HIF-1 signaling pathways. In addition, we also found that hypoxia promotes sumoylation in keloids and that HIF-1α is covalently modified by SUMO1 at Lys 391 and Lys 477 in HKFs. In summary, we elucidated the role and molecular mechanism of sumoylation in the formation of keloids, providing a new perspective for a potential therapeutic target of keloids.


Assuntos
Queloide/patologia , Proteína SUMO-1/genética , Proteínas Smad/metabolismo , Sumoilação/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Transdução de Sinais/fisiologia , Regulação para Cima
20.
Proc Natl Acad Sci U S A ; 117(26): 14958-14969, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32541040

RESUMO

Oxidative stress is a ubiquitous threat to all aerobic organisms and has been implicated in numerous pathological conditions such as cancer. Here we demonstrate a pivotal role for E2F1, a cell cycle regulatory transcription factor, in cell tolerance of oxidative stress. Cells lacking E2F1 are hypersensitive to oxidative stress due to the defects in cell cycle arrest. Oxidative stress inhibits E2F1 transcriptional activity, independent of changes in association with Rb and without decreasing its DNA-binding activity. Upon oxidative insult, SUMO2 is extensively conjugated to E2F1 mainly at lysine 266 residue, which specifically modulates E2F1 transcriptional activity to enhance cell cycle arrest for cell survival. We identify SENP3, a desumoylating enzyme, as an E2F1-interacting partner. Oxidative stress inhibits the interaction between E2F1 and SENP3, which leads to accumulation of sumoylated E2F1. SENP3-deficient cells exhibit hypersumoylation of E2F1 and are resistant to oxidative insult. High levels of SENP3 in breast cancer are associated with elevated levels of E2F targets, high tumor grade, and poor survival. Given the prevalence of elevated levels of SENP3 across numerous cancer types, the SENP3-E2F1 axis may serve as an avenue for therapeutic intervention in cancer.


Assuntos
Fator de Transcrição E2F1/metabolismo , Estresse Oxidativo , Motivos de Aminoácidos , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Fator de Transcrição E2F1/química , Fator de Transcrição E2F1/genética , Feminino , Humanos , Ligação Proteica , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação
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