Structural analysis of the major lightharvesting complex II by electron paramagnetic resonance (EPR) : comparison between monomers and trimers

Der Haupt-Lichtsammenkomplex II (LHCII) höherer Pflanzen ist das häufigsternMembranprotein der Welt und in die chloroplastidäre Thylakoidmembran integriert. DerrnLHCII kann als Modellsystem genutzt werden, um die Funktionsweise vonrnMembranproteinen besser zu verstehen, da 96 % seiner Struktur kristallografisch aufgelöstrnist und er in rekombinanter Form in vitro rückgefaltet werden kann. Hierbei entsteht einrnvoll funktionaler Protein-Pigment.Komplex, der nahezu identisch mit der in vivo Varianternist.rnElektronenparamagnetischen Resonanz (EPR) Spektroskopie ist eine hoch sensitive undrnideal geeignete Methode, um die Strukturdynamik von Proteinen zu untersuchen. Hierzurnist eine ortsspezifische Markierung mit Spinsonden notwendig, die kovalent an Cysteinernbinden. Möglich wird dies, indem sorgfältig ausgewählte Aminosäuren gegen Cysteinerngetauscht werden, ohne dass die Funktionsweise des LHCII beeinträchtigt wird.rnIm Rahmen dieser Arbeit wurden die Stabilität des verwendeten Spinmarkers und diernProbenqualität verbessert, indem alle Schritte der Probenpräparation untersucht wurden.rnMithilfe dieser Erkenntnisse konnte sowohl die Gefahr einer Proteinaggregation als auchrnein Verlust des EPR Signals deutlich vermindert werden. In Kombination mit derrngleichzeitigen Etablierung des Q-Band EPR können nun deutlich geringer konzentrierternProben zuverlässig vermessen werden. Darüber hinaus wurde eine reproduzierbarernMethode entwickelt, um heterogene Trimere herzustellen. Diese bestehen aus einemrndoppelt markierten Monomer und zwei unmarkierten Monomeren und erlauben es, diernkristallografisch unvollständig aufgelöste N-terminale Domäne im monomeren undrntrimeren Assemblierungsgrad zu untersuchen. Die Ergebnisse konnten einerseits diernVermutung bestätigen, dass diese Domäne im Vergleich zum starren Proteinkern sehrrnflexibel ist und andererseits, dass sie in Monomeren noch mobiler ist als in Trimeren.rnZudem wurde die lumenale Schleifenregion bei unterschiedlichen pH Werten undrnvariierender Pigmentzusammensetzung untersucht, da dieser Bereich sehr kontroversrndiskutiert wird. Die Messergebnisse offenbarten, dass diese Region starre und flexiblerernSektionen aufweist. Während der pH Wert keinen Einfluss auf die Konformation hatte,rnzeigte sich, dass die Abwesenheit von Neoxanthin zu einer Änderung der Konformationrnführt. Weiterführende Analysen der strukturellen Dynamik des LHCII in einerrnLipidmembran konnten hingegen nicht durchgeführt werden, da dies eine gerichteternInsertion des rückgefalteten Proteins in Liposomen erfordert, was trotz intensiverrnVersuche nicht zum Erfolg führte.

Chaperone BiP: Master regulator of the unfolded protein response (UPR) and its role in regulation of angiogenesis

Verschiedene Krankheiten gehen mit einer fehlerhaften Vaskularisierung einher. Allerdings ist der Erfolg der derzeitig vorhandenen Therapieansätze, die sich z.B. auf VEGF fokussieren, beschränkt. Aus diesem Grund ist es wichtig, neue Strategien zur Regulation der Angiogenese zu entwickeln. Hierbei stehen neue Signaltransduktions-wege im Fokus, die sich als vielversprechend erweisen, um Angiogenese zu fördern oder zu inhibieren. Die Blutgefäßneubildung ist ein hochregulierter Prozess, der mit einer hohen Proteinsyntheserate verknüpft ist. Die Angiogenese wurde bereits mit dem ER-Stress Signaltransduktionsweg, der Unfolded Protein Response (UPR), in Verbindung gebracht (Zeng et al., 2013; Bouvier et al., 2012). Eine im Rahmen der vorliegenden Studie durchgeführte histologische Untersuchung konnte eine Fehlregulierung der Expression von UPR beteiligten Proteinen in vivo unter pathologischen Bedingungen gezeigt werden. Bemerkenswerter Weise war BiP, der Hauptsensor der UPR, in Endothelzellen von Angiosarkomen sehr stark exprimiert. In in vitro Experimenten wurde gezeigt, dass das Herunterregulieren von BiP mittels RNAi Einfluss auf die inflammatorische Antwort und die Bildung angiogener Strukturen in Endothelzellen nimmt. Das Herunterregulieren des Proteins BiP verstärkte die inflammatorische Antwort von HUVEC, was sich in einer gesteigerten Bildung von IL-8 und ICAM-1 äußerte und wurde auf die Aktivierung der UPR durch die verringerte Menge an BiP zurückgeführt. Der Phänotyp BiP-herunterregulierter Zellen entsprach dem untransfizierter Zellen, welcher durch das Cytoskelett und die Expression des endothelspezifischen Markers CD31 charakterisiert wurde. Im Gegensatz dazu änderte sich der Grad der Glykosylierung in transfizierten Zellen. Im Hinblick auf die Blutgefäßbildung, zeigten sich eine gehemmte Migration und eine inhibierte Bildung Gefäß-ähnlicher Strukturen in BiP-herunterregulierten Zellen. In diesen Zellen war die Expression von KDR auffallend stark inhibiert, wohingegen die Flt-1 Expression sich als gleichbleibend herausstellte, was ebenfalls auf die Aktivierung der UPR zurückgeführt werden konnte. Alternativ wäre der reduzierte Level des Proteins BiP im Hinblick auf die Funktion als Helferenzym in der Proteinfaltung eine mögliche Erklärung für die gehemmte Expression von KDR. Die Ergebnisse dieser Studie deuten darauf hin, dass stabile Spiegel von BiP die Regulierung der Angiogenese durch die Kontrolle der UPR in physiologischen Prozessen unterstützen könnte. Eine Fehlregulierung von BiP durch Unterdrückung der UPR, wie z.B. in malignen Tumoren, könnte Tumorzellen und beteiligten Endothelzellen einen Vorteil verschaffen und zu einer gestörten Vaskularisierung führen. Somit stellt das Stresssensorprotein BiP und die UPR einen potentiellen Angriffspunkt für die Regulation der Angiogenese dar.

Leishmania major promastigote entry of an autophagy-like compartment and amastigote escape from the parasitophorous vacuole

In this thesis, we investigated the interaction of the obligate intracellular parasite Leishmania (L.) major with two phenotypes of human monocyte derived macrophages (hMDMs). Thereby we focused on the development and maturation of the parasitophorous vacuole (PV) and could show that compartment development is dependent on the parasite stage.rnFocusing on the ultrastructure of PVs containing axenic amastigotes, we demonstrated that the parasites are partially located in damaged PVs or in the cytoplasm of the host. Moreover, we visualized multiple amastigotes in a common PV 144 h p.i. in pro-inflammatory hMDM I but not in anti-inflammatory hMDM II indicating different PV development. rnRegarding the promastigote form, we demonstrated a different uptake of viable and apoptotic L. major promastigotes by hMDMs. Viable promastigotes are predominantly taken up via the flagellum tip whereas apoptotic promastigotes enter the cells via the parasite body. Analyzing compartment maturation, we found that 20-30% of the PVs get positive for the early maturation markers PI3P and EEA1 independent of the viability of the parasites and unaffected by the human macrophage type. Subsequently, 25-40% of the parasites acquire the autophagy marker LC3 on their PV, what is independent of the viability of the parasites as well. We quantified this and in hMDM II less LC3-positive compartments formed compared to hMDM I. Analyzing the ultrastructure, we investigated that the compartments consist of a single-membrane PV characteristic for LC3-associated phagocytosis (LAP). Involvement of LAP was confirmed by demonstrating that the protein kinase ULK1 is dispensable for LC3-compartment formation around Leishmania PVs. Visualizing compartment dynamics in real time showed that apoptotic promastigotes are degraded in LC3-positve compartments, whereas viable promastigotes are able to get rid of LC3-protein on their PV suggesting an involvement in parasite development and survival. In this thesis, we established a lentiviral based fluorescent imaging technique that we combined with High-Pressure-Freezing (HPF) and high-resolution 3D electron microscopy. We visualized a promastigote in a LC3-compartment whose ultrastructure showed an opening of the PV to the outside. To identify new LAP markers involved in Leishmania infection, we established an immuno-magnetic isolation protocol for the purification of Leishmania containing compartments.rnIn conclusion, this study suggests that L. major compartment biogenesis and maturation in pro- and anti-inflammatory human macrophages is dependent on the parasite stage and is different between axenic amastigotes, viable promastigotes and apoptotic promastigotes. Understanding the development and maturation of Leishmania parasites in human host cells is important to control and combat the neglected disease leishmaniasis in the future.rn

The contribution of surface residues to membrane binding and ligand transfer by the -tocopherol transfer protein ( -TTP)

Previous work has shown that the -tocopherol transfer protein ( -TTP) can bind to vesicular or immobilized phospholipid membranes. Revealing the molecular mechanisms by which -TTP associates with membranes is thought to be critical to understanding its function and role in the secretion of tocopherol from hepatocytes into the circulation. Calculations presented in the Orientations of Proteins in Membranes database have provided a testable model for the spatial arrangement of -TTP and other CRAL-TRIO family proteins with respect to the lipid bilayer. These calculations predicted that a hydrophobic surface mediates the interaction of -TTP with lipid membranes. To test the validity of these predictions, we used site-directed mutagenesis and examined the substituted mutants with regard to intermembrane ligand transfer, association with lipid layers and biological activity in cultured hepatocytes. Substitution of residues in helices A8 (F165A and F169A) and A10 (I202A, V206A and M209A) decreased the rate of intermembrane ligand transfer as well as protein adsorption to phospholipid bilayers. The largest impairment was observed upon mutation of residues that are predicted to be fully immersed in the lipid bilayer in both apo (open) and holo (closed) conformations such as Phe165 and Phe169. Mutation F169A, and especially F169D, significantly impaired -TTP-assisted secretion of -tocopherol outside cultured hepatocytes. Mutation of selected basic residues (R192H, K211A, and K217A) had little effect on transfer rates, indicating no significant involvement of nonspecific electrostatic interactions with membranes.

Elevated level of metabotropic glutamate receptor 2/3 in the prefrontal cortex in major depression

Clinical, postmortem and preclinical research strongly implicates dysregulation of glutamatergic neurotransmission in major depressive disorder (MDD). Recently, metabotropic glutamate receptors (mGluRs) have been proposed as attractive targets for the discovery of novel therapeutic approaches against depression. The aim of this study was to examine mGluR2/3 protein levels in the prefrontal cortex (PFC) from depressed subjects. In addition, to test whether antidepressants influence mGluR2/3 expression we also studied levels of mGluR2/3 in fluoxetine-treated monkeys. Postmortem human prefrontal samples containing Brodmann's area 10 (BA10) were obtained from 11 depressed and 11 psychiatrically healthy controls. Male rhesus monkeys were treated chronically with fluoxetine (dose escalated to 3mg/kg, p.o.; n=7) or placebo (n=6) for 39 weeks. The mGluR2/3 immunoreactivity was investigated using Western blot method. There was a robust (+67%) increase in the expression of the mGlu2/3 protein in the PFC of depressed subjects relative to healthy controls. The expression of mGlu2/3 was unchanged in the PFC of monkeys treated with fluoxetine. Our findings provide the first evidence that mGluR2/3 is elevated in the PFC in MDD. This observation is consistent with reports showing that mGluR2/3 antagonists exhibit antidepressant-like activity in animal models and demonstrates that these receptors are promising targets for the discovery of novel antidepressants.

On-line SPE LC-MS/MS for the quantification of Δ9-tetrahydrocannabinol (THC) and its two major metabolites in human peripheral blood by liquid chromatography tandem mass spectrometry

A universal and robust analytical method for the determination of Δ9-tetrahydrocannabinol (THC) and two of its metabolites Δ9-(11-OH)-tetrahydrocannabinol (11-OH-THC) and 11-nor-Δ9-carboxy-tetrahydrocannabinol (THC-COOH) in human whole blood was developed and validated for use in forensic toxicology. Protein precipitation, integrated solid phase extraction and on-line enrichment followed by high-performance liquid chromatography separation and detection with a triple quadrupole mass spectrometer were combined. The linear ranges used for the three cannabinoids were from 0.5 to 20 ng/mL for THC and 11-OH-THC and from 2.5 to 100 ng/mL for THC-COOH, therefore covering the requirements for forensic use. Correlation coefficients of 0.9980 or better were achieved for all three analytes. No relevant hydrolysis was observed for THC-COOH glucuronide with this procedure--in contrast to our previous GC-MS procedure, which obviously lead to an artificial increase of the THC-COOH concentration due to the hydrolysis of the glucuronide-conjugate occurring at high pH during the phase-transfer catalyzed methylation step.

Reduced metabotropic glutamate receptor 5 density in major depression determined by [(11)C]ABP688 PET and postmortem study

Clinical and preclinical evidence suggests a hyperactive glutamatergic system in clinical depression. Recently, the metabotropic glutamate receptor 5 (mGluR5) has been proposed as an attractive target for novel therapeutic approaches to depression. The goal of this study was to compare mGluR5 binding (in a positron emission tomography [PET] study) and mGluR5 protein expression (in a postmortem study) between individuals with major depressive disorder and psychiatrically healthy comparison subjects.

Caenorhabditis elegans Heterochromatin protein 1 (HPL-2) links developmental plasticity, longevity and lipid metabolism

Background Heterochromatin protein 1 (HP1) family proteins have a well-characterized role in heterochromatin packaging and gene regulation. Their function in organismal development, however, is less well understood. Here we used genome-wide expression profiling to assess novel functions of the Caenorhabditis elegans HP1 homolog HPL-2 at specific developmental stages. Results We show that HPL-2 regulates the expression of germline genes, extracellular matrix components and genes involved in lipid metabolism. Comparison of our expression data with HPL-2 ChIP-on-chip profiles reveals that a significant number of genes up- and down-regulated in the absence of HPL-2 are bound by HPL-2. Germline genes are specifically up-regulated in hpl-2 mutants, consistent with the function of HPL-2 as a repressor of ectopic germ cell fate. In addition, microarray results and phenotypic analysis suggest that HPL-2 regulates the dauer developmental decision, a striking example of phenotypic plasticity in which environmental conditions determine developmental fate. HPL-2 acts in dauer at least partly through modulation of daf-2/IIS and TGF-β signaling pathways, major determinants of the dauer program. hpl-2 mutants also show increased longevity and altered lipid metabolism, hallmarks of the long-lived, stress resistant dauers. Conclusions Our results suggest that the worm HP1 homologue HPL-2 may coordinately regulate dauer diapause, longevity and lipid metabolism, three processes dependent on developmental input and environmental conditions. Our findings are of general interest as a paradigm of how chromatin factors can both stabilize development by buffering environmental variation, and guide the organism through remodeling events that require plasticity of cell fate regulation.

Molecular analysis of HER2 signaling in human breast cancer by functional protein pathway activation mapping

Targeting of the HER2 protein in human breast cancer represents a major advance in oncology but relies on measurements of total HER2 protein and not HER2 signaling network activation. We used reverse-phase protein microarrays (RPMA) to measure total and phosphorylated HER2 in the context of HER family signaling to understand correlations between phosphorylated and total levels of HER2 and downstream signaling activity.

Molecular characterization of Neospora caninum MAG1, a dense granule protein secreted into the parasitophorous vacuole, and associated with the cyst wall and the cyst matrix

SUMMARY: In Neospora caninum and Toxoplasma gondii, the parasitophorous vacuole (PV) is synthesized at the time of infection. During tachyzoite-to-bradyzoite stage conversion, the PV is later transformed into a tissue cyst that allows parasites to survive in their host for extended periods of time. We report on the characterization of NcMAG1, the N. caninum orthologue of T. gondii MAG1 (matrix antigen 1; TgMAG1). The 456 amino acid predicted NcMAG1 protein is 54% identical to TgMAG1. By immunoblotting, a rabbit antiserum raised against recombinant NcMAG1 detected a major product of approximately 67 kDa in extracts of N. caninum tachyzoite-infected Vero cells, which was stained more prominently in extracts of infected Vero cells treated to induce in vitro bradyzoite conversion. Immunofluorescence and TEM localized the protein mainly within the cyst wall and the cyst matrix. In both tachyzoites and bradyzoites, NcMAG1 was associated with the parasite dense granules. Comparison between NcMAG1 and TgMAG1 amino acid sequences revealed that the C-terminal conserved regions exhibit 66% identity, while the N-terminal variable regions exhibit only 32% identity. Antibodies against NcMAG1-conserved region cross-reacted with the orthologuous protein in T. gondii but those against the variable region did not. This indicates that the variable region possesses unique antigenic characteristics.

Phylogenetic analysis of the gag region encoding the matrix protein of small ruminant lentiviruses: comparative analysis and molecular epidemiological applications

Little sequence information exists on the matrix-protein (MA) encoding region of small ruminant lentiviruses (SRLV). Fifty-two novel sequences were established and permitted a first phylogenetic analysis of this region of the SRLV genome. The variability of the MA encoding region is higher compared to the gag region encoding the capsid protein and surprisingly close to that reported for the env gene. In contrast to primate lentiviruses, the deduced amino acid sequences of the N- and C-terminal domains of MA are variable. This permitted to pinpoint a basic domain in the N-terminal domain that is conserved in all lentiviruses and likely to play an important functional role. Additionally, a seven amino acid insertion was detected in all MVV strains, which may be used to differentiate CAEV and MVV isolates. A molecular epidemiology analysis based on these sequences indicates that the Italian lentivirus strains are closely related to each other and to the CAEV-CO strain, a prototypic strain isolated three decades ago in the US. This suggests a common origin of the SRLV circulating in the monitored flocks, possibly related to the introduction of infected goats in a negative population. Finally, this study shows that the MA region is suitable for phylogenetic studies and may be applied to monitor SRLV eradication programs.

Therapeutic protein transduction of mammalian cells and mice by nucleic acid-free lentiviral nanoparticles

The straightforward production and dose-controlled administration of protein therapeutics remain major challenges for the biopharmaceutical manufacturing and gene therapy communities. Transgenes linked to HIV-1-derived vpr and pol-based protease cleavage (PC) sequences were co-produced as chimeric fusion proteins in a lentivirus production setting, encapsidated and processed to fusion peptide-free native protein in pseudotyped lentivirions for intracellular delivery and therapeutic action in target cells. Devoid of viral genome sequences, protein-transducing nanoparticles (PTNs) enabled transient and dose-dependent delivery of therapeutic proteins at functional quantities into a variety of mammalian cells in the absence of host chromosome modifications. PTNs delivering Manihot esculenta linamarase into rodent or human, tumor cell lines and spheroids mediated hydrolysis of the innocuous natural prodrug linamarin to cyanide and resulted in efficient cell killing. Following linamarin injection into nude mice, linamarase-transducing nanoparticles impacted solid tumor development through the bystander effect of cyanide.

Trypanosoma congolense procyclins: unmasking cryptic major surface glycoproteins in procyclic forms

In the tsetse fly, the protozoan parasite Trypanosoma congolense is covered by a dense layer of glycosylphosphatidylinositol (GPI)-anchored molecules. These include a protease-resistant surface molecule (PRS), which is expressed by procyclic forms early in infection, and a glutamic acid- and alanine-rich protein (GARP), which appears at later stages. Since neither of these surface antigens is expressed at intermediate stages, we investigated whether a GPI-anchored protein of 50 to 58 kDa, previously detected in procyclic culture forms, might constitute the coat of these parasites. We therefore partially purified the protein from T. congolense Kilifi procyclic forms, obtained an N-terminal amino acid sequence, and identified its gene. Detailed analyses showed that the mature protein consists almost exclusively of 13 heptapeptide repeats (EPGENGT). The protein is densely N glycosylated, with up to 13 high-mannose oligosaccharides ranging from Man(5)GlcNAc(2) to Man(9)GlcNAc(2) linked to the peptide repeats. The lipid moiety of the glycosylphosphatidylinositol is composed of sn-1-stearoyl-2-lyso-glycerol-3-HPO(4)-1-(2-O-acyl)-d-myo-inositol. Heavily glycosylated proteins with similar repeats were subsequently identified in T. congolense Savannah procyclic forms. Collectively, this group of proteins was named T. congolense procyclins to reflect their relationship to the EP and GPEET procyclins of T. brucei. Using an antiserum raised against the EPGENGT repeat, we show that T. congolense procyclins are expressed continuously in the fly midgut and thus form the surface coat of cells that are negative for both PRS and GARP.

Protein buffering in model systems and in whole human saliva

The aim of this study was to quantify the buffer attributes (value, power, range and optimum) of two model systems for whole human resting saliva, the purified proteins from whole human resting saliva and single proteins. Two model systems, the first containing amyloglucosidase and lysozyme, and the second containing amyloglucosidase and alpha-amylase, were shown to provide, in combination with hydrogencarbonate and di-hydrogenphosphate, almost identical buffer attributes as whole human resting saliva. It was further demonstrated that changes in the protein concentration as small as 0.1% may change the buffer value of a buffer solution up to 15 times. Additionally, it was shown that there was a protein concentration change in the same range (0.16%) between saliva samples collected at the time periods of 13:00 and others collected at 9:00 am and 17:00. The mode of the protein expression changed between these samples corresponded to the change in basic buffer power and the change of the buffer value at pH 6.7. Finally, SDS Page and Ruthenium II tris (bathophenantroline disulfonate) staining unveiled a constant protein expression in all samples except for one 50 kDa protein band. As the change in the expression pattern of that 50 kDa protein band corresponded to the change in basic buffer power and the buffer value at pH 6.7, it was reasonable to conclude that this 50 kDa protein band may contain the protein(s) belonging to the protein buffer system of human saliva.

Functional relevance of DNA polymorphisms within the promoter region of the prion protein gene and their association to BSE infection

Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative diseases that can occur spontaneously or can be caused by infection or mutations within the prion protein gene PRNP. Nonsynonymous DNA polymorphisms within the PRNP gene have been shown to influence susceptibility/resistance to infection in sheep and humans. Analysis of DNA polymorphisms within the core promoter region of the PRNP gene in four major German bovine breeds resulted in the identification of both SNPs and insertion/deletion (indel) polymorphisms. Comparative genotyping of both controls and animals that tested positive for bovine spongiform encephalopathy (BSE) revealed a significantly different distribution of two indel polymorphisms and two SNPs within Braunvieh animals, suggesting an association of these polymorphisms with BSE susceptibility. The functional relevance of these polymorphisms was analyzed using reporter gene constructs in neuronal cells. A specific haplotype near exon 1 was identified that exhibited a significantly lower expression level. Genotyping of nine polymorphisms within the promoter region and haplotype calculation revealed that the haplotype associated with the lowest expression level was underrepresented in the BSE group of all breeds compared to control animals, indicating a correlation of reduced PRNP expression and increased resistance to BSE.