RESUMO
SUMO conjugation is a conserved process of eukaryotes, and essential in metazoa. Similar to ubiquitylation, a SUMO-activating enzyme links to the SUMO carboxyl-terminal Gly in a thioester bond, and a SUMO-conjugating enzyme accepts activated SUMO and can transfer it to substrates. Unlike ubiquitylation, this transfer can also occur, in an unspecified number of cases, in the absence of ligase-like enzymes. Different isoforms of SUMO are present in eukaryotic genomes. Saccharomyces cerevisiae has only one SUMO protein, humans have four, and Arabidopsis thaliana has eight, the main isoforms being SUMO1 and SUMO2 with about 95% identity. Functionally similar to human SUMO2 and SUMO3, Arabidopsis SUMO1 and 2 can be transferred to substrates as single moieties, but can also form SUMO chains, a process enhanced by chain-forming ligases. By combined action with SUMO chain recognizing ubiquitin ligases, chains can channel substrates into the ubiquitin-dependent degradation pathway.A method is described to sumoylate substrates and to generate SUMO chains, using plant enzymes produced in E. coli. In vitro SUMO chain formation may serve for further analysis of SUMO chain functions. It can also provide an easy-to-synthesize substrate for SUMO-specific proteases.
Assuntos
Arabidopsis , Sumoilação , Humanos , Escherichia coli/metabolismo , Arabidopsis/metabolismo , Ligases/metabolismo , Isoformas de Proteínas/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
Structural maintenance of chromosomes (SMCs) complexes, cohesin, condensin, and Smc5/6, are essential for viability and participate in multiple processes, including sister chromatid cohesion, chromosome condensation, and DNA repair. Here we show that SUMO chains target all three SMC complexes and are antagonized by the SUMO protease Ulp2 to prevent their turnover. We uncover that the essential role of the cohesin-associated subunit Pds5 is to counteract SUMO chains jointly with Ulp2. Importantly, fusion of Ulp2 to kleisin Scc1 supports viability of PDS5 null cells and protects cohesin from proteasomal degradation mediated by the SUMO-targeted ubiquitin ligase Slx5/Slx8. The lethality of PDS5-deleted cells can also be bypassed by simultaneous loss of the proliferating cell nuclear antigen (PCNA) unloader, Elg1, and the cohesin releaser, Wpl1, but only when Ulp2 is functional. Condensin and Smc5/6 complex are similarly guarded by Ulp2 against unscheduled SUMO chain assembly, which we propose to time the availability of SMC complexes on chromatin.
Assuntos
Endopeptidases/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Genes Supressores , Mutação/genética , Subunidades Proteicas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Sumoilação , CoesinasRESUMO
Signaling by the ubiquitin-related SUMO pathway relies on coordinated conjugation and deconjugation events. SUMO-specific deconjugating enzymes counterbalance SUMOylation, but comprehensive insight into their substrate specificity and regulation is missing. By characterizing SENP6, we define an N-terminal multi-SIM domain as a critical determinant in targeting SENP6 to SUMO chains. Proteomic profiling reveals a network of SENP6 functions at the crossroads of chromatin organization and DNA damage response (DDR). SENP6 acts as a SUMO eraser at telomeric and centromeric chromatin domains and determines the SUMOylation status and chromatin association of the cohesin complex. Importantly, SENP6 is part of the hPSO4/PRP19 complex that drives ATR-Chk1 activation. SENP6 deficiency impairs chromatin association of the ATR cofactor ATRIP, thereby compromising the activation of Chk1 signaling in response to aphidicolin-induced replicative stress and sensitizing cells to DNA damage. We propose a general role of SENP6 in orchestrating chromatin dynamics and genome stability networks by balancing chromatin residency of protein complexes.
Assuntos
Cromatina/metabolismo , Cromossomos Humanos/metabolismo , Cisteína Endopeptidases/metabolismo , Genoma Humano , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Motivos de Aminoácidos , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/metabolismo , Quinase 1 do Ponto de Checagem/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cisteína Endopeptidases/química , Instabilidade Genômica , Células HEK293 , Células HeLa , Humanos , Proteínas Nucleares/metabolismo , Ligação Proteica , Sumoilação , Fatores de Transcrição/metabolismo , CoesinasRESUMO
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/metabolismoRESUMO
Covalent modification of proteins with the small ubiquitin-related modifier (SUMO) is found in all eukaryotes and is involved in many important processes. SUMO attachment may change interaction properties, subcellular localization, or stability of a modified protein. Usually, only a small fraction of a protein is modified at a given time because sumoylation is a highly dynamic process. The sumoylated state of a protein is controlled by the activity of the sumoylation enzymes that promote either their mono- or poly-sumoylation (SUMO chain formation), by SUMO proteases that reverse these modifications, and by SUMO-targeted ubiquitin ligases (STUbL, ULS) that mediate their degradation by the proteasome. While some organisms, such as humans, express multiple isoforms, budding yeast SUMO is encoded by a single and essential gene termed SMT3. The analysis of the simpler SUMO system in budding yeast has been instrumental in the identification of enzymes acting on this modification and controlling its dynamics. Sumoylation of proteins changes dramatically during the cell division cycle and under various stress conditions. Here we summarize various approaches that employ Saccharomyces cerevisiae as a model system to study the dynamics of sumoylation and how it is controlled.
Assuntos
Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Western Blotting/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Sumoilação , Ubiquitina/metabolismoRESUMO
The dynamic and reversible post-translational modification of proteins and protein complexes with the ubiquitin-related SUMO modifier regulates a wide variety of nuclear functions, such as transcription, replication and DNA repair. SUMO can be attached as a monomer to its targets, but can also form polymeric SUMO chains. While monoSUMOylation is generally involved in the assembly of protein complexes, multi- or polySUMOylation may have very different consequences. The evolutionary conserved paradigmatic signaling process initiated by multi- or polySUMOylation is the SUMO-targeted Ubiquitin ligase (StUbL) pathway, where the presence of multiple SUMO moieties primes ubiquitylation by the mammalian E3 ubiquitin ligases RNF4 or RNF111, or the yeast Slx5/8 heterodimer. The mammalian SUMO chain-specific isopeptidases SENP6 or SENP7, or yeast Ulp2, counterbalance chain formation thereby limiting StUbL activity. Many facets of SUMO chain signaling are still incompletely understood, mainly because only a limited number of polySUMOylated substrates have been identified. Here we summarize recent work that revealed a highly interconnected network of candidate polySUMO modified proteins functioning in DNA damage response and chromatin organization. Based on these datasets and published work on distinct polySUMO-regulated processes we discuss overarching concepts in SUMO chain function. We propose an evolutionary conserved role of polySUMOylation in orchestrating chromatin dynamics and genome stability networks by balancing chromatin-residency of protein complexes. This concept will be exemplified in processes, such as centromere/kinetochore organization, sister chromatid cohesion, DNA repair and replication.
RESUMO
Post-translational modifications by ubiquitin-related SUMO modifiers regulate cellular signaling networks and protein homeostasis. While SUMO1 is mainly conjugated to proteins as a monomer, SUMO2/3 can form polymeric chains. Poly-SUMOylation is best understood in the SUMO-targeted ubiquitin ligase (StUbL) pathway, where chains prime proteins for subsequent ubiquitylation by StUbLs. SUMO chains typically form in response to genotoxic or proteotoxic stress and are preferentially linked via lysine 11 of SUMO2/3. Here, we report that K11 of SUMO2/3 undergoes reversible acetylation with SIRT1 being the K11 deacetylase. In a purified in vitro system, acetylation of SUMO2/3 impairs chain formation and restricts chain length. In a cellular context, however, K11 acetyl-mimicking SUMO2 does not affect the StUbL pathway, indicating that in cells non-canonical chains are more prevalent. MS-based SUMO proteomics indeed identified non-canonical chain types under basal and stress conditions. Importantly, mimicking K11 acetylation alters chain architecture by favoring K5- and K35-linked chains, while inhibiting K7 and K21 linkages. These data provide insight into SUMO chain signaling and point to a role of K11 acetylation as a modulator of SUMO2/3 chains.
Assuntos
Lisina/metabolismo , Sirtuína 1/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Acetilação , Células HeLa , Resposta ao Choque Térmico , Humanos , Proteína da Leucemia Promielocítica/metabolismo , Transdução de Sinais , Sirtuína 1/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Ubiquitinas/metabolismoRESUMO
The small ubiquitin-related modifier (SUMO) conjugation apparatus usually attaches single SUMO moieties to its substrates, but SUMO chains have also been identified. To better define the biochemical requirements and characteristics of SUMO chain formation, mutations in surface-exposed Lys residues of Arabidopsis SUMO-conjugating enzyme (SCE) were tested for in vitro activity. Lys-to-Arg changes in the amino-terminal region of SCE allowed SUMO acceptance from SUMO-activating enzyme and supported substrate mono-sumoylation, but these mutations had significant effects on SUMO chain assembly. We found no indication that SUMO modification of SCE promotes chain formation. A substrate was identified that is modified by SUMO chain addition, showing that SCE can distinguish substrates for either mono-sumoylation or SUMO chain attachment. It is also shown that SCE with active site Cys mutated to Ser can accept SUMO to form an oxyester, but cannot transfer this SUMO moiety onto substrates, explaining a previously known dominant negative effect of this mutation.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/genética , Cisteína Endopeptidases/química , Regulação da Expressão Gênica de Plantas , Mutação , Processamento de Proteína Pós-Traducional , Motivos de Aminoácidos , Substituição de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sítios de Ligação , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais , Especificidade por Substrato , Sumoilação , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Like in most other areas of cellular metabolism, the functions of the ubiquitin-like modifier SUMO in the maintenance of genome stability are manifold and varied. Perturbations of global sumoylation causes a wide spectrum of phenotypes associated with defects in DNA maintenance, such as hypersensitivity to DNA-damaging agents, gross chromosomal rearrangements and loss of entire chromosomes. Consistent with these observations, many key factors involved in various DNA repair pathways have been identified as SUMO substrates. However, establishing a functional connection between a given SUMO target, the cognate SUMO ligase and a relevant phenotype has remained a challenge, mainly because of the difficulties involved in identifying important modification sites and downstream effectors that specifically recognize the target in its sumoylated state. This review will give an overview over the major pathways of DNA repair and genome maintenance influenced by the SUMO system and discuss selected examples of SUMO's actions in these pathways where the biological consequences of the modification have been elucidated.
Assuntos
Cromossomos/genética , Dano ao DNA , Reparo do DNA , DNA/metabolismo , Instabilidade Genômica , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Animais , Cromossomos/metabolismo , DNA/genética , Humanos , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Protein modification with the small ubiquitin-related modifier SUMO is a potent regulatory mechanism implicated in a variety of biological pathways. In vitro sumoylation reactions have emerged as a versatile tool to identify and characterize novel SUMO enzymes as well as their substrates. Here, we present detailed protocols for the purification and fluorescent labeling of mammalian SUMO paralogs for their application in sumoylation assays. These assays provide a fast readout for in vitro SUMO chain formation activity of E3 ligases in a paralog-specific manner. Finally, we critically analyze the application of fluorescent SUMO proteins to study substrate modification in vitro revealing also the drawbacks of the system.
Assuntos
Bioensaio , Processamento de Proteína Pós-Traducional , Proteína SUMO-1/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Corantes Fluorescentes/química , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Ligação Proteica , Proteínas Inibidoras de STAT Ativados/genética , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína SUMO-1/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Espectrometria de Fluorescência , Coloração e Rotulagem/métodos , Sumoilação , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinas/genéticaRESUMO
SUMO conjugation is a conserved process of eukaryotes, and essential in metazoa. Different isoforms of SUMO are present in eukaryotic genomes. Saccharomyces cerevisiae has only one SUMO protein, humans have four and Arabidopsis thaliana has eight, the main isoforms being SUMO1 and SUMO2 with about 95 % identity. Functionally similar to human SUMO2 and SUMO3, Arabidopsis SUMO1 and 2 can form chains, even though they do not possess a consensus SUMOylation motif. The surprising finding that plants have dedicated enzymes for chain synthesis implies a specific role for SUMO chains in plants. By the cooperative action with SUMO chain recognizing ubiquitin ligases, chains might channel substrates into the ubiquitin-dependent degradation pathway.A method is described to generate SUMO chains, using plant enzymes produced in E. coli. In vitro SUMO chain formation may serve for further analysis of SUMO chain functions. It can also provide an easy-to-synthesize substrate for SUMO-specific proteases.