New Interaction Partners For Nek4.1 And Nek4.2 Isoforms: From The Dna Damage Response To Rna Splicing.

Neks are serine-threonine kinases that are similar to NIMA, a protein found in Aspergillus nidulans which is essential for cell division. In humans there are eleven Neks which are involved in different biological functions besides the cell cycle control. Nek4 is one of the largest members of the Nek family and has been related to the primary cilia formation and in DNA damage response. However, its substrates and interaction partners are still unknown. In an attempt to better understand the role of Nek4, we performed an interactomics study to find new biological processes in which Nek4 is involved. We also described a novel Nek4 isoform which lacks a region of 46 amino acids derived from an insertion of an Alu sequence and showed the interactomics profile of these two Nek4 proteins. Isoform 1 and isoform 2 of Nek4 were expressed in human cells and after an immunoprecipitation followed by mass spectrometry, 474 interacting proteins were identified for isoform 1 and 149 for isoform 2 of Nek4. About 68% of isoform 2 potential interactors (102 proteins) are common between the two Nek4 isoforms. Our results reinforce Nek4 involvement in the DNA damage response, cilia maintenance and microtubule stabilization, and raise the possibility of new functional contexts, including apoptosis signaling, stress response, translation, protein quality control and, most intriguingly, RNA splicing. We show for the first time an unexpected difference between both Nek4 isoforms in RNA splicing control. Among the interacting partners, we found important proteins such as ANT3, Whirlin, PCNA, 14-3-3ε, SRSF1, SRSF2, SRPK1 and hNRNPs proteins. This study provides new insights into Nek4 functions, identifying new interaction partners and further suggests an interesting difference between isoform 1 and isoform 2 of this kinase. Nek4 isoform 1 may have similar roles compared to other Neks and these roles are not all preserved in isoform 2. Besides, in some processes, both isoforms showed opposite effects, indicating a possible fine controlled regulation.131

FEZ1 and formation of the flower-like nuclei

Orientador: Jorg KobargDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A proteína UNC-76 foi identificada como necessária para a fasciculação e elongação de axônios do verme Caenorhabditis elegans durante o desenvolvimento do sistema nervoso. A homóloga de mamíferos FEZ1 apresenta altos níveis de expressão em tecidos neuronais e camundongos knockout para o gene FEZ1 apresentam desvios de comportamento que remetem a desordens neurológicas. O papel de FEZ1 no desenvolvimento do sistema nervoso parece residir na sua associação com elementos do citoesqueleto e vias de sinalização (e.g., PKC?, E4B, DISC1) que conduzem o crescimento axonal e a polarização celular. Trabalhos do grupo mostram que FEZ1 é uma proteína multifuncional (hub), capaz de interagir com mais de 50 parceiros através de seus domínios coiled-coil. Além disso, a superexpressão de FEZ1 em células HEK293 provoca o aparecimento de núcleos multilobulados, um fenótipo comum em alguns tipos de leucemia. Nesse trabalho foi investigado o papel de FEZ1 nos mecanismos causadores dos núcleos multilobulados e as consequências funcionais de sua superexpressão na viabilidade celular, tentando extrapolar esse modelo para leucemias. Análises in silico de diversas leucemias mostraram que FEZ1 está superexpressa em LMAs e que isso pode se relacionar à ocorrência da fusão 11q23/MLL. A expressão de FEZ1 na linhagem leucêmica THP-1 foi detectada por Western blotting, mas, a expressão em PBMCs de pacientes ainda permanece sem provas empíricas. Para avaliar as consequências funcionais da superexpressão, uma linhagem com expressão estável e indutível foi obtida e utilizada em ensaios de proliferação e resistência a quimioterápicos. Porém, não foram observadas diferenças entre as linhagens expressando a fusão FLAG-FEZ1 e as que expressavam o FLAG tag apenas. Em um ensaio de IP-MS utilizando tais linhagens, foram identificadas proteínas cuja interação com FEZ1 pode ser modulada pela atividade de PKCs. Finalmente, a cotransfecção de FEZ1 inteira com coiled-coils C-terminais diminui a formação de núcleos multilobulados em quase 40%. A transfecção com o mutante FEZ1 nocys contendo 5 cisteínas mutadas não teve o mesmo efeito, mas, novos experimentos são necessários para determinar o potencial de sinergismo que esses dois componentes podem ter sobre a ocorrência desse fenômenoAbstract: The protein UNC-76 was identified as necessary for fasciculation and elongation of axons of the worm Caenorhabditis elegans during development of the nervous system. The mammalian homologue FEZ1 is mostly expressed in neuronal tissues and FEZ1 knockout mice present behavior abnormalities that resemble neurological disorders. The role of FEZ1 in the development of the nervous system seems to lie in its association with cytoskeletal elements and signaling pathways (e.g., PKC?, E4B, DISC1) regulating axon outgrowth and cell polarization. The studies of our group have shown that FEZ1 is a hub, able to interact with more than 50 partners through its coiled-coil domains. Furthermore, overexpression of FEZ1 in HEK293 cells causes the appearance of flower-like nuclei, a common phenotype to certain types of leukemia. In this work the role of FEZ1 in the mechanisms of flower-like nuclei formation and functional consequences of its overexpression on cell viability were investigated, attempting to extrapolate this model for leukemias. In silico analysis of several leukemias showed that FEZ1 is overexpressed in AML patients and that this may relate to the occurrence of 11q23/MLL genetic fusion. FEZ1 expression in leukemic THP-1 cells was detected by Western blotting, but the expression in PBMCs of leukemic patients still lacks empirical evidence. To assess the functional consequences of overexpression, cell lineage with stable and inducible expression of FEZ1 was obtained and used in proliferative assays. However, it was not observed any differences between lineages expressing FLAG-FEZ1 fusion protein or FLAG tag alone. IP-MS assay using these lineages identified proteins whose interaction with FEZ1 could be modulated by the activity of PKCs. Finally, cotransfection of C-terminal coiled-coils and FEZ1 full-length decreases flower-like nuclei formation to nearly 40%. Transfection with FEZ1nocys mutant containing five substituted cysteines did not play the same, but further experiments are needed to determine the potential synergism these two components may have on this phenomenonMestradoGenetica Animal e EvoluçãoMestre em Genética e Biologia Molecula

Structural Analysis of Intermolecular Interactions in the Kinesin Adaptor Complex Fasciculation and Elongation Protein Zeta 1/ Short Coiled-Coil Protein (FEZ1/SCOCO)

<div><p>Cytoskeleton and protein trafficking processes, including vesicle transport to synapses, are key processes in neuronal differentiation and axon outgrowth. The human protein FEZ1 (fasciculation and elongation protein zeta 1 / UNC-76, in <i>C. elegans</i>), SCOCO (short coiled-coil protein / UNC-69) and kinesins (e.g. kinesin heavy chain / UNC116) are involved in these processes. Exploiting the feature of FEZ1 protein as a bivalent adapter of transport mediated by kinesins and FEZ1 protein interaction with SCOCO (proteins involved in the same path of axonal growth), we investigated the structural aspects of intermolecular interactions involved in this complex formation by NMR (Nuclear Magnetic Resonance), cross-linking coupled with mass spectrometry (MS), SAXS (Small Angle X-ray Scattering) and molecular modelling. The topology of homodimerization was accessed through NMR (Nuclear Magnetic Resonance) studies of the region involved in this process, corresponding to FEZ1 (92-194). Through studies involving the protein in its monomeric configuration (reduced) and dimeric state, we propose that homodimerization occurs with FEZ1 chains oriented in an anti-parallel topology. We demonstrate that the interaction interface of FEZ1 and SCOCO defined by MS and computational modelling is in accordance with that previously demonstrated for UNC-76 and UNC-69. SAXS and literature data support a heterotetrameric complex model. These data provide details about the interaction interfaces probably involved in the transport machinery assembly and open perspectives to understand and interfere in this assembly and its involvement in neuronal differentiation and axon outgrowth.</p> </div

Purification of the FEZ1-SCOCO complex and GST-SCOCO and SAXS experimental

<p>data A) SDS-PAGE 10% of the 6His-FEZ1 (1-392) and GST-SCOCO (2-82) protein complex. The complex was analyzed by SAXS at 1.10 mg/mL in PBS buffer solution. The complex poly-dispersity was 28,0% according to DLS assay. B) SAXS (Small Angle X-ray Scattering) experiments of 6His-FEZ1 (1-392) interacting with GST-SCOCO (2-82). C) SDS-PAGE 10% of the GST-SCOCO (2-82) protein. D) The protein was analyzed by SAXS at 0.84 mg/mL in PBS buffer solution. The figure shows the experimental intensity points (empty symbols) and the theoretical fit (continuous line) obtained with the GNOM program package for both samples. Insets in both panels (B) and (C) show the linear behavior of the data in the Guinier region. R_g and D_max values obtained from the GNOM fitting for GST-SCOCO and FEZ1-SCOCO samples were (28.7 ±1, approx. 95 Ǻ) and (107±1, approx. 340 Ǻ), respectively. SAXS data of FEZ1 protein were previously published [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076602#B17" target="_blank">17</a>].</p

General scheme of UNC-76/FEZ1 (<i>C. elegans / H. sapiens</i>) and UNC-69/SCOCO (<i>C. elegans / H. sapiens</i>) proteins.

<p>Mutations observed in <i>C. elegans</i>, and the corresponding region in the human protein, are indicated by asterisks. Regions of interaction with other proteins are indicated. Coiled-coils: box, alpha-helix prediction: gray.</p

Interaction between FEZ1 and SCOCO.

<p>A) Purified recombinant proteins FEZ1 and SCOCO were incubated, chemically cross-linked, digested with trypsin, and analyzed by MS. MS/MS spectra were manually validated for b and y ion series of the α (peptide of FEZ1) and β (peptide of SCOCO) chains. B) General scheme of FEZ1 and SCOCO proteins cross-linked. Coiled-coils: box, alpha-helix prediction: gray. Amino acids 261-279 in FEZ1 correspond to the mininal interaction region of UNC-69/SCOCO in UNC-76/FEZ1.C) Best conformation based on both cross-link distance and energy value of the in silico modeled complex. FEZ1 is colored in green, and SCOCO is depicted in deep blue. The peptides identified in the MS analysis are shown in orange and the lysine residue in red. DSS is represented in yellow.</p

FEZ1 homodimerization involves few amino acids residues.

<p>A) Amino acid sequence of human FEZ1 protein (92-194). The numbers indicate the position of each amino acid residue within the full-length sequence. The first three residues, unnumbered, are generated by the recombinant protein’s cleavage with TEV protease. B) 15N-HSQC FEZ1 (92-194) Nuclear Magnetic Resonance (NMR) spectra. HSQC shows chemical shifts in reduced monomeric protein (black) and non-reduced dimeric protein (red). The spectrum was obtained in spectrometer 600 MHz. For the series of experiments, isotope 15N was introduced in minimal medium for growth of bacteria and induction of protein expression. C) steady-state heteronuclear NOE experiments with dimers and monomers. Heteronuclear NOEs intensities of the monomer were subtracted from those of the dimer, resulting in the differential pattern of relaxation corresponding to amino acids probably present in the region of homodimerization.</p

FEZ1 is an anti-parallel dimer.

<p>15N-edited NOESY-HSQC of dimers and monomers of FEZ1 (92-194) shows contacts between side chains of amino acids that occur in the homodimer, preferably in an anti-parallel topology. These contacts occur between residues 123 and 143, 127 and 139, 130 and 137, and are indicated in the sequence shown at the bottom of the figure.</p