RESUMO
Biorientation of chromosomes during cell division is necessary for precise dispatching of a mother cell's chromosomes into its two daughters. Kinetochores, large layered structures built on specialized chromosome loci named centromeres, promote biorientation by binding and sensing spindle microtubules. One of the outer layer main components is a ten-subunit assembly comprising Knl1C, Mis12C and Ndc80C (KMN) subcomplexes. The KMN is highly elongated and docks on kinetochores and microtubules through interfaces at its opposite extremes. Here, we combine cryogenic electron microscopy reconstructions and AlphaFold2 predictions to generate a model of the human KMN that reveals all intra-KMN interfaces. We identify and functionally validate two interaction interfaces that link Mis12C to Ndc80C and Knl1C. Through targeted interference experiments, we demonstrate that this mutual organization strongly stabilizes the KMN assembly. Our work thus reports a comprehensive structural and functional analysis of this part of the kinetochore microtubule-binding machinery and elucidates the path of connections from the chromatin-bound components to the force-generating components.
Assuntos
Microscopia Crioeletrônica , Cinetocoros , Proteínas Associadas aos Microtúbulos , Modelos Moleculares , Proteínas Nucleares , Humanos , Cinetocoros/metabolismo , Cinetocoros/ultraestrutura , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/química , Proteínas do Citoesqueleto/metabolismo , Proteínas do Citoesqueleto/química , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Ligação Proteica , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/química , Células HeLaRESUMO
State-subsidized programs develop medical data integration centers in Germany. To get infection disease (ID) researchers involved in the process of data sharing, common interests and minimum data requirements were prioritized. In 06/2019 we have initiated the German Infectious Disease Data Exchange (iDEx) project. We have developed and performed an online survey to determine prioritization of requests for data integration and exchange in ID research. The survey was designed with three sub-surveys, including a ranking of 15 data categories and 184 specific data items and a query of available 51 data collecting systems. A total of 84 researchers from 17 fields of ID research participated in the survey (predominant research fields: gastrointestinal infections n=11, healthcare-associated and antibiotic-resistant infections n=10, hepatitis n=10). 48% (40/84) of participants had experience as medical doctor. The three top ranked data categories were microbiology and parasitology, experimental data, and medication (53%, 52%, and 47% of maximal points, respectively). The most relevant data items for these categories were bloodstream infections, availability of biomaterial, and medication (88%, 87%, and 94% of maximal points, respectively). The ranking of requests of data integration and exchange is diverse and depends on the chosen measure. However, there is need to promote discipline-related digitalization and data exchange.
Assuntos
Doenças Transmissíveis , Hospitais , Alemanha/epidemiologia , Humanos , Armazenamento e Recuperação da Informação , Inquéritos e QuestionáriosRESUMO
P100, which is encoded by NF-kappa B2, inhibits Rel dimers. It can also be processed into p52, one of the DNA binding sub-units of NF-kappa B/Rel factors. Several p100 C-terminal truncations that result from gene rearrangements are associated with lymphomagenesis. Here, we characterized a new p100 mutant that we termed p100HB. It originates from a point-mutation that generates a premature stop-codon, and thus the protein lacks the last 125 amino acids. We have detected p100HB in several human tumor cell lines. The truncated protein is mainly unprocessed, and although it still binds Rel dimers, it has reduced inhibitory potency compared to p100 and translocates into the nucleus. Thus, p100HB may be associated with deregulated NF-kappa B/Rel functions.
Assuntos
Mutação , NF-kappa B/química , NF-kappa B/genética , Transporte Ativo do Núcleo Celular , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Bases , Western Blotting , Proteínas de Transporte/química , Códon , Dimerização , Éxons , Regulação da Expressão Gênica , Humanos , Proteínas I-kappa B/metabolismo , Células Jurkat , Linfócitos/metabolismo , Microscopia Confocal , Dados de Sequência Molecular , Subunidade p52 de NF-kappa B , Plasmídeos/metabolismo , Mutação Puntual , Testes de Precipitina , Estrutura Terciária de Proteína , Transporte Proteico , RNA Mensageiro/metabolismo , Fatores de Tempo , Transfecção , Células Tumorais CultivadasRESUMO
Tumor necrosis factor-alpha (TNF-alpha) and lymphotoxin-beta receptor (LTbetaR) signaling both play important roles in inflammatory and immune responses through activation of NF-kappaB. Using various deficient mouse embryonic fibroblast cells, we have compared the signaling pathways leading to NF-kappaB induction in response to TNF-alpha and LTbetaR activation. We demonstrate that LTbetaR ligation induces not only RelA/p50 dimers but also RelB/p50 dimers, whereas TNF-alpha induces only RelA/p50 dimers. LTbetaR-induced binding of RelB/p50 requires processing of p100 that is mediated by IKKalpha but is independent of IKKbeta, NEMO/IKKgamma, and RelA. Moreover, we show that RelB, p50, and p100 can associate in the same complex and that TNF-alpha but not LTbeta signaling increases the association of p100 with RelB/p50 dimers in the nucleus, leading to the specific inhibition of RelB DNA binding. These results suggest that the alternative NF-kappaB pathway based on p100 processing may account not only for the activation of RelB/p52 dimers but also for that of RelB/p50 dimers and that p100 regulates the binding activity of RelB/p50 dimers via at least two distinct mechanisms depending on the signaling pathway involved.
