Homology model of the CDK1/cyclin B complex

We describe a refined homology model of a CDK1/cyclin B complex that was previously used for the structure-based optimization of the Paullone class of inhibitors. The preliminary model was formed from the homologous regions of the deposited CDK2/cyclin A crystal structure. Further refinement of the CDK1/cyclin B complex was accomplished using molecular mechanics and hydropathic analysis with a protocol of constraints and local geometry searches. For the most part, our CKD1/cyclin B homology model is very similar to the high resolution CDK2/cyclin A crystal structure regarding secondary and tertiary features. However, minor discrepancies between the two kinase structures suggest the possibility that ligand design may be specifically tuned for either CDK1 or CDK2. Our examination of the CDK1/cyclin B model includes a comparison with the CDK2/cyclin A crystal structure in the PSTAIRE interface region, connecting portions to the ATP binding domain, as well as the ATP binding site itself.     

The biological activity of ubiquitinated BoNT/B light chain in vitro and in human SHSY‐5Y neuronal cells

BoNT/B light chain is a zinc‐dependent endopeptidase. After entering its target, the neuronal cell, BoNT/B is responsible for synaptobrevin‐2 (VAMP‐2) cleavage. This results in reduced neurotransmitter (acetylcholine) release from synaptic vesicles, yielding muscular paralysis. Since the toxin persists in neuronal cells for an extended period, regeneration of VAMP‐2 is prevented. We evaluated therapeutic targets to overcome botulinum persistence because early removal would rescue the neuronal cell. The ubiquitination/proteasome cellular pathway is responsible for removing “old” or undesirable proteins. Therefore, we assessed ubiquitination of BoNT/B light chain in vitro, and characterized the effects of ubiquitination modulating drugs, PMA (phorbol 12‐myristate 13‐acetate) and expoxomicin, on ubiquitination of BoNT/B light chain in neuronal cells. Both drugs altered BoNT/B light chain ubiquitination. Ubiquitination in vitro and in cells decreased the biological activity of BoNT/B light chain. These results further elucidate BoNT protein degradation pathways in intoxicated neuronal cells and mechanisms to enhance toxin removal. J. Cell. Biochem. 108: 660–667, 2009. Published 2009 Wiley‐Liss, Inc.     

Structure of the human laminin B2 chain gene reveals extensive divergence from the laminin B1 chain gene

The exon-intron structure of the human laminin B2 chain gene was elucidated from genomic lambda phage clones spanning 2 kilobase pairs (kb) of the 5'-flanking region, 58 kb of the structural gene and 10 kb of the 3'-flanking region. The entire gene was shown to contain 28 exons. The promoter region has no TATA or CAAT boxes whereas it contains five GC boxes and three AP-2-like binding sites. Comparison with the promoter region of the mouse gene revealed six highly conserved sequences of 14 to 42 base pairs in length. Sequencing of the last exon of the gene showed that the 3'-untranslated region of the mRNA can be up to 2797 nucleotides with five AATAAA potential polyadenylation signals. The similarity of the human 3'-untranslated sequence with that of mouse was shown to be 68.8%. The exon-intron structure of the laminin B2 chain gene demonstrated extensive divergence from the human laminin B1 chain gene, which has 34 exons. Only three intron locations are conserved in these two genes. The overall exon profile of the laminin B2 chain gene correlates only marginally with the pattern of structural domains and internal cysteine-rich repeats in the laminin B2 polypeptide chain.     

Inhibition of membrane fusion in vitro via cyclin B but not cyclin A.

It is now clear that complexes of cdc2 kinase with "mitotic" cyclins regulate the transition between the G2 phase of the cell cycle and mitosis and that membrane traffic in mammalian cells is arrested during mitosis. Using a cell-free assay, we have previously reported that the fusion of early endosomes is, in fact, inhibited via the cdc2 kinase (Tuomikoski, T., Felix, M.-A., Dorée, M., and Gruenberg, J. (1989) Nature 342, 942-945). In the present paper, we show that this in vitro inhibition occurs efficiently only when the kinase activity is specifically evoked by a cyclin of the B-type but not by cyclins of the A-type. In addition, high resolution two-dimensional gel analysis revealed that the kinases associated with A- and B-type cyclins exhibit different substrate preferences. These data suggest that the complexes of the cdc2 kinase with different cyclins may control specific events of the cell cycle.     

Control of division in Chlamydomonas by cyclin B/CDKB1 and the anaphase-promoting complex

In yeast and animals, cyclin B binds and activates the cyclin-dependent kinase (‘CDK’) CDK1 to drive entry into mitosis. We show that CYCB1, the sole cyclin B in Chlamydomonas, activates the plant-specific CDKB1 rather than the CDK1 ortholog CDKA1, confirming and extending previous results. Time-lapse microscopy shows that CYCB1 is synthesized before each division in the multiple fission cycle, then is rapidly degraded 3–5 minutes before division occurs. CYCB1 degradation is dependent on the anaphase-promoting complex (APC). Like CYCB1, CDKB1 is not synthesized until late G1; however, CDKB1 is not degraded with each division within the multiple fission cycle, but is degraded after all divisions have ceased. The microtubule plus-end-binding protein EB1 labeled with mNeonGreen allowed detection of mitotic events in live cells. The earliest detectable step in mitosis, splitting of polar EB1 signal into two foci, likely associated with future spindle poles, was dependent on CYCB1. CYCB1-GFP localized close to these foci immediately before spindle formation. Spindle breakdown, cleavage furrow formation and accumulation of EB1 in the furrow were dependent on the APC. In interphase, rapidly growing microtubules are marked by ‘comets’ of EB1; comets are absent in the absence of APC function. Thus CYCB1/CDKB1 and the APC modulate microtubule function and assembly while regulating mitotic progression. Genetic results suggest an independent additional role for the APC in regulating sister chromatid cohesion; this role is likely conserved across eukaryotes.     

SIC1 is ubiquitinated in vitro by a pathway that requires CDC4, CDC34, and cyclin/CDK activities.

Traversal from G1 to S-phase in cycling cells of budding yeast is dependent on the destruction of the S-phase cyclin/CDK inhibitor SIC1. Genetic data suggest that SIC1 proteolysis is mediated by the ubiquitin pathway and requires the action of CDC34, CDC4, CDC53, SKP1, and CLN/CDC28. As a first step in defining the functions of the corresponding gene products, we have reconstituted SIC1 multiubiquitination in DEAE-fractionated yeast extract. Multiubiquitination depends on cyclin/CDC28 protein kinase and the CDC34 ubiquitin-conjugating enzyme. Ubiquitin chain formation is abrogated in cdc4ts mutant extracts and assembly restored by the addition of exogenous CDC4, suggesting a direct role for this protein in SIC1 multiubiquitination. Deletion analysis of SIC1 indicates that the N-terminal 160 residues are both necessary and sufficient to serve as substrate for CDC34-dependent ubiquitination. The complementary C-terminal segment of SIC1 binds to the S-phase cyclin CLB5, indicating a modular structure for SIC1.     

Quantitative proteomic analysis reveals formation of an EscL-EscQ-EscN type III complex in enteropathogenic Escherichia coli

We characterized Orf5 and SepQ, two type III secretion (T3S) system proteins in enteropathogenic Escherichia coli, and showed that they are essential for T3S, associated with the bacterial membrane, and interact with EscN. Our findings suggest that Orf5 and SepQ are homologs of YscL and YscQ from Yersinia, respectively.     

Quantitative phosphoproteomic analysis reveals involvement of PD-1 in multiple T cell functions

Programmed cell death protein 1 (PD-1) is a critical inhibitory receptor that limits excessive T cell responses. Cancer cells have evolved to evade these immunoregulatory mechanisms by upregulating PD-1 ligands and preventing T cell–mediated anti-tumor responses. Consequently, therapeutic blockade of PD-1 enhances T cell–mediated anti-tumor immunity, but many patients do not respond and a significant proportion develop inflammatory toxicities. To improve anti-cancer therapy, it is critical to reveal the mechanisms by which PD-1 regulates T cell responses. We performed global quantitative phosphoproteomic interrogation of PD-1 signaling in T cells. By complementing our analysis with functional validation assays, we show that PD-1 targets tyrosine phosphosites that mediate proximal T cell receptor signaling, cytoskeletal organization, and immune synapse formation. PD-1 ligation also led to differential phosphorylation of serine and threonine sites within proteins regulating T cell activation, gene expression, and protein translation. In silico predictions revealed that kinase/substrate relationships engaged downstream of PD-1 ligation. These insights uncover the phosphoproteomic landscape of PD-1–triggered pathways and reveal novel PD-1 substrates that modulate diverse T cell functions and may serve as future therapeutic targets. These data are a useful resource in the design of future PD-1–targeting therapeutic approaches.     

Changes in cyclin B localisation during pig oocyte in vitro maturation

The localisation of cyclin B throughout in vitro maturation of pig oocytes was determined by indirect immunofluorescence using a monoclonal antibody specific for an epitope of the human cyclin B. Maturation of pig oocytes was induced by addition of Pergonal (2 UI/ml of FSH/LH) and beta-oestradiol to the medium where isolated ovarian follicles were cultured for up to 72 h. Immature gametes with an intact germinal vesicle were observed during the first 30 h of culture. Only 10% were competent to reinitiate meiosis and showed germinal vesicle breakdown (GVBD) after 36 h. However, after 48-72 h, 60% of the oocytes accomplished their maturation and showed metaphase chromosomes. Immature oocytes showed cyclin B immunofluorescent staining in the cytoplasm, whereas mature oocytes showed the immunofluorescent label concentrated in the nucleus. Metaphase chromosomes showed an intense immunofluorescence. The migration of cyclin B to the nucleus and its association with metaphase chromosomes in pig oocytes able to progress through meiosis resembled the subcellular localisation of cyclin B and the distribution of maturation promoting factor (MPF) in mitotic dividing cells.     

Quantitative 1H-NMR analysis reveals steric and electronic effects on the substrate specificity of benzoate dioxygenase in Ralstonia eutropha B9

The cis-dihydroxylation of arenes by Rieske dearomatizing dioxygenases (RDDs) represents a powerful tool for the production of chiral precursors in organic synthesis. Here, the substrate specificity of the RDD benzoate dioxygenase (BZDO) in Ralstonia eutropha B9 whole cells was explored using quantitative ¹H nuclear magnetic resonance spectroscopy (q¹H-NMR). The specific activity, specific carbon uptake, and regioselectivity of the dihydroxylation reaction were evaluated in resting cell cultures for a panel of 17 monosubstituted benzoates. Two new substrates of this dioxygenase system were identified (2-methyl- and 3-methoxybenzoic acid) and the corresponding cis-diol metabolites were characterized. Higher activities were observed for benzoates with smaller substituents, predominantly at the 3-position. Elevated activities were also observed in substrates bearing greater partial charge at the C-2 position of the benzoate ring. The regioselectivity of the reaction was directly measured using q¹H-NMR and found to have positive correlation with increasing substituent size. These results widen the pool of cis-diol metabolites available for synthetic applications and offer a window into the substrate traits that govern specificity for BZDO.     

The anaphase‐promoting complex initiates zygote division in Arabidopsis through degradation of cyclin B1

As the start of a new life cycle, activation of the first division of the zygote is a critical event in both plants and animals. Because the zygote in plants is difficult to access, our understanding of how this process is achieved remains poor. Here we report genetic and cell biological analyses of the zygote‐arrest 1 (zyg1) mutant in Arabidopsis, which showed zygote‐lethal and over‐accumulation of cyclin B1 D‐box‐GUS in ovules. Map‐based cloning showed that ZYG1 encodes the anaphase‐promoting complex/cyclosome (APC/C) subunit 11 (APC11). Live‐cell imaging studies showed that APC11 is expressed in both egg and sperm cells, in zygotes and during early embryogenesis. Using a GFP‐APC11 fusion construct that fully complements zyg1, we showed that GFP‐APC11 expression persisted throughout the mitotic cell cycle, and localized to cell plates during cytokinesis. Expression of non‐degradable cyclin B1 in the zygote, or mutations of either APC1 or APC4, also led to a zyg1‐like phenotype. Biochemical studies showed that APC11 has self‐ubiquitination activity and is able to ubiquitinate cyclin B1 and promote degradation of cyclin B1. These results together suggest that APC/C‐mediated degradation of cyclin B1 in Arabidopsis is critical for initiating the first division of the zygote.     

Bayesian phylogenetic analysis reveals two-domain topology of S-adenosylhomocysteine hydrolase protein sequences

S-Adenosylhomocysteine hydrolase (SahH) is involved in the degradation of the compound which inhibits methylation reactions. Using a Bayesian approach and other methods, we reconstructed a phylogenetic tree of amino acid sequences of this protein originating from all three major domains of living organisms. The SahH sequences formed two major branches: one composed mainly of Archaea and the other of eukaryotes and majority of bacteria, clearly contradicting the three-domain topology shown by small subunit rRNA gene. This topology suggests the occurrence of lateral transfer of this gene between the domains. Poor resolution of eukaryotes and bacteria excluded an ultimate conclusion in which out of the two domains this gene appeared first, however, the congruence of the secondary branches with SS rRNA and/or concatenated ribosomal protein datasets phylogenies suggested an "early" acquisition by some bacterial and eukaryotic phyla. Similarly, the branching pattern of Archaea reflected the phylogenies shown by SS rRNA and ribosomal proteins. SahH is widespread in Eucarya, albeit, due to reductive evolution, it is missing in the intracellular parasite Encephalitozoon cuniculi. On the other hand, the lack of affinity to the sequences from the alpha-Proteobacteria and cyanobacteria excludes a possibility of its acquisition in the course of mitochondrial or chloroplast endosymbioses. Unlike Archaea, most bacteria carry MTA/SAH nucleosidase, an enzyme involved also in metabolism of methylthioadenosine. However, the double function of MTA/SAH nucleosidase may be a barrier to ensure the efficient degradation of S-adenosylhomocysteine, specially when the intensity of methylation processes is high. This would explain the presence of S-adenosylhomocysteine hydrolase in the bacteria that have more complex metabolism. On the other hand, majority of obligate pathogenic bacteria due to simpler metabolism rely entirely on MTA/SAH nucleosidase. This could explain the observed phenetic pattern in which bacteria with larger (>6 Mb-million base pairs) genomes carry SAH hydrolase, whereas bacteria that have undergone reductive evolution usually carry MTA/SAH nucleosidase. This suggests that the presence or acquisition of S-adenosylhomocysteine hydrolase in bacteria may predispose towards higher metabolic, and in consequence, higher genomic complexity. The good examples are the phototrophic bacteria all of which carry this gene, however, the SahH phylogeny shows lack of congruence with SSU rRNA and photosyntethic genes, implying that the acquisition was independent and presumably preceded the acquisition of photosyntethic genes. The majority of cyanobacteria acquired this gene from Archaea, however, in some species the sahH gene was replaced by a copy from the beta- or gamma-Proteobacteria.     

Quaternary organization of the human eEF1B complex reveals unique multi-GEF domain assembly

Protein synthesis in eukaryotic cell is spatially and structurally compartmentalized that ensures high efficiency of this process. One of the distinctive features of higher eukaryotes is the existence of stable multi-protein complexes of aminoacyl-tRNA synthetases and translation elongation factors. Here, we report a quaternary organization of the human guanine-nucleotide exchange factor (GEF) complex, eEF1B, comprising α, β and γ subunits that specifically associate into a heterotrimeric form eEF1B(αβγ)₃. As both the eEF1Bα and eEF1Bβ proteins have structurally conserved GEF domains, their total number within the complex is equal to six. Such, so far, unique structural assembly of the guanine-nucleotide exchange factors within a stable complex may be considered as a ‘GEF hub’ that ensures efficient maintenance of the translationally active GTP-bound conformation of eEF1A in higher eukaryotes.     

The Drosophila PNG kinase complex regulates the translation of cyclin B

The Drosophila PAN GU (PNG) kinase complex regulates the developmental translation of cyclin B. cyclin B mRNA becomes unmasked during oogenesis independent of PNG activity, but PNG is required for translation from egg activation. We find that although polyadenylation of cyclin B augments translation, it is not essential, and a fully elongated poly(A) is not required for translation to proceed. In fact, changes in poly(A) tail length are not sufficient to account for PNG-mediated control of cyclin B translation and of the early embryonic cell cycles. We present evidence that PNG functions instead as an antagonist of PUMILIO-dependent translational repression. Our data argue that changes in poly(A) tail length are not a universal mechanism governing embryonic cell cycles, and that PNG-mediated derepression of translation is an important alternative mechanism in Drosophila.     

Quantitative analysis of cytokeratin network topology in the MCF7 cell line

BACKGROUND: In the MCF7 human breast cancer cell line, several patterns of cytokeratin networks are observed, depending on the intracellular localization. Our hypothesis is that architectural variations of cytokeratin networks depend on local tensions or forces appearing spontaneously in the cytoplasm. The aim of this work was to discriminate between the different patterns and to quantitate these variations. MATERIALS AND METHODS: Image analysis procedures were developed to extract cytokeratin filament networks visualized by immunofluorescence and confocal microscopy. Two methods were used to segment sets of curvilinear objects. The first, the "mesh-approach," based on classical methods of mathematical morphology, takes into account global network topology. The second, the "filament-approach" (novel), is meant to account for individual element morphology. These methods and their combination allow the computation of several features at two levels of geometry: global (network topology) and local (filament morphology). RESULTS: Variations in cytokeratin networks are characterized by their connectivity, density, mesh structure, and filament shape. The connectivity and the density of a network describe its location in a local "stress-force" zone or in a "relaxed" zone. The mesh structure characterizes the intracellular localization of the network. Moreover, the filament shape reflects the intracellular localization and the occurrence of a "stress-force" zone. CONCLUSIONS: These features permitted the quantitation of differences within the network patterns and within the specific filament shapes according to the intracellular localization. Further experiments on cells submitted to external forces will test the hypothesis that the architectural variations of intermediate filaments reflect intracytoplasmic tensions.     

Human laminin B2 chain. Comparison of the complete amino acid sequence with the B1 chain reveals variability in sequence homology between different structural domains

The complete amino acid sequence of the human laminin B2 chain has been determined by sequencing of cDNA clones. The six overlapping clones studied cover approximately 7.5 kilobases of which 5312 nucleotides were sequenced from the 5' end. The open reading frame codes for a 33-residue signal peptide and a 1576-residue B2 chain proper, which is 189 residues less than in the highly homologous B1 chain (Pikkarainen, T., Eddy, R., Fukushima, Y., Byers, M., Shows, T., Pihlajaniemi, T., Saraste, M., and Tryggvason, K. (1987) J. Biol. Chem. 262, 10454-10462). Computer analysis revealed that the B2 chain consists of distinct domains that contain helical structures, cysteine-rich repeats, and globular regions, as does the B1 chain. However, domain alpha and domain beta of the B1 chain have no counterpart in B2, and the number of cysteine-rich repeats is 12, or 1 less than in the B1 chain. The degree of homology between the two chains is highest in the cysteine repeat-containing domains III and V where 40% of the residues match. However, results demonstrate that the B1 and B2 chains of laminin are highly homologous proteins that are probably the products of related genes.     

HIV-1 Tat impairs cell cycle control by targeting the Tip60, Plk1 and cyclin B1 ternary complex

HIV-1 Tat triggers intrinsic and extrinsic apoptosis pathways in both infected and uninfected cells and plays an important role in the pathogenesis of AIDS. Knocking down Tip60, an interactive protein of Tat, leads to the impairment of cell cycle progression, indicating a key role of Tip60 in cell cycle control. We found that Tip60 interacts with Plk1 through its ZnFMYST domain, and that this interaction is enhanced in the G₂/M phase. In addition, cyclin B1 was confirmed to interact with the ZnF domain of Tip60. Immunofluorescence imaging showed that Tip60 co-localizes with both Plk1 and cyclin B1 at the centrosome during the mitotic phase and to the mid-body during cytokinesis. Further experiments revealed that Tip60 forms a ternary complex with Plk1 and cyclin B1 and acetylates Plk1 but not cyclin B1. HIV-1 Tat likely forms a quaternary complex with Tip60, cyclin B1 and Plk1. Fluorescent microscopy showed that Tat causes an unscheduled nuclear translocation of both cyclin B1 and Plk1, causing their co-localization with Tip60 in the nucleus. Tat, Tip60, cyclin B1 and Plk1 interactions provide new a mechanistic explanation for Tat-mediated cell cycle dysregulation and apoptosis.