Reet Kurg

Direct evidence of the cyclooxygenase pathway of prostaglandin synthesis in arthropods: genetic and biochemical characterization of two crustacean cyclooxygenases.

Prostaglandins, well-known lipid mediators in vertebrate animals, have also shown to play certain regulatory roles in insects and other arthropods acting on reproduction, immune system and ion transport. However, knowledge of their biosynthetic pathways in arthropods is lacking. In the present study, we report the cloning and expression of cyclooxygenase (COX) from amphipod crustaceans Gammarus spp and Caprella spp. The amphipod COX proteins contain key residues shown to be important for cyclooxygenase and peroxidase activities. Differently from all other known cyclooxygenases the N-terminal signal sequence of amphipod enzymes is not cleaved during protein expression in mammalian cells. The C-terminus of amphipod COX is shorter than that of mammalian isoforms and lacks the KDEL(STEL)-type endoplasmic reticulum retention/retrieval signal. Despite that, amphipod COX proteins are N-glycosylated and locate similarly to the vertebrate COX on the endoplasmic reticulum and nuclear envelope. Both amphipod COX mRNAs encode functional cyclooxygenases that catalyze the transformation of arachidonic acid into prostaglandins. Using bioinformatic analysis we identified a COX-like gene from the human body louse Pediculus humanus corporis genome that encodes a protein with about 30% sequence identity with human COX-1 and COX-2. Although the COX gene is known to be absent from genomes of Drosophila sp., Aedes aegypti, Bombyx mori, and other insects, our studies establish the existence of the COX gene in certain lineages within the insect world.

Characterization of the Functional Activities of the Bovine Papillomavirus Type 1 E2 Protein Single-Chain Heterodimers

ABSTRACT Papillomaviruses are small DNA viruses which establish persistent infection in the epithelial tissue of various animal species. Three papillomavirus proteins encoded by the bovine papillomavirus type 1 E2 open reading frame have a common C-terminal DNA binding and dimerization domain and function as dimeric proteins in the regulation of viral gene expression, genome replication, and maintenance. The full-length E2 protein, expressed usually at the lowest level of the three, is an activator, while shorter forms of E2, lacking the transactivation domain, serve as repressors of replication and transcription. In virally infected cells, the full-length E2 protein forms heterodimers with repressor forms of the E2 protein and the biological activities of such heterodimers are poorly known. In order to study the functionality of E2 heterodimers, we joined the full-length E2 protein and E2 repressor by a flexible polypeptide hinge so that they formed a single-chain intramolecular dimer. The single-chain E2 heterodimers folded correctly to form genuine pseudodimers capable of binding to the specific E2 protein binding site with high affinity. Characterization of the activities of this protein in transcription showed that it functions as an effective transcriptional activator, which is comparable to what was found for the full-length E2 protein. The single-chain heterodimer is dependent to some extent on Brd4 protein and is able to support papillomavirus origin replication; however, it does not support the partitioning of the multimeric E2 binding site containing plasmids in dividing cells. Our results suggest that E2 heterodimers serve as activators of transcription and replication during the viral life cycle.

Human papillomavirus E2 protein with single activation domain initiates HPV18 genome replication, but is not sufficient for long-term maintenance of virus genome

The papillomavirus life cycle is regulated by a family of proteins encoded by the E2 open reading frame; E2 proteins regulate viral gene expression, DNA replication and genome maintenance. We have previously shown that the bovine papillomavirus (BPV1) full-length E2 protein forms heterodimers with repressor forms of E2, and these E2 heterodimers serve as activators of transcription and replication during the viral life cycle. In the present study, using the single-chain E2 heterodimer as a model, we show that human papillomavirus (HPV) 11 and 18 E2 heterodimers with single activation domain are able to initiate replication of URR-containing plasmid in transient assay. Single-chain E2 heterodimer in the context of HPV18 genome initiates genome replication, but is not sufficient for long-term replication of HPV18 genome. We also show that HPV18 genome has a capacity to encode truncated E2 repressor E8/E2 which acts as a negative regulator of HPV18 genome replication.

Association of Bovine Papillomavirus E2 Protein with Nuclear Structures In Vivo

ABSTRACT Papillomaviruses are small DNA viruses which have the capacity to establish a persistent infection in mammalian epithelial cells. The papillomavirus E2 protein is a central coordinator of viral gene expression, genome replication, and maintenance. We have investigated the distribution of bovine papillomavirus E2 protein in nuclei of proliferating cells and found that E2 is associated with cellular chromatin. This distribution does not change during the entire cell cycle. The N-terminal transactivation domain, but not the C-terminal DNA-binding domain, of the E2 protein is responsible for this association. The majority of the full-length E2 protein can only be detected in chromatin-enriched fractions but not as a free protein in the nucleus. Limited micrococcal nuclease digestion revealed that the E2 protein partitioned to different chromatin regions. A fraction of the E2 protein was located at nuclear sites that are resistant against nuclease attack, whereas the remaining E2 resided on compact chromatin accessible to micrococcal nuclease. These data suggest that there are two pools of E2 in the cell nucleus: one that localizes on transcriptionally inactive compact chromatin and the other, which compartmentalizes to transcriptionally active nuclear structures of the cell. Our data also suggest that E2 associates with chromatin through cellular protein(s), which in turn is released from chromatin at 0.4 M salt.

Identification and analysis of papillomavirus E2 protein binding sites in the human genome

ABSTRACT Papillomavirus E2 protein is required for the replication and maintenance of viral genomes and transcriptional regulation of viral genes. E2 functions through sequence-specific binding to 12-bp DNA motifs—E2 binding sites (E2BS)—in the virus genome. Papillomaviruses are able to establish persistent infection in their host and have developed a long-term relationship with the host cell in order to guarantee the propagation of the virus. In this study, we have analyzed the occurrence and functionality of E2BSs in the human genome. Our computational analysis indicates that most E2BSs in the human genome are found in repetitive DNA regions and have G/C-rich spacer sequences. Using a chromatin immunoprecipitation approach, we show that human papillomavirus type 11 (HPV11) E2 interacts with a subset of cellular E2BSs located in active chromatin regions. Two E2 activities, sequence-specific DNA binding and interaction with cellular Brd4 protein, are important for E2 binding to consensus sites. E2 binding to cellular E2BSs has a moderate or no effect on cellular transcription. We suggest that the preference of HPV E2 proteins for E2BSs with A/T-rich spacers, which are present in the viral genomes and underrepresented in the human genome, ensures E2 binding to specific binding sites in the virus genome and may help to prevent extensive and possibly detrimental changes in cellular transcription in response to the viral protein.

The Human WBSCR22 Protein Is Involved in the Biogenesis of the 40S Ribosomal Subunits in Mammalian Cells

The human WBSCR22 protein was previously shown to be up-regulated in invasive breast cancer and its ectopic expression enhances tumor cell survival in the vasculature. In the current study, we show that the WBSCR22 protein is important for cell growth. Knock-down of WBSCR22 with siRNA results in slower growth of WBSCR22-depleted cells. Treatment with siWBSCR22 causes defects in the processing of pre-rRNAs and reduces the level of free 40S ribosomal subunit, suggesting that WBSCR22 is involved in ribosome small subunit biosynthesis. The human WBSCR22 partially complements the growth of WBSCR22 yeast homologue, bud23 deletion mutant suggesting that the human WBSCR22 is a functional homologue of yeast Bud23. WBSCR22 is localized throughout the cell nucleus and is not stably associated with ribosomal subunits within the cell nucleus. We also show that the WBSCR22 protein level is decreased in lymphoblastoid cell lines derived from William-Beuren Syndrome (WBS) patients compared to healthy controls. Our data suggest that the WBSCR22 protein is a ribosome biogenesis factor involved in the biosynthesis of 40S ribosomal particles in mammalian cells.

Coinfection of Chlamydia trachomatis, Ureaplasma urealyticum and human papillomavirus among patients attending STD clinics in Estonia

Women visiting Estonian STD clinics were subjected to PCR assay for human papillomavirus (HPV), Chlamydia trachomatis and Ureaplasma urealyticum biovar 2. The overall prevalence of coinfection was 8%. The chlamydial infection was found to be associated with HPV, especially with high-risk HPV (OR =2.5, p<0.005) and most significantly in women over 41 y of age. C. trachomatis infection also occurred more frequently in U. urealyticum-infected than in U. urealyticum-free patients (OR =2.6, p=0.02). U. urealyticum infection did not associate with HPV status. The clinical significance of the association between C. trachomatis and U. urelyticum infection remains to be elucidated.

Activation of 11R-Lipoxygenase Is Fully Ca2+-Dependent and Controlled by the Phospholipid Composition of the Target Membrane

Activation of some lipoxygenases (LOX) is found to be related to the selective membrane binding upon cell stimulation. In this study, a systematic analysis of the effect of the lipid composition on the membrane binding efficiency, Ca(2+) affinity, and enzymatic activity of 11R-LOX was performed. The analysis of the membrane targeting by fluorometric and surface plasmon resonance measurements in the absence of Ca(2+) showed an exclusive binding of 11R-LOX to the anionic phospholipids (phosphatidylinositol < phosphatidylglycerol ≈ phosphatidylserine) containing model membranes. The presence of Ca(2+) enhanced the rate of interaction and influenced its mode. The modulation of the activity of 11R-LOX indicated that (i) Ca(2+) binding is a prerequisite for productive membrane association, (ii) the reaction of 11R-LOX with arachidonic acid coincided with and was driven by its Ca(2+)-mediated membrane association, and (iii) phosphatidylethanolamine and anionic phospholipids had a synergistic effect on the Ca(2+) affinity, in line with a target-activated messenger affinity mechanism [Corbin, J. A., et al. (2007) Biochemistry 46, 4322-4336]. According to the mechanism proposed in this report, 11R-LOX can bind to the membranes in two different modes and the efficiency of productive membrane binding is determined by a concerted association of Ca(2+) and lipid headgroups.

Bovine papillomavirus type 1 E2 protein heterodimer is functional in papillomavirus DNA replication in vivo

Papillomaviruses are small DNA viruses that induce epithelial lesions in their host. The viral life cycle is regulated by the family of proteins encoded by the E2 open reading frame. In addition to the full-length E2 protein, the BPV-1 genome encodes two truncated E2 proteins, E2C and E8/E2, which maintain the DNA-binding-dimerization domains, but lack the activation domain. Heterodimers formed between the full-length E2 and truncated E2 proteins serve as activators of E2-dependent transcription and papillomavirus DNA replication. We show that the single activation domain of E2 is sufficient for interaction with viral helicase E1 and for initiation of DNA replication from different papillomavirus origins. Single-chain E2 heterodimer is able to activate papillomavirus DNA replication in the context of entire BPV genome in the absence of other E2 proteins. These data suggest that E2 heterodimers with single activation domain are functional in initiation of papillomavirus replication in vivo.

Roles of the hinge region and the DNA binding domain of the bovine papillomavirus type 1 E2 protein in initiation of DNA replication

The bovine papillomavirus (BPV-1) E2 protein is the regulator of extrachromosomal replication of papillomaviruses. The mutants with C-terminal truncations and in-frame internal deletions were constructed to study the role of structural domains of E2 in the initiation of DNA replication. We show that the replication initiation function of E2 is absolutely dependent on the ability of the protein to bind to DNA. Our study also confirms the borders of the functional domains of the E2 protein; residues 1-192 form the activation domain and residues 310-410 the DNA binding-dimerization domain. Some critical length and flexibility, but not the particular amino acid sequence between these two functional domains is required for the activity of the protein to support replication. The hinge region, including the major phosphorylation sites of E2, is also dispensable for the mediation of attachment of the BPV1 genome to the mitotic chromosomes.