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1.
Nat Commun ; 14(1): 355, 2023 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-36683055

RESUMO

Mitosis induces cellular rearrangements like spindle formation, Golgi fragmentation, and nuclear envelope breakdown. Similar to certain retroviruses, nuclear delivery during entry of human papillomavirus (HPV) genomes is facilitated by mitosis, during which minor capsid protein L2 tethers viral DNA to mitotic chromosomes. However, the mechanism of viral genome delivery and tethering to condensed chromosomes is barely understood. It is unclear, which cellular proteins facilitate this process or how this process is regulated. This work identifies crucial phosphorylations on HPV minor capsid protein L2 occurring at mitosis onset. L2's chromosome binding region (CBR) is sequentially phosphorylated by the master mitotic kinases CDK1 and PLK1. L2 phosphorylation, thus, regulates timely delivery of HPV vDNA to mitotic chromatin during mitosis. In summary, our work demonstrates a crucial role of mitotic kinases for nuclear delivery of viral DNA and provides important insights into the molecular mechanism of pathogen import into the nucleus during mitosis.


Assuntos
Proteínas do Capsídeo , Infecções por Papillomavirus , Humanos , Proteínas do Capsídeo/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , Internalização do Vírus , Mitose , Fosforilação , Genoma Viral , Proteínas de Ciclo Celular/metabolismo
2.
Mol Cell ; 82(11): 2113-2131.e8, 2022 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-35525244

RESUMO

Centromeres are specialized chromosome loci that seed the kinetochore, a large protein complex that effects chromosome segregation. A 16-subunit complex, the constitutive centromere associated network (CCAN), connects between the specialized centromeric chromatin, marked by the histone H3 variant CENP-A, and the spindle-binding moiety of the kinetochore. Here, we report a cryo-electron microscopy structure of human CCAN. We highlight unique features such as the pseudo GTPase CENP-M and report how a crucial CENP-C motif binds the CENP-LN complex. The CCAN structure has implications for the mechanism of specific recognition of the CENP-A nucleosome. A model consistent with our structure depicts the CENP-C-bound nucleosome as connected to the CCAN through extended, flexible regions of CENP-C. An alternative model identifies both CENP-C and CENP-N as specificity determinants but requires CENP-N to bind CENP-A in a mode distinct from the classical nucleosome octamer.


Assuntos
Cinetocoros , Nucleossomos , Centrômero/metabolismo , Proteína Centromérica A/metabolismo , Microscopia Crioeletrônica , Humanos , Cinetocoros/metabolismo , Nucleossomos/genética
3.
Mol Cell ; 81(1): 67-87.e9, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-33248027

RESUMO

Reflecting its pleiotropic functions, Polo-like kinase 1 (PLK1) localizes to various sub-cellular structures during mitosis. At kinetochores, PLK1 contributes to microtubule attachments and mitotic checkpoint signaling. Previous studies identified a wealth of potential PLK1 receptors at kinetochores, as well as requirements for various mitotic kinases, including BUB1, Aurora B, and PLK1 itself. Here, we combine ectopic localization, in vitro reconstitution, and kinetochore localization studies to demonstrate that most and likely all of the PLK1 is recruited through BUB1 in the outer kinetochore and centromeric protein U (CENP-U) in the inner kinetochore. BUB1 and CENP-U share a constellation of sequence motifs consisting of a putative PP2A-docking motif and two neighboring PLK1-docking sites, which, contingent on priming phosphorylation by cyclin-dependent kinase 1 and PLK1 itself, bind PLK1 and promote its dimerization. Our results rationalize previous observations and describe a unifying mechanism for recruitment of PLK1 to human kinetochores.


Assuntos
Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Histonas/metabolismo , Cinetocoros/metabolismo , Mitose , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteína Quinase CDC2/genética , Proteínas de Ciclo Celular/genética , Células HeLa , Histonas/genética , Humanos , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Quinase 1 Polo-Like
4.
Mol Cell ; 71(6): 923-939.e10, 2018 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-30174292

RESUMO

The approximately thirty core subunits of kinetochores assemble on centromeric chromatin containing the histone H3 variant CENP-A and connect chromosomes with spindle microtubules. The chromatin proximal 16-subunit CCAN (constitutive centromere associated network) creates a mechanically stable bridge between CENP-A and the kinetochore's microtubule-binding machinery, the 10-subunit KMN assembly. Here, we reconstituted a stoichiometric 11-subunit human CCAN core that forms when the CENP-OPQUR complex binds to a joint interface on the CENP-HIKM and CENP-LN complexes. The resulting CCAN particle is globular and connects KMN and CENP-A in a 26-subunit recombinant particle. The disordered, basic N-terminal tail of CENP-Q binds microtubules and promotes accurate chromosome alignment, cooperating with KMN in microtubule binding. The N-terminal basic tail of the NDC80 complex, the microtubule-binding subunit of KMN, can functionally replace the CENP-Q tail. Our work dissects the connectivity and architecture of CCAN and reveals unexpected functional similarities between CENP-OPQUR and the NDC80 complex.


Assuntos
Proteínas Cromossômicas não Histona/ultraestrutura , Cinetocoros/fisiologia , Cinetocoros/ultraestrutura , Centrômero/fisiologia , Proteína Centromérica A/metabolismo , Proteína Centromérica A/ultraestrutura , Proteínas Cromossômicas não Histona/metabolismo , Proteínas do Citoesqueleto , Células HeLa , Humanos , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Microtúbulos/fisiologia , Proteínas Nucleares/metabolismo
5.
Nature ; 537(7619): 249-253, 2016 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-27580032

RESUMO

Chromosomes are carriers of genetic material and their accurate transfer from a mother cell to its two daughters during cell division is of paramount importance for life. Kinetochores are crucial for this process, as they connect chromosomes with microtubules in the mitotic spindle. Kinetochores are multi-subunit complexes that assemble on specialized chromatin domains, the centromeres, that are able to enrich nucleosomes containing the histone H3 variant centromeric protein A (CENP-A). A group of several additional CENPs, collectively known as constitutive centromere associated network (CCAN), establish the inner kinetochore, whereas a ten-subunit assembly known as the KMN network creates a microtubule-binding site in the outer kinetochore. Interactions between CENP-A and two CCAN subunits, CENP-C and CENP-N, have been previously described, but a comprehensive understanding of CCAN organization and of how it contributes to the selective recognition of CENP-A has been missing. Here we use biochemical reconstitution to unveil fundamental principles of kinetochore organization and function. We show that cooperative interactions of a seven-subunit CCAN subcomplex, the CHIKMLN complex, determine binding selectivity for CENP-A over H3-nucleosomes. The CENP-A:CHIKMLN complex binds directly to the KMN network, resulting in a 21-subunit complex that forms a minimal high-affinity linkage between CENP-A nucleosomes and microtubules in vitro. This structural module is related to fungal point kinetochores, which bind a single microtubule. Its convolution with multiple CENP-A proteins may give rise to the regional kinetochores of higher eukaryotes, which bind multiple microtubules. Biochemical reconstitution paves the way for mechanistic and quantitative analyses of kinetochores.


Assuntos
Cinetocoros/química , Cinetocoros/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Autoantígenos/metabolismo , Centrômero/química , Centrômero/genética , Centrômero/metabolismo , Proteína Centromérica A , Proteínas Cromossômicas não Histona/metabolismo , Humanos , Microtúbulos/metabolismo , Nucleossomos/química , Nucleossomos/genética , Nucleossomos/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Fuso Acromático
6.
Curr Opin Struct Biol ; 37: 152-63, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27039078

RESUMO

Kinetochores are macromolecular complexes built on a specialized chromatin domain called the centromere. Kinetochores provide a site of attachment for spindle microtubules during mitosis. They also control a cell cycle checkpoint, the spindle assembly checkpoint, which coordinates mitotic exit with the completion of chromosome alignment on the mitotic spindle. Correct kinetochore operation is therefore indispensable for accurate chromosome segregation. With multiple copies of at least 30 structural core subunits and a myriad of regulatory subunits, kinetochores are among the largest known macromolecular machines. Biochemical reconstitution and structural analysis, together with functional studies, are bringing to light the organizational principles of these complex and fascinating structures. We summarize recent work and identify a few challenges for future work.


Assuntos
Cinetocoros/metabolismo , Sequência de Aminoácidos , Segregação de Cromossomos , Cinetocoros/química , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade
7.
J Mol Biol ; 390(5): 981-90, 2009 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-19481550

RESUMO

In honeybee (Apis mellifera) societies, the queen controls the development and the caste status of the members of the hive. Queen bees secrete pheromonal blends comprising 10 or more major and minor components, mainly hydrophobic. The major component, 9-keto-2(E)-decenoic acid (9-ODA), acts on the workers and male bees (drones), eliciting social or sexual responses. 9-ODA is captured in the antennal lymph and transported to the pheromone receptor(s) in the sensory neuron membranes by pheromone binding proteins (PBPs). A key issue is to understand how the pheromone, once tightly bound to its PBP, is released to activate the receptor. We report here on the structure at physiological pH of the main antennal PBP, ASP1, identified in workers and male honeybees, in its apo or complexed form, particularly with the main component of the queen mandibular pheromonal mixture (9-ODA). Contrary to the ASP1 structure at low pH, the ASP1 structure at pH 7.0 is a domain-swapped dimer with one or two ligands per monomer. This dimerization is disrupted by a unique residue mutation since Asp35 Asn and Asp35 Ala mutants remain monomeric at pH 7.0, as does native ASP1 at pH 4.0. Asp35 is conserved in only approximately 30% of medium-chain PBPs and is replaced by other residues, such as Asn, Ala and Ser, among others, thus excluding that they may perform domain swapping. Therefore, these different medium-chain PBPs, as well as PBPs from moths, very likely exhibit different mechanisms of ligand release or receptor recognition.


Assuntos
Abelhas/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Hierarquia Social , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Feromônios/metabolismo , Substituição de Aminoácidos , Animais , Cristalografia por Raios X , Feminino , Fluorescência , Concentração de Íons de Hidrogênio , Cinética , Ligantes , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
8.
J Mol Biol ; 380(1): 158-69, 2008 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-18508083

RESUMO

The behavior of insects and their perception of their surroundings are driven, in a large part, by odorants and pheromones. This is especially true for social insects, such as the honey bee, where the queen controls the development and the caste status of the other individuals. Pheromone perception is a complex phenomenon relying on a cascade of recognition events, initiated in antennae by pheromone recognition by a pheromone-binding protein and finishing with signal transduction at the axon membrane level. With to the objective of deciphering this initial step, we have determined the structures of the bee antennal pheromone-binding protein (ASP1) in the apo form and in complex with the main component of the queen mandibular pheromonal mixture, 9-keto-2(E)-decenoic acid (9-ODA) and with nonpheromonal components. In the apo protein, the C terminus obstructs the binding site. In contrast, ASP1 complexes have different open conformations, depending on the ligand shape, leading to different volumes of the binding cavity. The binding site integrity depends on the C terminus (111-119) conformation, which involves the interplay of two factors; i.e. the presence of a ligand and a low pH. Ligand binding to ASP1 is favored by low pH, opposite to what is observed with other pheromone-binding proteins, such as those of Bombyx mori and Anopheles gambiae.


Assuntos
Abelhas/química , Proteínas de Transporte/química , Proteínas de Insetos/química , Feromônios/química , Sequência de Aminoácidos , Animais , Apoproteínas/química , Ácidos Graxos Monoinsaturados/química , Concentração de Íons de Hidrogênio , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Ácido Palmítico/química , Conformação Proteica , Sulfonamidas/química
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