Inactivation and Disassembly of the Anaphase-Promoting Complex during Human Cytomegalovirus Infection Is Associated with Degradation of the APC5 and APC4 Subunits and Does Not Require UL97-Mediated Phosphorylation of Cdh1

Infection of quiescent cells by human cytomegalovirus (HCMV) elicits severe cell cycle deregulation, resulting in a G₁/S arrest, which can be partly attributed to the inactivation of the anaphase-promoting complex (APC). As we previously reported, the premature phosphorylation of its coactivator Cdh1 and/or the dissociation of the core complex can account for the inactivation. We have expanded on these results and further delineated the key components required for disabling the APC during HCMV infection. The viral protein kinase UL97 was hypothesized to phosphorylate Cdh1, and consistent with this, phosphatase assays utilizing a virus with a UL97 deletion mutation (ΔUL97 virus) indicated that Cdh1 is hypophosphorylated at early times in the infection. Mass spectrometry analysis demonstrated that UL97 can phosphorylate Cdh1 in vitro, and the majority of the sites identified correlated with previously characterized cyclin-dependent kinase (Cdk) consensus sites. Analysis of the APC core complex during ΔUL97 virus infection showed APC dissociation occurring at the same time as during infection with wild-type virus, suggesting that the UL97-mediated phosphorylation of Cdh1 is not required for this to occur. Further investigation of the APC subunits showed a proteasome-dependent loss of the APC5 and APC4 subunits that was temporally associated with the disassembly of the APC. Immediate early viral gene expression was not sufficient for the degradation of APC4 and APC5, indicating that a viral early gene product(s), possibly in association with a de novo-synthesized cellular protein(s), is involved.Human cytomegalovirus (HCMV), a highly prevalent β-herpesvirus, can cause serious birth defects and disease in immunocompromised individuals, and it may be associated with cancer and cardiovascular disease (53). Viral gene expression is temporally regulated and is dependent on many cellular factors for a productive infection. Immediate early (IE) genes are expressed by 2 h postinfection (p.i.) and transactivate the early genes required for viral DNA replication. The expression of the late genes, which encode proteins involved in virion maturation and egress, is dependent on viral DNA replication.The virus has adopted different strategies for altering the cellular environment to make it more conducive to productive infection, including the stimulation of host cell DNA replication pathways, cell cycle deregulation and arrest, immune evasion, and inhibition of apoptosis (53). Although HCMV encodes its own DNA polymerase, it is dependent on other cellular resources for DNA replication. Infection of quiescent cells induces passage toward S phase such that the host cell is stimulated to generate proteins and DNA precursors necessary for genome replication; however, entry into S phase and cellular DNA replication are subsequently blocked and the cell arrests in G₁/S (1, 10, 11, 14, 30, 45). Cellular resources are thereby presumably free to be efficiently utilized for viral replication. Cell cycle arrest by HCMV is achieved in part through the misregulation of several cell cycle proteins, including the phosphorylation and accumulation of the Rb family pocket proteins, upregulation of cyclins E and B and their associated kinase activities, inhibition of cyclin A expression, stabilization of p53, and accumulation of Cdc6 and geminin, which inhibits licensing of the cellular origins of DNA replication (8, 17, 30, 49, 54, 65). Some of these cell cycle defects can be attributed to a deregulation of the anaphase-promoting complex (APC) (8, 72, 79, 80), an E3 ubiquitin ligase that is responsible for the timely degradation of cell cycle proteins and mitotic cyclins to promote cycle progression from mitosis through G₁ to S phase (58, 74). As the APC also appears to be a common target among other viruses, including the chicken anemia virus, adenoviruses, and poxviruses (23, 36, 52, 70), understanding the mechanisms leading to its inactivation during viral infection has been of great interest.As we have previously reported, multiple mechanisms may be involved in disabling the APC during HCMV infection (72), which is not surprising given the complexity of its structure and regulation (for a review, see references 58 and 74). The APC is a large multisubunit complex consisting of at least 11 conserved core subunits, as well as other species-specific subunits. In metazoans, the APC2 and APC11 subunits form the catalytic core, and along with APC10, provide the platform for binding the E2 ubiquitin-conjugating enzyme. Each of the APC3, APC8, APC6, and APC7 subunits contain multiple copies of the tetratricopeptide repeat (TPR) motif and together make up the TPR subcomplex, which provides a platform of protein interaction surfaces for binding the coactivators (i.e., Cdh1 and Cdc20) and various substrates. These two subcomplexes are bridged by the large scaffolding subunit APC1, with the TPR subcomplex tethered to APC1 through APC4 and APC5. The binding between APC1, APC4, APC5, and APC8 is also interdependent, such that the loss of one subunit decreases the association of the other three (71).The APC is activated by either of its coactivators, Cdh1 or Cdc20, which also function in recruiting specific substrates to the APC during different phases of the cell cycle. The phosphorylation of several APC subunits at the onset of mitosis, including APC1 and the TPR subunits, by cyclin B/cyclin-dependent kinase 1 (Cdk1) and Plk1 allows the binding of Cdc20 and subsequent activation of the APC (APC^Cdc20) (19, 37), whereas the binding and activation of the complex by Cdh1 is inhibited through its phosphorylation by cyclin B/Cdk1 (9, 29, 38, 83). As cells pass the spindle assembly checkpoint, APC^Cdc20 ubiquitinates securin (to allow for sister chromatid separation) and cyclin B for degradation by the proteasome (42, 67). The subsequent inactivation of Cdk1 and activation of mitotic phosphatases during late anaphase relieves the inhibitory phosphorylation on Cdh1, presumably by Cdc14 (6, 38, 44), which then allows Cdh1 to bind and activate the APC (APC^Cdh1). APC^Cdh1 ubiquitinates Cdc20 and mitotic cyclins for degradation to facilitate mitotic exit and maintains their low levels, along with S-phase regulators (e.g., Cdc6, geminin, etc.), during G₁ (16, 50, 59, 63). The inactivation of APC^Cdh1 as cells enter S phase may be mediated in part through the phosphorylation of Cdh1 by cyclin A/Cdk2 (46) and Cdh1 binding to the inhibitor Emi1 (25). The inactivation of Cdh1 by phosphorylation has been shown in all organisms studied thus far (e.g., yeast, Drosophila, plants, mammals, etc.), and mutants mimicking constitutively phosphorylated Cdh1 on Cdk consensus sites can neither bind nor activate the APC in vivo or in vitro (9, 29, 38, 69, 83).During HCMV infection of fibroblasts in G₀/G₁, however, Cdh1 becomes prematurely phosphorylated in a Cdk-independent manner and no longer associates with the APC (72). This dissociation does not appear to be due to an overexpression of Emi1 (79). Cdc20 also can no longer associate with the APC (79), suggesting a defect in the APC core. We have further shown that the APC core complex disassembles during the infection, with the TPR subunits (i.e., APC3, APC7, and APC8) and APC10 localizing to the cytosol, while APC1 remains nuclear (72). Interestingly, both the phosphorylation of Cdh1 and the dissociation of the APC occur at similar times during HCMV infection. Although either of these mechanisms could render the APC inactive, it was unclear whether these processes are linked or represent independent (or redundant) pathways. The causative factor(s) in mediating these events and the question of whether such a factor(s) was of cellular or viral origin also remained unresolved.On the basis of the results of several recent studies (26, 32, 62), the viral protein kinase UL97 emerged as a likely candidate for involvement in the phosphorylation of Cdh1. Conserved among herpesviruses, UL97 functions in viral genome replication (7, 32, 81) and in nuclear egress of viral capsids (21, 39, 48). UL97 is present in the tegument of the virus particle (76) and is also expressed de novo with early kinetics (i.e., detectable by 5 h p.i. by Western blot assay), with increased expression at later times of the infection (51, 76, 77). UL97 is a serine/threonine (S/T) protein kinase (22), and recent studies have further characterized it as a Cdkl mimic, with predicted structural similarity to Cdk2 (64) and common substrates. UL97 has been shown to phosphorylate in vitro nuclear lamin A/C (21), the carboxyl-terminal domain of RNA polymerase II (5), the translation elongation factor 1δ (EF1δ) (33), and Rb (26, 62) on sites targeted by Cdks, and there is considerable evidence that UL97 phosphorylates lamin A/C, EF1δ, and Rb on these sites in infected cells as well (21, 26, 33, 62). Given that cyclin A/Cdk2 and cyclin B/Cdk1 complexes normally phosphorylate Cdh1, thus preventing its association with the APC, we hypothesized that UL97 phosphorylates Cdh1 during HCMV infection.In the present study, we provide further mechanistic details of the events and players involved in inactivating the APC during HCMV infection. Evidence that UL97 is the viral factor mediating the phosphorylation of Cdh1 was obtained. However, APC disassembly still occurred at similar times in ΔUL97 and wild-type virus infections, indicating that UL97-mediated phosphorylation of Cdh1 is not required for this event. The inactivation of the APC core complex is further attributed to the loss of the APC5 and APC4 subunits early during the infection. The degradation of these subunits is proteasome dependent and requires de novo synthesis of viral early or cellular proteins. While the primary mechanism of inactivation appears to be the dissociation of the complex and the targeted loss of APC5 and APC4, phosphorylation of Cdh1 may provide a small kinetic advantage and backup mechanism for disabling the APC.     

Immuno-proteasome subunit LMP7 is up-regulated in the ischemic kidney in an experimental model of renovascular hypertension

Immuno-proteasome is thought to be responsible for the processing of intracellular antigens and is induced when cells are treated with the inflammatory cytokines promoting cellular immunity. We tested the possibility that immuno-proteasome can be up-regulated in renal cells exposed to a long-lasting ischemia and inflammation in an experimental model of two-kidney, one-clip renovascular hypertension in the rat. Western blotting showed that immuno-proteasome subunit, LMP7, was up-regulated in the clipped ischemic kidney that was atrophic, but not in the contralateral unclipped kidney that underwent compensatory hypertrophy. Immunohistochemical analysis revealed that LMP7 was highly expressed in cortical epithelial and endothelial cells of the ischemic kidney. Surprisingly, the second immuno-subunit, LMP2, was almost undetectable, indicating that renal ischemia may induce exclusively the LMP7 subunit. We also found that renal ischemia neither reduced the SDS-stimulated proteasomal activity nor affected a high level of the PA28 activator. Thus, the results provide evidence that LMP7 immuno-subunit is induced in renal cells exposed to a long-lasting renal ischemia and inflammation, and that there is a direct link between LMP induction and renal atrophy. This opens an opportunity to study a role for LMP-containing proteasomes in the kidneys and other organs undergoing reduction in mass in diseases accompanied by a long-lasting ischemia and inflammatory responses.     

New species of Neophyllobius and Tycherobius (Acari, Camerobiidae) from turkey

Two new species of Neophyllobius (Acari, Camerobiidae) viz. N. populus sp. n., N. karabagiensis sp. n., and a new species of Tycherobius (Acari, Camerobiidae), Tycherobius dazkiriensis sp. n., are described from Turkey and illustrated based on females and a protonymph. A key to the known species of Tycherobius is presented.     

First principles calculations of thermodynamics and kinetic parameters and molecular dynamics simulations of acetylcholinesterase reactivators: can mouse data provide new insights into humans?

We have applied a theoretical methodology, previously developed to evaluate the association and kinetic reactivation constants of oximes, comparing theoretical data obtained for human acetylcholinesterase (HsAChE) with in vitro results from Mus musculus AChE (MmAChE) previously reported in the literature. Our results, further checked by additional molecular dynamics simulations steps, showed a good correlation between the theoretical and experimental data, supporting the methodology as appropriate for prediction of thermodynamic and kinetic parameters and corroborated MmAChE as a suitable model for studies with HsAChE.     

Sequence, structure and functional diversity of PD-(D/E)XK phosphodiesterase superfamily

Proteins belonging to PD-(D/E)XK phosphodiesterases constitute a functionally diverse superfamily with representatives involved in replication, restriction, DNA repair and tRNA–intron splicing. Their malfunction in humans triggers severe diseases, such as Fanconi anemia and Xeroderma pigmentosum. To date there have been several attempts to identify and classify new PD-(D/E)KK phosphodiesterases using remote homology detection methods. Such efforts are complicated, because the superfamily exhibits extreme sequence and structural divergence. Using advanced homology detection methods supported with superfamily-wide domain architecture and horizontal gene transfer analyses, we provide a comprehensive reclassification of proteins containing a PD-(D/E)XK domain. The PD-(D/E)XK phosphodiesterases span over 21 900 proteins, which can be classified into 121 groups of various families. Eleven of them, including DUF4420, DUF3883, DUF4263, COG5482, COG1395, Tsp45I, HaeII, Eco47II, ScaI, HpaII and Replic_Relax, are newly assigned to the PD-(D/E)XK superfamily. Some groups of PD-(D/E)XK proteins are present in all domains of life, whereas others occur within small numbers of organisms. We observed multiple horizontal gene transfers even between human pathogenic bacteria or from Prokaryota to Eukaryota. Uncommon domain arrangements greatly elaborate the PD-(D/E)XK world. These include domain architectures suggesting regulatory roles in Eukaryotes, like stress sensing and cell-cycle regulation. Our results may inspire further experimental studies aimed at identification of exact biological functions, specific substrates and molecular mechanisms of reactions performed by these highly diverse proteins.     

Metabolic Modeling of Dynamic Brain 13C NMR Multiplet Data: Concepts and Simulations with a Two-Compartment Neuronal-Glial Model

Metabolic modeling of dynamic ¹³C labeling curves during infusion of ¹³C-labeled substrates allows quantitative measurements of metabolic rates in vivo. However metabolic modeling studies performed in the brain to date have only modeled time courses of total isotopic enrichment at individual carbon positions (positional enrichments), not taking advantage of the additional dynamic ¹³C isotopomer information available from fine-structure multiplets in ¹³C spectra. Here we introduce a new ¹³C metabolic modeling approach using the concept of bonded cumulative isotopomers, or bonded cumomers. The direct relationship between bonded cumomers and ¹³C multiplets enables fitting of the dynamic multiplet data. The potential of this new approach is demonstrated using Monte-Carlo simulations with a brain two-compartment neuronal-glial model. The precision of positional and cumomer approaches are compared for two different metabolic models (with and without glutamine dilution) and for different infusion protocols ([1,6-¹³C₂]glucose, [1,2-¹³C₂]acetate, and double infusion [1,6-¹³C₂]glucose?+?[1,2-¹³C₂]acetate). In all cases, the bonded cumomer approach gives better precision than the positional approach. In addition, of the three different infusion protocols considered here, the double infusion protocol combined with dynamic bonded cumomer modeling appears the most robust for precise determination of all fluxes in the model. The concepts and simulations introduced in the present study set the foundation for taking full advantage of the available dynamic ¹³C multiplet data in metabolic modeling.     

Differential assembly of polypeptides of the light-harvesting 2 complex encoded by distinct operons during acclimation of Rhodobacter sphaeroides to low light intensity

In order to obtain an improved understanding of the assembly of the bacterial photosynthetic apparatus, we have conducted a proteomic analysis of pigment-protein complexes isolated from the purple bacterium Rhodobacter sphaeroides undergoing acclimation to reduced incident light intensity. Photoheterotrophically growing cells were shifted from 1,100 to 100?W/m(2) and intracytoplasmic membrane (ICM) vesicles isolated over 24-h were subjected to clear native polyacrylamide gel electrophoresis. Bands containing the LH2 and reaction center (RC)-LH1 complexes were excised and subjected to in-gel trypsin digestion followed by liquid chromatography (LC)-mass spectroscopy (MS)/MS. The results revealed that the LH2 band contained distinct levels of the LH2-α and -β polypeptides encoded by the two puc operons. Polypeptide subunits encoded by the puc2AB operon predominated under high light and in the early stages of acclimation to low light, while after 24?h, the puc1BAC components were most abundant. Surprisingly, the Puc2A polypeptide containing a 251 residue C-terminal extension not present in Puc1A, was a protein of major abundance. A predominance of Puc2A components in the LH2 complex formed at high light intensity is followed by a >2.5-fold enrichment in Puc1B levels between 3 and 24?h of acclimation, accompanied by a nearly twofold decrease in Puc2A levels. This indicates that the puc1BAC operon is under more stringent light control, thought to reflect differences in the puc1 upstream regulatory region. In contrast, elevated levels of Puc2 polypeptides were seen 48?h after the gratuitous induction of ICM formation at low aeration in the dark, while after 24?h of acclimation to low light, an absence of alterations in Puc polypeptide distributions was observed in the upper LH2-enriched gel band, despite an approximate twofold increase in overall LH2 levels. This is consistent with the origin of this band from a pool of LH2 laid down early in development that is distinct from subsequently assembled LH2-only domains, forming the LH2 gel band.     

Inhibition of Quorum Sensing Mediated Virulence Factors Production in Urinary Pathogen Serratia marcescens PS1 by Marine Sponges

The focal intent of this study was to find out an alternative strategy for the antibiotic usage against bacterial infections. The quorum sensing inhibitory (QSI) activity of marine sponges collected from Palk Bay, India was evaluated against acyl homoserine lactone (AHL) mediated violacein production in Chromobacterium violaceum (ATCC 12472), CV026 and virulence gene expressions in clinical isolate Serratia marcescens PS1. Out of 29 marine sponges tested, the methanol extracts of Aphrocallistes bocagei (TS 8), Haliclona (Gellius) megastoma (TS 25) and Clathria atrasanguinea (TS 27) inhibited the AHL mediated violacein production in C. violaceum (ATCC 12472) and CV026. Further, these sponge extracts inhibited the AHL dependent prodigiosin pigment, virulence enzymes such as protease, hemolysin production and biofilm formation in S. marcescens PS1. However, these sponge extracts were not inhibitory to bacterial growth, which reveals the fact that the QSI activity of these extracts was not related to static or killing effects on bacteria. Based on the obtained results, it is envisaged that the marine sponges could pave the way to prevent quorum sensing (QS) mediated bacterial infections.     

Stochastic frontier model approach for measuring stock market efficiency with different distributions

The stock market is considered essential for economic growth and expected to contribute to improved productivity. An efficient pricing mechanism of the stock market can be a driving force for channeling savings into profitable investments and thus facilitating optimal allocation of capital. This study investigated the technical efficiency of selected groups of companies of Bangladesh Stock Market that is the Dhaka Stock Exchange (DSE) market, using the stochastic frontier production function approach. For this, the authors considered the Cobb-Douglas Stochastic frontier in which the technical inefficiency effects are defined by a model with two distributional assumptions. Truncated normal and half-normal distributions were used in the model and both time-variant and time-invariant inefficiency effects were estimated. The results reveal that technical efficiency decreased gradually over the reference period and that truncated normal distribution is preferable to half-normal distribution for technical inefficiency effects. The value of technical efficiency was high for the investment group and low for the bank group, as compared with other groups in the DSE market for both distributions in time-varying environment whereas it was high for the investment group but low for the ceramic group as compared with other groups in the DSE market for both distributions in time-invariant situation.