Characterization of the cofactor-binding site in the SPOUT-fold methyltransferases by computational docking of S-adenosylmethionine to three crystal structures

BACKGROUND: There are several evolutionarily unrelated and structurally dissimilar superfamilies of S-adenosylmethionine (AdoMet)-dependent methyltransferases (MTases). A new superfamily (SPOUT) has been recently characterized on a sequence level and three structures of its members (1gz0, 1ipa, and 1k3r) have been solved. However, none of these structures include the cofactor or the substrate. Due to the strong evolutionary divergence and the paucity of experimental information, no confident predictions of protein-ligand and protein-substrate interactions could be made, which hampered the study of sequence-structure-function relationships in the SPOUT superfamily. RESULTS: We used the computational docking program AutoDock to identify the AdoMet-binding site on the surface of three MTase structures. We analyzed the sequence divergence in two distinct lineages of the SPOUT superfamily in the context of surface features and preferred cofactor binding mode to propose specific function for the conserved residues. CONCLUSION: Our docking analysis has confidently predicted the common AdoMet-binding site in three remotely related proteins structures. In the vicinity of the cofactor-binding site, subfamily-conserved grooves were identified on the protein surface, suggesting location of the target-binding/catalytic site. Functionally important residues were inferred and a general reaction mechanism, involving conformational change of a glycine-rich loop, was proposed

Cross-regulation between Aurora B and Citron kinase controls midbody architecture in cytokinesis.

Cytokinesis culminates in the final separation, or abscission, of the two daughter cells at the end of cell division. Abscission relies on an organelle, the midbody, which forms at the intercellular bridge and is composed of various proteins arranged in a precise stereotypic pattern. The molecular mechanisms controlling midbody organization and function, however, are obscure. Here we show that proper midbody architecture requires cross-regulation between two cell division kinases, Citron kinase (CIT-K) and Aurora B, the kinase component of the chromosomal passenger complex (CPC). CIT-K interacts directly with three CPC components and is required for proper midbody architecture and the orderly arrangement of midbody proteins, including the CPC. In addition, we show that CIT-K promotes Aurora B activity through phosphorylation of the INCENP CPC subunit at the TSS motif. In turn, Aurora B controls CIT-K localization and association with its central spindle partners through phosphorylation of CIT-K's coiled coil domain. Our results identify, for the first time, a cross-regulatory mechanism between two kinases during cytokinesis, which is crucial for establishing the stereotyped organization of midbody proteins.C.M. holds a Cambridge Cancer Centre PhD studentship, which is supported by Cancer Research UK and the MRC. Z.I.B. was supported by a Gwynaeth Pretty PhD studentship and the BlueGnome Molecular Genetics Fund from the Department of Pathology, University of Cambridge, and was also a recipient of a short-term EMBO fellowship and a fellowship from the Cambridge Philosophical Society. G.C. and M.G. are supported by a grant ‘Progetto di Ricerca di Interesse Nazionale’ (PRIN 2012) from the Ministero dell'Istruzione, Università e Ricerca. P.P.D. is the recipient of the Maplethorpe Fellowship from Murray Edwards College, Cambridge, UK.This is the final version of the article. It first appeared from Royal Society Publishing via https://doi.org/10.1098/rsob.16001

Suppression of Scant Identifies Endos as a Substrate of Greatwall Kinase and a Negative Regulator of Protein Phosphatase 2A in Mitosis

Protein phosphatase 2A (PP2A) plays a major role in dephosphorylating the targets of the major mitotic kinase Cdk1 at mitotic exit, yet how it is regulated in mitotic progression is poorly understood. Here we show that mutations in either the catalytic or regulatory twins/B55 subunit of PP2A act as enhancers of gwl^(Scant), a gain-of-function allele of the Greatwall kinase gene that leads to embryonic lethality in Drosophila when the maternal dosage of the mitotic kinase Polo is reduced. We also show that heterozygous mutant endos alleles suppress heterozygous gwl^(Scant); many more embryos survive. Furthermore, heterozygous PP2A mutations make females heterozygous for the strong mutation polo¹¹ partially sterile, even in the absence of gwl^(Scant). Heterozygosity for an endos mutation suppresses this PP2A/polo¹¹ sterility. Homozygous mutation or knockdown of endos leads to phenotypes suggestive of defects in maintaining the mitotic state. In accord with the genetic interactions shown by the gwl^(Scant) dominant mutant, the mitotic defects of Endos knockdown in cultured cells can be suppressed by knockdown of either the catalytic or the Twins/B55 regulatory subunits of PP2A but not by the other three regulatory B subunits of Drosophila PP2A. Greatwall phosphorylates Endos at a single site, Ser68, and this is essential for Endos function. Together these interactions suggest that Greatwall and Endos act to promote the inactivation of PP2A-Twins/B55 in Drosophila. We discuss the involvement of Polo kinase in such a regulatory loop

Multi-omic signatures of atherogenic dyslipidaemia : pre-clinical target identification and validation in humans

Dyslipidaemia is a major risk factor for atherosclerosis and cardiovascular diseases. The molecular mechanisms that translate dyslipidaemia into atherogenesis and reliable markers of its progression are yet to be fully elucidated. To address this issue, we conducted a comprehensive metabolomic and proteomic analysis in an experimental model of dyslipidaemia and in patients with familial hypercholesterolemia (FH)

Peroxiredoxin 6 Fails to Limit Phospholipid Peroxidation in Lung from Cftr-Knockout Mice Subjected to Oxidative Challenge

Oxidative stress plays a prominent role in the pathophysiology of cystic fibrosis (CF). Despite the presence of oxidative stress markers and a decreased antioxidant capacity in CF airway lining fluid, few studies have focused on the oxidant/antioxidant balance in CF cells. The aim of the current study was to investigate the cellular levels of reactive oxygen species (ROS), oxidative damage and enzymatic antioxidant defenses in the lung of Cftr-knockout mice in basal conditions and as a response to oxidative insult

The pentameric nucleoplasmin fold is present in Drosophila FKBP39 and a large number of chromatin-related proteins.

Nucleoplasmin is a histone chaperone that consists of a pentameric N-terminal domain and an unstructured C-terminal tail. The pentameric core domain, a doughnut-like structure with a central pore, is only found in the nucleoplasmin family. Here, we report the first structure of a nucleoplasmin-like domain (NPL) from the unrelated Drosophila protein, FKBP39, and we present evidence that this protein associates with chromatin. Furthermore, we show that two other chromatin proteins, Arabidopsis thaliana histone deacetylase type 2 (HD2) and Saccharomyces cerevisiae Fpr4, share the NPL fold and form pentamers, or a dimer of pentamers in the case of HD2. Thus, we propose a new family of proteins that share the pentameric nucleoplasmin-like NPL domain and are found in protists, fungi, plants and animals.We are grateful to Gunter Stier for providing the vector; Michael Nilges, Oleg Fedorov, Benjamin Bardiaux, Stefanie Hartmann and Wolfgang Rieping for helpful discussions; and Daniel Nietlispach for NMR expertise. We thank Renato Paro for generously providing us with an anti-FKBP39 antibody. We would like to thank the Wellcome Trust for financial support (grant 082010/Z/07/Z). V.T.F. and E.D.L. acknowledge support from Engineering and Physical Sciences Research Council under grants GR/R99393/01 and EP/C015452/1 for the creation of the Deuteration Laboratory platform operating within the Grenoble Partnership for Structural Biology. V.T.F. also acknowledges support from the European Union under contract RII3-CT-2003-505925. J.B.A. acknowledges the provision of a postdoctoral fellowship held at Keele University. M.R.P. and D.M.G. were supported by the Medical Research Council and Cancer Research UK grants to D.M.G. A.A.W. is a recipient of a Wellcome Trust Fellowship092441/Z/10/Z. J.D. and M.D. were supported by the Harmonia 5 Grant 2013/10/M/NZ2/00298 from the Polish National Science Center. The authors would like to thank the Institut Laue-Langevin (ILL), the European Synchrotron Radiation Facility (ESRF) and the European Molecular Biology Laboratory Hamburg outstation (EMBL-HH) for the provision of beamtime and access to the experimental facilities of D22, ID14eh3 and X33 respectively. We would also like to thank the local contacts at all the facilities for providing assistance in using the beam lines.This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.jmb.2015.03.01

Quantitative analysis of PPT1 interactome in human neuroblastoma cells

Peer reviewe

The Pentameric Nucleoplasmin Fold Is Present in Drosophila FKBP39 and a Large Number of Chromatin-Related Proteins

Nucleoplasmin is a histone chaperone that consists of a pentameric N-terminal domain and an unstructured C-terminal tail. The pentameric core domain, a doughnut-like structure with a central pore, is only found in the nucleoplasmin family. Here, we report the first structure of a nucleoplasmin-like domain (NPL) from the unrelated Drosophila protein, FKBP39, and we present evidence that this protein associates with chromatin. Furthermore, we show that two other chromatin proteins, Arabidopsis thaliana histone deacetylase type 2 (HD2) and Saccharomyces cerevisiae Fpr4, share the NPL fold and form pentamers, or a dimer of pentamers in the case of HD2. Thus, we propose a new family of proteins that share the pentameric nucleoplasmin-like NPL domain and are found in protists, fungi, plants and animals

Plk4 Phosphorylates Ana2 to Trigger Sas6 Recruitment and Procentriole Formation

Centrioles are 9-fold symmetrical structures at the core of centrosomes and base of cilia whose dysfunction has been linked to a wide range of inherited diseases and cancer [1]. Their duplication is regulated by a protein kinase of conserved structure, the C. elegans ZYG-1 or its Polo-like kinase 4 (Plk4) counterpart in other organisms [2, 3, 4]. Although Plk4’s centriolar partners and mechanisms that regulate its stability are known, its crucial substrates for centriole duplication have never been identified. Here we show that Drosophila Plk4 phosphorylates four conserved serines in the STAN motif of the core centriole protein Ana2 to enable it to bind and recruit its Sas6 partner. Ana2 and Sas6 normally load onto both mother and daughter centrioles immediately after their disengagement toward the end of mitosis to seed procentriole formation. Nonphosphorylatable Ana2 still localizes to the centriole but can no longer recruit Sas6 and centriole duplication fails. Thus, following centriole disengagement, recruitment of Ana2 and its phosphorylation by Plk4 are the earliest known events in centriole duplication to recruit Sas6 and thereby establish the architecture of the new procentriole engaged with its parent