RESUMEN
The study of the genome and the proteome of different species and representatives of distinct kingdoms, especially detection of proteome via wide-scaled analyses has various challenges and pitfalls. Attempts to combine all available information together and isolate some common features for determination of the pathway and their mechanism of action generally have a highly complicated nature. However, microtubule (MT) monomers are highly conserved protein structures, and microtubules are structurally conserved from Homo sapiens to Arabidopsis thaliana. The interaction of MT elements with microtubule-associated proteins and post-translational modifiers is fully dependent on protein interfaces, and almost all MT modifications are well described except acetylation. Crystallography and interactome data using different approaches were combined to identify conserved proteins important in acetylation of microtubules. Application of computational methods and comparative analysis of binding modes generated a robust predictive model of acetylation of the ϵ-amino group of Lys40 in α-tubulins. In turn, the model discarded some probable mechanisms of interaction between elements of interest. Reconstruction of unresolved protein structures was carried out with modeling by homology to the existing crystal structure (PDBID: 1Z2B) from B. taurus using Swiss-model server, followed by a molecular dynamics simulation. Docking of the human tubulin fragment with Lys40 into the active site of α-tubulin acetyltransferase, reproduces the binding mode of peptidomimetic from X-ray structure (PDBID: 4PK3).
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Lisina/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Procesamiento Proteico-Postraduccional , Tubulina (Proteína)/metabolismo , Acetilación , HumanosRESUMEN
According to the sequence and profile comparison with known catalytic domains, where identified protein phosphatases potentially involved in regulation of microtubule dynamics and structure from Arabidopsis thaliana, Nicotiana tabacum, Medicago sativa, Oryza sativa subsp. japonica, Zea mays, and Triticum aestivum. Selected proteins were related to classical non-receptor, serine/threonine-specific and dual protein phosphatases. By application of template structures of human protein phosphatases, it was performed homology modelling of the catalytic domains of 17 plant protein phosphatases. Based on the results of the structural alignment, molecular dynamics, and conservatism in positions of functionally importance, it was confirmed homology of selected plant proteins and known protein phosphatases regulating structure and dynamics of microtubules.
Asunto(s)
Microtúbulos/metabolismo , Fosfoproteínas Fosfatasas/química , Proteínas de Plantas/química , Plantas/enzimología , Dominio Catalítico , Humanos , Fosfoproteínas Fosfatasas/genética , Proteínas de Plantas/genética , Homología Estructural de ProteínaRESUMEN
The results of computer modeling of plant kinesin-8/αß-tubulin complexes with such αß-tubulins' modified amino acid residues as phosphorylated Tyr262 and Tyr107 are reported in this paper. The molecular dynamics of these modified complexes in comparison with the dynamics of non-modified ones suggests that the phosphorylation of both α- and ß-tubulins reveals stabilizing effect on the protein structure around the modified residue. It was found also that the phosphorylation of Tyr107 in ß-tubulin molecule favors to more advantageous kinesin-8 binding with the phosphorylated microtubule surface in terms of energy.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cinesinas/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Simulación de Dinámica Molecular , Fosforilación , Unión Proteica , Procesamiento Proteico-PostraduccionalRESUMEN
MAIN CONCLUSION: The similarity of IREH1 (Incomplete Root Hair Elongation 1) and animal MAST kinases was confirmed; IREH1cDNA was cloned while expressing in cultured animal cells co-localized with the centrosome. In mammals and fruit flies, microtubule-associated serine/threonine-protein kinases (MAST) are strongly involved in the regulation of the microtubule system. Higher plants also possess protein kinases homologous to MASTs, but their function and interaction with the cytoskeleton remain unclear. Here, we confirmed the sequence and structural similarity of MAST-related putative protein kinase IREH1 (At3g17850) and known animal MAST kinases. We report the first cloning of full-length cDNA of the IREH1 from Arabidopsis thaliana. Recombinant GFP-IREH1 protein was expressed in different cultured animal cells. It revealed co-localization with the centrosome without influencing cell morphology and microtubule arrangement. Structural N-terminal region of the IREH1 molecule co-localized with centrosome as well.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Centrosoma/metabolismo , Chlorocebus aethiops , Clonación Molecular , Citoesqueleto/metabolismo , ADN Complementario/genética , Drosophila/genética , Proteínas de Drosophila/genética , Genes Reporteros , Células HEK293 , Humanos , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Recombinantes de Fusión , Células VeroRESUMEN
The spindle assembly checkpoint (SAC) is a refined surveillance mechanism which ensures that chromosomes undergoing mitosis do not segregate until they are properly attached to the spindle microtubules (MT). The SAC has been extensively studied in metazoans and yeast, but little is known about its role in plants. We identified proteins interacting with a MT-associated protein MAP65-3, which plays a critical role in organising mitotic MT arrays, and carried out a functional analysis of previously and newly identified SAC components. We show that Arabidopsis SAC proteins BUB3.1, MAD2, BUBR1/MAD3s and BRK1 interact with each other and with MAP65-3. We found that two BUBR1/MAD3s interacted specifically at centromeres. When stably expressed in Arabidopsis, BRK1 localised to the kinetochores during all stages of the mitotic cell cycle. Early in mitosis, BUB3.1 and BUBR1/MAD3.1 localise to the mitotic spindle, where MAP65-3 organises spindle MTs. A double-knockout mad3.1 mad3.2 mutant presented spindle MT abnormalities, chromosome misalignments on the metaphase plate and the production of lagging chromosomes and micronuclei during mitosis. We conclude that BRK1 and BUBR1/MAD3-related proteins play a key role in ensuring faithful chromosome segregation during mitosis and that their interaction with MAP65-3 may be important for the regulation of MT-chromosome attachment.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citología , Arabidopsis/metabolismo , Puntos de Control de la Fase M del Ciclo Celular , Anafase , Animales , Arabidopsis/genética , Arabidopsis/parasitología , Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Proteínas Fluorescentes Verdes/metabolismo , Cinetocoros , Proteínas Mad2/genética , Proteínas Mad2/metabolismo , Metafase , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Datos de Secuencia Molecular , Mutación , Nematodos , Fenotipo , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Unión Proteica , Subunidades de Proteína/metabolismo , Transporte de Proteínas , Huso Acromático , Fracciones Subcelulares/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
A bioinformatic search was carried for plant homologues of human serine-threonine protein kinases involved in regulation of cell division and microtubule protein phosphorylation (SLK, PAK6, PAK7, MARK1, MAST2, TTBK1, TTBK2, AURKA, PLK1, PLK4 and PASK). A number of SLK, MAST2 and AURKA plant homologues were identified. The closest identified homologue of human AURKA kinase was a protein of unknown function, A7PY12/GSVIVT00026259001 from Vitis vinifera (herein named as "STALK", Serine-Threonine Aurora-Like Kinase). Analysis of STALK's three-dimensional structure confirmed its relationship to the subgroup of AURKA-like protein kinases.