Mutational analysis of ligand - LDL-receptor interactions

Lipoproteinrezeptoren sind Plasmamembranproteine, welche für die spezifische Bindung und Aufnahme der im Blutkreislauf zirkulierenden Lipoproteine in hepatische und extrahepatische Gewebe verantwortlich sind. Dieser physiologische Vorgang wird als rezeptorvermittelte Endozytose bezeichnet und basiert auf der Interaktion zwischen Mitgliedern der sogenannten Low Density Lipoprotein (LDL) Rezeptor Familie und Lipoproteinen im Plasma. Der als erster entdeckte und am besten charakterisierte Lipoproteinrezeptor ist der LDL Rezeptor (LDLR). Dieser Rezeptor spielt eine wichtige Rolle in der Regulation der Cholesterinspiegel durch die Bindung und Internalisierung von Lipoproteinen, welche Apolipoprotein- B (apoB) und /oder -E (apoE) als Proteinkomponente besitzen. In der vorliegenden Arbeit wurde das legende Huhn, ein gut etablierter Modellorganismus zur Erforschung des Lipidstoffwechsels, herangezogen um die Interaktion des LDL Rezeptors mit Apolipoprotein B- und -E hältigen Lipoproteinen näher zu charakterisieren. Früheren Studien zufolge gibt es zahlreiche Oberflächenrezeptoren im Huhn, die eine deutliche Homologie zur Familie der LDL Rezeptorproteine von Säugern aufweisen. Der zum menschlichen LDL Rezeptor homologe Rezeptor im Huhn wurde erstmals im Jahr 2003 beschrieben und weist die klassischen Strukturmerkmale der Säuger-LDL Rezeptoren auf, was auf eine hohe evolutionäre Konservierung deutet. Alle Mitglieder der LDL Rezeptorfamilie besitzen einen charakteristischen modularen Aufbau aus vier bis fünf unterschiedlichen Domänen. Die amino-terminale Region enthält die Ligandenbindungsdomäne des Rezeptors und besteht aus mehreren cysteinreichen Abschnitten, den sogenannten LA-repeats, welche die Interaktion mit verschiedenen Proteinen, wie Apolipoprotein, Proteasen und Signalproteinen vermitteln. Mutationsanalysen zeigen, dass die einzelnen Repeats unterschiedliche funktionelle Bedeutungen bei der Ligandenbindung besitzen. Bindungsstudien im Hühnersystem ergaben, dass einige Rezeptoren der LDLR Familie Apolipoprotein E binden, ein in Säugern, jedoch nicht im Huhn produziertes Protein. Andere Rezeptoren wiederum besitzen diese Bindungseigenschaft nicht. Dieses unterschiedliche Verhalten weist darauf hin, dass bestimmte Variationen in der ligandenbindenden Domäne des Rezeptors einen entscheidenden Einfluss auf die Ligandenbindung besitzen. In der vorliegenden Arbeit habe ich mich damit beschäftigt, die strukturellen Unterschiede zwischen ApoE-bindenden Rezeptoren und Rezeptoren, welche die Fähigkeit ApoE zu binden nicht besitzen, zu ermitteln. Gemäß den Ergebnissen aus früheren Mutationsanalysen wurde die Ligandenbindungsdomäne des Hühner- LDLR an vermeintlich wichtigen Bindungsregionen mutiert. Die bakteriell exprimierten mutanten Rezeptorproteine wurden durch Affninitätschromatographie gereinigt und, um die physiologische Funktionsweise zu erlangen, einem Faltungsprozess unterzogen, welcher durch das molekulare Chaperon RAP (Receptor-associated Protein) unterstützt wurde. Schließlich wurden die Rezeptormutanten hinsichtlich der Ligandenbindung von Apolipoprotein B- und -E -hältigen Lipoproteinen untersucht. Diese Bindungsanalysen ergaben, dass das erste cysteinreiche LA Repeat (LA1) nicht an der Binding von Apolipoprotein B beteiligt scheint. Indessen führten Mutationen in den Repeats 4 und 5 (LA4, LA5), sowie in der Linker-Region zwischen diesen Repeats zu erheblichen Einschränkung bezüglich der Bindung von ApoB-hältigen Lipoproteinen. Bindungsanalysen zur Erforschung der Interaktion zwischen ApoE und den Rezeptormutanten lieferten bisher keine reproduzierbaren Ergebnisse, und erfordern daher weitere Untersuchungen. Die Daten, die im Zuge dieser Arbeit durch Experimente im Huhn ermittelt wurden, tragen zum Verständnis des molekularen Mechanismus der LDL Rezeptor-Ligand Interaktion bei und stehen im Einklang mit Untersuchungsergebnissen am menschlichen LDL Rezeptor, welche eine große Bedeutung des fünften LA Repeats (LA5) für die Ligandenbindung beschreiben.Lipoprotein receptors are cell surface-exposed receptors that are specific for the binding and uptake of lipoproteins from the circulation into hepatic and extrahepatic tissues. This process is termed receptor-mediated endocytosis and is largely dependent upon the interaction between members of the so-called Low Density Lipoprotein (LDL) receptor family and plasma lipoproteins. The best characterized receptor of this family is the LDL receptor (LDLR). It plays a major role in the regulation of plasma cholesterol levels by mediating the binding and uptake of apolipoprotein B- (apoB) and -E (apoE) -containing plasma lipoprotein particles. To gain further insight into the molecular mechanism of LDL receptor- ligand interactions, I performed the present study in the domesticated chicken (Gallus gallus), which serves as an excellent model organism for research on lipid metabolism. Various surface receptors homologous to members of the mammalian LDLR family have been discovered in this species, and are recognized as important in the maintenance of systemic lipid homeostasis and embryongenesis including follicle development and oocyte growth. In 2003, the first avian LDL receptor ortholog was characterized and revealed a high degree of conservation during evolution, since the LDLR’s hallmark properties are already present in the chicken protein. The numerous avian and mammalian relatives of the receptor family share characteristic similarities, both in structural and functional terms. Via highly conserved cysteine-rich repeat elements in the extracellular ligand-binding domain the receptors are able to bind many unrelated proteins, such as apolipoproteins, proteases, signaling molecules, and several other groups of proteins. Acting as cargo transporters, members of this receptor family are not only capable of transporting macromolecules, but also of signal transduction. According to previous studies, certain chicken receptors belonging to the LDLR family are able to bind apolipoprotein E, a protein of mammalian origin, which is not produced in the avian species, while other receptors like the chicken LDLR do not have binding affinity for apoE. In agreement with previous investigations, this observation indicates that certain variations in the ligand-binding domain of the receptor contribute to the recognition of a variety of heterogeneous ligands. In the present study I focused on the investigation of structural differences between apoE-binding competent and apoE-binding incompetent receptors that are pivotal for ligand-binding. To address these issues I generated receptor fragments of the chicken LDLR consisting of the ligand-binding region. Based on previous findings derived from extensive studies of the human LDLR, mutations in putatively important receptor domains that were characterized as critical for apoE binding were introduced. Following bacterial expression, the receptor mutants were purified using affinity chromatography. Since the functionality of the LDLR depends on the correct folding including disulfide bond formation, the main challenge was to re-fold the recombinant receptor fragments into a binding-active conformation. This was achieved by the assistance of the receptor-associated protein (RAP), a molecular chaperone especially acting on LDLR family members. The refolding procedure was performed in the presence of RAP in an environment that allows disulfide bond formation and Ca2+ incorporation. Finally, the mutated receptors were characterized with regards to the ligand-binding properties towards apolipoprotein B and -E containing lipid particles. The data obtained from solid phase binding assays revealed that the first LA repeat is not required to bind apoB- containing lipoproteins. However, mutations in the linker region separating repeat 4 and 5 as well as mutations in LA repeat 5 dramatically impaired the binding of apoB-containing lipoproteins. Initial investigations examining the binding activity of the mutant receptors towards apoE-containing particles did not lead to reproducible results and thus require further investigations. These data obtained in an oviparous species contribute to the understanding of LDLR-ligand interactions and are in agreement with previous investigations, in which the importance of LA5 for ligand recognition of the human LDLR was established

Lysosomal acid lipase regulates VLDL synthesis and insulin sensitivity in mice

AIMS/HYPOTHESIS: Lysosomal acid lipase (LAL) hydrolyses cholesteryl esters and triacylglycerols (TG) within lysosomes to mobilise NEFA and cholesterol. Since LAL-deficient (Lal (-/-) ) mice suffer from progressive loss of adipose tissue and severe accumulation of lipids in hepatic lysosomes, we hypothesised that LAL deficiency triggers alternative energy pathway(s). METHODS: We studied metabolic adaptations in Lal (-/-) mice. RESULTS: Despite loss of adipose tissue, Lal (-/-) mice show enhanced glucose clearance during insulin and glucose tolerance tests and have increased uptake of [(3)H]2-deoxy-D-glucose into skeletal muscle compared with wild-type mice. In agreement, fasted Lal (-/-) mice exhibit reduced glucose and glycogen levels in skeletal muscle. We observed 84% decreased plasma leptin levels and significantly reduced hepatic ATP, glucose, glycogen and glutamine concentrations in fed Lal (-/-) mice. Markedly reduced hepatic acyl-CoA concentrations decrease the expression of peroxisome proliferator-activated receptor α (PPARα) target genes. However, treatment of Lal (-/-) mice with the PPARα agonist fenofibrate further decreased plasma TG (and hepatic glucose and glycogen) concentrations in Lal (-/-) mice. Depletion of hepatic nuclear factor 4α and forkhead box protein a2 in fasted Lal (-/-) mice might be responsible for reduced expression of microsomal TG transfer protein, defective VLDL synthesis and drastically reduced plasma TG levels. CONCLUSIONS/INTERPRETATION: Our findings indicate that neither activation nor inactivation of PPARα per se but rather the availability of hepatic acyl-CoA concentrations regulates VLDL synthesis and subsequent metabolic adaptations in Lal (-/-) mice. We conclude that decreased plasma VLDL production enhances glucose uptake into skeletal muscle to compensate for the lack of energy supply

Stratospheric aerosol - Observations, processes, and impact on climate

Interest in stratospheric aerosol and its role in climate have increased over the last decade due to the observed increase in stratospheric aerosol since 2000 and the potential for changes in the sulfur cycle induced by climate change. This review provides an overview about the advances in stratospheric aerosol research since the last comprehensive assessment of stratospheric aerosol was published in 2006. A crucial development since 2006 is the substantial improvement in the agreement between in situ and space-based inferences of stratospheric aerosol properties during volcanically quiescent periods. Furthermore, new measurement systems and techniques, both in situ and space based, have been developed for measuring physical aerosol properties with greater accuracy and for characterizing aerosol composition. However, these changes induce challenges to constructing a long-term stratospheric aerosol climatology. Currently, changes in stratospheric aerosol levels less than 20% cannot be confidently quantified. The volcanic signals tend to mask any nonvolcanically driven change, making them difficult to understand. While the role of carbonyl sulfide as a substantial and relatively constant source of stratospheric sulfur has been confirmed by new observations and model simulations, large uncertainties remain with respect to the contribution from anthropogenic sulfur dioxide emissions. New evidence has been provided that stratospheric aerosol can also contain small amounts of nonsulfate matter such as black carbon and organics. Chemistry-climate models have substantially increased in quantity and sophistication. In many models the implementation of stratospheric aerosol processes is coupled to radiation and/or stratospheric chemistry modules to account for relevant feedback processes

Emissions from a modern log wood masonry heater and wood pellet boiler : Composition and biological impact on air-liquid interface exposed human lung cancer cells

The consumption of wood fuel is markedly increasing in developing and industrialized countries. Known side effects of wood smoke inhalation manifest in proinflammatory signaling, oxidative stress, DNA damage and hence increased cancer risk. In this study, the composition and acute biological impact of emissions of state-of-the-art wood combustion compliances: masonry heater (MH) and pellet boiler (PB) were investigated. Therefore A549 cells were exposed to emission aerosols in an automated air-liquid interface exposure station followed by cytotoxicity, transcriptome and proteome analyses. In parallel, aerosols were subjected to a chemical and physical haracterization. Compared to PB, the MH combustion at the same dilution ratio resulted in a 3-fold higher particle mass concentration (PM2.5) and deposited dose (PB: 27 $\pm$ 2 ng/cm2, MH; 73 $\pm$ 12 ng/cm2). Additionally, the MH aerosol displayed a substantially larger concentration of aldehydes, polycyclic aromatic hydrocarbons (PAH) or oxidized PAH. Gene ontology analysis of transcriptome of A549 cells exposed to MH emissions revealed the activation of proinflammatory response and key signaling cascades MAP kinase and JAK-STAT. Furthermore, CYP1A1, an essential enzyme in PAH metabolism, was induced. PB combustion aerosol activated the proinflammatory marker IL6 and different transport processes. The proteomics data uncovered induction of DNA damage-associated proteins in response to PB and DNA doublestrand break processing proteins in response to MH emissions. Taking together, the MH produces emissions with a higher particle dose and more toxic compounds while causing only mild biological responses. This finding points to a significant mitigating effect of antioxidative compounds in MH wood smoke

Acyl-CoA:Diacylglycerol Acyltransferase 1 Expression Level in the Hematopoietic Compartment Impacts Inflammation in the Vascular Plaques of Atherosclerotic Mice.

The final step of triacylglycerol synthesis is catalyzed by acyl-CoA:diacylglycerol acyltransferases (DGATs). We have previously shown that ApoE-/-Dgat1-/- mice are protected from developing atherosclerosis in association with reduced foam cell formation. However, the role of DGAT1, specifically in myeloid and other hematopoietic cell types, in determining this protective phenotype is unknown. To address this question, we reconstituted the bone marrow of irradiated Ldlr-/-mice with that from wild-type (WT→ Ldlr-/-) and Dgat1-/-(Dgat1-/-→ Ldlr-/-) donor mice. We noted that DGAT1 in the hematopoietic compartment exerts a sex-specific effect on systemic cholesterol homeostasis. However, both male and female Dgat1-/-→ Ldlr-/-mice had higher circulating neutrophil and lower lymphocyte counts than control mice, suggestive of a classical inflammatory phenotype. Moreover, specifically examining the aortae of these mice revealed that Dgat1-/-→ Ldlr-/-mice have atherosclerotic plaques with increased macrophage content. This increase was coupled to a reduced plaque collagen content, leading to a reduced collagen-to-macrophage ratio. Together, these findings point to a difference in the inflammatory contribution to plaque composition between Dgat1-/-→ Ldlr-/-and control mice. By contrast, DGAT1 deficiency did not affect the transcriptional responses of cultured macrophages to lipoprotein treatment in vitro, suggesting that the alterations seen in the plaques of Dgat1-/-→ Ldlr-/-mice in vivo do not reflect a cell intrinsic effect of DGAT1 in macrophages. We conclude that although DGAT1 in the hematopoietic compartment does not impact the overall lipid content of atherosclerotic plaques, it exerts reciprocal effects on inflammation and fibrosis, two processes that control plaque vulnerability

Acyl-CoA:Diacylglycerol Acyltransferase 1 Expression Level in the Hematopoietic Compartment Impacts Inflammation in the Vascular Plaques of Atherosclerotic Mice.

The final step of triacylglycerol synthesis is catalyzed by acyl-CoA:diacylglycerol acyltransferases (DGATs). We have previously shown that ApoE-/-Dgat1-/- mice are protected from developing atherosclerosis in association with reduced foam cell formation. However, the role of DGAT1, specifically in myeloid and other hematopoietic cell types, in determining this protective phenotype is unknown. To address this question, we reconstituted the bone marrow of irradiated Ldlr-/-mice with that from wild-type (WT→ Ldlr-/-) and Dgat1-/-(Dgat1-/-→ Ldlr-/-) donor mice. We noted that DGAT1 in the hematopoietic compartment exerts a sex-specific effect on systemic cholesterol homeostasis. However, both male and female Dgat1-/-→ Ldlr-/-mice had higher circulating neutrophil and lower lymphocyte counts than control mice, suggestive of a classical inflammatory phenotype. Moreover, specifically examining the aortae of these mice revealed that Dgat1-/-→ Ldlr-/-mice have atherosclerotic plaques with increased macrophage content. This increase was coupled to a reduced plaque collagen content, leading to a reduced collagen-to-macrophage ratio. Together, these findings point to a difference in the inflammatory contribution to plaque composition between Dgat1-/-→ Ldlr-/-and control mice. By contrast, DGAT1 deficiency did not affect the transcriptional responses of cultured macrophages to lipoprotein treatment in vitro, suggesting that the alterations seen in the plaques of Dgat1-/-→ Ldlr-/-mice in vivo do not reflect a cell intrinsic effect of DGAT1 in macrophages. We conclude that although DGAT1 in the hematopoietic compartment does not impact the overall lipid content of atherosclerotic plaques, it exerts reciprocal effects on inflammation and fibrosis, two processes that control plaque vulnerability

Biocatalytic and Bioelectrocatalytic Approaches for the Reduction of Carbon Dioxide using Enzymes

In the recent decade, CO2 has increasingly been regarded not only as a greenhouse gas but even more as a chemical feedstock for carbon-based materials. Different strategies have evolved to realize CO2 utilization and conversion into fuels and chemicals. In particular, biological approaches have drawn attention, as natural CO2 conversion serves as a model for many processes. Microorganisms and enzymes have been studied extensively for redox reactions involving CO2. In this review, we focus on monitoring nonliving biocatalyzed reactions for the reduction of CO2 by using enzymes. We depict the opportunities but also challenges associated with utilizing such biocatalysts. Besides the application of enzymes with co-factors, resembling natural processes, and co-factor recovery, we also discuss implementation into photochemical and electrochemical techniques

Altered circulating immune cell numbers in <i>Dgat1</i>^{<i>–/–</i>}<i>→ Ldlr</i>^{<i>–/–</i>}mice fed a WTD.

<p>(A) Total leukocyte counts, and relative (B) monocyte, (C) eosinophil, (D) basophil, (E) lymphocyte, and (F) neutrophil counts in both <i>WT→ Ldlr</i>^{<i>–/–</i>}and <i>Dgat1</i>^{<i>–/–</i>}<i>→ Ldlr</i>^{<i>–/–</i>}after 13 weeks (females, ♀) and 19 weeks (males, ♂) of WTD feeding (n = 9 per group), showing reduced lymphocyte and increased neutrophil counts in male mice, with a similar trend in female mice. Data are presented as mean ± SEM. **, p ≤ 0.01; ***, p ≤ 0.001.</p

Influence of molecular designs on polaronic and vibrational transitions in a conjugated push-pull copolymer

Electron-phonon interactions of free charge-carriers in doped pi-conjugated polymers are conceptually described by 1-dimensional (1D) delocalization. Thereby, polaronic transitions fit the 1D-Froehlich model in quasi-confined chains. However, recent developments in conjugated polymers have diversified the backbones to become elaborate heterocylcic macromolecules. Their complexity makes it difficult to investigate the electron-phonon coupling. In this work we resolve the electron-phonon interactions in the ground and doped state in a complex push-pull polymer. We focus on the polaronic transitions using in-situ spectroscopy to work out the differences between single-unit and push-pull systems to obtain the desired structural- electronic correlations in the doped state. We apply the classic 1D-Froehlich model to generate optical model fits. Interestingly, we find the 1D-approach in push-pull polarons in agreement to the model, pointing at the strong 1D-character and plain electronic structure of the push-pull structure. In contrast, polarons in the single-unit polymer emerge to a multi-dimensional problem difficult to resolve due to their anisotropy. Thus, we report an enhancement of the 1D-character by the push-pull concept in the doped state - an important view in light of the main purpose of push-pull polymers for photovoltaic devices