Untersuchung des Scrapie assoziierten Prion Protein PrP27-30 und Stammdifferenzierung von transmissiblen spongiformen Enzephalopathien mittels Fourier-Transform-Infrarotspektroskopie

Title page, Contents 1\. Introduction1 1.1. Literature overview 1 1.2. Aims 20 1.3. Fourier-transform infrared spectroscopy 21 1.4. Cluster analysis 33 2\. Materials and methods35 2.1. TSE agents 35 2.2. Extraction and purification of PrP27-30 36 2.3. Protein analysis 38 2.4. FT-IR spectroscopy 42 3\. Results49 3.1. Purification and spectroscopic properties of PrP27-30 samples from Syrian Hamsters 49 3.2. Strain differences 56 3.3. Cluster analysis 64 3.4. Temperature gradient experiments 66 3.5. Kinetic measurements of 13C Urea induced unfolding of PrP27-30 from 263K 73 3.6. Discrimination of sCJD disease and its strains from blinded human brain samples 76 4\. Discussion93 4.1. Secondary structure characteristics of PrP27-30 from different TSE strains 93 4.2. The structure of different TSE-strains show a different temperature dependent behavior 102 4.3. Urea induced unfolding of PrP27-30 aggregates 103 4.4. Protective function of the non protein constituents of prions 104 4.5. Molecular implications for the structural variations among the TSEs 105 4.6. Structural constrains from FT-IR spectroscopy for model building of PrP27-30 106 4.7. FT-IR characteristics of isolated PrP27-30 versus prions in infected tissue or recombinant SHaPrP90-232 108 4.8. Conclusions 109 5\. Abstract113 Zusammenfassung115 6\. References 119 Publications and presentations135 Acknowledgments137 Curriculum Vitae 139 Appendix141The transmissible spongiform encephalopathies (TSEs) exhibit strain variations similar to genuine microorganisms. Strain diversity in TSEs has been suggested to be encoded by differences in the structure of the misfolded prion protein isoform (PrPSc). PrPSc is derived from the cellular prion protein (PrPC) by posttranslational modifications and it is assumed to be the only component of the infectious unit called prion. The development of a reliable method for discrimination of different TSE strains causing scrapie, BSE, or Creutzfeldt- Jakob disease is a big challenge and remains an open issue. In the presented work, this problem was addressed using the Fourier transform-infrared (FT-IR) spectroscopy of PrP27-30, the proteinase K resistant core of PrPSc. Various FT-IR techniques were used to explore the secondary structure, the structural stability, and hydrogen-deuterium exchange characteristics of PrP27-30 of four different TSE isolates (263K, ME7-H, 22A-H, and BSE-H) adapted to Syrian hamsters. Each of these variants showed characteristic incubation times and disease-specific symptoms. The strain differentiation capacity of the FT-IR approach was objectively proven for the first time by multivariate cluster analysis. The secondary structure of PrP27-30 was compared using samples suspended in H2O or D2O and samples dried for FT-IR microscopic measurements. Temperature induced secondary structure changes and H/D-exchange characteristics in PrP27-30 samples from 263K, ME7-H, and 22A-H were investigated in D2O. Urea induced changes in PrP27-30 samples from strain 263K was investigated in the presence of 6M urea. The results demonstrated that the second derivative FT-IR spectra obtained from dried protein films or samples hydrated in H2O or D2O consistently showed strain-specific infrared characteristics in the secondary structure sensitive amide I region. The secondary structure analysis of these spectra reveal strain dependent conformational diversities, assigned mainly to differences in the -sheet structure but also to other structural components present in PrPSc such as turns and -helices. These strain-specific hallmarks were complimented by strain-dependent spectral traits in the amide II and amide A absorption regions, and the different H/D-exchange behaviour of the various PrP27-30 samples. These variations were due to diversity in functional groups of the peptide backbone that are exposed to the solvent and/or to different structural flexibilities of sub-structures within the PrP27-30 aggregate. Such strain-specific spectra could serve as fingerprints for the determination and classification of the TSE-isolates. The multivariate cluster analysis suggested that the strain-specific structural variations could be best investigated in D2O. This approach was applied as well to differentiate between cases of sporadic CJD and its strains from a set of human brain samples including different forms of sporadic CJD and controls. This study showed that the discrimination of the CJD cases and the distinction between the sporadic CJD strains is hindered due to the heterogeneous structural composition of the purified samples. The latter was most probably due to the presence of protein contaminants, co-propagation of PrPSc with a different structure, or possibly both. However, before a final evaluation estimate of the potential of the FT-IR technique to discriminate human TSEs can be given, an analysis on PrP27-30 virtually free from contaminant proteins is required. FT-IR spectra of PrP27-30 samples from 263K, ME7-H and 22A-H heated to 90°C showed that the specific resistance to heat treatment is associated with the thermal stability of different secondary structure components providing additional means for TSEs strain discrimination. Similar characteristics were observed in the time course of the urea-induced unfolding/denaturation of PrP27-30 aggregates from 263K. This strongly suggests that the spectral changes induced by the chaotropic agent should be sufficient to discriminate between TSEs strains. The temperature experiments suggested a possible protective role of the specific non-protein constituent of the prions. This was associated with the existence of tightly packed parts of the aggregates related with the structural stability of the prion rods exposed to 90°C and characterised by complete resistance to H/D exchange. The spectroscopic information extracted from temperature gradient and urea-induced unfolding/denaturation experiments suggested non-cooperative unfolding of the PrP27-30 aggregates. Prolonged treatment of PrP27-30 with 6 M urea caused the unfolding of most of the prion structural components. However, spectral features characteristic of aggregated protein indicated the presence of some PrP27-30 resistant to urea denaturation. In conclusion, the FT-IR technique can be used for a reliable discrimination of the four TSE agents adapted to Syrian hamster, based on the secondary structure characteristics of purified strain specific PrP27-30 samples. Temperature gradient experiments and/or urea induced unfolding of PrP27-30, can also reveal differences between the TSE strains. Thus the presented results strongly suggest that FT-IR spectroscopy has a significant diagnostic potential for TSE strain differentiation.Die transmissiblen spongiformen Enzephalopathien (TSE) zeigen Stammvariationen ähnlich wie normale Mikroorganismen. Diese Stammunterschiede werden als Folge von Strukturunterschiede bei der Faltung der PrPSc-Isoform des Prionproteins betrachtet. PrPSc entsteht durch posttranslationale Modifizierungen des zellulären Prionproteins (PrPC) wobei angenommen wird, dass die einzige Komponente der Infektionseinheit das Prion darstellt. Die Entwicklung einer zuverlässigen Methode für die Diskriminierung der einzelnen TSE-Erreger wie Scrapie, BSE und Creutzfeldt-Jakob- Krankheit (CJK) ist eine wichtige, noch immer ungelöste Aufgabe. In der vorliegenden Arbeit wurden die Möglichkeiten der Fouriertransform-Infrarot (FT-IR) Spektroskopie für die Charakterisierungen des PrP27-30, des Proteinase K resistenten Kernbereichs von PrPSc untersucht. Dabei wurden verschiedene FT-IR-Techniken eingesetzt, um die Sekundärstrukturunterschiede, Stabilität und das Wasserstoff-Deuterium Austauschverhalten des PrP27 30 von verschiedenen, auf den Goldhamster adaptierten TSE-Stämme zu untersuchen (263K, ME7-H, 22A-H und BSE-H). Jede dieser Varianten zeigte charakteristische Inkubationszeiten und spezifische Krankheitssymptome. Erstmals wurde die Möglichkeit der FT-IR-Spektroskopie, zwischen den einzelnen Stämmen unterscheiden zu können durch multivariate Clusteranalyse objektiv nachgewiesen. Darüber hinaus wurde die Sekundärstruktur des PrP27-30 anhand von Proben, die in H2O oder D2O resuspendiert waren, oder als Trockenproben vorlagen mit der FT-IR Technik vergleichend untersucht. Die temperaturabhängigen Sekundärstruktur- veränderungen und das H/D- Austauschverhalten von PrP27-30 der Stämme 263K, ME7-H und 22A-H wurden dabei in D2O untersucht, und die Harnstoff-induzierten Strukturveränderungen des PrP27-30 vom 263K in Anwesenheit von 6M Harnstoff charakterisiert. Die erarbeiteten Ergebnisse zeigten, dass die zweiten Ableitungen der FT-IR-Spektren der getrocknete, oder in H2O bzw. D2O suspendierten Proteinproben reproduzierbar stammspezifische spektrale Muster in der Sekundärstruktur-sensitiven Amid-I-Region aufweisen. Die Sekundärstrukturanalyse dieser Spektren belegte die Existenz stammspezifischer Konformationsunterschiede, die hauptsächlich auf unterschiedliche ß Faltblatt Strukturen zurückzuführen sind. Diese spektroskopischen Signaturen konnten durch zusätzliche stammspezifische, spektrale Charakteristika in den Amid-II- und Amid-A-Absorptionsregionen sowie durch verschiedene H/D-Austauschraten der einzelnen PrP27-30-Varianten ergänzt werden, wobei die Letzteren auf die unterschiedliche Flexibilität der Substrukturen des PrP27-30-Aggregates zurückgeführt wurden. Die Spektren dieser Prionaggregaten können somit als stammspezifische Fingerabdrücke für die Bestimmung und Klassifizierung von TSE-Isolaten dienen. Die multivariate Clusteranalyse zeigte insbesondere, dass die stammspezifischen spektralen Unterschiede am besten in D2O untersucht werden können. Das hier entwickelte Vorgehen wurde schließlich auch für die Untersuchung von Unterschieden zwischen CJK-Proben in einem Set humaner Gehirnproben, bestehend aus Proben von sporadischer CJK und Kontrollen festzustellen angewendet. Diese Studien zeigten, dass eine sichere Unterscheidung zwischen den CJK-Stämmen infolge der unterschiedlichen Reinheit der Proben nicht möglich war. Eine Erklärung hierfür könnte eine ungenügende Homogenität der PrP27-30 Proben sein. Um den Einfluss von Proteinkontaminationen auf das Spektrum bei CJD-Proben auszuschließen, wären praktisch reine PrP27-30 Proben erforderlich. FT-IR-Untersuchungen von PrP27-30 Proben der Stämme 263K, ME7-H und 22A-H, die bis auf 90oC erhitzt wurden, zeigten, dass die Hitzeresistenz der Proben von der thermischen Stabilität der Sekundärstruktur abhängt. Derartige Untersuchungen liefern eine zusätzliche Möglichkeit zur Differenzierung von einzelnen TSE-Stämmen. Zusätzlich strukturelle Unterschiede zwischen den Stämmen könnten aus der Zeitabhängigkeit der Harnstoff-Induzierten Entfaltung, der PrP27-30- Aggregate erhalten werden. Erste Ergebnisse am TSE-Stamm 263K deuteten darauf hin, dass Spektrenunterschiede, die durch das chaotrope Reagenz verursacht werden, ausreichend sein könnten, um zwischen verschiedenen TSE-Formen zu unterscheiden. Darüber hinaus weisen die Temperaturexperimente auch auf eine Schutzfunktion von spezifischen Nichtproteinkomponenten des Prions hin, was offensichtlich auch im Zusammenhang mit der dichten Packung der Proteinaggregate, eine Erklärung für die hohe Stabilität die Prionaggregate bei 90oC liefern könnte, die u.a. durch eine vollständige Resistenz gegenüber dem H/D-Austausch gekennzeichnet ist. Eine längere Behandlung des PrP27-30 mit 6 M Harnstoff verursachte die Entfaltung des größten Teils der Prionproteinaggregate, wobei die spektralen Muster, die für Proteinaggregate charakteristisch sind, auf gegenüber Harnstoffdenaturierung persistierendes PrP27-30 hinweisen. Die FT-IR-Technik konnte folglich zur Unterscheidung zwischen vier im Goldhamster adaptierte TSE-Stämme erfolgreich eingesetzt werden, wobei die Differenzierung auf stammesspezifische Sekundärstruktur- Charakteristika der gereinigten PrP27-30-Isolaten basierte. Die Temperatur- abhängigen Experimente mit und/oder die Harnstoff-induzierte Denaturierung von PrP27-30 können ebenfalls für die Analyse von Stammunterschieden genutzt werden. Die vorliegende Arbeit belegt eindrucksvoll, dass die FT-IR- Spektroskopie ein großes diagnostisches Potential für die Unterscheidung von TSE-Stämmen hat

Profiling Distinctive Inflammatory and Redox Responses to Hydrogen Sulfide in Stretched and Stimulated Lung Cells

Hydrogen sulfide (H2S) protects against stretch-induced lung injury. However, the impact of H2S on individual cells or their crosstalk upon stretch remains unclear. Therefore, we addressed this issue in vitro using relevant lung cells. We have explored (i) the anti-inflammatory properties of H2S on epithelial (A549 and BEAS-2B), macrophage (RAW264.7) and endothelial (HUVEC) cells subjected to cycling mechanical stretch; (ii) the intercellular transduction of inflammation by co-culturing epithelial cells and macrophages (A549 and RAW264.7); (iii) the effect of H2S on neutrophils (Hoxb8) in transmigration (co-culture setup with HUVECs) and chemotaxis experiments. In stretched epithelial cells (A549, BEAS-2B), the release of interleukin-8 was not prevented by H2S treatment. However, H2S reduced macrophage inflammatory protein-2 (MIP-2) release from unstretched macrophages (RAW264.7) co-cultured with stretched epithelial cells. In stretched macrophages, H2S prevented MIP-2 release by limiting nicotinamide adenine dinucleotide phosphate oxidase-derived superoxide radicals (ROS). In endothelial cells (HUVEC), H2S inhibited interleukin-8 release and preserved endothelial integrity. In neutrophils (Hoxb8), H2S limited MIP-2-induced transmigration through endothelial monolayers, ROS formation and their chemotactic movement. H2S induces anti-inflammatory effects in a cell-type specific manner. H2S limits stretch- and/or paracrine-induced inflammatory response in endothelial, macrophage, and neutrophil cells by maintaining redox homeostasis as underlying mechanism

Hydrogen Sulfide Prevents Formation of Reactive Oxygen Species through PI3K/Akt Signaling and Limits Ventilator-Induced Lung Injury

The development of ventilator-induced lung injury (VILI) is still a major problem in mechanically ventilated patients. Low dose inhalation of hydrogen sulfide (H2S) during mechanical ventilation has been proven to prevent lung damage by limiting inflammatory responses in rodent models. However, the capacity of H2S to affect oxidative processes in VILI and its underlying molecular signaling pathways remains elusive. In the present study we show that ventilation with moderate tidal volumes of 12 ml/kg for 6 h led to an excessive formation of reactive oxygen species (ROS) in mice lungs which was prevented by supplemental inhalation of 80 parts per million of H2S. In addition, phosphorylation of the signaling protein Akt was induced by H2S. In contrast, inhibition of Akt by LY294002 during ventilation reestablished lung damage, neutrophil influx, and proinflammatory cytokine release despite the presence of H2S. Moreover, the ability of H2S to induce the antioxidant glutathione and to prevent ROS production was reversed in the presence of the Akt inhibitor. Here, we provide the first evidence that H2S-mediated Akt activation is a key step in protection against VILI, suggesting that Akt signaling limits not only inflammatory but also detrimental oxidative processes that promote the development of lung injury

Sevoflurane posttreatment prevents oxidative and inflammatory injury in ventilator-induced lung injury.

Mechanical ventilation is a life-saving clinical treatment but it can induce or aggravate lung injury. New therapeutic strategies, aimed at reducing the negative effects of mechanical ventilation such as excessive production of reactive oxygen species, release of pro-inflammatory cytokines, and transmigration as well as activation of neutrophil cells, are needed to improve the clinical outcome of ventilated patients. Though the inhaled anesthetic sevoflurane is known to exert organ-protective effects, little is known about the potential of sevoflurane therapy in ventilator-induced lung injury. This study focused on the effects of delayed sevoflurane application in mechanically ventilated C57BL/6N mice. Lung function, lung injury, oxidative stress, and inflammatory parameters were analyzed and compared between non-ventilated and ventilated groups with or without sevoflurane anesthesia. Mechanical ventilation led to a substantial induction of lung injury, reactive oxygen species production, pro-inflammatory cytokine release, and neutrophil influx. In contrast, sevoflurane posttreatment time dependently reduced histological signs of lung injury. Most interestingly, increased production of reactive oxygen species was clearly inhibited in all sevoflurane posttreatment groups. Likewise, the release of the pro-inflammatory cytokines interleukin-1β and MIP-1β and neutrophil transmigration were completely prevented by sevoflurane independent of the onset of sevoflurane administration. In conclusion, sevoflurane posttreatment time dependently limits lung injury, and oxidative and pro-inflammatory responses are clearly prevented by sevoflurane irrespective of the onset of posttreatment. These findings underline the therapeutic potential of sevoflurane treatment in ventilator-induced lung injury

Pre- and posttreatment with hydrogen sulfide prevents ventilator-induced lung injury by limiting inflammation and oxidation

<div><p>Although essential in critical care medicine, mechanical ventilation often results in ventilator-induced lung injury. Low concentrations of hydrogen sulfide have been proven to have anti-inflammatory and anti-oxidative effects in the lung. The aim of this study was to analyze the kinetic effects of pre- and posttreatment with hydrogen sulfide in order to prevent lung injury as well as inflammatory and oxidative stress upon mechanical ventilation. Mice were either non-ventilated or mechanically ventilated with a tidal volume of 12 ml/kg for 6 h. Pretreated mice inhaled hydrogen sulfide in low dose for 1, 3, or 5 h prior to mechanical ventilation. Posttreated mice were ventilated with air followed by ventilation with hydrogen sulfide in various combinations. In addition, mice were ventilated with air for 10 h, or with air for 5 h and subsequently with hydrogen sulfide for 5 h. Histology, interleukin-1β, neutrophil counts, and reactive oxygen species formation were examined in the lungs. Both pre-and posttreatment with hydrogen sulfide time-dependently reduced or even prevented edema formation, gross histological damage, neutrophil influx and reactive oxygen species production in the lung. These results were also observed in posttreatment, when the experimental time was extended and hydrogen sulfide administration started as late as after 5 h air ventilation. In conclusion, hydrogen sulfide exerts lung protection even when its application is limited to a short or delayed period. The observed lung protection is mediated by inhibition of inflammatory and oxidative signaling.</p></div

Effect of H₂S pretreatment on ventilator-induced lung injury.

<p>Mice spontaneously breathed air (control) or for 6 h, or they were mechanically ventilated with 12 ml/kg for 6 h either with air alone (6 h air) or air supplemented with 80 ppm H₂S (6 h H₂S). All other mice spontaneously breathed air supplemented with 80 ppm H₂S 1, 3, or 5 h prior to mechanical ventilation with air for another 6 h as indicated. Lung sections were stained with H&E. Representative pictures are shown for each experimental group as indicated (A). Alveolar wall thickness was measured (B) and ventilator-induced lung injury (VILI) score was calculated (C). Data represent means ± SEM for n = 7/group. ANOVA (Tukey`s post hoc test), *<i>P</i><0.05 vs. control group; ^#<i>P</i><0.05 vs. 6h air vent group; ^&<i>P</i><0.05 vs. 1h H₂S pre + 6h air vent group.</p

Effect of expanded H₂S posttreatment on ventilator-induced lung damage.

<p>Mice spontaneously breathed air (control) or for 6 h, or they were mechanically ventilated with 12 ml/kg either with air alone (6 h air, 10 h air) or air supplemented with 80 ppm H₂S (6 h H₂S). Another group of mice was first mechanically ventilated with air alone for 5 h, followed by ventilation with 80 ppm H₂S for another 5 h. Lung sections were stained with H&E. Representative pictures are shown for each experimental group as indicated (A). Alveolar wall thickness was measured (B) and ventilator-induced lung injury (VILI) score was calculated (C). Data represent means ± SEM for n = 6/group. ANOVA (Tukey`s post hoc test), *<i>P</i><0.05 vs. control group; ^#<i>P</i><0.05 vs. 6h air vent group; ^&<i>P</i><0.05 vs. 10h air vent group.</p