Dephosphorylation of Orc2 by protein phosphatase 1 promotes the binding of the origin recognition complex to chromatin

Phosphorylation of Orc2, one of the six subunits of the origin recognition complex (ORC), by cyclin A/CDK2 during S phase leads to the dissociation of Orc2, Orc3, Orc4, and Orc5 subunits (Orc2–5) from human chromatin and replication origins. Dephosphorylation of the phosphorylated Orc2 by protein phosphatase 1 (PP1) is accompanied by the binding of the dissociated subunits to chromatin. Here we show that PP1 physically interacts with Orc2. The binding of PP1 to Orc2 and the dephosphorylation of Orc2 by PP1 occurred in a cell cycle-dependent manner through an interaction with 119-KSVSF-123, which is the consensus motif for the binding of PP1, of Orc2. The dephosphorylation of Orc2 by PP1 is required for the binding of Orc2 to chromatin. These results support that PP1 dephosphorylates Orc2 to promote the binding of ORC to chromatin and replication origins for the subsequent round of the cell cycle.     

Molecular determinants of nuclear protein phosphatase-1 regulation by NIPP-1.

NIPP-1 is a subunit of the major nuclear protein phosphatase-1 (PP-1) in mammalian cells and potently inhibits PP-1 activity in vitro. Using yeast two-hybrid and co-sedimentation assays, we mapped a PP-1-binding site and the inhibition function to the central one-third domain of NIPP-1. Full-length NIPP-1 (351 residues) and the central domain, NIPP-1(143-217), were equally potent PP-1 inhibitors (IC50 = 0.3 nM). Synthetic peptides spanning the central domain of NIPP-1 further narrowed the PP-1 inhibitory function to residues 191-200. A second, noninhibitory PP-1-binding site was identified by far-Western assays with digoxygenin-conjugated catalytic subunit (PP-1C) and included a consensus RVXF motif (residues 200-203) found in many other PP-1-binding proteins. The substitutions, V201A and/or F203A, in the RVXF motif, or phosphorylation of Ser199 or Ser204, which are established phosphorylation sites for protein kinase A and protein kinase CK2, respectively, prevented PP-1C-binding by NIPP-1(191-210) in the far-Western assay. NIPP-1(191-210) competed for PP-1 inhibition by full-length NIPP-1(1-351), inhibitor-1 and inhibitor-2, and dissociated PP-1C from inhibitor-1- and NIPP-1(143-217)-Sepharose but not from full-length NIPP-1(1-351)-Sepharose. Together, these data identified some of the key elements in the central domain of NIPP-1 that regulate PP-1 activity and suggested that the flanking sequences stabilize the association of NIPP-1 with PP-1C.     

Molecular characterization of phosphoglycerate mutase in archaea

The interconversion of 3-phosphoglycerate and 2-phosphoglycerate during glycolysis and gluconeogenesis is catalyzed by phosphoglycerate mutase (PGM). In bacteria and eukaryotes two structurally distinct enzymes have been found, a cofactor-dependent and a cofactor-independent (iPGM) type. Sequence analysis of archaeal genomes did not find PGMs of either kind, but identified a new family of proteins, distantly related to iPGMs. In this study, these predicted archaeal PGMs from Pyrococcus furiosus and Methanococcus jannaschii have been functionally produced in Escherichia coli, and characterization of the purified proteins has confirmed that they are iPGMs. Analysis of the available microbial genomes indicates that this new type of iPGM is widely distributed among archaea and also encoded in several bacteria. In addition, as has been demonstrated in certain bacteria, some archaea appear to possess an alternative, cofactor-dependent PGM.     

Molecular determinants of vanilloid sensitivity in TRPV1

Vanilloid receptor 1 (TRPV1), a membrane-associated cation channel, is activated by the pungent vanilloid from chili peppers, capsaicin, and the ultra potent vanilloid from Euphorbia resinifera, resiniferatoxin (RTX), as well as by physical stimuli (heat and protons) and proposed endogenous ligands (anandamide, N-arachidonyldopamine, N-oleoyldopamine, and products of lipoxygenase). Only limited information is available in TRPV1 on the residues that contribute to vanilloid activation. Interestingly, rabbits have been suggested to be insensitive to capsaicin and have been shown to lack detectable [(3)H]RTX binding in membranes prepared from their dorsal root ganglia. We have cloned rabbit TRPV1 (oTRPV1) and report that it exhibits high homology to rat and human TRPV1. Like its mammalian orthologs, oTRPV1 is selectively expressed in sensory neurons and is sensitive to protons and heat activation but is 100-fold less sensitive to vanilloid activation than either rat or human. Here we identify key residues (Met(547) and Thr(550)) in transmembrane regions 3 and 4 (TM3/4) of rat and human TRPV1 that confer vanilloid sensitivity, [(3)H]RTX binding and competitive antagonist binding to rabbit TRPV1. We also show that these residues differentially affect ligand recognition as well as the assays of functional response versus ligand binding. Furthermore, these residues account for the reported pharmacological differences of RTX, PPAHV (phorbol 12-phenyl-acetate 13-acetate 20-homovanillate) and capsazepine between human and rat TRPV1. Based on our data we propose a model of the TM3/4 region of TRPV1 bound to capsaicin or RTX that may aid in the development of potent TRPV1 antagonists with utility in the treatment of sensory disorders.     

Localized melting of duplex DNA by Cdc6/Orc1 at the DNA replication origin in the hyperthermophilic archaeon Pyrococcus furiosus

The initiation step is a key process to regulate the frequency of DNA replication. Although recent studies in Archaea defined the origin of DNA replication (oriC) and the Cdc6/Orc1 homolog as an origin recognition protein, the location and mechanism of duplex opening have remained unclear. We have found that Cdc6/Orc1 binds to oriC and unwinds duplex DNA in the hyperthermophilic archaeon Pyrococcus furiosus, by means of a P1 endonuclease assay. A primer extension analysis further revealed that this localized unwinding occurs in the oriC region at a specific site, which is 12-bp long and rich in adenine and thymine. This site is different from the predicted duplex unwinding element (DUE) that we reported previously. We also discovered that Cdc6/Orc1 induces topological changes in supercoiled oriC DNA, and that this process is dependent on the AAA+ domain. These results indicate that topological alterations of oriC DNA by Cdc6/Orc1 introduce a single-stranded region at the 12-mer site, that could possibly serve as an entry point for Mcm helicase.     

Molecular determinants of CaV2.1 channel regulation by calcium-binding protein-1

Presynaptic Ca(V)2.1 channels, which conduct P/Q-type Ca(2+) currents, initiate synaptic transmission at most synapses in the central nervous system. Regulation of Ca(V)2.1 channels by CaM contributes significantly to short term facilitation and rapid depression of synaptic transmission. Short term synaptic plasticity is diverse in form and function at different synapses, yet CaM is ubiquitously expressed. Differential regulation of Ca(V)2.1 channels by CaM-like Ca(2+) sensor (CaS) proteins differentially affects short term synaptic facilitation and rapid synaptic depression in transfected sympathetic neuron synapses. Here, we define the molecular determinants for differential regulation of Ca(V)2.1 channels by the CaS protein calcium-binding protein-1 (CaBP1) by analysis of chimeras in which the unique structural domains of CaBP1 are inserted into CaM. Our results show that the N-terminal domain, including its myristoylation site, and the second EF-hand, which is inactive in Ca(2+) binding, are the key molecular determinants of differential regulation of Ca(V)2.1 channels by CaBP1. These findings give insight into the molecular code by which CaS proteins differentially regulate Ca(V)2.1 channel function and provide diversity of form and function of short term synaptic plasticity.     

Cyclin-Mediated Export of Human Orc1

Viral cyclin/cdk6 complexes interact with and phosphorylate human Orc1, a component of the origin recognition complex (ORC) that functions in DNA replication. Here we assess the effect that viral cyclin has on the intracellular location of human Orc1, which is present in both nuclear and cytoplasmic pools. Overexpression of K cyclin or cyclin A results in Crm1-dependent export of Orc1 to the cytoplasm, and this process is dependent on the phosphorylation status of several cdk target sites in Orc1. These findings support a model where S phase promoting cyclin activity drives the export of a component of replication complexes.     

Molecular identification of a mechanosensitive channel in archaea

The TM1 domain of the large conductance mechanosensitive (MS) channel of Escherichia coli was used as a genetic probe to search the genomic database of the archaeon Methanoccoccus jannashii for MscL homologs. We report that the hypothetical protein MJ0170 of M. jannashii exhibited 38.5% sequence identity with the TM1 domain of Eco-MscL. Moreover, MJ0170 was found to be a conserved homolog of MscS, the second type of E. coli MS channel encoded by the yggB gene. Furthermore, we identified a cluster of charged residues KIKEE in the C-terminus of MJ0170 that strikingly resembled the charged C-terminal amino acid cluster present in Eco-MscL (RKKEE). We cloned and expressed MJ0170 in E. coli, which when reconstituted into liposomes or expressed in the cell membrane of giant E. coli spheroplasts, exhibited similar activity to the bacterial MS channels. Our study suggests that the M. jannashii MS channel and its homologs evolved as a result of gene duplication of the ancestral MscL-like molecule with the TM1 domain remaining the most conserved structural motif among prokaryotic MS channels.     

Mechanism for the degradation of origin recognition complex containing Orc5p with a defective Walker A motif and its suppression by over-production of Orc4p in yeast cells

Orc5p is one of six subunits constituting the ORC (origin recognition complex), a possible initiator of chromosomal DNA replication in eukaryotes. Orc5p contains a Walker A motif. We recently reported that a strain of Saccharomyces cerevisiae having a mutation in Orc5p's Walker A motif (orc5-A), showed cell-cycle arrest at G2/M and degradation of ORC at high temperatures (37 degrees C). Over-production of Orc4p, another subunit of ORC, specifically suppressed these phenotypes [Takahashi, Yamaguchi, Yamairi, Makise, Takenaka, Tsuchiya and Mizushima (2004) J. Biol. Chem. 279, 8469-8477]. In the present study, we examined the mechanisms of ORC degradation and of its suppression by Orc4p over-production. In orc5-A, at high temperatures, ORC is degraded by proteasomes; either addition of a proteasome inhibitor, or introduction of a mutation of either tan1-1 or nob1-4 that inhibits proteasomes, prevented ORC degradation. Introduction of the tan1-1 mutation restored cell cycle progression, suggesting that the defect was due to ORC degradation by proteasomes. Yeast two-hybrid and co-immunoprecipitation analyses suggested that Orc5p interacts preferentially with Orc4p and that the orc5-A mutation diminishes this interaction. We suggest that this interaction is mediated by the C-terminal region of Orc4p, and the N-terminal region of Orc5p. Based on these observations, we consider that ATP binding to Orc5p is required for efficient interaction with Orc4p and that, in orc5-A, loss of this interaction at higher temperatures allows proteasomes to degrade ORC, causing growth defects. This model could also explain why over-production of Orc4p suppresses the orc5-A strain's phenotype.     

Regulation of the functional interactions between archaeal eukaryote-like Cdc6/Orc1 proteins on the replication origin by two different mechanisms

The crenarchaeon Sulfolobus solfataricus contains three active origins of replication and three eukaryote-like Cdc6/Orc1 proteins known as SsoCdc6 proteins. It has the potential to become a powerful model system in understanding the central mechanism of the eukaryotic DNA replication. In this research, we designed a group of duplex DNA substrates containing specific origin recognition boxes (ORBs) of the archaeon and identified the DNA-binding activities of different SsoCdc6 proteins. Furthermore, we showed that the DNA-protein interaction between the DNA substrate and the SsoCdc6-1 or SsoCdc6-3 strikingly regulated their DNA-binding activities of each other on the origin. On the other hand, the protein-protein interactions between SsoCdc6-1 and SsoCdc6-2 were observed to mutually modulate the stimulating or inhibitive effects on the DNA-binding activities of each other. Thus, two different mechanisms were demonstrated to be involved in the regulations of the functions of the SsoCdc6 proteins on the replication origins. The results of this study imply that the interactions between multiple SsoCdc6 proteins and origin DNA collectively contribute to the positive or negative regulation of DNA replication initiation in the archaeon species.     

Biochemical characterization of Cdc6/Orc1 binding to the replication origin of the euryarchaeon Methanothermobacter thermoautotrophicus

Archaeal cell division cycle protein 6 (Cdc6)/Origin Replication Complex subunit 1 (Orc1) proteins share sequence homology with eukaryotic DNA replication initiation factors but are also structurally similar to the bacterial initiator DnaA. To better understand whether Cdc6/Orc1 functions in an eukaryotic or bacterial-like manner, we have characterized the interaction of two Cdc6/Orc1 paralogs (mthCdc6-1 and mthCdc6-2) with the replication origin from Methanothermobacter thermoautotrophicus. We show that while both proteins display a low affinity for a small dsDNA of random sequence, mthCdc6-1 binds tightly to a short duplex containing a single copy of a 13 bp sequence that is repeated throughout the origin. Surprisingly, sequence comparisons show that this 13 bp sequence is a minimized version of the Origin Recognition Box element found in many euryarchaeotal origins. Analysis of mthCdc6-1 mutants demonstrates that the helix–turn–helix motif in the winged-helix domain mediates the interaction with this sequence. Association of both mthCdc6/Orc1 paralogs with the duplex containing the minimized Origin Recognition Box fits to an independent binding sites model, but their interaction with longer DNA ligands is cooperative. Together, our data provide the first detailed biophysical characterization of the association of an archaeal DNA replication initiator with its origin. Our observations also indicate that the origin-binding properties of Cdc6/Orc1 proteins closely resemble those of bacterial DnaA.     

Selective instability of Orc1 protein accounts for the absence of functional origin recognition complexes during the M-G(1) transition in mammals

To investigate the events leading to initiation of DNA replication in mammalian chromosomes, the time when hamster origin recognition complexes (ORCs) became functional was related to the time when Orc1, Orc2 and Mcm3 proteins became stably bound to hamster chromatin. Functional ORCs, defined as those able to initiate DNA replication, were absent during mitosis and early G(1) phase, and reappeared as cells progressed through G(1) phase. Immunoblotting analysis revealed that hamster Orc1 and Orc2 proteins were present in nuclei at equivalent concentrations throughout the cell cycle, but only Orc2 was stably bound to chromatin. Orc1 and Mcm3 were easily eluted from chromatin during mitosis and early G(1) phase, but became stably bound during mid-G(1) phase, concomitant with the appearance of a functional pre-replication complex at a hamster replication origin. Since hamster Orc proteins are closely related to their human and mouse homologs, the unexpected behavior of hamster Orc1 provides a novel mechanism in mammals for delaying assembly of pre-replication complexes until mitosis is complete and a nuclear structure has formed.     

Molecular determinants of acute single-cell lysis by human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) infection of CD4-positive lymphocytes is accompanied by acute cytopathic effects, i.e., syncytium formation and single-cell lysis. Syncytium formation involves cell-cell fusion mediated by viral envelope glycoproteins on the surface of infected cells and by CD4 glycoproteins on adjacent cells. The molecular basis for the lysis of single-HIV-1 infected cells is unclear. Here we report that the expression of functional envelope glycoproteins from primary and laboratory-adapted HIV-1 isolates resulted in the lysis of single CD4-positive lymphocytes. As was previously observed in HIV-1 infected cultures, single-cell lysis in this system primarily involved necrosis and was not inhibited by soluble CD4. Binding of the viral envelope glycoproteins to the CD4 glycoprotein facilitated, but was not sufficient for, cytolysis. Importantly, the ability of the HIV-1 envelope glycoproteins to mediate membrane fusion was essential for single-cell killing. By contrast, the long cytoplasmic tail of the gp41 transmembrane envelope glycoprotein was neither necessary nor sufficient for single-cell lysis. These results suggest that intracellular envelope glycoprotein-CD4 interactions initiate autofusion events that disrupt cell membrane integrity, leading to single-cell lysis by HIV-1.     

Molecular identification of methanogenic archaea from sheep in Queensland, Australia reveal more uncultured novel archaea

Molecular diversity of rumen methanogens in sheep in Queensland, Australia was investigated using 16S rRNA gene libraries prepared from pooled rumen contents from nine merino sheep. A total of 78 clones were identified revealing 26 different sequences. Of these 26 sequences, eight sequences (15 clones) were 95-100% similar to cultivated methanogens belonging to the orders Methanobacteriales and Methanomicrobiales, and the remaining 18 phylotypes (63 clones) were 72-75% similar to Thermoplasma acidophilum and Thermoplasma volcanium. These unique sequences clustered within a distinct and strongly supported (100% bootstrap support) phylogenetic group, exclusively composed of sequences from uncharacterized archaea from very diverse anaerobic environments. Members of this unique group that were previously considered atypical for the rumen environment were the predominant clones.     

Binding of AlF-C, an Orc1-binding transcriptional regulator, enhances replicator activity of the rat aldolase B origin

A region encompassing the rat aldolase B gene (aldB) promoter acts as a chromosomal origin of DNA replication (origin) in rat aldolase B-nonexpressing hepatoma cells. To examine replicator function of the aldB origin, we constructed recombinant mouse cell lines in which the rat aldB origin and the mutant derivatives were inserted into the same position at the mouse chromosome 8 by cre-mediated recombination. Nascent strand abundance assays revealed that the rat origin acts as a replicator at the ectopic mouse locus. Mutation of site C in the rat origin, which binds an Orc1-binding protein AlF-C in vitro, resulted in a significant reduction of the replicator activity in the mouse cells. Chromatin immunoprecipitation (ChIP) assays indicated that the reduction of replicator activity was paralleled with the reduced binding of AlF-C and Orc1, suggesting that sequence-specific binding of AlF-C to the ectopic rat origin leads to enhanced replicator activity in cooperation with Orc1. Involvement of AlF-C in replication in vivo was further examined for the aldB origin at its original rat locus and for a different rat origin identified in the present study, which contained an AlF-C-binding site. ChIP assays revealed that both replication origins bind AlF-C and Orc1. We think that the results presented here may represent one mode of origin recognition in mammalian cells.     

Molecular determinants for virulence in coxsackievirus B1 infection.

Studies demonstrated that a strain derived from an infectious clone of coxsackievirus B1 (CVB1N) (N. Iizuka, H. Yonekawa, and A. Nomoto, J. Virol. 65:4867-4873, 1991) was 3 to 4 log10 less virulent than the myotropic Tucson strain of CVB1 (CVB1T) following intraperitoneal inoculation of newborn mice. Replacement of nucleotides (nt) 69 to 804 from the 5' untranslated region (5' UTR) and 1A coding region of CVB1N or nt 117 to 161 from the 5' UTR with the corresponding part from CVB1T restored greater than 90% of the virulence. Sequencing of the 5' UTR of CVB1T demonstrated areas with a greater similarity to particular echoviruses than to CVB1N, suggesting that recombination events might have occurred, perhaps influencing the virulence phenotype.     

Molecular determinants of sugar transport regulation by ATP

Intracellular ATP inhibits human erythrocyte net sugar transport by binding cooperatively to the glucose transport protein (GluT1). ATP binding produces altered transporter affinity for substrate and promotes substrate occlusion within a post-translocation vestibule formed by GluT1 cytosolic domains. The accompanying paper (Cloherty, E. K., Levine, K. B., Graybill, C., and Carruthers, A. (2002) Biochemistry 41, 12639-12651) demonstrates that reduced intracellular pH promotes high-affinity ATP binding to GluT1 but inhibits ATP-modulation of GluT1-mediated sugar transport. The present study explores the role of GluT1 residues 326-343 (a proposed GluT1 ATP-binding site subdomain) in GluT1 ATP binding by using alanine scanning mutagenesis. Cos-7 and HEK cells were transfected with a cDNA encoding full-length human GluT1 terminating in a carboxyl-terminal hemagglutinin (HA)-His6 epitope. The transporter (GluT1.HA.H6) is expressed at the surface of both cell-types and is catalytically active. In HEK cells, both parental GluT1- and GluT1.HA.H6-mediated sugar transport are acutely sensitive to cellular metabolic inhibition. Isolated, detergent-solubilized GluT1.HA.H6 is photolabeled by [gamma-32P]-azidoATP in an ATP-protectable manner. Alanine substitution of E329 or G332/R333/R334 enhances GluT1.HA.H6 [gamma-32P]azidoATP photoincorporation but blocks acute modulation of net sugar transport by cellular metabolic inhibition. These actions resemble those of reduced pH on ATP binding to and modulation of red cell GluT1. It is proposed that cooperative nucleotide binding to GluT1 and nucleotide modulation of GluT1-mediated sugar transport are regulated by a proton-sensitive saltbridge (Glu329-Arg333/334).     

Molecular determinants for α-tubulin methylation by SETD2

Post-translational modifications to tubulin are important for many microtubule-based functions inside cells. It was recently shown that methylation of tubulin by the histone methyltransferase SETD2 occurs on mitotic spindle microtubules during cell division, with its absence resulting in mitotic defects. However, the catalytic mechanism of methyl addition to tubulin is unclear. We used a truncated version of human wild type SETD2 (tSETD2) containing the catalytic SET and C-terminal Set2–Rpb1–interacting (SRI) domains to investigate the biochemical mechanism of tubulin methylation. We found that recombinant tSETD2 had a higher activity toward tubulin dimers than polymerized microtubules. Using recombinant single-isotype tubulin, we demonstrated that methylation was restricted to lysine 40 of α-tubulin. We then introduced pathogenic mutations into tSETD2 to probe the recognition of histone and tubulin substrates. A mutation in the catalytic domain (R1625C) allowed tSETD2 to bind to tubulin but not methylate it, whereas a mutation in the SRI domain (R2510H) caused loss of both tubulin binding and methylation. Further investigation of the role of the SRI domain in substrate binding found that mutations within this region had differential effects on the ability of tSETD2 to bind to tubulin versus the binding partner RNA polymerase II for methylating histones in vivo, suggesting distinct mechanisms for tubulin and histone methylation by SETD2. Finally, we found that substrate recognition also requires the negatively charged C-terminal tail of α-tubulin. Together, this study provides a framework for understanding how SETD2 serves as a dual methyltransferase for both histone and tubulin methylation.