Dreidimensionale Strukturbestimmung des Glycin-Betain Transporters BetP mittels Kryo-Elektronen Kristallographie

The soil bacterium Corynebacterium glutamicum has five secondary transporters for compatible solutes allowing it to cope with osmotic stress. The most abundant of them, the transporter BetP, performs a high affinity uptake of glycine-betain when encountering hyperosmotic stress. BetP belongs to the betaine/carnitine/choline/transporter (BCCT) family, and is predicted to have twelve transmembrane helices with both termini facing the cytoplasm. The goal of this thesis is to facilitate understanding of BetP function by determining a three dimensional (3D) model of its structure. Two-dimensional (2D) crystallization of wild-type (WT) BetP has been successfully performed by reconstitution into a mixture of E. coli lipids and bovine cardiolipin, which resulted in vesicular crystals diffracting to 7.5 Å resolution (Ziegler, Morbach et al. 2004). Diffraction patterns of these crystals however showed unfocused spots, generally due to high mosaicity. Better results were obtained by using the constitutively active mutant BetPdeltaC45 in which the first 45 amino acids of the positively charged C-terminus were removed. BetPdeltaC45 crystals obtained under the same conditions for BetP WT were concluded to be pseudo crystals, based on the inconsistence of symmetry. These crystals had BetPdeltaC45 molecules randomly up/downwards inserted into membrane crystals, and cannot be used for structure determination, even though they diffracted up to 7 Å. The problem of pseudo crystal formation could be solved by changing the lipids used for 2D crystallization to a native lipid extract from C. glutamicum cells. This change of lipids improved the crystals to well-ordered packing with exclusive p121_b symmetry. To understand the role of lipids in crystal packing and order, lipids were extracted at different stages during crystallization, and identified by using multiple precursor ion scanning mass spectrometry. The results show that phosphatidyl glycerol (PG) 16:0-18:1 is the most dominant lipid species in C. glutamicum membranes, and that BetP has a preference for the fatty acid moieties 16:0-18:1. Crystallization with synthetic PG 16:0-18:1 proved that an excess of this lipid prevents pseudo crystal formation, but these crystals did not reach the quality as previously achieved by using the C. glutamicum lipids. Apart from the effect of lipids in crystallinity, the concentration and type of salts influenced crystal growth and morphology. High salt conditions (>400 mM LiCl or KCl) yielded tubular crystals, whereas low salt conditions (<300 mM LiCl, NaCl or KCl) led to formation of up to 10 µm large sheet-like crystals. The intermediate concentration gave a mixture of sheet-like and tubular crystals. In terms of resolution, sheets diffracted better than tubes. The sheet-like crystals used for 3D map reconstruction were obtained from a dialysis buffer containing 200 mM NaCl combined with using C. glutamicum lipids. Electron microscopic images were taken from frozen-hydrated crystals using a helium-cooled JEOL 300 SFF microscope or a liquid nitrogen-cooled FEI Tecnai G2 microscope at 300 kV, which allowed optimal data collection and minimized radiation damage to the sample. More than 1000 images of tilt angles up to 50° were taken and evaluated using optical diffraction of a laser beam. The best 200 images were processed with the MRC image processing software package, and 79 images from different tilt angles were merged to the final data set used for calculation of a 3D map at a planar resolution of 8 Å. The structure shows BetPdeltaC45 as a trimer with each monomer consisting of 12 transmembrane alpha-helices. Protein termini and loop regions could not be determined due to the limited resolution of the map. Six of the twelve helices line a central cavity forming a potential substrate-binding chamber. Each monomer shows a central cavity in different sizes and shapes. Thus, the constitutively active BetPdeltaC45 thus forms an unusual asymmetric homotrimer. BetP most likely reflects three different conformational states of secondary transporters: the cytoplasmically open (C), the occluded (O), and the periplasmically open (P) states. The C and O states are similar to BetP WT projection structure, while the P state is discrepant and highly flexible due to the shape and size of the central cavity as well as the lowest intensity of the density. The observation of the P state corresponds well to the constitutively active property of BetPdeltaC45. For the high resolution structure of the C and O states are available, this work presents the first structural information of the P state of a secondary transporter.Thema dieser Arbeit sind die Struktur/Funktionsprinzipien des osmoaktiven Betaintransporters BetP aus Corynebacterium glutamicum, eines Proteins aus der Familie der Betain/Carnitine/Cholin Transporter (BCCT) welches das kompatible Solut Betain unter Ausnutzung eines Natriumgradienten in die Zelle importiert. Gleichzeitig dient BetP auch als Osmolaritäts- und Temperatursensor der die Menge an importierten Solut ohne Umwege anpassen kann, wenn es z.B. als Folge von Überflutungen oder Dürren zu starken Schwankungen in der Wasseraktivität des bakteriellen Lebensraums kommt. Zum besseren Verständnis von osmoregulatorischen Sekundärtransportern und der BCCT Proteinfamilie wurde die dreidimensionale (3D) Struktur von BetP mittels Elektronenkristallographie bestimmt. Der BetP Wildtyp und eine konstitutiv aktive Mutante mit einem um 45 Aminosäuren gekürzten C-Terminus (BetPdeltaC45) wurden in E. coli überexprimiert und mittels Streptavidin-Affinitätschromatographie aufgereinigt. Die C-terminal gekürzte Mutante wurde gewählt da sie konstitutiv aktiv ist und Betain weitestgehend unabhängig von der Osmolarität der Umgebung über die Membran transportiert, was sie hervorragend geeignet macht den aktiven Zustand von BetP strukturell zu charakterisieren. Sowohl der BetP Wildtyp als auch die BetPdeltaC45 Mutante formten die für die Elektronenkristallographie nötigen zweidimensionalen (2D) Kristalle. Die Entfernung des positiv geladenen C-Terminus in der BetPdeltaC45 Mutante hatte jedoch einen starken Effekt auf die Kristallisierung und führte reproduzierbar zur Bildung von besser geordneten Kristallen mit niedrigerer Mosaizität. Die BetPdeltaC45 Mutante wurde daher für die weitere Optimierung und die 3D Strukturaufklärung verwendet. Neben dem Protein, sind die verwendeten Lipide für die Bildung von geordneten 2D Kristallen von entscheidender Bedeutung. Während der Optimierung wurde BetPdeltaC45 mittels dreier verschiedener Lipidpreparationen kristallisiert. Zum besseren Verständnis des Lipideinflusses auf das Kristallisationsverhalten wurden außerdem Lipidextrakte aus verschiedenen Stadien der Kristallisation hergestellt und die enthaltenen Lipide mittels quantitativer Massenspektrometrie bestimmt. Die Ergebnisse zeigen die Präferenz von BetP für 16:0-18:1 Fettsäurereste und das eine an diesen Resten reiche Lipidmischung die Bildung von Pseudokristallen verhindert. Die besten Kristallisationsergebnisse wurden mit dem nativen C. glutamicum Lipidextrakt erzielt. Die mit dieser Lipidmischung gezüchteten Kristalle formten großflächig kristallin geordnete Platten, die besonders für die Aufnahme von 3D Daten geeignet sind. Wichtiger noch als die Größe war der höhere Ordnungsgrad, welcher die Datenaufnahme bis zu einer Auflösung von 7-8 Å erlaubte und damit die Identifizierung von Transmembranhelices, den grundsätzlichen Strukturbausteinen von Membranproteinen ermöglicht. Zur Bestimmung der 3D Struktur von BetPdeltaC45 wurden elektronenkristallographische Aufnahmen von mehr als 200 Kristallen aufgenommen und die zur Strukturaufklärung nötigen Phasen und Amplituden mittels Bildbearbeitung extrahiert. Die 3D Information wurde aus zwischen 0 und 50 Grad gekippten Aufnahmen der 2D Kristalle gewonnen. Alle Daten wurden unter Einhaltung der p121_b Symmetrie zu einem einheitlichen Datensatz vereinigt, der mit einem mittlerem Phasenresidual von 20.4° eine hohe Qualität aufzeigt. Amplituden und Phasen dieses Datensatzes wurden für die Errechnung einer 3D Dichtekarte verwendet. Die bestimmte Dichte hat eine planare Auflösung von 8 Å und zeigt BetP als Trimer mit 12 länglichen Dichten pro Monomer, welche den 12 mittels Hydrophobizitätsplot vorhergesagten Transmembranhelices zugeordnet wurden. Interessanterweise ist die Anordnung dieser Dichten unterschiedlich für alle drei Monomere, wie eindeutig mittels Analyse einer Kreuzkorrelations-Dichtekarte zwischen den Monomeren und anhand der möglichen Kristallsymmetrien gezeigt werden konnte. Die Unterschiede dieses äußerst ungewöhnlichen asymmetrischen Homotrimers konzentrieren sich um einen von vier Helices umrahmten Hohlraum mit unterschiedlich großen Eintrittsöffnungen für jedes Monomer. Basierend auf der Anordnung des BetP asymmetrischen Homotrimers und bekannten biochemischen Daten wurde ein Modell erstellt in dem sich die Substratbindungstellen in den zentralen Hohlräumen befinden und in dem jedes Monomer des asymmetrischen Homotrimers einen unterschiedlichen Aktivierungszustand einnimmt. Die drei Aktivierungszustände wurden als die cytoplasmatisch offene, die geschlossene und periplastisch offene Konformation von sekundären Transportern interpretiert. Die vorliegende Arbeit liefert die ersten Strukturinformationen für einen Transporter der BCCT Familie und die periplastisch offene Konformation eines Sekundärtransporters und liefert damit wichtige Bausteine für das bessere Verständnis dieser für alle Zellen überlebenswichtigen Klasse von Transportern

Healthcare Information and the Utilization of Pap-smear Testing amongst Taiwanese Women

This study is to investigate the determinants of healthcare information amongst women in Taiwan aged between 25 and 69 years, and the association with cervical cancer screening.A two-stage estimation model was adopted for this investigation. In the first stage, the determinants of healthcare information were estimated by the OLS method, with the predicted values of the healthcare information then being linked to the decision to undergo Pap-smear testing. The nationwide survey dataset was obtained from the 2002‘Health Promotion of Knowledge, Attitudes and Practice' (HPKAP) in Taiwan, provided by the Bureau of Health Promotion. A total of 9,106 individuals were included in the analysis. The results reveal that the variations in the level of healthcare information are an important contributory factor to the utilization of cervical cancer screening in Taiwan. Therefore, in addition to providing free screening under the NHI, it is important for the healthcare authorities to place greater effort into strengthening the knowledge and information on cervical cancer screening and Pap-smear testing, for those who are currently less informed, so as to enhance the overall efficiency of the screening program.Health information; Pap-smear testing; Cervical cancer; National Health Insurance; Taiwan

Effects of human parvovirus B19 VP1 unique region protein on macrophage responses

<p>Abstract</p> <p>Background</p> <p>Activity of secreted phospholipase A (sPLA2) has been implicated in a wide range of cellular responses. However, little is known about the function of human parvovirus B19-VP1 unique region (VP1u) with sPLA2 activity on macrophage.</p> <p>Methods</p> <p>To investigate the roles of B19-VP1u in response to macrophage, phospholipase A2 activity, cell migration assay, phagocytosis activity, metalloproteinase assay, RT-PCR and immunoblotting were performed.</p> <p>Results</p> <p>In the present study, we report that migration, phagocytosis, IL-6, IL-1β mRNA, and MMP9 activity are significantly increased in RAW264.7 cells by B19-VP1u protein with sPLA2 activity, but not by B19-VP1uD175A protein that is mutated and lacks sPLA2 activity. Additionally, significant increases of phosphorylated ERK1/2 and JNK proteins were detected in macrophages that were treated with B19-VP1u protein, but not when they were treated with B19-VP1uD175A protein.</p> <p>Conclusion</p> <p>Taken together, our experimental results suggest that B19-VP1u with sPLA2 activity affects production of IL-6, IL-1β mRNA, and MMP9 activity, possibly through the involvement of ERK1/2 and JNK signaling pathways. These findings could provide clues in understanding the role of B19-VP1u and its sPLA2 enzymatic activity in B19 infection and B19-related diseases.</p

Computational and Experimental Studies of Substrate Binding, Conformational Change and Importance of the Trimeric State in the Glycine Betaine Transporter BetP

The glycine betaine/sodium symporter BetP responds to changes in external osmolality by regulation of its transport activity. A recent X-ray structure of BetP confirms that it is a homotrimer and in this structure each protomer adopts an identical conformation, in which the pathway is occluded from both sides. Despite the availability of a wealth of experimental data for BetP, the structures of the alternate states (e.g., open to the outside of the cell), molecular mechanisms of substrate and Na⁺ binding and transport, as well as the functional implications of the trimeric state remain poorly understood. To address these questions, we carried out computational studies using a range of techniques to derive hypotheses that were then tested experimentally. First, to identify structural features of the alternate states, we developed a procedure for flexible fitting of the X-ray structure of BetP into a lower-resolution cryo-EM map of BetP in a more native lipid environment, in which the three protomers have different conformations. These results suggest that: (i) the protomers adopt distinct conformational states relevant to the transport cycle; and (ii) there is conformational coupling between the protomers. Second, we performed all-atom molecular dynamics simulations and in silico alanine scanning of BetP trimers in order to identify interface residues crucial for maintaining the trimeric state. Mutations of these residues to alanine were introduced experimentally revealing that the isolated monomers are functional, and that the trimeric state is important for the regulation and higher activity of the protein. Finally, using molecular modeling and biochemical experiments we identified two Na⁺ binding sites in BetP that could not be resolved in the 3.35 Å resolution X-ray structure

The Effectiveness of Traditional Chinese Medicine in Treating Patients with Leukemia

Leukemia is the most common malignancy among all childhood cancers and is associated with a low survival rate in adult patients. Since 1995, the National Health Insurance (NHI) program in Taiwan has been offering insurance coverage for Traditional Chinese Medicine (TCM), along with conventional Western medicine (WM). This study analyzes the status of TCM utilization in Taiwan, in both pediatric and adult patients with leukemia. A retrospective cohort study was conducted using population-based National Health Insurance Research Database of Registry of Catastrophic Illness, involving patient data from 2001 to 2010 and follow-up data through 2011. The effectiveness of TCM use was evaluated. Relevant sociodemographic data showed that both pediatric and adult patients who were TCM users one year prior to leukemia diagnosis were more likely to utilize TCM services for cancer therapy. A greater part of medical expenditure of TCM users was lower than that of TCM nonusers, except little discrepancy in drug fee of adult patients. The survival rate is also higher in TCM users. Altogether, these data show that TCM has the potential to serve as an adjuvant therapy when combined with conventional WM in the treatment of patients with leukemia

EBV-positive Hodgkin lymphoma is associated with suppression of p21cip1/waf1 and a worse prognosis

<p>Abstract</p> <p>Background</p> <p>About 30-50% of Hodgkin lymphomas (HLs) harbor the Epstein-Barr virus (EBV), but the impact of EBV infection on clinical outcomes has been unclear. EBV-encoded small RNAs (<it>EBER</it>s) are presented in all EBV-infected cells, but their functions are still less understood.</p> <p>Results</p> <p><it>EBER1 </it>was transfected into two HL cell lines, KMH2 and L428, and microarrays were used to screen for <it>EBER1</it>-induced changes. We found that <it>EBER1 </it>suppressed <it>p21</it>^cip1/waf1transcription in HL cell lines. In addition, positive regulators of <it>p21</it>^cip1/waf1transcription, such as p53, EGR1, and STAT1, were decreased. Suppression of <it>p21</it>^cip1/waf1in the <it>EBER1</it>⁺HL cell lines was associated with increased resistance to histone deacetylase inhibitors or proteasome inhibitors, drugs known to cause apoptosis by increasing p21^cip1/waf1levels. On biopsy specimens, EBV⁺HLs had weaker expression of both p21^cip1/waf1and active caspase 3. Clinically, suppression of p21^cip1/waf1in EBV⁺HLs was associated with a worse 2-year disease-free survival rate (45% for EBV⁺HLs <it>vs</it>. 77% for EBV^-HLs, <it>p </it>= 0.002).</p> <p>Conclusion</p> <p>Although the underlying mechanisms are still relatively unclear, <it>EBER1 </it>inhibits <it>p21</it>^cip1/waf1transcription and prevents apoptosis through down-regulation of p53, EGR1, and STAT1. The anti-apoptotic activity of <it>EBER1 </it>may be important in the rescue of Reed-Sternberg cells from drug-induced apoptosis and in the clinical behaviors of EBV⁺HLs.</p