Long-term exposure to inorganic arsenic (iAs) through drinking water has been associated with cognitive impairment in children and adults; however, the related pathogenic mechanisms have not been completely described. Increased or chronic inflammation in the brain is linked to impaired cognition and neurodegeneration; iAs induces strong inflammatory responses in several cells, but this effect has been poorly evaluated in central nervous system (CNS) cells. Because astrocytes are the most abundant cells in the CNS and play a critical role in brain homeostasis, including regulation of the inflammatory response, any functional impairment in them can be deleterious for the brain. We propose that iAs could induce cognitive impairment through inflammatory response activation in astrocytes. In the present work, rat cortical astrocytes were acutely exposed in vitro to the monomethylated metabolite of iAs (MMA(III)), which accumulates in glial cells without compromising cell viability. MMA(III) LD50 in astrocytes was 10.52 μM, however, exposure to sub-toxic MMA(III) concentrations (50-1000 nM) significantly increased IL-1β, IL-6, TNF-α, COX-2, and MIF-1 gene expression. These effects were consistent with amyloid precursor protein (APP) and β-secretase (BACE-1) increased gene expression, mainly for those MMA(III) concentrations that also induced TNF-α over-expression. Other effects of MMA(III) on cortical astrocytes included increased proliferative and metabolic activity. All tested MMA(III) concentrations led to an inhibition of intracellular lactate dehydrogenase (LDH) activity. Results suggest that MMA(III) induces important metabolic and functional changes in astrocytes that may affect brain homeostasis and that inflammation may play a major role in cognitive impairment-related pathogenicity in As-exposed populations.

Alzheimer’s disease (AD) is a highly complex and rapidly progressive neurodegenerative disorder characterized by the systemic collapse of cognitive function and formation of dense amyloid-β (Aβ) plaques and neurofibrillary tangles (NFTs). AD pathology is derived from the cholinergic, amyloid and tau hypotheses, respectively. Current pharmacotherapy with known anti-cholinesterases, such as Aricept ® and Exelon ®, only offer symptomatic relief without any disease-modifying effects (DMEs). It is now clear that in order to prevent the rapid progression of AD, new therapeutic treatments should target multiple AD pathways as opposed to the traditional “one drug, one target” approach. This research project employed medicinal chemistry tools to develop multifunctional small organic molecules against three key targets of AD pathology – the cholinesterases (AChE and BuChE), AChE-induced and self-induced Aβ1-40 aggregation and generation (β-secretase). A chemical library composed of 112 derivatives was generated to gather structure-activity relationship (SAR) data. The derivatives were based on a novel, non-fused, 2,4-disubstituted pyrimidine ring (2,4-DPR) template with substituents at the C-2 and C-4 position varying in size, steric and electronic properties. Molecular modeling was utilized to investigate their binding modes within the target enzymes and along with the acquired SAR, the chemical library was screened to identify lead multifunctional candidates.

Alzheimer’s disease (AD) is a highly complex and rapidly progressive neurodegenerative disorder characterized by the systemic collapse of cognitive function and formation of dense amyloid-β (Aβ) plaques and neurofibrillary tangles (NFTs). AD pathology is derived from the cholinergic, amyloid and tau hypotheses, respectively. Current pharmacotherapy with known anti-cholinesterases, such as Aricept ® and Exelon ®, only offer symptomatic relief without any disease-modifying effects (DMEs). It is now clear that in order to prevent the rapid progression of AD, new therapeutic treatments should target multiple AD pathways as opposed to the traditional “one drug, one target” approach. This research project employed medicinal chemistry tools to develop multifunctional small organic molecules against three key targets of AD pathology – the cholinesterases (AChE and BuChE), AChE-induced and self-induced Aβ1-40 aggregation and generation (β-secretase). A chemical library composed of 112 derivatives was generated to gather structure-activity relationship (SAR) data. The derivatives were based on a novel, non-fused, 2,4-disubstituted pyrimidine ring (2,4-DPR) template with substituents at the C-2 and C-4 position varying in size, steric and electronic properties. Molecular modeling was utilized to investigate their binding modes within the target enzymes and along with the acquired SAR, the chemical library was screened to identify lead multifunctional candidates.

This work reports on the successful formation of supported multicomponent lipid bilayer membranes (sLBMs) from natural occurring lipids as well as synthetic lipids on a set of polymer cushions consisting of alternating maleic acid copolymers. Maleic acid copolymers provide a versatile platform to adjust the physico-chemical behaviour by the choice of the comonomer unit. The formation of sLBMs was triggered by a transient reduction of the electrostatic repulsion between the polymer cushions and the lipid vesicles by lowering the solutions pH to 4. Upon formation the stability of sLBMs was not affected by subsequent variations of the environmental pH to 7.2. Even drastic changes in the environmental pH (between pH 2 and pH 9) did not lead to delamination and proved the stability of the polymer sLBM. The degree of hydrophilicity and swelling of the anionic polymer cushions was found to determine both the kinetics of the membrane formation and the mobility of the lipid bilayer with lipid diffusion coefficients in the range from 0.26 to 2.6 µm2 s-1. An increase in cushion hydrophilicity correlated with a strong increase in the diffusion coefficient of the lipids. This trend was found to correlate with the kinetics of bilayer formation in the process of vesicle spreading. The observations strongly support the important role of the support’s polarity for the fluidity of the sLBM, which is probably related to the presence of a water layer between support and bilayer. The investigated polymer cushions are considered to open new options for the in situ modulation of lipid bilayer membranes characteristics to match the requirements for the successful integration of functional transmembrane proteins (TMPs). As each cushion exhibits different physico-chemical properties, the resulting behaviour of the sLBMs and TMPs could be exactly adjusted to the specific requirements of biological samples. This is exemplarily shown by the integration of the TMP beta amyloid precursor protein cleaving enzyme (BACE). Integrated BACE was observed to be mobile on all polymer cushions. On the contrary, no lateral mobility of BACE was found in solid sLBM. Furthermore, the activity of integrated BACE was analysed by the cleavage of an amyloid precursor protein analogue. Remarkably, the polymer cushions did not only enhance the mobility but were also found to increase the activity of BACE by a factor of 1.5 to 2.5 in comparison to solid sLBM. From the obtained results it is obvious that even small cytoplasmic domains of transmembrane proteins might not be preserved upon the integration in silica sLBM. The observed beneficial effects of the utilised polymer cushions on the mobility and activity of transmembrane proteins motivate further studies to clarify the general applicability of the polymer platform. Altogether, this polymer platform provides valuable options to form sLBM with varying characteristics to reconstitute transmembrane proteins for a wide range of possible future applications in biology. / Die vorliegende Arbeit beschreibt die Bildung von polymer unterstützten Lipiddoppelschichten zur Integration von transmembranen Proteinen. Das Polymerkissensystem besteht aus alternierenden Maleinsäurecopolymeren. Lipiddoppelschichten wurden durch die Steuerung der elektrostatischen Repulsion erzeugt: die Verringerung des pH-Wertes auf 4 wurde eine Erhöhung der adsorbierten Vesikelmenge auf den Polymeroberflächen induziert. Nach der erfolgten Bildung der Lipiddoppelschichten kann der pH-Wert beliebig variiert werden, ohne dass die Stabilität der Lipiddoppelschichten beeinflusst wird. Auch drastische Veränderungen des pH-Milieus (pH 2 - pH 9) führten zu keinen Veränderungen in der Membranintegrität. Der Grad der Hydrophilie und der Quellung der anionischen Polymerschichten beeinflusst sowohl die Bildung der Modellmembranen als auch die Mobilität der integrierten Lipidmoleküle. Dabei reichen die erzielten Lipiddiffusionskoeffizienten von 0.26 bis 2.6 µm2 s-1. Dabei ist die Mobilität direkt von der Hydrophilie des Substrates abhängig. Die beobachteten Ergebnisse zeigen deutlich die entscheidende Rolle der Polarität der verwendeten Substratoberflächen auf die Lipidmobilität, die sehr wahrscheinlich mit der Präsenz einer variablen Wasserschicht zusammenhängt. Die untersuchten Polymerkissen eröffnen neue Möglichkeiten für die insitu Modulierung der Charakteristika von Lipidschichten, um funktionale transmembrane Proteine zu integrieren. Aufgrund der unterschiedlichen physiko-chemischen Eigenschaften kann das Verhalten der Lipidschichten und der transmembranen Proteine nach den spezifischen Anforderungen des Modellsystems angepasst werden. Die funktionale Integration wurde am Beispiel des transmembranen Proteins BACE nachempfunden. Die Mobilität des integrierten BACE wurde auf allen Polymerkissen beobachtet. Im Gegensatz dazu wurde auf harten Substraten keine BACE Mobilität gefunden. Die Aktivität des integrierten BACE wurde durch die enzymatische Spaltung eines APP-Analogons nachgewiesen. Bemerkenswerteweise wurde ein Anstieg der BACE Aktivität auf den Polymerkissen um den Faktor 1,5 bis 2,5 im Vergleich zu den auf harten Substraten integrierten BACE beobachtet. Zusammenfassend, die verwendeten Polymerkissen bieten vielfältige Möglichkeiten Lipidschichten mit variierenden Eigenschaften für die Integration von transmembranen Proteinen zu erzeugen.

No description available.

This work reports on the successful formation of supported multicomponent lipid bilayer membranes (sLBMs) from natural occurring lipids as well as synthetic lipids on a set of polymer cushions consisting of alternating maleic acid copolymers. Maleic acid copolymers provide a versatile platform to adjust the physico-chemical behaviour by the choice of the comonomer unit. The formation of sLBMs was triggered by a transient reduction of the electrostatic repulsion between the polymer cushions and the lipid vesicles by lowering the solutions pH to 4. Upon formation the stability of sLBMs was not affected by subsequent variations of the environmental pH to 7.2. Even drastic changes in the environmental pH (between pH 2 and pH 9) did not lead to delamination and proved the stability of the polymer sLBM. The degree of hydrophilicity and swelling of the anionic polymer cushions was found to determine both the kinetics of the membrane formation and the mobility of the lipid bilayer with lipid diffusion coefficients in the range from 0.26 to 2.6 µm2 s-1. An increase in cushion hydrophilicity correlated with a strong increase in the diffusion coefficient of the lipids. This trend was found to correlate with the kinetics of bilayer formation in the process of vesicle spreading. The observations strongly support the important role of the support’s polarity for the fluidity of the sLBM, which is probably related to the presence of a water layer between support and bilayer. The investigated polymer cushions are considered to open new options for the in situ modulation of lipid bilayer membranes characteristics to match the requirements for the successful integration of functional transmembrane proteins (TMPs). As each cushion exhibits different physico-chemical properties, the resulting behaviour of the sLBMs and TMPs could be exactly adjusted to the specific requirements of biological samples. This is exemplarily shown by the integration of the TMP beta amyloid precursor protein cleaving enzyme (BACE). Integrated BACE was observed to be mobile on all polymer cushions. On the contrary, no lateral mobility of BACE was found in solid sLBM. Furthermore, the activity of integrated BACE was analysed by the cleavage of an amyloid precursor protein analogue. Remarkably, the polymer cushions did not only enhance the mobility but were also found to increase the activity of BACE by a factor of 1.5 to 2.5 in comparison to solid sLBM. From the obtained results it is obvious that even small cytoplasmic domains of transmembrane proteins might not be preserved upon the integration in silica sLBM. The observed beneficial effects of the utilised polymer cushions on the mobility and activity of transmembrane proteins motivate further studies to clarify the general applicability of the polymer platform. Altogether, this polymer platform provides valuable options to form sLBM with varying characteristics to reconstitute transmembrane proteins for a wide range of possible future applications in biology. / Die vorliegende Arbeit beschreibt die Bildung von polymer unterstützten Lipiddoppelschichten zur Integration von transmembranen Proteinen. Das Polymerkissensystem besteht aus alternierenden Maleinsäurecopolymeren. Lipiddoppelschichten wurden durch die Steuerung der elektrostatischen Repulsion erzeugt: die Verringerung des pH-Wertes auf 4 wurde eine Erhöhung der adsorbierten Vesikelmenge auf den Polymeroberflächen induziert. Nach der erfolgten Bildung der Lipiddoppelschichten kann der pH-Wert beliebig variiert werden, ohne dass die Stabilität der Lipiddoppelschichten beeinflusst wird. Auch drastische Veränderungen des pH-Milieus (pH 2 - pH 9) führten zu keinen Veränderungen in der Membranintegrität. Der Grad der Hydrophilie und der Quellung der anionischen Polymerschichten beeinflusst sowohl die Bildung der Modellmembranen als auch die Mobilität der integrierten Lipidmoleküle. Dabei reichen die erzielten Lipiddiffusionskoeffizienten von 0.26 bis 2.6 µm2 s-1. Dabei ist die Mobilität direkt von der Hydrophilie des Substrates abhängig. Die beobachteten Ergebnisse zeigen deutlich die entscheidende Rolle der Polarität der verwendeten Substratoberflächen auf die Lipidmobilität, die sehr wahrscheinlich mit der Präsenz einer variablen Wasserschicht zusammenhängt. Die untersuchten Polymerkissen eröffnen neue Möglichkeiten für die insitu Modulierung der Charakteristika von Lipidschichten, um funktionale transmembrane Proteine zu integrieren. Aufgrund der unterschiedlichen physiko-chemischen Eigenschaften kann das Verhalten der Lipidschichten und der transmembranen Proteine nach den spezifischen Anforderungen des Modellsystems angepasst werden. Die funktionale Integration wurde am Beispiel des transmembranen Proteins BACE nachempfunden. Die Mobilität des integrierten BACE wurde auf allen Polymerkissen beobachtet. Im Gegensatz dazu wurde auf harten Substraten keine BACE Mobilität gefunden. Die Aktivität des integrierten BACE wurde durch die enzymatische Spaltung eines APP-Analogons nachgewiesen. Bemerkenswerteweise wurde ein Anstieg der BACE Aktivität auf den Polymerkissen um den Faktor 1,5 bis 2,5 im Vergleich zu den auf harten Substraten integrierten BACE beobachtet. Zusammenfassend, die verwendeten Polymerkissen bieten vielfältige Möglichkeiten Lipidschichten mit variierenden Eigenschaften für die Integration von transmembranen Proteinen zu erzeugen.

Hepatocyte Growth Factor (HGF)–induced Epithelial–Mesenchymal Transition (EMT) is a complex cellular pathway that causes epithelial cell scattering by breaking cell–cell contacts, eliminating apical–basal polarity, and replacing epithelial markers and characteristics with mesenchymal markers. Early EMT events include a brief period of cell spreading, followed by cell compaction and cell–cell contact breaks. A forward chemical genetics drug screen of 50,000 unique compounds measuring HGF–induced cell scattering identified 26 novel EMT inhibitors, including 2 proteolytic inhibitors. Here, we show that B5500–4, one of the EMT inhibitors from the screen, blocks HGF–induced EMT by a predicted blocking of the protease furin, in addition to secondarily blocking Beta–Secretase (BACE).We also show that MMP–12 and MMP–9 are required for HGF–induced EMT to progress. MMP–12 is required for cell contraction, and its inhibition produces a continuous cell spreading phenotype.We also demonstrate that both furin and BACE activity are required for HGF–induced EMT to proceed, but that they are involved in separate pathways. We show that BACE inhibition leads to a failure of cell spreading in early EMT, and that EphA2 is a member of this pathway. We also demonstrate that it is likely BACE2, and not BACE1 that is responsible for early cell spreading. Furin is also required for HGF–induced cell scattering, but does not play a role in the cell spreading process. These findings highlight the importance of proteolytic activity at the earliest stages of HGF–induced EMT.

Dans l’épiderme, les mélanocytes participent à la protection de la peau contre les rayons ionisants du soleil en synthétisant un pigment, la mélanine, dans des compartiments apparentés aux lysosomes appelés melanosomes. La mélanogenèse est un processus séquentiel initié par la production de fibres amyloïdes dont la composante principale est la protéine PMEL. Ces fibres séquestrent la mélanine et permettent l’élimination d’intermédiaires toxiques produits lors de sa synthèse. La mélanogenèse et le phénotype pigmenté sont affectés lorsque le processus de formation des fibres est altéré. Les fibres résultent du clivage de PMEL dans les endosomes précurseurs des mélanosomes mais les protéases impliquées dans ce processus restent peu ou pas caractérisées. Afin de mieux comprendre les mécanismes de formation des fibres amyloïdes dérivées de PMEL, j’ai étudié le rôle de deux protéases : les Bêta-sécrétases BACE1 et BACE2. En combinant des techniques de biochimie, d’immunocytochimie et d’imagerie photonique et électronique, j’ai montré que la perte de l’expression de Bace2 in vivo (souris KO BACE2) ou sa déplétion (siRNA) dans une lignée de mélanocytes inhibent le clivage amyloïdogénique de PMEL et affectent à la fois la formation de fibres de PMEL dans les mélanosomes et la pigmentation. J’ai pu notamment reproduire in vitro le clivage spécifique de PMEL en utilisant une forme recombinante de BACE2. En parallèle, j’ai également étudié le rôle de BACE1 dans la mélanogenèse. Mes résultats indiquent que BACE1, bien que n’étant pas impliquée dans le clivage de PMEL, régulerait la maturation des mélanosomes précoces in vivo et in cellulo, en modulant les contacts entre mélanosomes et réticulum endoplasmique (RE). Dans les mélanocytes, BACE1 est présente dans le RE et interagit avec des protéines impliquées dans les contacts RE-endosomes. Ces contacts seraient cruciaux pour le transfert de molécules nécessaires à la maturation des mélanosomes. L’ensemble de ces résultats démontre un rôle pour chacune des Bêta-sécrétases dans le processus de mélanogenèse, levant le voile sur des processus clés liés à la biogenèse des mélanosomes. Par ailleurs, les fibres de PMEL constituant le modèle le plus abouti de l’amyloïdogenèse physiologique chez les mammifères, ces études pourraient à plus long terme aider à la compréhension de la formation des fibres amyloïdes pathologiques ; notamment dans la maladie d’Alzheimer où l’amyloïdogenèse d’APP est très similaire à celle de PMEL. / In the epidermis, melanocytes synthetize a pigment called melanin, in lysosome-related-organelles called melanosomes, in order to protect the skin against the ionizing radiations of the sun. Melanogenesis is a sequential process initiated by the formation of amyloid fibrils whose principal component is the protein PMEL. Those fibrils sequester the melanin pigment and allow the removal of toxic intermediates formed during its synthesis. Melanogenesis and the pigmented phenotype are affected when the process of fibrils formation is altered. Fibrils come from the processing of PMEL in endosome precursors of melanosomes but the proteases implicated in this process are not well characterized. In order to better understand the mechanisms implicated in the formation of the PMEL amyloid fibrils, I studied the role of two proteases: the Beta-secretases BACE1 and BACE2. Using a combination of biochemical, immunocytochemical methods and photonic and electronic imaging, I have shown that the loss of Bace2 expression in vivo (BACE2 KO mice) or its depletion (siRNA), in a melanocyte cell line, inhibit the amyloidogenic processing of PMEL and affect both the formation of the PMEL fibrils in melanosomes and pigmentation. I could reproduce in vitro the specific cleavage of PMEL by using a recombinant form of BACE2. In parallel, I have also studied the role of BACE1 in melanogenesis. My results indicate that BACE1, even though it is not implicated in PMEL processing, could regulate the maturation of early melanosomes in vivo and in cellulo, by modulating the contacts between melanosomes and endoplasmic reticulum (ER). In melanocytes, BACE1 is present in the ER and interacts with proteins implicated in ER-endosomes contacts. Those contacts would be crucial for the transfer of molecules that are necessary for melanosome maturation. All together those results demonstrate the role of both Beta-secretases in melanogenesis, and reveal key processes involved in melanosome biogenesis. Moreover, because PMEL fibrils are the most completed model of physiological amyloidogenesis in mammals, theses studies could help in the future the understanding of the formation of pathological amyloid fibrils; in particular in the Alzheimer’s disease where the amyloidogenesis of APP is very similar to the one of PMEL.

Die Alzheimer Krankheit ist die häufigste Altersdemenz. Ein spezifisches pathologisches Merkmal der Alzheimer Krankheit ist die Amyloid-Ablagerung im Gehirn. Die Hauptkomponente der so genannten Amyloid-Plaques ist das Amyloid beta-Peptid (A-beta). A-beta entsteht durch sequenzielle proteolytische Spaltung aus einem membrangebundenen Vorläuferprotein, dem beta-APP (betaamyloid precursor protein). Die kürzlich identifizierte beta-Sekretase (BACE, beta-site APPcleaving enzyme) generiert den Schnitt am N-Terminus von A-beta. Es entsteht ein C-terminales, membrangebundenes beta-APP-Fragment, das beta-APP-CTF. Beta-APP-CTF ist das direkte Substrat für die gamma-Sekretase, die innerhalb der Membrandomäne schneidet, wodurch A-beta freigesetzt wird. In der vorliegenden Arbeit kann erstmalig gezeigt werden, dass BACE auf dem sekretorischen Transportweg aus dem Endoplasmatischen Retikulum (ER), über den Golgi-Apparat zur Zelloberfläche transportiert wird. Auf dem Transport wird BACE durch N-Glycosylierung und Propeptidabspaltung posttranslational modifiziert. BACE wird im ER N-glycosyliert und die mannosereichen Zucker werden auf dem Transport durch den Golgi-Apparat in Endoglycosidase H resistente Zucker des komplexen Typs modifiziert. Die Propeptidabspaltung, durch Furin oder furinähnliche Propeptidkonvertasen, findet unmittelbar vor dem Aufbau der komplexen Zucker statt. Ferner konnte gezeigt werden, dass der Transport von BACE die A-beta-Entstehung limitieren kann. In polarisierten Madin-Darby canine kidney (MDCK) Zellen wird BACE überwiegend zur apikalen Plasmamembran transportiert und damit entgegengesetzt zu seinem Substrat beta-APP. Der gegensätzliche Transport von BACE und beta-APP begrenzt die A-beta Entstehung. Wird der apikale Transport von beta-APP durch Deletion seines basolateralen Sortierungssignals erhöht, entsteht vermehrt A-beta. Der differenzielle Transport von BACE und beta-APP könnte ein Hinweis darauf sein, dass beta-APP nicht das physiologische Substrat von BACE ist. / Alzheimer`s disease is the most common cause of progressive cognitive decline in the aged population. Pathologically Alzheimer`s disease is characterized by the invariant accumulation of senile plaques. Senile plaques are predominantly composed of the amyloid beta-peptide (A-beta), which is derived from the membrane bound beta-amyloid precursor protein (beta-APP) by sequential proteolytic cleavage. The recently identified beta-secretase (BACE) is responsible for the cleavage at the N-terminus of the A-beta domain. This cleavage generates membrane-bound beta-APP-Cterminal fragments (beta-APP-CTF) which are the immediate precursor for gamma-secretase cleavage and therefore for liberation of A-beta. The present work shows that BACE moves along the secretory pathway, while it undergoes post-translational modifications, which can be monitored by a significant increase in the molecular mass and cleavage of its pro-peptide. BACE becomes N-glycosylated within the ER and the increase in molecular mass is caused by complex N-glycosylation. The mature form of BACE is resistant to endoglycosidase H treatment; this indicates that BACE traffics through the Golgi. Furthermore the mature form of BACE does not contain the pro-peptide anymore. Pro-BACE is predominantly located within the endoplasmic reticulum. Pro-peptide cleavage occurs immediately before full maturation by furin or a furin-like proprotein convertase. Moreover traffic of BACE can limit A-beta generation. In the well established model system of polarized Madin-Darby canine kidney (MDCK) cells, the majority of BACE is sorted to the apical domain. Interestingly it has been shown previously that the substrate of BACE, beta-APP is transported to the basolateral surface of MCDK cells. Therefore, substantial amounts of BACE are targeted away from beta-APP to a non-amyloidogenic compartment, a cellular mechanism that limits A-beta generation. Upon deletion of the basolateral sorting signal of beta-APP, apically missorted beta-APP is processed by BACE. The differential targeting of BACE and its substrate beta-APP suggest that beta-APP might not be the major physiological substrate of BACE.

Proteolytic processing of the amyloid precursor protein (APP) may lead to the formation of the Abeta peptide, the major constituent of amyloid plaques in Alzheimer`s disease. The full-length APP is a substrate for at least 2 different (alpha and beta) proteases ("secretases"). The beta-secretase, BACE, cleaves APP in the first step of processing leading to the formation of the neurotoxic Abeta. BACE competes for APP with alpha-secretase, which cleaves APP within its Abeta sequence, thus precluding Abeta formation. It is thus important to understand how is the access of the alpha- and beta-secretase to APP regulated and how are the individual activities of these secretases modulated. Both these regulatory mechanisms, access to substrate and direct activity modulation, can be determined by the lipid composition of the membrane. Integral membrane proteins (like APP and BACE), can be viewed as solutes in a two-dimensional liquid membrane, and as such their state, and biological activity, critically depend on the physico-chemical character (fluidity, curvature, surface charge distribution, lateral domain heterogeneity etc.) of the lipid bilayer. These collective membrane properties will influence the activity of embedded membrane proteins. In addition, activity regulation may involve a direct interaction with a specific lipid (cofactor or co-structure function). Interactions of membrane proteins are furthermore affected by lateral domain organization of the membrane. Previous results had suggested that the regulation of the activity of the alpha- and beta-secretases and of their access to APP is lipid dependent, and involves lipid rafts. Using the baculovirus expression system, we have purified recombinant human full-length APP and BACE to homogeneity, and reconstituted them in large (~100nm, LUVs) and giant (10-150microm, GUVs) unilamellar vesicles. Using a soluble peptide substrate mimicking the beta-cleavage site of APP, we have examined the involvement of individual lipid species in modulating BACE activity in LUVs of various lipid compositions. We have identified 3 groups of lipids that stimulate proteolytic activity of BACE: 1.cerebrosides, 2.anionic glycerophospholipids, 3. cholesterol. Furthermore, we have co-reconstituted APP and BACE together in LUVs and demonstrated that BACE cleaves APP at the correct site, generating the beta-cleaved ectodomain identical to that from cells. We have developed an assay to quantitatively follow the beta-cleavage in proteoliposomes, and we have shown that the rate of cleavage in total brain lipid proteoliposomes is higher than in phosphatidylcholine vesicles. We have also studied partitioning of APP and BACE in GUVs between liquid ordered (lo) and liquid disordered (ld) phases. In this system, significant part of the BACE pool (about 20%) partitions into the lo phase, and its partitioning into lo phase can be further enhanced by cross-linking of membrane components. Only negligible fraction of APP can be found in the lo phase. We continue to study the behavior of co-reconstituted APP and BACE in GUVs The work presented in this thesis has yielded some interesting results and raised further questions. One of the important assignments of this project will in the next stage be the characterization of the impact of membrane domain organization on the beta-cleavage. Different domain arrangements that can be hypothesized in cell membranes can be modeled by varying the degree of phase fragmentation in proteoliposomes comprising reconstituted APP and BACE.