RESUMO
Selective ubiquitin-dependent autophagy plays a pivotal role in the elimination of protein aggregates, assemblies, or organelles and counteracts the cytotoxicity of proteins linked to neurodegenerative diseases. Following substrate ubiquitylation, the cargo is delivered to autophagosomes involving adaptors like human p62 that bind ubiquitin and the autophagosomal ubiquitin-like protein Atg8/LC3; however, whether similar pathways exist in lower eukaryotes remained unclear. Here, we identify by a screen in yeast a new class of ubiquitin-Atg8 adaptors termed CUET proteins, comprising the ubiquitin-binding CUE-domain protein Cue5 from yeast and its human homolog Tollip. Cue5 collaborates with Rsp5 ubiquitin ligase, and the corresponding yeast mutants accumulate aggregation-prone proteins and are vulnerable to polyQ protein expression. Similarly, Tollip depletion causes cytotoxicity toward polyQ proteins, whereas Tollip overexpression clears human cells from Huntington's disease-linked polyQ proteins by autophagy. We thus propose that CUET proteins play a critical and ancient role in autophagic clearance of cytotoxic protein aggregates.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Agregação Patológica de Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Família da Proteína 8 Relacionada à Autofagia , Humanos , Doença de Huntington/metabolismo , Peptídeos/metabolismo , UbiquitinaçãoRESUMO
The alternative PCNA loader containing CTF18-DCC1-CTF8 facilitates sister chromatid cohesion (SCC) by poorly defined mechanisms. Here we found that in DT40 cells, CTF18 acts complementarily with the Warsaw breakage syndrome DDX11 helicase in mediating SCC and proliferation. We uncover that the lethality and cohesion defects of ctf18 ddx11 mutants are associated with reduced levels of chromatin-bound cohesin and rescued by depletion of WAPL, a cohesin-removal factor. On the contrary, high levels of ESCO1/2 acetyltransferases that acetylate cohesin to establish SCC do not rescue ctf18 ddx11 phenotypes. Notably, the tight proximity of sister centromeres and increased anaphase bridges characteristic of WAPL-depleted cells are abrogated by loss of both CTF18 and DDX11 The results reveal that vertebrate CTF18 and DDX11 collaborate to provide sufficient amounts of chromatin-loaded cohesin available for SCC generation in the presence of WAPL-mediated cohesin-unloading activity. This process modulates chromosome structure and is essential for cellular proliferation in vertebrates.
Assuntos
Cromátides , Proteínas Cromossômicas não Histona , Animais , Proteínas de Ciclo Celular/genética , Cromátides/genética , Proteínas Cromossômicas não Histona/genética , Vertebrados/genética , CoesinasRESUMO
Protein modification by SUMO affects a wide range of protein substrates. Surprisingly, although SUMO pathway mutants display strong phenotypes, the function of individual SUMO modifications is often enigmatic, and SUMOylation-defective mutants commonly lack notable phenotypes. Here, we use DNA double-strand break repair as an example and show that DNA damage triggers a SUMOylation wave, leading to simultaneous multisite modifications of several repair proteins of the same pathway. Catalyzed by a DNA-bound SUMO ligase and triggered by single-stranded DNA, SUMOylation stabilizes physical interactions between the proteins. Notably, only wholesale elimination of SUMOylation of several repair proteins significantly affects the homologous recombination pathway by considerably slowing down DNA repair. Thus, SUMO acts synergistically on several proteins, and individual modifications only add up to efficient repair. We propose that SUMOylation may thus often target a protein group rather than individual proteins, whereas localized modification enzymes and highly specific triggers ensure specificity.
Assuntos
Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Reparo de DNA por Recombinação , Saccharomyces cerevisiae/metabolismo , Sumoilação , Quebras de DNA de Cadeia Simples , Proteína SUMO-1/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
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
Reversible modification of proteins by SUMO (small ubiquitin-like modifier) affects a large number of cellular processes. In striking contrast to the related ubiquitin pathway, only a few enzymes participate in the SUMO system, although this pathway has numerous substrates as well. Emerging evidence suggests that SUMOylation frequently targets entire groups of physically interacting proteins rather than individual proteins. Protein-group SUMOylation appears to be triggered by recruitment of SUMO ligases to preassembled protein complexes. Because SUMOylation typically affects groups of proteins that bear SUMO-interaction motifs (SIMs), protein-group SUMOylation may foster physical interactions between proteins through multiple SUMO-SIM interactions. Individual SUMO modifications may act redundantly or additively, yet they may mediate dedicated functions as well. In this review, we focus on the unorthodox principles of this pathway and give examples for SUMO-controlled nuclear activities. We propose that collective SUMOylation is typical for nuclear assemblies and argue that SUMO serves as a distinguishing mark for functionally engaged protein fractions.
Assuntos
Núcleo Celular/metabolismo , Proteínas/metabolismo , Sumoilação/fisiologia , Adenosina Trifosfatases/metabolismo , Motivos de Aminoácidos , Animais , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/ultraestrutura , Reparo do DNA/fisiologia , Enzimas/metabolismo , Humanos , Lisina/metabolismo , Modelos Biológicos , Complexos Multiproteicos , Proteínas Nucleares/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Mapeamento de Interação de Proteínas , Proteômica , Ribossomos/metabolismo , Especificidade por Substrato , Sumoilação/genética , Telômero/metabolismo , Homeostase do Telômero/fisiologia , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteína com ValosinaRESUMO
Modification by the ubiquitin-like protein SUMO affects hundreds of cellular substrate proteins and regulates a wide variety of physiological processes. While the SUMO system appears to predominantly target nuclear proteins and, to a lesser extent, cytosolic proteins, hardly anything is known about the SUMOylation of proteins targeted to membrane-enclosed organelles. Here, we identify a large set of structurally and functionally unrelated mitochondrial proteins as substrates of the SUMO pathway in yeast. We show that SUMO modification of mitochondrial proteins does not rely on mitochondrial targeting and, in fact, is strongly enhanced upon import failure, consistent with the modification occurring in the cytosol. Moreover, SUMOylated forms of mitochondrial proteins particularly accumulate in HSP70- and proteasome-deficient cells, suggesting that SUMOylation participates in cellular protein quality control. We therefore propose that SUMO serves as a mark for nonfunctional mitochondrial proteins, which only sporadically arise in unstressed cells but strongly accumulate upon defective mitochondrial import and impaired proteostasis. Overall, our findings provide support for a role of SUMO in the cytosolic response to aberrant proteins.
Assuntos
Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Transporte Biológico/fisiologia , Microscopia de Fluorescência , Proteostase , Saccharomyces cerevisiae/metabolismo , SumoilaçãoRESUMO
DNA replication is sensitive to damage in the template. To bypass lesions and complete replication, cells activate recombination-mediated (error-free) and translesion synthesis-mediated (error-prone) DNA damage tolerance pathways. Crucial for error-free DNA damage tolerance is template switching, which depends on the formation and resolution of damage-bypass intermediates consisting of sister chromatid junctions. Here we show that a chromatin architectural pathway involving the high mobility group box protein Hmo1 channels replication-associated lesions into the error-free DNA damage tolerance pathway mediated by Rad5 and PCNA polyubiquitylation, while preventing mutagenic bypass and toxic recombination. In the process of template switching, Hmo1 also promotes sister chromatid junction formation predominantly during replication. Its C-terminal tail, implicated in chromatin bending, facilitates the formation of catenations/hemicatenations and mediates the roles of Hmo1 in DNA damage tolerance pathway choice and sister chromatid junction formation. Together, the results suggest that replication-associated topological changes involving the molecular DNA bender, Hmo1, set the stage for dedicated repair reactions that limit errors during replication and impact on genome stability.
Assuntos
Cromossomos Fúngicos/ultraestrutura , Dano ao DNA , DNA Fúngico/genética , Proteínas de Grupo de Alta Mobilidade/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Cromátides/genética , Cromátides/ultraestrutura , Cromatina/ultraestrutura , Cromossomos Fúngicos/genética , DNA Helicases/metabolismo , Replicação do DNA , DNA Cruciforme , DNA Fúngico/efeitos dos fármacos , Instabilidade Genômica , Proteínas de Grupo de Alta Mobilidade/química , Proteínas de Grupo de Alta Mobilidade/genética , Metanossulfonato de Metila/farmacologia , Mutagênicos/farmacologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteína de Replicação A/metabolismo , Fase S , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Sister chromatid cohesion, established during replication by the ring-shaped multiprotein complex cohesin, is essential for faithful chromosome segregation. Replisome-associated proteins are required to generate cohesion by two independent pathways. One mediates conversion of cohesins bound to unreplicated DNA ahead of replication forks into cohesive entities behind them, while the second promotes cohesin de novo loading onto newly replicated DNA. The latter process depends on the cohesin loader Scc2 (NIPBL in vertebrates) and the alternative PCNA loader CTF18-RFC. However, the mechanism of de novo cohesin loading during replication is unknown. Here we show that PCNA physically recruits the yeast cohesin loader Scc2 via its C-terminal PCNA-interacting protein motif. Binding to PCNA is crucial, as the scc2-pip mutant deficient in Scc2-PCNA interaction is defective in cohesion when combined with replisome mutants of the cohesin conversion pathway. Importantly, the role of NIPBL recruitment to PCNA for cohesion generation is conserved in vertebrate cells.
Assuntos
Cromátides , Segregação de Cromossomos , Animais , Antígeno Nuclear de Célula em Proliferação/genética , Cromátides/genética , Proteínas de Ciclo Celular/genética , Saccharomyces cerevisiae/genética , CoesinasRESUMO
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
Accurate chromosomal DNA replication is fundamental for optimal cellular function and genome integrity. Replication perturbations activate DNA damage tolerance pathways, which are crucial to complete genome duplication as well as to prevent formation of deleterious double strand breaks. Cells use two general strategies to tolerate lesions: recombination to a homologous template, and trans-lesion synthesis with specialized polymerases. While key players of these processes have been outlined, much less is known on their choreography and regulation. Recent advances have uncovered principles by which DNA damage tolerance is regulated locally and temporally - in relation to replication timing and cell cycle stage -, and are beginning to elucidate the DNA dynamics that mediate lesion tolerance and influence chromosome structure during replication.
Assuntos
Reparo do DNA , Células Eucarióticas/metabolismo , Animais , Dano ao DNA , Replicação do DNA , HumanosRESUMO
Protein modification by conjugation to the ubiquitin-related protein SUMO (SUMOylation) regulates numerous cellular functions and is reversible. However, unlike typical posttranslational modifications, SUMOylation often targets and regulates proteins of functionally and physically linked protein groups, rather than individual proteins. Functional studies of protein-group SUMOylation are thus particularly challenging, as they require the identification of ideally all members of a modified protein group. Here, we describe mass spectrometric approaches to detect SUMOylated protein groups in Saccharomyces cerevisiae, yet the protocols can be readily adapted for studies of SUMOylation in mammalian cells.
Assuntos
Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Cromatografia de Afinidade , Marcação por Isótopo , Espectrometria de Massas , Ligação Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Especificidade por Substrato , SumoilaçãoRESUMO
Selective ubiquitin-dependent autophagy mediates the disposal of superfluous cellular structures and clears cells of protein aggregates such as polyQ proteins linked to Huntington disease. Crucial selectivity factors of this pathway are ubiquitin-Atg8 receptors such as human SQSTM1/p62, which recognize ubiquitinated cargoes and deliver them to phagophores for degradation. Contrasting previous beliefs, we recently showed that ubiquitin-dependent autophagy is not restricted to higher eukaryotes but also exists in yeast with Cue5, harboring a ubiquitin-binding CUE domain, being a ubiquitin-Atg8 receptor. Notably, the human CUE domain protein TOLLIP is functionally similar to yeast Cue5, indicating that Cue5/TOLLIP (CUET) proteins represent a new and conserved class of autophagy receptors. Remarkably, both Cue5 in yeast and TOLLIP in human cells mediate efficient clearance of aggregation-prone polyQ proteins derived from human HTT/huntingtin. Indeed, TOLLIP is potentially more potent in polyQ clearance than SQSTM1/p62 in HeLa cells, suggesting that TOLLIP, also implicated in innate immunity, may be significant for human health and disease.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Agregação Patológica de Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , HumanosRESUMO
The tumour microenvironment regulates tumour progression and the spread of cancer in the body. Targeting the stromal cells that surround cancer cells could, therefore, improve the effectiveness of existing cancer treatments. Here, we show that magnetic nanoparticle clusters encapsulated inside a liposome can, under the influence of an external magnet, target both the tumour and its microenvironment. We use the outstanding T2 contrast properties (r2=573-1,286 s(-1) mM(-1)) of these ferri-liposomes, which are â¼95 nm in diameter, to non-invasively monitor drug delivery in vivo. We also visualize the targeting of the tumour microenvironment by the drug-loaded ferri-liposomes and the uptake of a model probe by cells. Furthermore, we used the ferri-liposomes to deliver a cathepsin protease inhibitor to a mammary tumour and its microenvironment in a mouse, which substantially reduced the size of the tumour compared with systemic delivery of the same drug.