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1.
Cell Mol Life Sci ; 77(1): 81-91, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31728578

RESUMO

The compaction of DNA and the continuous action of DNA transactions, including transcription and DNA replication, create complex DNA topologies that require Type IIA Topoisomerases, which resolve DNA topological strain and control genome dynamics. The human TOP2 enzymes catalyze their reactions via formation of a reversible covalent enzyme DNA-protein crosslink, the TOP2 cleavage complex (TOP2cc). Spurious interactions of TOP2 with DNA damage, environmental toxicants and chemotherapeutic "poisons" perturbs the TOP2 reaction cycle, leading to an accumulation of DNA-protein crosslinks, and ultimately, genomic instability and cell death. Emerging evidence shows that TOP2-DNA protein crosslink (DPC) repair entails multiple strand break repair activities, such as removal of the poisoned TOP2 protein and rejoining of the DNA ends through homologous recombination (HR) or non-homologous end joining (NHEJ). Herein, we discuss the molecular mechanisms of TOP2-DPC resolution, with specific emphasis on the recently uncovered ZATTZnf451-licensed TDP2-catalyzed TOP2-DPC reversal mechanism.


Assuntos
Quebras de DNA , Reparo do DNA , DNA Topoisomerases Tipo II/metabolismo , DNA/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Aminoaciltransferases/química , Aminoaciltransferases/metabolismo , Animais , DNA/química , DNA/genética , DNA Topoisomerases Tipo II/química , Humanos , Proteínas de Ligação a Poli-ADP-Ribose/química , Conformação Proteica , Sumoilação , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
2.
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
3.
Medicina (B Aires) ; 79(Spec 6/1): 570-575, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31864228

RESUMO

Post-translational modifications (PTMs) are covalent modifications in proteins during or after their synthesis. Among them, the best known are phosphorylation, methylation, acetylation, and also cleavage or binding of small peptides (ubiquitination, SUMOylation and NEDDylation). Often the protein is modified in multiple sites and these modifications are coordinated generating a PTMs crosstalk. Altered patterns of PTMs have been related to several pathologies. Currently, advances in mass spectrometry have made it possible to study multiple PTMs simultaneously. Oncology is one of the disciplines that incorporated these technologies for the need to better characterize tumors. In cancer, several alterations related to the ubiquitinlike PTMs have been described, such as SUMOylation. In particular, the interaction between different PTMs with SUMOylation has been studied in the context of the von Hippel Lindau (VHL) multitumoral syndrome, generating new putative biomarkers for the evolution of these tumors. RSUME or RWDD3, an enhancer of SUMOylation that acts on VHL and HIF proteins, shows a correlation with malignant parameters in this type of tumors, such as angiogenesis. Regulators of PTMs are becoming relevant as biomarkers in cancer.


Assuntos
Neoplasias/metabolismo , Processamento de Proteína Pós-Traducional , Proteoma/metabolismo , Humanos , Neoplasias/fisiopatologia , Fosforilação/fisiologia , Proteoma/fisiologia , Sumoilação/fisiologia , Fatores de Transcrição/metabolismo , Ubiquitinação/fisiologia
4.
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
5.
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
6.
Adv Exp Med Biol ; 1206: 237-259, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31776989

RESUMO

Protein modification refers to the chemical modification of proteins after their biosynthesis, which is also called posttranslational modification (PTM). PTM causes changes in protein properties and functions. PTM includes an attachment of addition of functional groups, such as methylation, acetylation, glycosylation and phosphorylation; a covalent coupling of small peptides or proteins, such as ubiquitination and SUMOylation; or chemical changes in amino acids, such as citrullination (conversion of arginine to citrulline). Protein modification plays an important role in cellular processes. Since a protein can be modified in different ways, such as acetylation, methylation and phosphorylation, the functions of proteins are different under different modification states. Moreover, the same modification at different sites may have completely different effects on protein function. For example, phosphorylation at some sites in a protein may lead to a functional activation, while phosphorylation at other sites may cause an inhibition of the functions. Thus, different modifications, combinations and sites changes lead to different functional regulations of a protein, resulting in different effects in the cells. In autophagy, PTMs are widely involved in the regulation of autophagy, including ubiquitination, phosphorylation and acetylation. Ubiquitination is the covalent conjugation of ubiquitin to the substrates through a series of enzymes. Phosphorylation refers to an attachment of a phosphoryl group into a protein, primarily on serine, threonine and tyrosine, which is catalyzed by the kinases. Phosphorylation, a common modification, regulates protein function and localization. Phosphorylation in autophagy regulates the activity of autophagy-associated proteins and the initiation and progression of autophagy by regulating signaling pathways. Acetylation means the addition of acetyl groups onto lysine or N-terminal segment of target proteins through acetyltransferases. Acetylation and deacetylation are both involved in the regulation of autophagy initiation and selective autophagy by controlling the acetylation level of important proteins in the autophagy process. In this chapter, we will focus on the regulation of ubiquitination and phosphorylation in autophagy.


Assuntos
Autofagia , Processamento de Proteína Pós-Traducional , Acetilação , Fosforilação , Sumoilação , Ubiquitinação
7.
Biochem Soc Trans ; 47(6): 1815-1831, 2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31754722

RESUMO

FKBP51 and FKBP52 are two iconic members of the family of peptidyl-prolyl-(cis/trans)-isomerases (EC: 5.2.1.8), which comprises proteins that catalyze the cis/trans isomerization of peptidyl-prolyl peptide bonds in unfolded and partially folded polypeptide chains and native state proteins. Originally, both proteins have been studied as molecular chaperones belonging to the steroid receptor heterocomplex, where they were first discovered. In addition to their expected role in receptor folding and chaperoning, FKBP51 and FKBP52 are also involved in many biological processes, such as signal transduction, transcriptional regulation, protein transport, cancer development, and cell differentiation, just to mention a few examples. Recent studies have revealed that both proteins are subject of post-translational modifications such as phosphorylation, SUMOlyation, and acetylation. In this work, we summarize recent advances in the study of these immunophilins portraying them as scaffolding proteins capable to organize protein heterocomplexes, describing some of their antagonistic properties in the physiology of the cell, and the putative regulation of their properties by those post-translational modifications.


Assuntos
Processamento de Proteína Pós-Traducional , Proteínas de Ligação a Tacrolimo/fisiologia , Acetilação , Humanos , Fosforilação , Ligação Proteica , Transporte Proteico , Sumoilação , Proteínas de Ligação a Tacrolimo/metabolismo , Fatores de Transcrição/metabolismo
8.
PLoS Genet ; 15(11): e1008477, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31747400

RESUMO

The kinetochore is the central molecular machine that drives chromosome segregation in all eukaryotes. Genetic studies have suggested that protein sumoylation plays a role in regulating the inner kinetochore; however, the mechanism remains elusive. Here, we show that Saccharomyces cerevisiae Ulp2, an evolutionarily conserved SUMO specific protease, contains a previously uncharacterized kinetochore-targeting motif that recruits Ulp2 to the kinetochore via the Ctf3CENP-I-Mcm16CENP-H-Mcm22CENP-K complex (CMM). Once recruited, Ulp2 selectively targets multiple subunits of the kinetochore, specifically the Constitutive Centromere-Associated Network (CCAN), via its SUMO-interacting motif (SIM). Mutations that impair the kinetochore recruitment of Ulp2 or its binding to SUMO result in an elevated rate of chromosome loss, while mutations that affect both result in a synergistic increase of chromosome loss rate, hyper-sensitivity to DNA replication stress, along with a dramatic accumulation of hyper-sumoylated CCAN. Notably, sumoylation of CCAN occurs at the kinetochore and is perturbed by DNA replication stress. These results indicate that Ulp2 utilizes its dual substrate recognition to prevent hyper-sumoylation of CCAN, ensuring accurate chromosome segregation during cell division.


Assuntos
Centrômero/genética , Segregação de Cromossomos/genética , Endopeptidases/genética , Proteínas de Saccharomyces cerevisiae/genética , Sumoilação/genética , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Cinetocoros/metabolismo , Saccharomyces cerevisiae/genética
9.
Genes Dev ; 33(19-20): 1346-1354, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31575678

RESUMO

The homologous recombination (HR) machinery plays multiple roles in genome maintenance. Best studied in the context of DNA double-stranded break (DSB) repair, recombination enzymes can cleave, pair, and unwind DNA molecules, and collaborate with regulatory proteins to execute multiple DNA processing steps before generating specific repair products. HR proteins also help to cope with problems arising from DNA replication, modulating impaired replication forks or filling DNA gaps. Given these important roles, it is not surprising that each HR step is subject to complex regulation to adjust repair efficiency and outcomes as well as to limit toxic intermediates. Recent studies have revealed intricate regulation of all steps of HR by the protein modifier SUMO, which has been increasingly recognized for its broad influence in nuclear functions. This review aims to connect established roles of SUMO with its newly identified effects on recombinational repair and stimulate further thought on many unanswered questions.


Assuntos
Recombinação Homóloga/genética , Proteína SUMO-1/metabolismo , Animais , Regulação da Expressão Gênica/genética , Humanos , Rad51 Recombinase/metabolismo , Sumoilação
10.
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
11.
Mol Cell ; 76(4): 632-645.e6, 2019 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-31519521

RESUMO

Similar to ubiquitin, SUMO forms chains, but the identity of SUMO-chain-modified factors and the purpose of this modification remain largely unknown. Here, we identify the budding yeast SUMO protease Ulp2, able to disassemble SUMO chains, as a DDK interactor enriched at replication origins that promotes DNA replication initiation. Replication-engaged DDK is SUMOylated on chromatin, becoming a degradation-prone substrate when Ulp2 no longer protects it against SUMO chain assembly. Specifically, SUMO chains channel DDK for SUMO-targeted ubiquitin ligase Slx5/Slx8-mediated and Cdc48 segregase-assisted proteasomal degradation. Importantly, the SUMOylation-defective ddk-KR mutant rescues inefficient replication onset and MCM activation in cells lacking Ulp2, suggesting that SUMO chains time DDK degradation. Using two unbiased proteomic approaches, we further identify subunits of the MCM helicase and other factors as SUMO-chain-modified degradation-prone substrates of Ulp2 and Slx5/Slx8. We thus propose SUMO-chain/Ulp2-protease-regulated proteasomal degradation as a mechanism that times the availability of functionally engaged SUMO-modified protein pools during replication and beyond.


Assuntos
Replicação do DNA , DNA Fúngico/biossíntese , Endopeptidases/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Origem de Replicação , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Sumoilação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA Fúngico/genética , Endopeptidases/genética , Regulação Fúngica da Expressão Gênica , Mutação , Complexo de Endopeptidases do Proteassoma/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Tempo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteína com Valosina/genética , Proteína com Valosina/metabolismo
12.
Nat Commun ; 10(1): 3678, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31417085

RESUMO

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


Assuntos
Proteínas Nucleares/metabolismo , Sumoilação , Ubiquitina-Proteína Ligases/metabolismo , Escherichia coli , Células HeLa , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proteína SUMO-1/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ubiquitinação , Ubiquitinas/metabolismo
13.
Eur J Pharmacol ; 861: 172617, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31430457

RESUMO

Mitochondrial dysfunctions contribute to brain injury in ischemic stroke while disturbance of mitochondrial dynamics results in mitochondrial dysfunction. Mitochondrial E3 ubiquitin ligase 1 (Mul1) involves in regulation of mitochondrial fission and fusion. This study aims to explore whether Mul1 contributes to brain injury in ischemic stroke and the underlying mechanisms. First, a rat ischemic stroke model was established by middle cerebral artery occlusion (MCAO), which showed ischemic injuries (increase in neurological deficit score and infarct volume) and upregulation of Mul1 in brain tissues. Next, Mul1 siRNAs were injected intracerebroventricularly to knockdown Mul1 expression, which evidently attenuated brain injuries (decrease in neurological deficit score, infarct volume and caspase-3 activity), restored mitochondrial dynamics and functions (decreases in mitochondrial fission and cytochrome c release while increase in ATP production), and restored protein levels of dynamin-related protein 1 (Drp1, a mitochondrial fission protein) and mitofusin2 (Mfn2, a mitochondrial fusion protein) through suppressing their sumoylation and ubiquitination, respectively. Finally, PC12 cells were cultured under hypoxic condition to mimic the ischemic stroke. Consistently, knockdown of Mul1 significantly reduced hypoxic injuries (decrease in apoptosis and LDH release), restored protein levels of Drp1 and Mfn2, recovered mitochondrial dynamics and functions (decreases in mitochondrial fission, mitochondrial membrane potential, reactive oxygen species production and cytochrome c release while increase in ATP production). Based on these observations, we conclude that upregulation of Mul1 contributes to brain injury in ischemic stroke rats and disturbs mitochondrial dynamics through sumoylation of Drp1 and ubiquitination of Mfn2.


Assuntos
Isquemia Encefálica/complicações , Encéfalo/patologia , Dinâmica Mitocondrial , Proteínas Mitocondriais/metabolismo , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Apoptose , Hipóxia Celular , Modelos Animais de Doenças , Dinaminas/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Técnicas de Silenciamento de Genes , Masculino , Proteínas Mitocondriais/deficiência , Proteínas Mitocondriais/genética , Células PC12 , Ratos , Ratos Sprague-Dawley , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/enzimologia , Sumoilação , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genética , Regulação para Cima
14.
Nat Commun ; 10(1): 3812, 2019 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-31444354

RESUMO

Acute myeloid leukemia (AML) is a genetically heterogeneous malignant disorder of the hematopoietic system, characterized by the accumulation of DNA-damaged immature myeloid precursors. Here, we find that hCINAP is involved in the repair of double-stranded DNA breaks (DSB) and that its expression correlates with AML prognosis. Following DSB, hCINAP is recruited to damage sites where it promotes SENP3-dependent deSUMOylation of NPM1. This in turn results in the dissociation of RAP80 from the damage site and CTIP-dependent DNA resection and homologous recombination. NPM1 SUMOylation is required for recruitment of DNA repair proteins at the early stage of DNA-damage response (DDR), and SUMOylated NPM1 impacts the assembly of the BRCA1 complex. Knockdown of hCINAP also sensitizes a patient-derived xenograft (PDX) mouse model to chemotherapy. In clinical AML samples, low hCINAP expression is associated with a higher overall survival rate in patients. These results provide mechanistic insight into the function of hCINAP during the DNA-damage response and its role in AML resistance to therapy.


Assuntos
Adenilato Quinase/metabolismo , Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Leucemia Mieloide Aguda/genética , Reparo de DNA por Recombinação , Adenilato Quinase/genética , Adenilato Quinase/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antineoplásicos/uso terapêutico , Proteína BRCA1/metabolismo , Cisteína Endopeptidases/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Feminino , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Células HEK293 , Células HeLa , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Masculino , Camundongos , Pessoa de Meia-Idade , Proteínas Nucleares/metabolismo , Sumoilação , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto Jovem
15.
Oxid Med Cell Longev ; 2019: 1232146, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31428220

RESUMO

Background: Zinc plays a role in mitophagy and protects cardiomyocytes from ischemia/reperfusion injury. This study is aimed at investigating whether SUMOylation of Drp1 is involved in the protection of zinc ion on cardiac I/R injury. Methods: Mouse hearts were subjected to 30 minutes of regional ischemia followed by 2 hours of reperfusion (ischemia/reoxygenation (I/R)). Infarct size and apoptosis were assessed. HL-1 cells were subjected to 24 hours of hypoxia and 6 hours of reoxygenation (hypoxia/reoxygenation (H/R)). Zinc was given 5 min before reperfusion for 30 min. SENP2 overexpression plasmid (Flag-SENP2), Drp1 mutation plasmid (Myc-Drp1 4KR), and SUMO1 siRNA were transfected into HL-1 cells for 48 h before hypoxia. Effects of zinc on SUMO family members were analyzed by Western blotting. SUMOylation of Drp1, apoptosis and the collapse of mitochondrial membrane potential (ΔΨm), and mitophagy were evaluated. Results: Compared with the control, SUMO1 modification level of proteins in the H/R decreased, while this effect was reversed by zinc. In the setting of H/R, zinc attenuated myocardial apoptosis, which was reversed by SUMO1 siRNA. Similar effects were observed in SUMO1 KO mice exposed to H/R. In addition, the dynamin-related protein 1 (Drp1) is a target protein of SUMO1. The SUMOylation of Drp1 induced by zinc regulated mitophagy and contributed to the protective effect of zinc on H/R injury. Conclusions: SUMOylation of Drp1 played an essential role in zinc-induced cardio protection against I/R injury. Our findings provide a promising therapeutic approach for acute myocardial I/R injury.


Assuntos
Dinaminas/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Substâncias Protetoras/farmacologia , Zinco/farmacologia , Animais , Apoptose/efeitos dos fármacos , Hipóxia Celular , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Dinaminas/genética , Coração/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutagênese , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Substâncias Protetoras/uso terapêutico , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína SUMO-1/antagonistas & inibidores , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , Sumoilação/efeitos dos fármacos
16.
Int J Mol Sci ; 20(16)2019 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-31405039

RESUMO

SUMO (Small Ubiquitin-related MOdifier) is a post-translational modifier of the ubiquitin family controlling the function and fate of thousands of proteins. SUMOylation is deregulated in various hematological malignancies, where it participates in both tumorigenesis and cancer cell response to therapies. This is the case for Acute Promyelocytic Leukemias (APL) where SUMOylation, and subsequent destruction, of the PML-RARα fusion oncoprotein are triggered by arsenic trioxide, which is used as front-line therapy in combination with retinoic acid to cure APL patients. A similar arsenic-induced SUMO-dependent degradation was also documented for Tax, a human T-cell lymphotropic virus type I (HTLV1) viral protein implicated in Adult T-cell Leukemogenesis. SUMOylation also participates in Acute Myeloid Leukemia (AML) response to both chemo- and differentiation therapies, in particular through its ability to regulate gene expression. In Multiple Myeloma, many enzymes of the SUMO pathway are overexpressed and their high expression correlates with lower response to melphalan-based chemotherapies. B-cell lymphomas overexpressing the c-Myc oncogene also overexpress most components of the SUMO pathway and are highly sensitive to SUMOylation inhibition. Targeting the SUMO pathway with recently discovered pharmacological inhibitors, alone or in combination with current therapies, might therefore constitute a powerful strategy to improve the treatment of these cancers.


Assuntos
Leucemia/metabolismo , Linfoma/metabolismo , Mieloma Múltiplo/metabolismo , Proteína SUMO-1/metabolismo , Animais , Antineoplásicos/uso terapêutico , Neoplasias Hematológicas/tratamento farmacológico , Neoplasias Hematológicas/metabolismo , Humanos , Leucemia/tratamento farmacológico , Linfoma/tratamento farmacológico , Terapia de Alvo Molecular , Mieloma Múltiplo/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Sumoilação/efeitos dos fármacos
17.
Mol Cell ; 76(1): 11-26.e7, 2019 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-31400850

RESUMO

Alternative lengthening of telomeres (ALT) is a homology-directed repair (HDR) mechanism of telomere elongation that controls proliferation in aggressive cancers. We show that the disruption of RAD51-associated protein 1 (RAD51AP1) in ALT+ cancer cells leads to generational telomere shortening. This is due to RAD51AP1's involvement in RAD51-dependent homologous recombination (HR) and RAD52-POLD3-dependent break induced DNA synthesis. RAD51AP1 KO ALT+ cells exhibit telomere dysfunction and cytosolic telomeric DNA fragments that are sensed by cGAS. Intriguingly, they activate ULK1-ATG7-dependent autophagy as a survival mechanism to mitigate DNA damage and apoptosis. Importantly, RAD51AP1 protein levels are elevated in ALT+ cells due to MMS21 associated SUMOylation. Mutation of a single SUMO-targeted lysine residue perturbs telomere dynamics. These findings indicate that RAD51AP1 is an essential mediator of the ALT mechanism and is co-opted by post-translational mechanisms to maintain telomere length and ensure proliferation of ALT+ cancer cells.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Neoplasias/metabolismo , Proteínas de Ligação a RNA/metabolismo , Homeostase do Telômero , Telômero/metabolismo , Autofagia , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proliferação de Células , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células HeLa , Recombinação Homóloga , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ligases/genética , Ligases/metabolismo , Lisina , Neoplasias/genética , Neoplasias/patologia , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Estabilidade Proteica , Proteínas de Ligação a RNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Transdução de Sinais , Sumoilação , Telômero/genética , Telômero/patologia
18.
Nat Commun ; 10(1): 3789, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31439836

RESUMO

ProMyelocyticLeukemia (PML) protein can polymerize into a mega-Dalton nuclear assembly of 0.1-2 µm in diameter. The mechanism of PML nuclear body biogenesis remains elusive. Here, PMLRBCC is successfully purified. The gel filtration and ultracentrifugation analysis suggest a previously unrecognized sequential oligomerization mechanism via PML monomer, dimer, tetramer and N-mer. Consistently, PML B1-box structure (2.0 Å) and SAXS characterization reveal an unexpected networking by W157-, F158- and SD1-interfaces. Structure-based perturbations in these B1 interfaces not only impair oligomerization in vitro but also abolish PML sumoylation and nuclear body biogenesis in HeLaPml-/- cell. More importantly, as demonstrated by in vivo study using transgenic mice, PML-RARα (PR) F158E precludes leukemogenesis. In addition, single cell RNA sequencing analysis shows that B1 oligomerization is an important regulator in PML-RARα-driven transactivation. Altogether, these results not only define a previously unrecognized B1-box oligomerization in PML, but also highlight oligomerization as an important factor in carcinogenesis.


Assuntos
Carcinogênese , Leucemia Promielocítica Aguda/patologia , Proteína da Leucemia Promielocítica/metabolismo , Multimerização Proteica , Animais , Técnicas de Inativação de Genes , Células HeLa , Humanos , Leucemia Promielocítica Aguda/genética , Camundongos , Camundongos Transgênicos , Mutação , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteínas de Fusão Oncogênica/ultraestrutura , Proteína da Leucemia Promielocítica/genética , Proteína da Leucemia Promielocítica/ultraestrutura , Domínios Proteicos/genética , Receptor alfa de Ácido Retinoico/genética , Receptor alfa de Ácido Retinoico/metabolismo , Receptor alfa de Ácido Retinoico/ultraestrutura , Espalhamento a Baixo Ângulo , Análise de Sequência de RNA , Análise de Célula Única , Sumoilação , Difração de Raios X
19.
Methods Mol Biol ; 1934: 223-233, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31256382

RESUMO

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


Assuntos
Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Cromatografia de Afinidade , Humanos , Processamento de Proteína Pós-Traducional , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/isolamento & purificação , Especificidade por Substrato , Sumoilação
20.
Plant Cell Physiol ; 60(10): 2282-2292, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31290980

RESUMO

Brassinosteroids (BRs), a group of plant steroid hormones, participate in the regulation of plant growth and developmental processes. BR functions through the BES1/BZR1 family of transcription factors, however, the regulation of the BES1 activity by post-translational modifications remains largely unknown. Here, we present evidence that the SUMO E3 ligase SIZ1 negatively regulates BR signaling pathway. T-DNA insertion mutant siz1-2 shows BL (Brassinolide, the most active BR) hypersensitivity and BRZ (Brassinazole, a BR biosynthesis inhibitor) insensitivity during hypocotyl elongation. In addition, expression of BES1-dependent BR-response genes is hyper-regulated in siz1-2 seedlings. The siz1-2bes1-D double mutant exhibits longer hypocotyl than bes1-D. Moreover, SIZ1 physically interacts with BES1 in vivo and in vitro and mediates the sumoylation of BES1. A K302R substitution in BES1 blocks its sumoylation mediated by SIZ1 in plants, indicating that K302 is the principal site for SUMO conjugation. Consistently, we find that sumoylation inhibits BES1 protein stability and activity. Taken together, our data show that the sumoylation of BES1 via SIZ1 negatively regulates the BR signaling pathway.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Brassinosteroides/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ligases/metabolismo , Reguladores de Crescimento de Planta/metabolismo , Transdução de Sinais , Esteroides Heterocíclicos/metabolismo , Arabidopsis/enzimologia , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Ligação a DNA/genética , Hipocótilo/enzimologia , Hipocótilo/genética , Hipocótilo/fisiologia , Ligases/genética , Plântula/enzimologia , Plântula/genética , Plântula/fisiologia , Sumoilação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
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