RESUMO
Mucosal-associated invariant T (MAIT) cells use canonical semi-invariant T cell receptors (TCR) to recognize microbial riboflavin precursors displayed by the antigen-presenting molecule MR1. The extent of MAIT TCR crossreactivity toward physiological, microbially unrelated antigens remains underexplored. We describe MAIT TCRs endowed with MR1-dependent reactivity to tumor and healthy cells in the absence of microbial metabolites. MAIT cells bearing TCRs crossreactive toward self are rare but commonly found within healthy donors and display T-helper-like functions in vitro. Experiments with MR1-tetramers loaded with distinct ligands revealed significant crossreactivity among MAIT TCRs both ex vivo and upon in vitro expansion. A canonical MAIT TCR was selected on the basis of extremely promiscuous MR1 recognition. Structural and molecular dynamic analyses associated promiscuity to unique TCRß-chain features that were enriched within self-reactive MAIT cells of healthy individuals. Thus, self-reactive recognition of MR1 represents a functionally relevant indication of MAIT TCR crossreactivity, suggesting a potentially broader role of MAIT cells in immune homeostasis and diseases, beyond microbial immunosurveillance.
Assuntos
Células T Invariantes Associadas à Mucosa , Humanos , Membrana Celular , Comunicação Celular , Reações Cruzadas , Reparo do DNA , Antígenos de Histocompatibilidade Classe I , Antígenos de Histocompatibilidade MenorRESUMO
Cohesin's association with and translocation along chromosomal DNAs depend on an ATP hydrolysis cycle driving the association and subsequent release of DNA. This involves DNA being 'clamped' by Scc2 and ATP-dependent engagement of cohesin's Smc1 and Smc3 head domains. Scc2's replacement by Pds5 abrogates cohesin's ATPase and has an important role in halting DNA loop extrusion. The ATPase domains of all SMC proteins are separated from their hinge dimerisation domains by 50-nm-long coiled coils, which have been observed to zip up along their entire length and fold around an elbow, thereby greatly shortening the distance between hinges and ATPase heads. Whether folding exists in vivo or has any physiological importance is not known. We present here a cryo-EM structure of the apo form of cohesin that reveals the structure of folded and zipped-up coils in unprecedented detail and shows that Scc2 can associate with Smc1's ATPase head even when it is fully disengaged from that of Smc3. Using cysteine-specific crosslinking, we show that cohesin's coiled coils are frequently folded in vivo, including when cohesin holds sister chromatids together. Moreover, we describe a mutation (SMC1D588Y) within Smc1's hinge that alters how Scc2 and Pds5 interact with Smc1's hinge and that enables Scc2 to support loading in the absence of its normal partner Scc4. The mutant phenotype of loading without Scc4 is only explicable if loading depends on an association between Scc2/4 and cohesin's hinge, which in turn requires coiled coil folding.
Assuntos
Proteínas de Ciclo Celular/química , Proteínas Cromossômicas não Histona/química , Cromossomos/química , Proteínas de Saccharomyces cerevisiae/química , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromátides , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos/metabolismo , Microscopia Crioeletrônica , DNA/metabolismo , Dimerização , Regulação Fúngica da Expressão Gênica , Hidrólise , Domínios Proteicos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , CoesinasRESUMO
Cohesin and other members of the structural maintenance of chromosomes (SMC)-kleisin family such as condensin and Smc5-6, as well as central players in genome function and structure such as topoisomerases, DNA and RNA polymerases, and DNA repair enzymes contain nucleotide binding domains (NBD) which bind and eventually cleave ATP. The released energy is harnessed in various ways by these enzymes in order to fulfill their essential functions. However, unlike other enzymes, Smc-kleisin complexes-well sized, elongated and multisubunit in nature-have only recently been purified as holocomplexes. This progress offers both the opportunity and the challenge to determine in detail the potency of the ATPase activity of these large protein assemblies-typically exceeding 0.5 MDa in molecular weight-and examine its mechanistic features. We describe here in further detail a combined comprehensive protocol which we have successfully employed before for assaying the ATPase activity of recombinant budding yeast cohesin holocomplexes. We believe that with small and appropriate modifications the methods described here should be applicable to other ATPase complexes.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos/metabolismo , Animais , Células Cultivadas , Cromossomos Fúngicos/genética , Proteínas Fúngicas/metabolismo , Insetos/metabolismo , Saccharomycetales/metabolismo , CoesinasRESUMO
Cohesin organizes DNA into chromatids, regulates enhancer-promoter interactions, and confers sister chromatid cohesion. Its association with chromosomes is regulated by hook-shaped HEAT repeat proteins that bind Scc1, namely Scc3, Pds5, and Scc2. Unlike Pds5, Scc2 is not a stable cohesin constituent but, as shown here, transiently replaces Pds5. Scc1 mutations that compromise its interaction with Scc2 adversely affect cohesin's ATPase activity and loading. Moreover, Scc2 mutations that alter how the ATPase responds to DNA abolish loading despite cohesin's initial association with loading sites. Lastly, Scc2 mutations that permit loading in the absence of Scc4 increase Scc2's association with chromosomal cohesin and reduce that of Pds5. We suggest that cohesin switches between two states: one with Pds5 bound that is unable to hydrolyze ATP efficiently but is capable of release from chromosomes and another in which Scc2 replaces Pds5 and stimulates ATP hydrolysis necessary for loading and translocation from loading sites.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/genética , Proteínas de Ciclo Celular/genética , Cromátides/genética , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos , DNA Fúngico/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , CoesinasRESUMO
As predicted by the notion that sister chromatid cohesion is mediated by entrapment of sister DNAs inside cohesin rings, there is perfect correlation between co-entrapment of circular minichromosomes and sister chromatid cohesion. In most cells where cohesin loads without conferring cohesion, it does so by entrapment of individual DNAs. However, cohesin with a hinge domain whose positively charged lumen is neutralized loads and moves along chromatin despite failing to entrap DNAs. Thus, cohesin engages chromatin in non-topological, as well as topological, manners. Since hinge mutations, but not Smc-kleisin fusions, abolish entrapment, DNAs may enter cohesin rings through hinge opening. Mutation of three highly conserved lysine residues inside the Smc1 moiety of Smc1/3 hinges abolishes all loading without affecting cohesin's recruitment to CEN loading sites or its ability to hydrolyze ATP. We suggest that loading and translocation are mediated by conformational changes in cohesin's hinge driven by cycles of ATP hydrolysis.
Assuntos
Proteínas de Ciclo Celular/química , Cromátides/química , Proteínas Cromossômicas não Histona/química , DNA/química , Trifosfato de Adenosina/química , Animais , Sítios de Ligação , Cromatina/química , Humanos , Hidrólise , Lisina/química , Camundongos , Mutação , Proteínas Nucleares/genética , Conformação Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , CoesinasRESUMO
Cohesin and condensin are Smc-kleisin complexes responsible for shaping our chromosomes. Despite extensive genetic and genomic information available on their function, their biochemistry has been hard to study. Two recent studies finally bring exciting new insights into their enzymology.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona , Cromossomos , DNA , CoesinasRESUMO
The focus of modern molecular biology on protein structure and function has reached unparalleled levels. Whether interacting with nucleic acids or other proteins, protein contacts are the basis for fine-tuning all cellular processes. It is for this reason imperative that protein interactions are studied in ways that reflect actual events taking place inside living cells.Here, we describe in detail a method that combines the residue-level resolution provided by structural biology with physiological studies inside living cells, with the overall goal of explaining the contribution of protein-protein interactions in cellular processes. We use as a powerful example our experience with the DNA exit gate interface of the chromosomal cohesin complex, and we argue that this methodology may be followed to address similar questions within any protein complex and in various model systems.
Assuntos
Proteínas de Ciclo Celular/química , Cromatina/química , Proteínas Cromossômicas não Histona/química , Cromossomos Fúngicos , Reagentes de Ligações Cruzadas/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/metabolismo , CoesinasRESUMO
Chromosomes are shaped by the combined function of the condensin and cohesin Smc-kleisin complexes. After more than two decades of research in this field, a new study finally sheds light on how these machines might interact with their DNA substrates.
Assuntos
Adenosina Trifosfatases , Proteínas de Ligação a DNA/genética , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona , Cromossomos , DNA , Complexos MultiproteicosRESUMO
Mitotic chromosome condensation, sister chromatid cohesion, and higher order folding of interphase chromatin are mediated by condensin and cohesin, eukaryotic members of the SMC (structural maintenance of chromosomes)-kleisin protein family. Other members facilitate chromosome segregation in bacteria [1]. A hallmark of these complexes is the binding of the two ends of a kleisin subunit to the apices of V-shaped Smc dimers, creating a tripartite ring capable of entrapping DNA (Figure 1A). In addition to creating rings, kleisins recruit regulatory subunits. One family of regulators, namely Kite dimers (Kleisin interacting winged-helix tandem elements), interact with Smc-kleisin rings from bacteria, archaea and the eukaryotic Smc5-6 complex, but not with either condensin or cohesin [2]. These instead possess proteins containing HEAT (Huntingtin/EF3/PP2A/Tor1) repeat domains whose origin and distribution have not yet been characterized. Using a combination of profile Hidden Markov Model (HMM)-based homology searches, network analysis and structural alignments, we identify a common origin for these regulators, for which we propose the name Hawks, i.e. HEAT proteins associated with kleisins.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Eucariotos/metabolismo , Evolução Molecular , Mitose , Complexos Multiproteicos/metabolismo , Segregação de Cromossomos , Cadeias de Markov , CoesinasRESUMO
Cohesin facilitates sister chromatid cohesion through the formation of a large ring structure that encircles DNA. Its function relies on two structural maintenance of chromosomes (Smc) proteins, which are found in almost all organisms tested, from bacteria to humans. In accordance with their ubiquity, Smc complexes, such as cohesin, condensin, Smc5-6, and the dosage compensation complex, affect almost all processes of DNA homeostasis. Although their precise molecular mechanism remains enigmatic, here we provide an overview of the architecture of eukaryotic Smc complexes with a particular focus on cohesin, which has seen the most progress recently. Given the evident conservation of many structural features between Smc complexes, it is expected that architecture and topology will have a significant role when deciphering their precise molecular mechanisms.
Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Complexos Multiproteicos/metabolismo , Animais , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/química , Células Eucarióticas/metabolismo , Humanos , Modelos Biológicos , CoesinasRESUMO
Sister chromatid cohesion conferred by entrapment of sister DNAs within a tripartite ring formed between cohesin's Scc1, Smc1, and Smc3 subunits is created during S and destroyed at anaphase through Scc1 cleavage by separase. Cohesin's association with chromosomes is controlled by opposing activities: loading by Scc2/4 complex and release by a separase-independent releasing activity as well as by cleavage. Coentrapment of sister DNAs at replication is accompanied by acetylation of Smc3 by Eco1, which blocks releasing activity and ensures that sisters remain connected. Because fusion of Smc3 to Scc1 prevents release and bypasses the requirement for Eco1, we suggested that release is mediated by disengagement of the Smc3/Scc1 interface. We show that mutations capable of bypassing Eco1 in Smc1, Smc3, Scc1, Wapl, Pds5, and Scc3 subunits reduce dissociation of N-terminal cleavage fragments of Scc1 (NScc1) from Smc3. This process involves interaction between Smc ATPase heads and is inhibited by Smc3 acetylation.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Acetilação , Sítios de Ligação , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , DNA Fúngico/metabolismo , Modelos Moleculares , Mutação , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , CoesinasRESUMO
Through their association with a kleisin subunit (Scc1), cohesin's Smc1 and Smc3 subunits are thought to form tripartite rings that mediate sister chromatid cohesion. Unlike the structure of Smc1/Smc3 and Smc1/Scc1 interfaces, that of Smc3/Scc1 is not known. Disconnection of this interface is thought to release cohesin from chromosomes in a process regulated by acetylation. We show here that the N-terminal domain of yeast Scc1 contains two α helices, forming a four-helix bundle with the coiled coil emerging from Smc3's adenosine triphosphatase head. Mutations affecting this interaction compromise cohesin's association with chromosomes. The interface is far from Smc3 residues, whose acetylation prevents cohesin's dissociation from chromosomes. Cohesin complexes holding chromatids together in vivo do indeed have the configuration of hetero-trimeric rings, and sister DNAs are entrapped within these.
Assuntos
Proteínas de Ciclo Celular/química , Proteínas Cromossômicas não Histona/química , Proteínas de Saccharomyces cerevisiae/química , Adenosina Trifosfatases/química , Sequência de Aminoácidos , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Sequência Conservada , Reagentes de Ligações Cruzadas/química , Cristalografia por Raios X , DNA/química , Mutação , Multimerização Proteica , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/genética , CoesinasRESUMO
Cohesin's Smc1 and Smc3 subunits form V-shaped heterodimers, the nucleotide binding domains (NBDs) of which bind the C- and N-terminal domains, respectively, of the α-kleisin subunit, forming a large tripartite ring within in which sister DNAs are entrapped, and thereby held together (sister chromatid cohesion). During replication, establishment of stable cohesion is dependent on Eco1-mediated acetylation of Smc3's NBD, which is thought to prevent dissociation of α-kleisin from Smc3, thereby locking shut a "DNA exit gate." How Scc3 and Pds5, regulatory subunits bound to α-kleisin, regulate cohesion establishment and maintenance is poorly understood. We show here that by binding to α-kleisin adjacent to its Smc3 nucleotide binding N-terminal domain, Pds5 not only promotes cohesin's release from chromatin but also mediates de novo acetylation of Smc3 by Eco1 during S phase and subsequently prevents de-acetylation by the deacetylase Hos1/HDAC8. By first promoting cohesin's release from chromosomes and subsequently creating and guarding the chemical modification responsible for blocking release, Pds5 enables chromosomal cohesin to switch during S phase from a state of high turnover to one capable of tenaciously holding sister chromatids together for extended periods of time, a duality that has hitherto complicated analysis of this versatile cohesin subunit.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetilação , Acetiltransferases/genética , Acetiltransferases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/genética , Western Blotting , Proteínas de Ciclo Celular/genética , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Cromossomos Fúngicos/genética , Cromossomos Fúngicos/metabolismo , DNA Fúngico/genética , DNA Fúngico/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Fase S , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , CoesinasRESUMO
It has been postulated that chromatin modifications can persist through mitosis and meiosis, thereby securing memory of transcriptional states. Whether these chromatin marks can self-propagate in progeny independently of relevant trans-acting factors is an important question in phenomena related to epigenesis. "Adaptive cellular memory" displayed by yeast cells offers a convenient system to address this question. The yeast GAL genes are slowly activated by Gal4 when cells are first exposed to galactose, but their progeny, grown in glucose media, exhibit a fast activation mode upon re-exposure to this sugar. This "galactose memory" persists for several generations and was recently proposed to involve chromatin modifications and perinuclear topology of the GAL genes cluster. Here, we perform a heterokaryon assay demonstrating that this memory does not have a chromatin basis but is maintained by cytoplasmic factor(s) produced upon previous galactose induction. We show that Gal3, the cytoplasmic rate-limiting factor that releases the Gal4 activator, is dispensable for preserving galactose memory. Instead, the important memory determinant is a close Gal3 homolog, the highly expressed Gal1 galactokinase, the residual activity of which preserves memory in progeny cells by rapidly turning on the Gal4 activator upon cells' re-exposure to galactose.
Assuntos
Galactoquinase/metabolismo , Galactose/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transcrição Gênica , Meios de Cultura , Galactoquinase/genética , Deleção de Genes , Glucose/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The SWR1 complex (SWR1-C)-dependent deposition of the histone variant Htz1 on promoter nucleosomes is typical of Saccharomyces cerevisiae genes whose expression is frequently reprogrammed. Although this epigenetic marking is of significant physiological importance, the determinants of Htz1 deposition, the conditions that set off SWR1-C occupancy, and the implications of Htz1 in transcriptional initiation are issues that remain unresolved. In this report, we addressed these questions by investigating the GAL1 promoter. We show that Htz1 is required for efficient Mediator recruitment and transcription only when the GAL1 promoter is under the influence of the Tup1 corepressor. In fact, we show that it is Tup1 that specifies Htz1 deposition for the promoter nucleosome covering the transcription start site. This deposition occurs rapidly following transcriptional repression, and it correlates with a Tup1-independent transient recruitment of the SWR1 complex. We propose that Tup1 cooperates with SWR1-C and specifies Htz1 deposition at GAL1, thereby marking the promoter for rapid neutralization from its repressive effects.
Assuntos
Galactoquinase/metabolismo , Regulação Fúngica da Expressão Gênica , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Animais , Galactoquinase/genética , Glucose/metabolismo , Histonas/genética , Complexos Multiproteicos , Proteínas Nucleares/genética , Proteínas Repressoras/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transcrição GênicaRESUMO
Among lower eukaryotes, glucose repression is a conserved, widely spread mechanism regulating carbon catabolism. The yeast Snf1 kinase, the Mig1 DNA-binding repressor and the Mig1-interacting co-repressor complex Cyc8(Ssn6)-Tup1 are central components of this pathway. Previous experiments suggested that cytoplasmic translocation of Mig1, upon its phosphorylation by Snf1 in the nucleus, is the key regulatory step for releasing glucose repression. In this report we re-evaluate this model. We establish the coordinated repressive action of Mig1 and Cyc8-Tup1 on GAL1 transcription, but we find that Cyc8-Tup1 is not tethered by Mig1 to the promoter DNA. We demonstrate that both negative regulators occupy GAL1 continuously under either repression or activation conditions, although the majority of the Mig1 is redistributed to the cytoplasm upon activation. We show that Snf1-dependent phosphorylation of Mig1 abolishes interaction with Cyc8-Tup1, and we propose that regulation of this interaction, not the Mig1 cytoplasmic localization, is the molecular switch that controls transcriptional repression/de-repression.