Altered subunit communication in subfamilies of trimeric dUTPases

The enzyme dUTPase is essential in preventing uracil incorporation into DNA. Design of antagonists against this novel chemotherapeutic target requires identification of species-specific differences in the structure and mechanism of the enzyme. This task is now approached via comparisons of available crystallographic structures of dUTPases from Homo sapiens, Escherichia coli, and retroviruses. The eukaryotic protein uniquely displays polar and charged amino acid residues participating in threefold intersubunit interactions. In bacterial and retroviral dUTPases, threefold interactions are mainly hydrophobic. The residues responsible for this contrast are mapped in multiple sequence alignment to positions differently and characteristically conserved in distinct evolutionary branches. The general feature of this contrast is further strengthened by a second eukaryotic model structure constructed using comparative modeling. The dUTPase cDNA from Drosophila melanogaster was identified, sequenced, and the model structure of the encoded polypeptide displayed a polar hydrogen-bonding network of threefold interactions, identically to the human structure. Results allow clear distinction between two subfamilies of trimeric dUTPases where altered subunit communication may account for a functional difference in the catalytic cycle.     

Kinetic properties and specificity of trimeric Plasmodium falciparum and human dUTPases

Deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase, EC 3.6.1.23) catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate, and plays important roles in nucleotide metabolism and DNA replication. Hydrolysis of other nucleotides similar in structure to dUTP would be physiologically negative and therefore high substrate specificity is essential. Binding and hydrolysis of nucleotides different to dUTP by the dUTPases from Plasmodium falciparum (PfdUTPase) and human (hdUTPase) was evaluated by applying isothermal titration calorimetry (ITC). The ribo and deoxyribonucleoside triphosphates dGTP, dATP, dCTP, dTTP, UTP, FdUTP and IdUTP have been analysed. dUTP and FdUTP were the most specific substrates for both enzymes. The specificity constants (k_cat/K_m) for the remaining ones, except for the IdUTP, were very similar for both enzymes, although PfdUTPase showed a slightly higher specificity for dCTP and UTP and the human enzyme for dTTP and dCTP. PfdUTPase was very efficient in using FdUTP as substrate indicating that small size substituents in the 5′ position are well tolerated. In addition product inhibition was assessed by binding studies with the nucleoside monophosphate derivatives and thermodynamic parameters were established. When FdUTP hydrolysis was monitored, Plasmodium dUTPase was more sensitive to end-product inhibition by FdUMP than the human enzyme. Taken together these results highlight further significant differences between the human and Plasmodium enzymes that may be exploitable in selective inhibitor design.     

Latent membrane protein 1 of Epstein-Barr virus mimics a constitutively active receptor molecule.

Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is an integral membrane protein which has transforming potential and is necessary but not sufficient for B-cell immortalization by EBV. LMP1 molecules aggregate in the plasma membrane and recruit tumour necrosis factor receptor (TNF-R) -associated factors (TRAFs) which are presumably involved in the signalling cascade leading to NF-kappaB activation by LMP1. Comparable activities are mediated by CD40 and other members of the TNF-R family, which implies that LMP1 could function as a receptor. LMP1 lacks extended extracellular domains similar to beta-adrenergic receptors but, in contrast, it also lacks any motifs involved in ligand binding. By using LMP1 mutants which can be oligomerized at will, we show that the function of LMP1 in 293 cells and B cells is solely dependent on oligomerization of its carboxy-terminus. Biochemically, oligomerization is an intrinsic property of the transmembrane domain of wild-type LMP1 and causes a constitutive phenotype which can be conferred to the signalling domains of CD40 or the TNF-2 receptor. In EBV, immortalized B cells cross-linking in conjunction with membrane targeting of the carboxy-terminal signalling domain of LMP1 is sufficient for its biological activities. Thus, LMP1 acts like a constitutively activated receptor whose biological activities are ligand-independent.     

Hexameric,trimeric, dimeric,and monomeric forms of inorganic pyrophosphatase from Escherichia coli

The conditions were found for obtaining trimeric, dimeric, and monomeric forms of the Escherichia coli inorganic pyrophosphatase from its native hexameric form. Interconversions of the oligomers were studied, and rate constants for their dissociation and association were determined. All forms were found to be catalytically active, with the activity decreasing in the order: hexamer-trimer-dimer-monomer. The activity of trimeric and dimeric forms was high enough to study and to compare their catalytic properties. The monomeric form of the enzyme was unstable.     

Evidence for the existence of trimeric and monomeric Photosystem I complexes in thylakoid membranes from cyanobacteria

In cyanobacteria, solubilization of thylakoid membranes by detergents yields both monomeric and trimeric Photosystem I (PS I) complexes in variable amounts. We present evidence for the existence of both monomeric and trimeric PS I in cyanobacterial thylakoid membranes with the oligomeric state depending in vitro on the ion concentration. At low salt concentrations (i.e.10 mM MgSO₄) PS I is mainly extracted as a trimer from these membranes and at high salt concentrations (i.e.150 mM MgSO₄) nearly exclusively as a monomer, irrespective of the type of salt used (i.e. mono- or bivalent ions) and the temperature (i.e. 4°C or 20°C). Once solubilized, the PS I trimer is stable over a wide range of ion concentrations (i.e. beyond 0.5 M). A model is presented which suggests a monomer-oligomer equilibrium of PS I, but also of PS II and the cyt. b6/f-complex in the cyanobacterial thylakoid membrane. The possible physiological role of this equilibrium in the regulation of state transitions is discussed.Abbreviations -DM dodecyl--D-maltoside - Chl chlorophyll - cyt. b6f cytochrome b6f complex - EM electron microscopy - HPLC high performance liquid chromatography - LDAO N, N-dimethyl-N-dodecyl amine oxide - MES 4-morpholino ethane sulfonic acid - PAGE polyacrylamide gel electrophoresis - PBS phycobilisome - PS photosystem - SDS sodium dodecyl sulfate - 2D two dimensional - 3D three dimensional     

病毒dUTPase研究进展

脱氧尿苷焦磷酸酶（dUTP pyrophosphatase,dUTPasc）广泛存在于真核、原核细胞和病毒等生物有机体中,通过催化水解脱氧尿苷三磷酸（dUTP）,减少尿嘧啶在DNA合成中的错误掺入,降低细胞中的dUTP／dTTP比例,保证DNA复制的正确性和顺利进行。病毒编码的dUTPasc具有种属特异性,且与病毒的毒力和高效复制密切相关。本文就dUTPase的生物学功能、分类特征、表达调控、分布定位及病毒dUTPase功能的研究进展进行了概述,为深入开展dUTPase功能研究提供理论基础。     

Identification and characterization of pseudorabies virus dUTPase.

Sequence analysis within the long segment of the pseudorabies virus (PrV) genome identified an open reading frame of 804 bp whose deduced protein product of 268 amino acids exhibited homology to dUTPases of other herpesviruses. The gene was designated UL50 because of its colinearity with the homologous gene of herpes simplex virus type 1. An antiserum raised against a bacterially expressed fragment of PrV UL50 specifically detected a 33-kDa protein in lysates of infected cells, which is in agreement with the predicted molecular mass of the PrV UL50 protein. A UL50-negative PrV mutant (PrV UL50-) was constructed by the insertion of a beta-galactosidase expression cassette into the UL50 coding sequence. A corresponding rescuant (PrV UL50resc) was also isolated. The interruption of the UL50 gene led to the disappearance of the 33-kDa protein, whereas restoration of UL50 gene expression restored detection of the 33-kDa protein. Enzyme activity assays confirmed that UL50 of PrV codes for a dUTPase which copurifies with nuclei of infected cells. PrV UL50- replicated with an only slightly reduced efficiency in epithelial cells in culture compared with that of its parental wild-type virus strain. Our results thus demonstrate that UL50 of PrV encodes a protein of 33 kDa with dUTPase activity which copurifies with nuclei of infected cells and is dispensable for replication in cultured epithelial cells.     

Characterization of the multiple conformational States of free monomeric and trimeric human immunodeficiency virus envelope glycoproteins after fixation by cross-linker

The human immunodeficiency virus type 1 (HIV-1) gp120 exterior and gp41 transmembrane envelope glycoproteins assemble into trimers on the virus surface that represent potential targets for antibodies. Potent neutralizing antibodies bind the monomeric gp120 glycoprotein with small changes in entropy, whereas unusually large decreases in entropy accompany gp120 binding by soluble CD4 and less potent neutralizing antibodies. The high degree of conformational flexibility in the free gp120 molecule implied by these observations has been suggested to contribute to masking the trimer from antibodies that recognize the gp120 receptor-binding regions. Here we use cross-linking and recognition by antibodies to investigate the conformational states of gp120 monomers and soluble and cell surface forms of the trimeric HIV-1 envelope glycoproteins. The fraction of monomeric and trimeric envelope glycoproteins able to be recognized after fixation was inversely related to the entropic changes associated with ligand binding. In addition, fixation apparently limited the access of antibodies to the V3 loop and gp41-interactive surface of gp120 only in the context of trimeric envelope glycoproteins. The results support a model in which the unliganded monomeric and trimeric HIV-1 envelope glycoproteins sample several different conformations. Depletion of particular fixed conformations by antibodies allowed characterization of the relationships among the conformational states. Potent neutralizing antibodies recognize the greatest number of conformations and therefore can bind the virion envelope glycoproteins more rapidly and completely than weakly neutralizing antibodies. Thus, the conformational flexibility of the HIV-1 envelope glycoproteins creates thermodynamic and kinetic barriers to neutralization by antibodies directed against the receptor-binding regions of gp120.     

The functional significance of the monomeric and trimeric states of the photosystem II light harvesting complexes

The main light harvesting complex of photosystem II in plants, LHCII, exists in a trimeric state. To understand the biological significance of trimerization, a comparison has been made been LHCII trimers and LHCII monomers prepared by treatment with phospholipase. The treatment used caused no loss of chlorophyll, but there was a difference in carotenoid composition, together with the previously observed alterations in absorption spectrum. It was found that, when compared to monomers, LHCII trimers showed increased thermal stability and a reduced structural flexibility as determined by the decreased rate and amplitude of fluorescence quenching in low-detergent concentration. It is suggested that LHCII should be considered as having two interacting domains: the lutein 1 domain, the site of fluorescence quenching [Wentworth et al. (2003) J. Biol. Chem. 278, 21845-21850], and the lutein 2 domain. The lutein 2 domain faces the interior of the trimer, the differences in absorption spectrum and carotenoid binding in trimers compared to monomers indicating that the trimeric state modulates the conformation of this domain. It is suggested that the lutein 2 domain controls the conformation of the lutein 1 domain, thereby providing allosteric control of fluorescence quenching in LHCII. Thus, the pigment configuration and protein conformation in trimers is adapted for efficient light harvesting and enhanced protein stability. Furthermore, trimers exhibit the optimum level of control of energy dissipation by modulating the development of the quenched state of the complex.     

Characterization of antibody responses elicited by human immunodeficiency virus type 1 primary isolate trimeric and monomeric envelope glycoproteins in selected adjuvants

We previously reported that soluble, stable YU2 gp140 trimeric human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein immunogens could elicit improved breadth of neutralization against HIV-1 isolates compared to monomeric YU2 gp120 proteins. In this guinea pig immunization study, we sought to extend these data and determine if adjuvant could quantitatively or qualitatively alter the neutralizing response elicited by trimeric or monomeric immunogens. Consistent with our earlier studies, the YU2 gp140 immunogens elicited higher-titer neutralizing antibodies against homologous and heterologous isolates than those elicited by monomeric YU2 gp120. Additionally, the GlaxoSmithKline family of adjuvants AS01B, AS02A, and AS03 induced higher levels of neutralizing antibodies compared to emulsification of the same immunogens in Ribi adjuvant. Further analysis of vaccine sera indicated that homologous virus neutralization was not mediated by antibodies to the V3 loop, although V3 loop-directed neutralization could be detected for some heterologous isolates. In most gp120-inoculated animals, the homologous YU2 neutralization activity was inhibited by a peptide derived from the YU2 V1 loop, whereas the neutralizing activity elicited by YU2 gp140 trimers was much less sensitive to V1 peptide inhibition. Consistent with a less V1-focused antibody response, sera from the gp140-immunized animals more efficiently neutralized heterologous HIV-1 isolates, as determined by two distinct neutralization formats. Thus, there appear to be qualitative differences in the neutralizing antibody response elicited by YU2 gp140 compared to YU2 monomeric gp120. Further mapping analysis of more conserved regions of gp120/gp41 may be required to determine the neutralizing specificity elicited by the trimeric immunogens.     

Investigation of the structure of trimeric and monomeric photosystem I reaction centre complexes

Electron microscopy of monomeric and trimeric forms of the reaction centre of photosystem I from the thermophilic cyanobacterium Phormidium laminosum has allowed the construction of a three-dimensional model describing the shape of the complex. The trimeric form of the Photosystem I reaction centre complex was found to have a very regular shape corresponding to a rounded equilateral triangle with edges ˜18 nm long and a thickness of ˜6 nm. A distinctive chiral arrangement of the three reaction centres in the trimer could be observed on one face of the complex, whereas the opposing face appeared to be smooth with no distinctive internal features. The monomeric reaction centre is roughly pearshaped, with a length of ˜15 nm and a width of ˜9 nm. A thickness of 6 nm is assumed from comparison with the trimer. It is predicted to lie with its shortest axis spanning the membrane. A double-lobed structure, with one lobe larger than the other, was occasionally observed for the monomeric reaction centre. No experimental evidence could be obtained for the existence of the trimeric form in the membrane. The formation of the trimeric form after detergent extraction is suggested. The trimeric form was found to be more stable than the monomeric form in solutions containing anionic and non-ionic detergents.     

The immune response to Epstein-Barr virus

Epstein-Barr virus is a gammaherpes virus that establishes persistent infection in the majority of humans. Despite the potent oncogenic potential of the virus it is relatively rarely associated with malignant disease. This review focuses on the cellular immune responses that successfully control Epstein-Barr virus infection in most individuals.     

The immunology of Epstein-Barr virus infection

Epstein-Barr virus is a classic example of a persistent human virus that has caught the imagination of immunologists, virologists and oncologists because of the juxtaposition of a number of important properties. First, the ability of the virus to immortalize B lymphocytes in vitro has provided an antigen presenting cell in which all the latent antigens of the virus are displayed and are available for systematic study. Second, the virus presents an ideal system for studying the immune parameters that maintain latency and the consequences of disturbing this cell-virus relationship. Third, this wealth of immunological background has provided a platform for elucidating the role of the immune system in protection from viral-associated malignancies of B cell and epithelial cell origin. Finally, attention is now being directed towards the development of vaccine formulations which might have broad application in the control of human malignancies.     

Epstein-Barr virus serology

Because of the ubiquitous nature of EBV, most people are infected with this virus by the time they are adults. People acquire the virus at an early age, earlier in developing countries and in socioeconomically deprived areas of the United States, where about 80% of 5-year-old children are seropositive. In economically privileged areas, only about 40–50% of children are seropositive by age 5. Infections during childhood are usually asymptomatic. In contrast, 50% of adolescents who become infected with EBV develop the fatigue, fever, pharyngitis, and atypical lymphocytosis characteristic of acute infectious mononucleosis (IM). Heterophil antibodies, which are the basis for screening tests for IM, usually appear in the serum of these patients. However, approximately 10% of patients (more commonly children) with EBV induced IM do not develop heterophil antibodies. For this reason, tests for specific antibody-mediated immune responses to EBV may be necessary for diagnosis.     

Epstein-Barr virus recombinants from overlapping cosmid fragments.

Five overlapping type 1 Epstein-Barr virus (EBV) DNA fragments constituting a complete replication- and transformation-competent genome were cloned into cosmids and transfected together into P3HR-1 cells, along with a plasmid encoding the Z immediate-early activator of EBV replication. P3HR-1 cells harbor a type 2 EBV which is unable to transform primary B lymphocytes because of a deletion of DNA encoding EBNA LP and EBNA 2, but the P3HR-1 EBV can provide replication functions in trans and can recombine with the transfected cosmids. EBV recombinants which have the type 1 EBNA LP and 2 genes from the transfected EcoRI-A cosmid DNA were selectively and clonally recovered by exploiting the unique ability of the recombinants to transform primary B lymphocytes into lymphoblastoid cell lines. PCR and immunoblot analyses for seven distinguishing markers of the type 1 transfected DNAs identified cell lines infected with EBV recombinants which had incorporated EBV DNA fragments beyond the transformation marker-rescuing EcoRI-A fragment. Approximately 10% of the transforming virus recombinants had markers mapping at 7, 46 to 52, 93 to 100, 108 to 110, 122, and 152 kbp from the 172-kbp transfected genome. These recombinants probably result from recombination among the transfected cosmid-cloned EBV DNA fragments. The one recombinant virus examined in detail by Southern blot analysis has all the polymorphisms characteristic of the transfected type 1 cosmid DNA and none characteristic of the type 2 P3HR-1 EBV DNA. This recombinant was wild type in primary B-lymphocyte infection, growth transformation, and lytic replication. Overall, the type 1 EBNA 3A gene was incorporated into 26% of the transformation marker-rescued recombinants, a frequency which was considerably higher than that observed in previous experiments with two-cosmid EBV DNA cotransfections into P3HR-1 cells (B. Tomkinson and E. Kieff, J. Virol. 66:780-789, 1992). Of the recombinants which had incorporated the marker-rescuing cosmid DNA fragment and the fragment encoding the type 1 EBNA 3A gene, most had incorporated markers from at least two other transfected cosmid DNA fragments, indicating a propensity for multiple homologous recombinations. The frequency of incorporation of the nonselected transfected type 1 EBNA 3C gene, which is near the end of two of the transfected cosmids, was 26% overall, versus 3% in previous experiments using transfections with two EBV DNA cosmids. In contrast, the frequency of incorporation of a 12-kb EBV DNA deletion which was near the end of two of the transfected cosmids was only 13%.(ABSTRACT TRUNCATED AT 400 WORDS)