RESUMO
Chromosomes pair and synapse with their homologous partners to segregate correctly at the first meiotic division. Association of telomeres with the LINC (Linker of Nucleoskeleton and Cytoskeleton) complex composed of SUN1 and KASH5 enables telomere-led chromosome movements and telomere bouquet formation, facilitating precise pairwise alignment of homologs. Here, we identify a direct interaction between SUN1 and Speedy A (SPDYA) and determine the crystal structure of human SUN1-SPDYA-CDK2 ternary complex. Analysis of meiosis prophase I process in SPDYA-binding-deficient SUN1 mutant mice reveals that the SUN1-SPDYA interaction is required for the telomere-LINC complex connection and the assembly of a ring-shaped telomere supramolecular architecture at the nuclear envelope, which is critical for efficient homologous pairing and synapsis. Overall, our results provide structural insights into meiotic telomere structure that is essential for meiotic prophase I progression.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Prófase Meiótica I , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Telômero/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/isolamento & purificação , Proteínas de Ciclo Celular/ultraestrutura , Linhagem Celular Tumoral , Cristalografia por Raios X , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/isolamento & purificação , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 2 Dependente de Ciclina/ultraestrutura , Feminino , Células HEK293 , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/ultraestrutura , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/isolamento & purificação , Proteínas Associadas aos Microtúbulos/ultraestrutura , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/ultraestrutura , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestruturaRESUMO
The formation of the chromosome axis is key to meiotic recombination and hence the correct distribution of chromosomes to meiotic products. A key component of the axis in Arabidopsis is the HORMA domain protein (HORMAD) ASY1, the homolog of Hop1 in yeast and HORMAD1/2 in mammals. The chromosomal association of ASY1 is dynamic, i.e., ASY1 is recruited to the axis at early prophase and later largely removed when homologous chromosomes synapse. PCH2/TRIP13 proteins are well-known regulators of meiotic HORMADs and required for their depletion from synapsed chromosomes. However, no direct interaction has been found between PCH2/TRIP13 and the presumptive HORMAD substrates in any organism other than in budding yeast. Thus, it remains largely elusive how the dynamics of ASY1 and other meiotic HORMADs are controlled. Here, we have identified COMET, the Arabidopsis homolog of human p31comet, which is known for its function in the spindle assembly checkpoint (SAC), as a central regulator of ASY1 dynamics in meiosis. We provide evidence that COMET controls ASY1 localization by serving as an adaptor for PCH2. Because ASY1 accumulates in the cytoplasm in early prophase and is persistently present on chromosomes in comet, we conclude that COMET is required for both the recruitment of ASY1 to the nucleus and the subsequent removal from the axis. The here-revealed function of COMET as an adaptor for PCH2 remarkably resembles the regulation of another HORMAD, Mad2, in the SAC in yeast and animals, revealing a conserved regulatory module of HORMA-domain-containing protein complexes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/isolamento & purificação , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/isolamento & purificação , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/isolamento & purificação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/isolamento & purificação , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Meiose , Plantas Geneticamente Modificadas , Prófase , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
A long-standing conundrum is how mitotic chromosomes can compact, as required for clean separation to daughter cells, while maintaining close parallel alignment of sister chromatids. Pursuit of this question, by high resolution 3D fluorescence imaging of living and fixed mammalian cells, has led to three discoveries. First, we show that the structural axes of separated sister chromatids are linked by evenly spaced "mini-axis" bridges. Second, when chromosomes first emerge as discrete units, at prophase, they are organized as co-oriented sister linear loop arrays emanating from a conjoined axis. We show that this same basic organization persists throughout mitosis, without helical coiling. Third, from prophase onward, chromosomes are deformed into sequential arrays of half-helical segments of alternating handedness (perversions), accompanied by correlated kinks. These arrays fluctuate dynamically over <15 s timescales. Together these discoveries redefine the foundation for thinking about the evolution of mitotic chromosomes as they prepare for anaphase segregation.
Assuntos
Proteínas de Ciclo Celular/genética , Cromossomos/genética , Proteínas de Ligação a DNA/genética , Mitose/genética , Adenosina Trifosfatases/genética , Anáfase/genética , Animais , Proteínas de Ciclo Celular/isolamento & purificação , Cromátides/genética , Proteínas Cromossômicas não Histona , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/isolamento & purificação , Imageamento Tridimensional , Mamíferos , Metáfase/genética , Prófase/genéticaRESUMO
The protein-folding chaperone Hsp90 enables the maturation and stability of various oncogenic signaling proteins and is thus pursued as a cancer drug target. Folding in particular of protein kinases is assisted by the co-chaperone Cdc37. Several inhibitors against the Hsp90 ATP-binding site have been developed. However, they displayed significant toxicity in clinical trials. By contrast, the natural product conglobatin A has an exceptionally low toxicity in mice. It targets the protein-protein interface (PPI) of Hsp90 and Cdc37, suggesting that interface inhibitors have an interesting drug development potential. In order to identify inhibitors of the Hsp90/Cdc37 PPI, we have established a mammalian cell lysate-based, medium-throughput amenable split Renilla luciferase assay. This assay employs N-terminal and C-terminal fragments of Renilla luciferase fused to full-length human Hsp90 and Cdc37, respectively. We expect that our assay will allow for the identification of novel Hsp90/Cdc37 interaction inhibitors. Such tool compounds will help to evaluate whether the toxicity profile of Hsp90/Cdc37 PPI inhibitors is in general more favorable than that of ATP-competitive Hsp90 inhibitors. Further development of such tool compounds may lead to new classes of Hsp90 inhibitors with applications in cancer and other diseases.
Assuntos
Bioensaio , Proteínas de Ciclo Celular/isolamento & purificação , Chaperoninas/isolamento & purificação , Proteínas de Choque Térmico HSP90/isolamento & purificação , Mapas de Interação de Proteínas/genética , Animais , Antineoplásicos/farmacologia , Sítios de Ligação/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Chaperoninas/genética , Proteínas de Choque Térmico HSP90/genética , Humanos , Luciferases de Renilla/química , Luciferases de Renilla/genética , Camundongos , Chaperonas Moleculares/genética , Chaperonas Moleculares/isolamento & purificação , Neoplasias/tratamento farmacológico , Neoplasias/genética , Ligação Proteica/efeitos dos fármacosRESUMO
Many cell stressors block protein translation, inducing formation of cytoplasmic aggregates. These aggregates, named stress granules (SGs), are composed by translationally stalled ribonucleoproteins and their assembly strongly contributes to cell survival. Composition and dynamics of SGs are thus important starting points for identifying critical factors of the stress response. In the present study we link components of the H/ACA snoRNP complexes, highly concentrated in the nucleoli and the Cajal bodies, to SG composition. H/ACA snoRNPs are composed by a core of four highly conserved proteins -dyskerin, Nhp2, Nop10 and Gar1- and are involved in several fundamental processes, including ribosome biogenesis, RNA pseudouridylation, stabilization of small nucleolar RNAs and telomere maintenance. By taking advantage of cells overexpressing a dyskerin splice variant undergoing a dynamic intracellular trafficking, we were able to show that H/ACA snoRNP components can participate in SG formation, this way contributing to the stress response and perhaps transducing signals from the nucleus to the cytoplasm. Collectively, our results show for the first time that H/ACA snoRNP proteins can have additional non-nuclear functions, either independently or interacting with each other, thus further strengthening the close relationship linking nucleolus to SG composition.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Grânulos Citoplasmáticos/metabolismo , Proteínas Nucleares/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/isolamento & purificação , Células HeLa , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/isolamento & purificação , Células Tumorais CultivadasRESUMO
The cell is constructed by higher-order structures and organelles through complex interactions among distinct structural constituents. The centrosome is a membraneless organelle composed of two microtubule-derived structures called centrioles and an amorphous mass of pericentriolar material. Super-resolution microscopic analyses in various organisms revealed that diverse pericentriolar material proteins are concentrically localized around a centriole in a highly organized manner. However, the molecular nature underlying these organizations remains unknown. Here we show that two human pericentriolar material scaffolds, Cep63 and Cep152, cooperatively generate a heterotetrameric α-helical bundle that functions in conjunction with its neighboring hydrophobic motifs to self-assemble into a higher-order cylindrical architecture capable of recruiting downstream components, including Plk4, a key regulator for centriole duplication. Mutations disrupting the self-assembly abrogate Plk4-mediated centriole duplication. Because pericentriolar material organization is evolutionarily conserved, this work may offer a paradigm for investigating the assembly and function of centrosomal scaffolds in various organisms.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Centríolos/metabolismo , Proteínas de Neoplasias/metabolismo , Multimerização Proteica/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Motivos de Aminoácidos/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/isolamento & purificação , Linhagem Celular Tumoral , Cristalografia por Raios X , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Microscopia de Fluorescência , Mutação , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/isolamento & purificação , Conformação Proteica em alfa-Hélice , Proteínas Serina-Treonina Quinases/isolamento & purificação , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Imagem com Lapso de TempoRESUMO
In eukaryotes, homologous recombination plays a pivotal role in both genome maintenance and generation of genetic diversity. Eukaryotic RecA homologues, RAD51 and DMC1, are key proteins in homologous recombination that promote pairing between homologous DNA sequences. Arabidopsis thaliana is a prominent model plant for studying eukaryotic homologous recombination. However, A. thaliana RAD51 and DMC1 have not been biochemically characterized. In the present study, we purified A. thaliana RAD51 (AtRAD51) and DMC1 (AtDMC1). Biochemical analyses revealed that both AtRAD51 and AtDMC1 possess ATP hydrolyzing activity, filament formation activity and homologous pairing activity in vitro. We then compared the homologous pairing activities of AtRAD51 and AtDMC1 with those of the Oryza sativa and Homo sapiens RAD51 and DMC1 proteins.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Ciclo Celular/metabolismo , Rad51 Recombinase/metabolismo , Recombinases Rec A/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Proteínas de Arabidopsis/isolamento & purificação , Proteínas de Ciclo Celular/isolamento & purificação , Hidrólise , Rad51 Recombinase/isolamento & purificação , Recombinases Rec A/isolamento & purificação , Alinhamento de SequênciaRESUMO
Homologous recombination (HR) is an essential pathway to restart stalled replication forks, repair spontaneous DNA double-strand breaks, and generate genetic diversity. Together with genetic studies in model organisms, the development of purification protocols and biochemical assays has allowed investigators to begin to understand how the complex machinery of HR functions. At the core of the HR process is the recombination enzyme RecA in bacteria or RAD51 and DMC1 in eukaryotes. The main steps of HR can be reconstituted in vitro and involve: (1) The formation of a ssDNA-RAD51 complex into a helical structure termed the nucleoprotein filament after one DNA strand has been resected at the site of the break. (2) The homologous DNA pairing with an intact copy of the damaged chromatid to form a joint molecule also called displacement loop (D-loop). (3) The exchange of DNA strands and de novo DNA synthesis to restore the damaged/lost DNA. (4) The resolution of joint molecules by nucleolytic cleavage. The human tumor suppressor BRCA2 is a mediator of HR as it actively facilitates the DNA transactions of the recombination proteins RAD51 and DMC1 in a variety of ways: It stabilizes ssDNA-RAD51/DMC1 nucleoprotein filaments. It limits the assembly of RAD51 on dsDNA. It facilitates the replacement of replication protein A by RAD51. The result of these activities is a net increase of DNA strand exchange products as observed in vitro. Here, we describe some of the biochemical assays used to dissect the mediator activities of BRCA2.
Assuntos
Proteína BRCA2/metabolismo , DNA de Cadeia Simples/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Reparo de DNA por Recombinação , Proteína BRCA2/química , Proteína BRCA2/isolamento & purificação , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/isolamento & purificação , Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , DNA de Cadeia Simples/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Eletroforese em Gel de Ágar/métodos , Eletroforese em Gel de Poliacrilamida/instrumentação , Eletroforese em Gel de Poliacrilamida/métodos , Ensaio de Desvio de Mobilidade Eletroforética/instrumentação , Rad51 Recombinase/química , Rad51 Recombinase/isolamento & purificação , Rad51 Recombinase/metabolismo , Coloração e Rotulagem/instrumentação , Coloração e Rotulagem/métodos , Especificidade por SubstratoRESUMO
DNA end resection initiates the largely accurate repair of DNA double-strand breaks (DSBs) by homologous recombination. Specifically, recombination requires the formation of 3' overhangs at DSB sites, which is carried out by nucleases that specifically degrade 5'-terminated DNA. In most cases, DNA end resection is a two-step process, comprising of initial short-range followed by more processive long-range resection. In this chapter, we describe selected assays that reconstitute both the short- and long-range pathways. First, we define methods to study the exonuclease and endonuclease activities of the MRE11-RAD50-NBS1 (MRN) complex in conjunction with phosphorylated cofactor CtIP. This reaction is particularly important to initiate processing of DNA breaks and to recruit components belonging to the subsequent long-range pathway. Next, we describe assays that reconstitute the concerted reactions of Bloom (BLM) or Werner (WRN) helicases that function together with the DNA2 nuclease-helicase, and which are as a complex capable to resect DNA of kilobases in length. The reconstituted reactions allow us to understand how the resection pathways function at the molecular level. The assays will be invaluable to define regulatory mechanisms and to identify inhibitory compounds, which may be valuable in cancer therapy.
Assuntos
Técnicas de Cultura de Células/métodos , Quebras de DNA de Cadeia Dupla , Ensaios Enzimáticos/métodos , Proteínas Recombinantes/isolamento & purificação , Reparo de DNA por Recombinação , Hidrolases Anidrido Ácido , Animais , Baculoviridae/genética , Soluções Tampão , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Técnicas de Cultura de Células/instrumentação , Proteínas de Ciclo Celular/isolamento & purificação , Proteínas de Ciclo Celular/metabolismo , DNA Helicases/isolamento & purificação , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/isolamento & purificação , Enzimas Reparadoras do DNA/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Eletroforese em Gel de Poliacrilamida/instrumentação , Eletroforese em Gel de Poliacrilamida/métodos , Endodesoxirribonucleases , Ensaios Enzimáticos/instrumentação , Humanos , Proteína Homóloga a MRE11/isolamento & purificação , Proteína Homóloga a MRE11/metabolismo , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Oligonucleotídeos/metabolismo , RecQ Helicases/isolamento & purificação , RecQ Helicases/metabolismo , Proteínas Recombinantes/metabolismo , Proteína de Replicação A/isolamento & purificação , Proteína de Replicação A/metabolismo , Células Sf9 , Spodoptera , Transfecção/métodos , Helicase da Síndrome de Werner/isolamento & purificação , Helicase da Síndrome de Werner/metabolismoRESUMO
The Cdc48 ATPase and its cofactors Ufd1/Npl4 (UN) extract polyubiquitinated proteins from membranes or macromolecular complexes, but how they perform these functions is unclear. Cdc48 consists of an N-terminal domain that binds UN and two stacked hexameric ATPase rings (D1 and D2) surrounding a central pore. Here, we use purified components to elucidate how the Cdc48 complex processes substrates. After interaction of the polyubiquitin chain with UN, ATP hydrolysis by the D2 ring moves the polypeptide completely through the double ring, generating a pulling force on the substrate and causing its unfolding. ATP hydrolysis by the D1 ring is important for subsequent substrate release from the Cdc48 complex. This release requires cooperation of Cdc48 with a deubiquitinase, which trims polyubiquitin to an oligoubiquitin chain that is then also translocated through the pore. Together, these results lead to a new paradigm for the function of Cdc48 and its mammalian ortholog p97/VCP.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/isolamento & purificação , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/isolamento & purificação , Endopeptidases/metabolismo , Modelos Moleculares , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Proteína com ValosinaRESUMO
In global proteomic analysis, it is estimated that proteins span from millions to less than 100 copies per cell. The challenge of protein quantitation by classic shotgun proteomic techniques relies on the presence of missing values in peptides belonging to low-abundance proteins that lowers intraruns reproducibility affecting postdata statistical analysis. Here, we present a new analytical workflow MvM (missing value monitoring) able to recover quantitation of missing values generated by shotgun analysis. In particular, we used confident data-dependent acquisition (DDA) quantitation only for proteins measured in all the runs, while we filled the missing values with data-independent acquisition analysis using the library previously generated in DDA. We analyzed cell cycle regulated proteins, as they are low abundance proteins with highly dynamic expression levels. Indeed, we found that cell cycle related proteins are the major components of the missing values-rich proteome. Using the MvM workflow, we doubled the number of robustly quantified cell cycle related proteins, and we reduced the number of missing values achieving robust quantitation for proteins over â¼50 molecules per cell. MvM allows lower quantification variance among replicates for low abundance proteins with respect to DDA analysis, which demonstrates the potential of this novel workflow to measure low abundance, dynamically regulated proteins.
Assuntos
Proteínas de Ciclo Celular/isolamento & purificação , Peptídeos/isolamento & purificação , Proteoma/genética , Proteômica , Proteínas de Ciclo Celular/genética , Peptídeos/genética , Espectrometria de Massas em TandemRESUMO
The cohesin complex is involved in a broad range of chromosomal biology, including DNA repair, gene transcription as well as sister chromatid cohesion. Cohesin is a large, ring-shaped protein complex and is thought to entrap DNA molecules inside of its ring. The unique DNA association is central to cohesin function and requires its ATPase and another heterodimer complex called the cohesin loader. Here we describe the biochemical reconstitution of topological cohesin loading onto DNA using the purified fission yeast cohesin proteins.
Assuntos
Proteínas de Ciclo Celular/isolamento & purificação , Proteínas Cromossômicas não Histona/isolamento & purificação , DNA/genética , Biologia Molecular/métodos , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Adenosina Trifosfatases/genética , Proteínas de Ciclo Celular/genética , Cromátides/genética , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/genética , Troca de Cromátide Irmã/genética , CoesinasRESUMO
During the cell cycle, duplicated sister chromatids become physically connected during S phase through a process called sister-chromatid cohesion. Cohesion is terminated during the metaphase-to-anaphase transition to trigger sister-chromatid segregation. The establishment and dissolution of cohesion are highly regulated by the cohesin complex and its multitude of regulators. In particular, the cohesin regulator Wapl promotes the release of cohesin from chromosomes during both interphase and mitosis. Here, we describe in vitro protein binding assays between Wapl and a cohesin subcomplex, and cellular assays in human cells that probe the functions of Wapl in cohesin release.
Assuntos
Proteínas de Transporte/isolamento & purificação , Proteínas de Ciclo Celular/isolamento & purificação , Proteínas Cromossômicas não Histona/isolamento & purificação , Segregação de Cromossomos/genética , Biologia Molecular/métodos , Proteínas Nucleares/isolamento & purificação , Proteínas Proto-Oncogênicas/isolamento & purificação , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Cromátides/genética , Proteínas Cromossômicas não Histona/genética , Humanos , Mitose/genética , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas/genética , Saccharomyces cerevisiae/genética , CoesinasRESUMO
Polo-like kinaseâ 1 (Plk1), a validated cancer target, harbors a protein-protein interaction domain referred to as the polo-box domain (PBD), in addition to its enzymatic domain. Although functional inhibition either of the enzymatic domain or of the PBD has been shown to inhibit Plk1, so far there have been no reports of bifunctional agents with the potential to target both protein domains. Here we report the development of Plk1 inhibitors that incorporate both an ATP-competitive ligand of the enzymatic domain, derived from BIâ 2536, and a functional inhibitor of the PBD, based either on the small molecule poloxin-2 or on a PBD-binding peptide. Although these bifunctional agents do not seem to bind both protein domains simultaneously, the most potent compound displays low-nanomolar activity against the Plk1 PBD, with excellent selectivity over the PBDs of Plk2 and Plk3. Our data provide insights into challenges and opportunities relating to the optimization of Plk1 PBD ligands as potent Plk1 inhibitors.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Peptídeos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas de Ciclo Celular/isolamento & purificação , Proteínas de Ciclo Celular/metabolismo , Relação Dose-Resposta a Droga , Polarização de Fluorescência , Humanos , Estrutura Molecular , Peptídeos/síntese química , Peptídeos/química , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/isolamento & purificação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/isolamento & purificação , Proteínas Proto-Oncogênicas/metabolismo , Relação Estrutura-Atividade , Quinase 1 Polo-LikeRESUMO
Centrosomes are major microtubule-organizing centers in eukaryotic cells and play a critical role in embryonic development and asymmetric cell division. Centrosomes comprise a pair of centrioles surrounded by an amorphous proteinaceous meshwork called the pericentriolar material (PCM). Robust deposition of PCM around the centrioles is essential for a centrosome to achieve full microtubule nucleating potential. Despite the wealth of information on PCM composition and function, the mechanism and regulation of PCM assembly have been difficult to ascertain, due in part to the lack of an in vitro system. Here, we describe methods to establish an in vitro system to study PCM assembly in Caenorhabditis elegans. Specifically, we describe (1) how to express and purify the C. elegans PCM proteins SPD-5, SPD-2, and PLK-1 from baculovirus-infected insect cells, (2) how to assemble these proteins into PCM-like structures in vitro, and (3) how to quantify this assembly process in a semiautomated fashion.
Assuntos
Proteínas de Caenorhabditis elegans/química , Proteínas de Ciclo Celular/química , Centríolos/química , Animais , Proteínas de Caenorhabditis elegans/isolamento & purificação , Proteínas de Ciclo Celular/isolamento & purificação , Cromatografia de Afinidade , Proteínas de Drosophila/química , Proteínas de Drosophila/isolamento & purificação , Proteínas de Fluorescência Verde/química , Microscopia de Fluorescência , Multimerização Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/isolamento & purificação , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/isolamento & purificação , Células Sf9 , Quinase 1 Polo-LikeRESUMO
PURPOSE: Cancer of the upper digestive tract (uGI) is a major contributor to cancer-related death worldwide. Due to a rise in occurrence, together with poor survival rates and a lack of diagnostic or prognostic clinical assays, there is a clear need to establish molecular biomarkers. EXPERIMENTAL DESIGN: Initial assessment was performed on urine samples from 60 control and 60 uGI cancer patients using MS to establish a peak pattern or fingerprint model, which was validated by a further set of 59 samples. RESULTS: We detected 86 cluster peaks by MS above frequency and detection thresholds. Statistical testing and model building resulted in a peak profiling model of five relevant peaks with 88% overall sensitivity and 91% specificity, and overall correctness of 90%. High-resolution MS of 40 samples in the 2-10 kDa range resulted in 646 identified proteins, and pattern matching identified four of the five model peaks within significant parameters, namely programmed cell death 6 interacting protein (PDCD6IP/Alix/AIP1), Rabenosyn-5 (ZFYVE20), protein S100A8, and protein S100A9, of which the first two were validated by Western blotting. CONCLUSIONS AND CLINICAL RELEVANCE: We demonstrate that MS analysis of human urine can identify lead biomarker candidates in uGI cancers, which makes this technique potentially useful in defining and consolidating biomarker patterns for uGI cancer screening.
Assuntos
Biomarcadores Tumorais/urina , Proteínas de Ligação ao Cálcio/urina , Proteínas de Ciclo Celular/urina , Complexos Endossomais de Distribuição Requeridos para Transporte/urina , Neoplasias Esofágicas/urina , Neoplasias Gástricas/urina , Proteínas de Transporte Vesicular/urina , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/isolamento & purificação , Proteínas de Ligação ao Cálcio/isolamento & purificação , Estudos de Casos e Controles , Proteínas de Ciclo Celular/isolamento & purificação , Cromatografia de Afinidade , Complexos Endossomais de Distribuição Requeridos para Transporte/isolamento & purificação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas de Transporte Vesicular/isolamento & purificação , Adulto JovemRESUMO
In yeasts, small intrinsically disordered proteins (IDPs) modulate ribonucleotide reductase (RNR) activity to ensure an optimal supply of dNTPs for DNA synthesis. The Schizosaccharomyces pombe Spd1 protein can directly inhibit the large RNR subunit (R1), import the small subunit (R2) into the nucleus and induce an architectural change in the R1-R2 holocomplex. Here, we report the characterization of Spd2, a protein with sequence similarity to Spd1. We show that Spd2 is a CRL4(Cdt2)-controlled IDP that functions together with Spd1 in the DNA damage response and in modulation of RNR architecture. However, Spd2 does not regulate dNTP pools and R2 nuclear import. Furthermore, deletion of spd2 only weakly suppresses the Rad3(ATR) checkpoint dependency of CRL4(Cdt2) mutants. However, when we raised intracellular dNTP pools by inactivation of RNR feedback inhibition, deletion of spd2 could suppress the checkpoint dependency of CRL4(Cdt2) mutant cells to the same extent as deletion of spd1. Collectively, these observations suggest that Spd1 on its own regulates dNTP pools, whereas in combination with Spd2 it modulates RNR architecture and sensitizes cells to DNA damage.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Ribonucleotídeo Redutases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Regulação Alostérica/genética , Sequência de Aminoácidos , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/isolamento & purificação , Quinase do Ponto de Checagem 2/metabolismo , Reparo do DNA/genética , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/isolamento & purificação , Dados de Sequência Molecular , Mutação/genética , Nucleotidases/metabolismo , Conformação Proteica , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/isolamento & purificação , Homologia de Sequência de AminoácidosRESUMO
Cisplatin is widely used as an antineoplastic drug, but its ototoxic and nephrotoxic side-effects, as well as the inherent or acquired resistance of some cancers to cisplatin, remain significant clinical problems. Cisplatin's selectivity in killing rapidly proliferating cancer cells is largely dependent on covalent binding to DNA via cisplatin's chloride sites that had been aquated. We hypothesized that cisplatin's toxicity in slowly proliferating or terminally differentiated cells is primarily due to drug-protein interactions, instead of drug-DNA binding. To identify proteins that bind to cisplatin, we synthesized two different platinum-agarose conjugates, one with two amino groups and another with two chlorides attached to platinum that are available for protein binding, and conducted pull-down assays using cochlear and kidney cells. Mass spectrometric analysis on protein bands after gel electrophoresis and Coomassie blue staining identified several proteins, including myosin IIA, glucose-regulated protein 94 (GRP94), heat shock protein 90 (HSP90), calreticulin, valosin containing protein (VCP), and ribosomal protein L5, as cisplatin-binding proteins. Future studies on the interaction of these proteins with cisplatin will elucidate whether these drug-protein interactions are involved in ototoxicity and nephrotoxicity, or contribute to tumor sensitivity or resistance to cisplatin treatment.
Assuntos
Antineoplásicos/química , Extratos Celulares/química , Cisplatino/química , Glicoconjugados/química , Sefarose/química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/isolamento & purificação , Animais , Antineoplásicos/síntese química , Calreticulina/química , Calreticulina/isolamento & purificação , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/isolamento & purificação , Linhagem Celular , Cisplatino/análogos & derivados , Cisplatino/síntese química , Células Epiteliais/química , Células Epiteliais/citologia , Glicoconjugados/síntese química , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/isolamento & purificação , Túbulos Renais Proximais/química , Túbulos Renais Proximais/citologia , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/isolamento & purificação , Camundongos , Miosina não Muscular Tipo IIA/química , Miosina não Muscular Tipo IIA/isolamento & purificação , Órgão Espiral/química , Órgão Espiral/citologia , Ligação Proteica , Proteínas Ribossômicas/química , Proteínas Ribossômicas/isolamento & purificação , Proteína com ValosinaRESUMO
The role of Dmc1 as a meiosis-specific general recombinase was first demonstrated in Saccharomyces cerevisiae. Progress in understanding the biochemical mechanism of ScDmc1 has been hampered by its tendency to form inactive aggregates. We have found that the inclusion of ATP during protein purification prevents Dmc1 aggregation. ScDmc1 so prepared is capable of forming D-loops and responsive to its accessory factors Rad54 and Rdh54. Negative staining electron microscopy and iterative helical real-space reconstruction revealed that the ScDmc1-ssDNA nucleoprotein filament harbors 6.5 protomers per turn with a pitch of â¼106Å. The ScDmc1 purification procedure and companion molecular analyses should facilitate future studies on this recombinase.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Proteínas de Ligação a DNA/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/enzimologia , Trifosfato de Adenosina/química , Cálcio/química , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/isolamento & purificação , Cromatografia em Gel , DNA Helicases/química , Enzimas Reparadoras do DNA/química , DNA Topoisomerases/química , DNA Fúngico/química , DNA Fúngico/ultraestrutura , DNA de Cadeia Simples/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/isolamento & purificação , Recombinação Homóloga , Humanos , Hidrólise , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/isolamento & purificaçãoRESUMO
Methylation of lysine 4 on histone H3 (H3K4) at promoters is tightly linked to transcriptional regulation in human cells. At least six different COMPASS-like multisubunit (SET1/MLL) complexes that contain methyltransferase activity for H3K4 have been described, but a comprehensive and quantitative analysis of these SET1/MLL complexes is lacking. We applied label-free quantitative mass spectrometry to determine the subunit composition and stoichiometry of the human SET1/MLL complexes. We identified both known and novel, unique and shared interactors and determined their distribution and stoichiometry over the different SET1/MLL complexes. In addition to being a core COMPASS subunit, the Dpy30 protein is a genuine subunit of the NURF chromatin remodeling complex. Furthermore, we identified the Bod1 protein as a discriminator between the SET1B and SET1A complexes, and we show that the H3K36me-interactor Psip1 preferentially binds to the MLL2 complex. Finally, absolute protein quantification in crude lysates mirrors many of the observed SET1/MLL complex stoichiometries. Our findings provide a molecular framework for understanding the diversity and abundance of the different SET1/MLL complexes, which together establish the H3K4 methylation landscape in human cells.