Whole-Genome Characterization and Genotyping of Global WU Polyomavirus Strains

Exploration of the genetic diversity of WU polyomavirus (WUV) has been limited in terms of the specimen numbers and particularly the sizes of the genomic fragments analyzed. Using whole-genome sequencing of 48 WUV strains collected in four continents over a 5-year period and 16 publicly available whole-genome sequences, we identified three main WUV clades and five subtypes, provisionally termed Ia, Ib, Ic, II, IIIa, and IIIb. Overall nucleotide variation was low (0 to 1.2%). The discriminatory power of the previous VP2 fragment typing method was found to be limited, and a new, larger genotyping region within the VP2/1 interface was proposed.In 2007, two new human polyomaviruses isolated from respiratory samples of pediatric patients suffering from respiratory disease were discovered, with one being KI polyomavirus (KIV) (2) and the other being WU polyomavirus (WUV) (8).WU polyomavirus shares most of the genomic characteristics of other polyomaviruses, with a noncoding control region (NCCR) separating the early and late coding regions on opposite strands. However, unlike for JCV and BKV, but similar to what was observed for KIV, a late-region-residing agnoprotein gene has not been identified in WUV (8).Despite being frequently detected in respiratory samples of ill patients, no distinct disease associations have so far been conclusively identified for WUV (1, 2, 4, 8, 10, 27). There have been some suggestions that sequence variation plays a role in disease severity and pathogenesis in other polyomaviruses (6, 24). Unfortunately, due to the early nature of research into WUV, there has been a dearth of available complete genomic sequences.In this study, we set out to investigate a large sample set of whole WUV genomes from diverse geographical, temporal, and clinical origins. Incorporating existing WUV genomes with this data set allowed us to investigate global WUV genomic diversity, to characterize the WUV genome, and to propose a new robust typing scheme.     

Only one independent genetic association with rheumatoid arthritis within the KIAA1109-TENR-IL2-IL21 locus in Caucasian sample sets: confirmation of association of rs6822844 with rheumatoid arthritis at a genome-wide level of significance

Introduction

To investigate whether accelerated hand bone mineral density (BMD) loss is associated with progressive joint damage in hands and feet in the first year of rheumatoid arthritis (RA) and whether it is an independent predictor of subsequent progressive total joint damage after 4 years.

Methods

In 256 recent-onset RA patients, baseline and 1-year hand BMD was measured in metacarpals 2-4 by digital X-ray radiogrammetry. Joint damage in hands and feet were scored in random order according to the Sharp-van der Heijde method at baseline and yearly up to 4 years.

Results

68% of the patients had accelerated hand BMD loss (>-0.003 g/cm²) in the first year of RA. Hand BMD loss was associated with progressive joint damage after 1 year both in hands and feet with odds ratios (OR) (95% confidence intervals [CI]) of 5.3 (1.3-20.9) and 3.1 (1.0-9.7). In univariate analysis, hand BMD loss in the first year was a predictor of subsequent progressive total joint damage after 4 years with an OR (95% CI) of 3.1 (1.3-7.6). Multivariate analysis showed that only progressive joint damage in the first year and anti-citrullinated protein antibody positivity were independent predictors of long-term progressive joint damage.

Conclusions

In the first year of RA, accelerated hand BMD loss is associated with progressive joint damage in both hands and feet. Hand BMD loss in the first year of recent-onset RA predicts subsequent progressive total joint damage, however not independent of progressive joint damage in the first year.     

Modulatory and catalytic modes of ATP binding by the calcium pump

We present crystal structures of the calcium-free E2 state of the sarcoplasmic reticulum Ca2+ -ATPase, stabilized by the inhibitor thapsigargin and the ATP analog AMPPCP. The structures allow us to describe the ATP binding site in a modulatory mode uncoupled from the Asp351 phosphorylation site. The Glu439 side chain interacts with AMPPCP via an Mg2+ ion in accordance with previous Fe2+ -cleavage studies implicating this residue in the ATPase cycle and in magnesium binding. Functional data on Ca2+ mediated activation indicate that the crystallized state represents an initial stage of ATP modulated deprotonation of E2, preceding the binding of Ca2+ ions in the membrane from the cytoplasmic side. We propose a mechanism of Ca2+ activation of phosphorylation leading directly from the compact E2-ATP form to the Ca2E1-ATP state. In addition, a role of Glu439 in ATP modulation of other steps of the functional cycle is suggested.     

Community-acquired MRSA and pig-farming

Background

Female genital tuberculosis is an uncommon disease that is rarely diagnosed in developed countries.

Case presentation

A 61-year-old postmenopausal woman who had undergone surgery and treated with adjuvant chemotherapy for infiltrating ductal carcinoma of the breast five years ago, presented with bloody vaginal discharge, fatigue, weight loss, and low grade fevers at night for two months. Histological examination of the endometrium, done based on the suspicion of a second primary cancer due to the tamoxifen therapy, revealed a granulomatous reaction. Liquid and solid mycobacterial cultures of the tissues were performed. Although the acid fast staining was negative, the liquid culture was positive for Mycobacterium tuberculosis. Involvement of other systems was not detected. The patient was treated with a three-drug antituberculosis regimen for 9 months and recovered fully.

Conclusion

Female genital tuberculosis is a rare but curable disease that should be included in the differential diagnosis of women with menstrual problems. Early diagnosis is important and may prevent unnecessary invasive procedures for the patient.     

Natural products as starting materials for development of second-generation SERCA inhibitors targeted towards prostate cancer cells

An analysis of the binding of the 8-O-N-tert-butoxycarbonyl-12-aminododecanoyl derivative of 8-O-debutanoylthapsigargin to the target molecule, the SERCA pump, has revealed the importance of the length and flexibility of the side chain attached to O-8. Based on the analysis a series of analogues to the 2-unsubstituted analogue trilobolide has been constructed and shown to be equipotent with thapsigargin as SERCA inhibitors. Only the 12-Boc-aminododecaonoyl derivative, however, was found to be apoptotic.     

Cyclopiazonic Acid Is Complexed to a Divalent Metal Ion When Bound to the Sarcoplasmic Reticulum Ca2+-ATPase

We have determined the structure of the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) in an E2·P_i-like form stabilized as a complex with , an ATP analog, adenosine 5′-(β,γ-methylene)triphosphate (AMPPCP), and cyclopiazonic acid (CPA). The structure determined at 2.5Å resolution leads to a significantly revised model of CPA binding when compared with earlier reports. It shows that a divalent metal ion is required for CPA binding through coordination of the tetramic acid moiety at a characteristic kink of the M1 helix found in all P-type ATPase structures, which is expected to be part of the cytoplasmic cation access pathway. Our model is consistent with the biochemical data on CPA function and provides new measures in structure-based drug design targeting Ca²⁺-ATPases, e.g. from pathogens. We also present an extended structural basis of ATP modulation pinpointing key residues at or near the ATP binding site. A structural comparison to the Na⁺,K⁺-ATPase reveals that the Phe⁹³ side chain occupies the equivalent binding pocket of the CPA site in SERCA, suggesting an important role of this residue in stabilization of the potassium-occluded E2 state of Na⁺,K⁺-ATPase.The Ca²⁺-ATPase from sarco(endo)plasmic reticulum of rabbit skeletal muscle (SERCA,⁵ isoform 1a) is a thoroughly studied member of the P-type ATPase family (1). SERCA possesses 10 transmembrane helices (M1 through M10) with both the N terminus and the C terminus facing the cytoplasmic side and three cytoplasmic domains, inserted in loops between M2 and M3 (A-domain) and between M4 and M5 (P- and N-domain) (2). The enzyme mediates the uptake of Ca²⁺ ions into the lumen of the sarcoplasmic reticulum (SR) after their release into the cytoplasm through calcium release channels during muscle contraction (3). SERCA, plasma membrane Ca²⁺-ATPase, and a third, Golgi-located secretory pathway Ca²⁺-ATPase are important factors in calcium and manganese homeostasis, transport, signaling, and regulation (4, 5).Crystal structures of all major states in the reaction cycle of SERCA have been determined. These include the Ca₂E₁·ATP state (6, 7) with high affinity Ca²⁺ binding sites accessible from the cytoplasmic side of the SR membrane, the calcium-occluded transition state (6), the open E2P state with luminal facing ion binding sites that have low affinity for Ca²⁺ and high affinity for protons (8) and the proton-occluded H_2–3E2[ATP] state with a bound modulatory ATP (9). This considerable amount of structural information has turned the Ca²⁺-ATPase into a valuable model system for studies on structural rearrangements that take place during the catalytic cycle of P-type ATPases. SERCA is considered a promising drug target in medical research, with a particular focus on prostate cancer and infectious diseases. Several compounds have already been shown to bind and inhibit SERCA by stabilizing the enzyme in a particular conformational state. Thapsigargin (TG), cyclopiazonic acid (CPA), and 2,5-di-(tert-butyl) hydroquinone (BHQ) stabilize an E2-like state, and 1,3-dibromo-2,4,6-tri (methylisothiouronium)benzene stabilizes an E1-P-like conformation (10–13). CPA is a toxic indole tetramic acid first isolated from Penicillium cyclopium (14) and later found to be produced by Aspergillus versicolor and Aspergillus flavus. Like TG, CPA specifically binds to and inhibits SERCA with nanomolar affinity (15). Indeed, CPA is widely used in biochemical and physiological studies on Ca²⁺ signaling and muscle function, where it causes Ca²⁺ store depletion due to specific inhibition of Ca²⁺ reuptake by SERCA. CPA and TG were originally proposed to bind to similar sites on SERCA (16), but recent crystal structures have shown a distinct site of interaction (17, 18). Despite these structural insights, a previously demonstrated magnesium dependence of CPA binding (19) remained unexplained, and opposing CPA binding modes were observed (see below).Tetramic acids are synthesized naturally, and more than 150 natural derivatives have been isolated from bacterial and fungal species (reviewed in Ref. 20). Tetramic acids possessing a 3-acyl group have the ability to chelate divalent metal ions. For instance, tenuazonic acid from the fungus Phoma sorghina has been shown to form complexes with Ca²⁺ and Mg²⁺ (21), as well as heavier metals such as Cu(II), Ni(II), and Fe(III) (22).Previously published crystallographic structures of the SERCA·CPA complex (PDB ID 2O9J and 2EAS) demonstrated that CPA binds within the proposed calcium access channel of SERCA. However, the structures did not reveal a role for magnesium, and the orientation of CPA within this binding site differed in the two studies (17, 18). To address these ambiguities, we have determined the crystal structure of SERCA in complex with , AMPPCP (an ATP analog), and Mn²⁺·CPA. The structure reveals novel insight into CPA binding, which we find to be mediated by a divalent cation, as demonstrated by means of the anomalous scattering properties of Mn²⁺. Further and improved refinement using previously deposited data (PDB ID 2O9J and 2OA0), in light of our new findings, also revealed a strong plausibility for a magnesium ion bound at this site. Furthermore, we find a new configuration of the bound AMPPCP nucleotide, addressing the modulatory role of ATP binding to the E2·P_i occluded conformation of SERCA.     

Critical Roles of Hydrophobicity and Orientation of Side Chains for Inactivation of Sarcoplasmic Reticulum Ca2+-ATPase with Thapsigargin and Thapsigargin Analogs

Thapsigargin (Tg), a specific inhibitor of sarco/endoplasmic Ca²⁺-ATPases (SERCA), binds with high affinity to the E2 conformation of these ATPases. SERCA inhibition leads to elevated calcium levels in the cytoplasm, which in turn induces apoptosis. We present x-ray crystallographic and intrinsic fluorescence data to show how Tg and chemical analogs of the compound with modified or removed side chains bind to isolated SERCA 1a membranes. This occurs by uptake via the membrane lipid followed by insertion into a resident intramembranous binding site with few adaptative changes. Our binding data indicate that a balanced hydrophobicity and accurate positioning of the side chains, provided by the central guaianolide ring structure, defines a pharmacophore of Tg that governs both high affinity and access to the protein-binding site. Tg analogs substituted with long linkers at O-8 extend from the binding site between transmembrane segments to the putative N-terminal Ca²⁺ entry pathway. The long chain analogs provide a rational basis for the localization of the linker, the presence of which is necessary for enabling prostate-specific antigen to cleave peptide-conjugated prodrugs targeting SERCA of cancer cells (Denmeade, S. R., Jakobsen, C. M., Janssen, S., Khan, S. R., Garrett, E. S., Lilja, H., Christensen, S. B., and Isaacs, J. T. (2003) J. Natl. Cancer Inst. 95, 990–1000). Our study demonstrates the usefulness of a simple in vitro system to test and direct development toward the formulation of new Tg derivatives with improved properties for SERCA targeting. Finally, we propose that the Tg binding pocket may be a regulatory site that, for example, is sensitive to cholesterol.     

Tuftsin signals through its receptor neuropilin‐1 via the transforming growth factor beta pathway

Tuftsin (Thr‐Lys‐Pro‐Arg) is a natural immunomodulating peptide found to stimulate phagocytosis in macrophages/microglia. Tuftsin binds to the receptor neuropilin‐1 (Nrp1) on the surface of cells. Nrp1 is a single‐pass transmembrane protein, but its intracellular C‐terminal domain is too small to signal independently. Instead, it associates with a variety of coreceptors. Despite its long history, the pathway through which tuftsin signals has not been described. To investigate this question, we employed various inhibitors to Nrp1's coreceptors to determine which route is responsible for tuftsin signaling. We use the inhibitor EG00229, which prevents tuftsin binding to Nrp1 on the surface of microglia and reverses the anti‐inflammatory M2 shift induced by tuftsin. Furthermore, we demonstrate that blockade of transforming growth factor beta (TGFβ) signaling via TβR1 disrupts the M2 shift similar to EG00229. We report that tuftsin promotes Smad3 phosphorylation and reduces Akt phosphorylation. Taken together, our data show that tuftsin signals through Nrp1 and the canonical TGFβ signaling pathway.

     

Structure of the RACK1 dimer from Saccharomyces cerevisiae

Receptor for activated C-kinase 1 (RACK1) serves as a scaffolding protein in numerous signaling pathways involving kinases and membrane-bound receptors from different cellular compartments. It exists simultaneously as a cytosolic free form and as a ribosome-bound protein. As part of the 40S ribosomal subunit, it triggers translational regulation by establishing a direct link between protein kinase C and the protein synthesis machinery. It has been suggested that RACK1 could recruit other signaling molecules onto the ribosome, providing a signal-specific modulation of the translational process. RACK1 is able to dimerize both in vitro and in vivo. This homodimer formation has been observed in several processes including the regulation of the N-methyl-d-aspartate receptor by the Fyn kinase in the brain and the oxygen-independent degradation of hypoxia-inducible factor 1. The functional relevance of this dimerization is, however, still unclear and the question of a possible dimerization of the ribosome-bound protein is still pending. Here, we report the first structure of a RACK1 homodimer, as determined from two independent crystal forms of the Saccharomyces cerevisiae RACK1 protein (also known as Asc1p) at 2.9 and 3.9 Å resolution. The structure reveals an atypical mode of dimerization where monomers intertwine on blade 4, thus exposing a novel surface of the protein to potential interacting partners. We discuss the significance of the dimer structure for RACK1 function.