P-I class metalloproteinase from Bothrops moojeni venom is a post-proline cleaving peptidase with kininogenase activity: Insights into substrate selectivity and kinetic behavior

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Dimerization of β-site β-amyloid precursor protein-cleaving enzyme

Cleavage of the beta-amyloid precursor protein (APP) by the aspartyl protease beta-site APP-cleaving enzyme (BACE) is the first step in the generation of the amyloid beta-peptide, which is deposited in the brain of Alzheimer's disease patients. Whereas the subsequent cleavage by gamma-secretase was shown to originate from the cooperation of a multicomponent complex, it is currently unknown whether in a cellular environment BACE is enzymatically active as a monomer or in concert with other proteins. Using blue native gel electrophoresis we found that endogenous and overexpressed BACE has a molecular mass of 140 kDa instead of the expected mass of 70 kDa under denaturing conditions. This suggests that under native conditions BACE exists as a homodimer. Homodimerization was confirmed by co-immunoprecipitation of full-length BACE carrying different epitope tags. In contrast, the soluble active BACE ectodomain was exclusively present as a monomer both under native and denaturing conditions. A domain analysis revealed that the BACE ectodomain dimerized as long as it was attached to the membrane, whereas the cytoplasmic domain and the transmembrane domain were dispensable for dimerization. By adding a KKXX-endoplasmic reticulum retention signal to BACE, we demonstrate that dimerization of BACE occurs already before full maturation and pro-peptide cleavage. Furthermore, kinetic analysis of the purified native BACE dimer revealed a higher affinity and turnover rate in comparison to the monomeric soluble BACE. Dimerization of BACE might, thus, facilitate binding and cleavage of physiological substrates.

Lactone-ring-cleaving enzyme: Genetic analysis, novel RNA editing, and evolutionary implications

A lactonohydrolase from Fusarium oxysporum AKU 3702 is an enzyme catalyzing the hydrolysis of aldonate lactones to the corresponding aldonic acids. The amino acid sequences of the NH2 terminus and internal peptide fragments of the enzyme were determined to prepare synthetic oligonucleotides as primers for the PCR. An approximate 1,000-base genomic DNA fragment thus amplified was used as the probe to clone both genomic DNA and cDNA for the enzyme. The lactonohydrolase genomic gene consists of six exons separated by five short introns. A novel type of RNA editing, in which lactonohydrolase mRNA included the insertion of guanosine and cytidine residues, was observed. The predicted amino acid sequence of the cloned lactonohydrolase cDNA showed significant similarity to those of the gluconolactonase from Zymomonas mobilis, and paraoxonases from human and rabbit, forming a unique superfamily consisting of C-O cleaving enzymes and P-O cleaving enzymes. Lactonohydrolase was expressed under the control of the lac promoter in Escherichia coli.

Peptides from Lactobacillus hydrolysates of bovine milk caseins inhibit prolyl-peptidases of human colon cells.

Prolyl-rich peptides derived from hydrolysates of bovine caseins have been previously shown to inhibit angiotensin converting enzyme (ACE) activity, suggesting that they may also be able to inhibit the enzymatic activities of prolyl-specific peptidases. This study shows that peptides derived from α(S1)-casein and β-casein inhibited the enzymatic activities of purified recombinant matrix metalloprotease (MMP)-2, MMP-7, and MMP-9. The inhibitory efficacy was sequence-dependent. These peptides also selectively inhibited the enzymatic activities of prolyl-amino-peptidases, prolyl-amino-dipeptidases, and prolyl-endopeptidases in extracts of HT-29 and SW480 human colon carcinoma cells, but not in intact cells. They were not cytotoxic or growth inhibitory for these cells. Thus, the prolyl-rich selected peptides were good and selective inhibitors of MMPs and post-proline-cleaving proteases, demonstrating their potential to control inadequate proteolytic activity in the human digestive tract, without inducing cytotoxic effects.

Design and synthesis of novel non peptidomimtic beta-secretase inhibitors in the treatment of Alzheimer's disease

The aspartic protease BACE1 (β-amyloid precursor protein cleaving enzyme, β-secretase) is recognized as one of the most promising targets in the treatment of Alzheimer's disease (AD). The accumulation of β-amyloid peptide (Aβ) in the brain is a major factor in the pathogenesis of AD. Aβ is formed by initial cleavage of β-amyloid precursor protein (APP) by β-secretase, therefore BACE1 inhibition represents one of the therapeutic approaches to control progression of AD, by preventing the abnormal generation of Aβ. For this reason, in the last decade, many research efforts have focused at the identification of new BACE1 inhibitors as drug candidates. Generally, BACE1 inhibitors are grouped into two families: substrate-based inhibitors, designed as peptidomimetic inhibitors, and non-peptidomimetic ones. The research on non-peptidomimetic small molecules BACE1 inhibitors remains the most interesting approach, since these compounds hold an improved bioavailability after systemic administration, due to a good blood-brain barrier permeability in comparison to peptidomimetic inhibitors. Very recently, our research group discovered a new promising lead compound for the treatment of AD, named lipocrine, a hybrid derivative between lipoic acid and the AChE inhibitor (AChEI) tacrine, characterized by a tetrahydroacridinic moiety. Lipocrine is one of the first compounds able to inhibit the catalytic activity of AChE and AChE-induced amyloid-β aggregation and to protect against reactive oxygen species. Due to this interesting profile, lipocrine was also evaluated for BACE1 inhibitory activity, resulting in a potent lead compound for BACE1 inhibition. Starting from this interesting profile, a series of tetrahydroacridine analogues were synthesised varying the chain length between the two fragments. Moreover, following the approach of combining in a single molecule two different pharmacophores, we designed and synthesised different compounds bearing the moieties of known AChEIs (rivastigmine and caproctamine) coupled with lipoic acid, since it was shown that dithiolane group is an important structural feature of lipocrine for the optimal inhibition of BACE1. All the tetrahydroacridines, rivastigmine and caproctamine-based compounds, were evaluated for BACE1 inhibitory activity in a FRET (fluorescence resonance energy transfer) enzymatic assay (test A). With the aim to enhancing the biological activity of the lead compound, we applied the molecular simplification approach to design and synthesize novel heterocyclic compounds related to lipocrine, in which the tetrahydroacridine moiety was replaced by 4-amino-quinoline or 4-amino-quinazoline rings. All the synthesized compounds were also evaluated in a modified FRET enzymatic assay (test B), changing the fluorescent substrate for enzymatic BACE1 cleavage. This test method guided deep structure-activity relationships for BACE1 inhibition on the most promising quinazoline-based derivatives. By varying the substituent on the 2-position of the quinazoline ring and by replacing the lipoic acid residue in lateral chain with different moieties (i.e. trans-ferulic acid, a known antioxidant molecule), a series of quinazoline derivatives were obtained. In order to confirm inhibitory activity of the most active compounds, they were evaluated with a third FRET assay (test C) which, surprisingly, did not confirm the previous good activity profiles. An evaluation study of kinetic parameters of the three assays revealed that method C is endowed with the best specificity and enzymatic efficiency. Biological evaluation of the modified 2,4-diamino-quinazoline derivatives measured through the method C, allow to obtain a new lead compound bearing the trans-ferulic acid residue coupled to 2,4-diamino-quinazoline core endowed with a good BACE1 inhibitory activity (IC50 = 0.8 mM). We reported on the variability of the results in the three different FRET assays that are known to have some disadvantages in term of interference rates that are strongly dependent on compound properties. The observed results variability could be also ascribed to different enzyme origin, varied substrate and different fluorescent groups. The inhibitors should be tested on a parallel screening in order to have a more reliable data prior to be tested into cellular assay. With this aim, preliminary cellular BACE1 inhibition assay carried out on lipocrine confirmed a good cellular activity profile (EC50 = 3.7 mM) strengthening the idea to find a small molecule non-peptidomimetic compound as BACE1 inhibitor. In conclusion, the present study allowed to identify a new lead compound endowed with BACE1 inhibitory activity in submicromolar range. Further lead optimization to the obtained derivative is needed in order to obtain a more potent and a selective BACE1 inhibitor based on 2,4-diamino-quinazoline scaffold. A side project related to the synthesis of novel enzymatic inhibitors of BACE1 in order to explore the pseudopeptidic transition-state isosteres chemistry was carried out during research stage at Università de Montrèal (Canada) in Hanessian's group. The aim of this work has been the synthesis of the δ-aminocyclohexane carboxylic acid motif with stereochemically defined substitution to incorporating such a constrained core in potential BACE1 inhibitors. This fragment, endowed with reduced peptidic character, is not known in the context of peptidomimetic design. In particular, we envisioned an alternative route based on an organocatalytic asymmetric conjugate addition of nitroalkanes to cyclohexenone in presence of D-proline and trans-2,5-dimethylpiperazine. The enantioenriched obtained 3-(α-nitroalkyl)-cyclohexanones were further functionalized to give the corresponding δ-nitroalkyl cyclohexane carboxylic acids. These intermediates were elaborated to the target structures 3-(α-aminoalkyl)-1-cyclohexane carboxylic acids in a new readily accessible way.

Neuronal and nonneuronal quantitative BACE immunocytochemical expression in the entorhinohippocampal and frontal regions in Alzheimer's disease.

In this study, we quantitatively investigated the expression of beta-site amyloid precursor protein cleaving enzyme (BACE) in the entorhinohippocampal and frontal cortex of Alzheimer's disease (AD) and old control subjects. The semiquantitative estimation indicated that the intensity of BACE overall immunoreactivity did not differ significantly between AD and controls, but that a significantly stronger staining was observed in the hippocampal regions CA3-4 compared to other regions in both AD patients and controls. The quantitative estimation confirmed that the number of BACE-positive neuronal profiles was not significantly decreased in AD. However, some degeneration of BACE-positive profiles was attested by the colocalization of neurons expressing BACE and exhibiting neurofibrillary tangles (NFT), as well as by a decrease in the surface area of BACE-positive profiles. In addition, BACE immunocytochemical expression was observed in and around senile plaques (SP), as well as in reactive astrocytes. BACE-immunoreactive astrocytes were localized in the vicinity or close to the plaques and their number was significantly increased in AD entorhinal cortex. The higher amount of beta-amyloid SP and NFT in AD was not correlated with an increase in BACE immunoreactivity. Taken together, these data accent that AD progression does not require an increased neuronal BACE protein level, but suggest an active role of BACE in immunoreactive astrocytes. Moreover, the strong expression in controls and regions less vulnerable to AD puts forward the probable existence of alternate BACE functions.

Tau hyperphosphorylation and increased BACE1 and RAGE levels in the cortex of PPARβ/δ-null mice.

The role of peroxisome proliferator activator receptor (PPAR)β/δ in the pathogenesis of Alzheimer's disease has only recently been explored through the use of PPARβ/δ agonists. Here we evaluated the effects of PPARβ/δ deficiency on the amyloidogenic pathway and tau hyperphosphorylation. PPARβ/δ-null mice showed cognitive impairment in the object recognition task, accompanied by enhanced DNA-binding activity of NF-κB in the cortex and increased expression of IL-6. In addition, two NF-κB-target genes involved in β-amyloid (Aβ) synthesis and deposition, the β site APP cleaving enzyme 1 (Bace1) and the receptor for advanced glycation endproducts (Rage), respectively, increased in PPARβ/δ-null mice compared to wild type animals. The protein levels of glial fibrillary acidic protein (GFAP) increased in the cortex of PPARβ/δ-null mice, which would suggest the presence of astrogliosis. Finally, tau hyperphosphorylation at Ser199 and enhanced levels of PHF-tau were associated with increased levels of the tau kinases CDK5 and phospho-ERK1/2 in the cortex of PPARβ/δ(-/-) mice. Collectively, our findings indicate that PPARβ/δ deficiency results in cognitive impairment associated with enhanced inflammation, astrogliosis and tau hyperphosphorylation in the cortex.

A common BACE1 polymorphism is a risk factor for sporadic Creutzfeldt-Jakob disease

The β site APP cleaving enzyme 1 (BACE1) is the rate-limiting β-secretase enzyme in the amyloidogenic processing of APP and Aβ formation, and therefore it has a prominent role in Alzheimer"s disease (AD) pathology. Recent evidence suggests that the prion protein (PrP) interacts directly with BACE1 regulating its β-secretase activity. Moreover, PrP has been proposed as the cellular receptor involved in the impairment of synaptic plasticity and toxicity caused by Aβ oligomers. Provided that common pathophysiologic mechanisms are shared by Alzheimer"s and Creutzfeldt-Jakob (CJD) diseases, we investigated for the first time to the best of our knowledge a possible association of a common synonymous BACE1 polymorphism (rs638405) with sporadic CJD (sCJD). Our results indicate that BACE1 C-allele is associated with an increased risk for developing sCJD, mainly in PRNP M129M homozygous subjects with early onset. These results extend the very short list of genes (other than PRNP) involved in the development of human prion diseases; and support the notion that similar to AD, in sCJD several loci may contribute with modest overall effects to disease risk. These findings underscore the interplay in both pathologies of APP, Aβ oligomers, ApoE, PrP and BACE1, and suggest that aging and perhaps vascular risk factors may modulate disease pathologies in part through these key players

Genotypic variation in Actinidia deliciosa fruit size and carbohydrate content

In a global and increasingly competitive fresh produce market, more attention is being given to fruit quality traits and consumer satisfaction. Kiwifruit occupies a niche position in the worldwide market, when compared to apples, oranges or bananas. It is a fruit with extraordinarily good nutritional traits, and its benefits to human health have been widely described. Until recently, international trade in kiwifruit was restricted to a single cultivar, but different types of kiwifruit are now becoming available in the market. Effective programmes of kiwifruit improvement start by considering the requirements of consumers, and recent surveys indicate that sweeter fruit with better flavour are generally preferred. There is a strong correlation between at-harvest dry matter and starch content, and soluble solid concentration and flavour when fruit are eating ripe. This suggests that carbon accumulation strongly influences the development of kiwifruit taste. The overall aim of the present study was to determine what factors affect carbon accumulation during Actinidia deliciosa berry development. One way of doing this is by comparing kiwifruit genotypes that differ greatly in their ability to accumulate dry matter in their fruit. Starch is the major component of dry matter content. It was hypothesized that genotypes were different in sink strength. Sink strength, by definition, is the effect of sink size and sink activity. Chapter 1 reviews fruit growth, kiwifruit growth and development and carbon metabolism. Chapter 2 describes the materials and methods used. Chapter 3, 4, 5 and 6 describes different types of experimental work. Chapter 7 contains the final discussions and the conclusions Three Actinidia deliciosa breeding populations were analysed in detail to confirm that observed differences in dry matter content were genetically determined. Fruit of the different genotypes differed in dry matter content mainly because of differences in starch concentrations and dry weight accumulation rates, irrespective of fruit size. More detailed experiments were therefore carried out on genotypes which varied most in fruit starch concentrations to determine why sink strengths were so different. The kiwifruit berry comprises three tissues which differ in dry matter content. It was initially hypothesised that observed differences in starch content could be due to a larger proportion of one or other of these tissues, for example, of the central core which is highest in dry matter content. The study results showed that this was not the case. Sink size, intended as cell number or cell size, was then investigated. The outer pericarp makes up about 60% of berry weight in ‘Hayward’ kiwifruit. The outer pericarp contains two types of parenchyma cells: large cells with low starch concentration, and small cells with high starch concentration. Large cell, small cell and total cell densities in the outer pericarp were shown to be not correlated with either dry matter content or fruit size but further investigation of volume proportion among cell types seemed justified. It was then shown that genotypes with fruit having higher dry matter contents also had a higher proportion of small cells. However, the higher proportion of small cell volume could only explain half of the observed differences in starch content. So, sink activity, intended as sucrose to starch metabolism, was investigated. In transiently starch storing sinks, such as tomato fruit and potato tubers, a pivotal role in carbon metabolism has been attributed to sucrose cleaving enzymes (mainly sucrose synthase and cell wall invertase) and to ADP-glucose pyrophosphorylase (the committed step in starch synthesis). Studies on tomato and potato genotypes differing in starch content or in final fruit soluble solid concentrations have demonstrated a strong link with either sucrose synthase or ADP-glucose pyrophosphorylase, at both enzyme activity and gene expression levels, depending on the case. Little is known about sucrose cleaving enzyme and ADP-glucose pyrophosphorylase isoforms. The HortResearch Actinidia EST database was then screened to identify sequences putatively encoding for sucrose synthase, invertase and ADP-glucose pyrophosphorylase isoforms and specific primers were designed. Sucrose synthase, invertase and ADP-glucose pyrophosphorylase isoform transcript levels were anlayzed throughout fruit development of a selection of four genotypes (two high dry matter and two low dry matter). High dry matter genotypes showed higher amounts of sucrose synthase transcripts (SUS1, SUS2 or both) and higher ADP-glucose pyrophosphorylase (AGPL4, large subunit 4) gene expression, mainly early in fruit development. SUS1- like gene expression has been linked with starch biosynthesis in several crop (tomato, potato and maize). An enhancement of its transcript level early in fruit development of high dry matter genotypes means that more activated glucose (UDP-glucose) is available for starch synthesis. This can be then correlated to the higher starch observed since soon after the onset of net starch accumulation. The higher expression level of AGPL4 observed in high dry matter genotypes suggests an involvement of this subunit in drive carbon flux into starch. Changes in both enzymes (SUSY and AGPse) are then responsible of higher starch concentrations. Low dry matter genotypes showed generally higher vacuolar invertase gene expression (and also enzyme activity), early in fruit development. This alternative cleavage strategy can possibly contribute to energy loss, in that invertases’ products are not adenylated, and further reactions and transport are needed to convert carbon into starch. Although these elements match well with observed differences in starch contents, other factors could be involved in carbon metabolism control. From the microarray experiment, in fact, several kinases and transcription factors have been found to be differentially expressed. Sink strength is known to be modified by application of regulators. In ‘Hayward’ kiwifruit, the synthetic cytokinin CPPU (N-(2-Chloro-4-Pyridyl)-N-Phenylurea) promotes a dramatic increase in fruit size, whereas dry matter content decreases. The behaviour of CPPU-treated ‘Hayward’ kiwifruit was similar to that of fruit from low dry matter genotypes: dry matter and starch concentrations were lower. However, the CPPU effect was strongly source limited, whereas in genotype variation it was not. Moreover, CPPU-treated fruit gene expression (at sucrose cleavage and AGPase levels) was similar to that in high dry matter genotypes. It was therefore concluded that CPPU promotes both sink size and sink activity, but at different “speeds” and this ends in the observed decrease in dry matter content and starch concentration. The lower “speed” in sink activity is probably due to a differential partitioning of activated glucose between starch storage and cell wall synthesis to sustain cell expansion. Starch is the main carbohydrate accumulated in growing Actinidia deliciosa fruit. Results obtained in the present study suggest that sucrose synthase and AGPase enzymes contribute to sucrose to starch conversion, and differences in their gene expression levels, mainly early in fruit development, strongly affect the rate at which starch is therefore accumulated. This results are interesting in that starch and Actinidia deliciosa fruit quality are tightly connected.

Gamma-secretase modulators and inhibitors in Alzheimer's disease: influence of genetic background on efficacy and mechanism of action

According to the amyloid hypothesis, Alzheimer’s disease (AD) is caused by aberrant production or clearance of the amyloid-β (Aβ) peptides, and in particular of the longer more aggregation-prone Aβ42. The Aβ peptides are generated through successive proteolytic cleavage of the amyloid precursor protein (APP) by the β-site APP cleaving enzyme (BACE) and γ-secretase. γ-secretase produces Aβ peptides with variable C-termini ranging from Aβ34 to Aβ48, presumably by sequential trimming of longer into shorter peptides. γ-secretase is a multiprotein complex consisting of at least four different proteins and the presenilin proteins (PS1 or PS2) contain the catalytic center of the complex. In 2001 several non-steroidal anti-inflammatory drugs were identified as the founding members of a new class of γ-secretase modulators (GSMs) that can selectively reduce production of Aβ42. Concomitantly, these GSMs increase Aβ38 production indicating closely coordinated generation of Aβ42 and Aβ38 and a potential precursor-product relationship between these peptides. GSMs seem to exert their activity by direct modulation of γ-secretase. Support for this hypothesis is drawn from the finding that some PS mutations associated with early-onset familial AD (FAD) can modulate the cellular response to GSMs and to γ-secretase inhibitors (GSIs), which inhibit production of all Aβ peptides and are known to directly interact with PS. A particularly interesting FAD PS mutation is PS1-ΔExon9, a complex deletion mutant that blocks endoproteolysis of PS1 and renders cells completely non-responsive to GSMs. Studies presented in this thesis show that the diminished response of PS1-ΔExon9 to GSMs is mainly caused by its lack of endoproteolytic cleavage. Furthermore, we were able to demonstrate that a reduced response to GSMs and GSIs is not limited to PS1-ΔExon9 but is a common effect of aggressive FAD-associated PS1 mutations. Surprisingly, we also found that while the Aβ42 response to GSMs is almost completely abolished by these PS1 mutations, the accompanying Aβ38 increase was indistinguishable to wild-type PS1. Finally, the reduced response to GSIs was confirmed in a mouse model with transgenic expression of an aggressive FAD-associated PS1 mutation as a highly potent GSI failed to reduce Aβ42 levels in brain of these mice. Taken together, our findings provide clear evidence for independent generation of Aβ42 and Aβ38 peptides, and argue that the sequential cleavage model might be an oversimplification of the molecular mechanism of γ-secretase. Most importantly, our results highlight the significance of genetic background in drug discovery efforts aimed at γ-secretase, and indicate that the use of cellular models with transgenic expression of FAD-associated PS mutations might confound studies of the potency and efficacy of GSMs and GSIs. Therefore, such models should be strictly avoided in the ongoing preclinical development of these promising and potentially disease-modifying therapeutics for AD.

Molecular interaction of the human metalloprotease meprin beta with the amyloid precursor protein, ADAM10, and ADAM17 based on proteomics

Die Zinkendopeptidasen Meprin α und β sind Schlüsselkomponenten in patho(physiologischen) Prozessen wie Entzündung, Kollagenassemblierung und Angiogenese. Nach ihrer Entdeckung in murinen Bürstensaummembranen und humanen Darmepithelien, wurden weitere Expressionsorte identifiziert, z.B. Leukozyten, Krebszellen und die humane Haut. Tiermodelle, Zellkulturen und biochemische Analysen weisen auf Funktionen der Meprine in der Epithelialdifferenzierung, Zellmigration, Matrixmodellierung, Angiogenese, Bindegewebsausbildung und immunologische Prozesse hin. Dennoch sind ihre physiologischen Substrate weitgehend noch unbekannt. Massenspektrometrisch basierte Proteomics-Analysen enthüllten eine einzigartige Spaltspezifität für saure Aminosäurereste in der P1´ Position und identifizierten neue biologische Substratkandidaten. Unter den 269 extrazellulären Proteinen, die in einem Substratscreen identifiziert wurden, stellten sich das amyloid precursor protein (APP) and ADAM10 (a disintegrin and metalloprotease 10) als sehr vielversprechende Kandidaten heraus. Mehrere Schnittstellen innerhalb des APP Proteins, hervorgerufen durch verschiedenen Proteasen, haben unterschiedlichen Auswirkungen zur Folge. Die β-Sekretase BACE (β-site APP cleaving enzyme) prozessiert APP an einer Schnittstelle, welche als initialer Schritt in der Entwicklung der Alzheimer Erkrankung gilt. Toxische Aβ (Amyloid β)-Peptide werden in den extrazellulären Raum freigesetzt und aggregieren dort zu senilen Plaques. Membran verankertes Meprin β hat eine β-Sekretase Aktivität, die in einem Zellkultur-basierten System bestätigt werden konnte. Die proteolytische Effizienz von Meprin β wurde in FRET (Fluorescence Resonance Energy Transfer)-Analysen bestimmt und war um den Faktor 104 höher als die von BACE1. Weiterhin konnte gezeigt werden, dass Meprin β die ersten zwei Aminosäuren prozessiert und somit aminoterminal einen Glutamatrest freisetzt, welcher nachfolgend durch die Glutaminylzyklase in ein Pyroglutamat zykliert werden kann. Trunkierte Aβ-Peptide werden nur in Alzheimer Patienten generiert. Aufgrund einer erhöhten Hydrophobie weisen diese Peptide eine höhere Tendenz zur Aggregation auf und somit eine erhöhte Toxizität. Bis heute wurde keine Protease identifiziert, welche diese Schnittstelle prozessiert. Die Bildung der Meprin vermittelten N-terminalen APP Fragmenten wurde in vitro und in vivo detektiert. Diese N-APP Peptide hatten keine cytotoxischen Auswirkungen auf murine und humane Gehirnzellen, obwohl zuvor N-APP als Ligand für den death receptor (DR) 6 identifiziert wurde, der für axonale Degenerationsprozesse verantwortlich ist. rnIm nicht-amyloidogenen Weg prozessiert ADAM10 APP und entlässt die Ektodomäne von der Zellmembran. Wir konnten das ADAM10 Propeptid als Substrat von Meprin β identifizieren und in FRET Analysen, in vitro und in vivo zeigen, dass die Meprin vermittelte Prozessierung zu einer erhöhten ADAM10 Aktivität führt. Darüber hinaus wurde ADAM10 als Sheddase für Meprin β identifiziert. Shedding konnte durch Phorbol 12-myristate 13-acetate (PMA) oder durch das Ionophor A23187 hervorgerufen werden, sowie durch ADAM10 Inhibitoren blockiert werden. rnDiese Arbeit konnte somit ein komplexes proteolytisches Netwerk innerhalb der Neurophysiologie aufdecken, welches für die Entwicklung der Alzheimer Demenz wichtig sein kann.rn

Proteolytic maturation of protein C upon engineering the mouse mammary gland to express furin.

Endoproteolytic processing of the human protein C (HPC) precursor to its mature form involves cleavage of the propeptide after amino acids Lys-2-Arg-1 and removal of a Lys156-Arg157 dipeptide connecting the light and heavy chains. This processing was inefficient in the mammary gland of transgenic mice and pigs. We hypothesized that the protein processing capacity of specific animal organs may be improved by the coexpression of selected processing enzymes. We tested this by targeting expression of the human proprotein processing enzyme, named paired basic amino acid cleaving enzyme (PACE)/furin, or an enzymatically inactive mutant, PACEM, to the mouse mammary gland. In contrast to mice expressing HPC alone, or to HPC/PACEM bigenic mice, coexpression of PACE with HPC resulted in efficient conversion of the precursor to mature protein, with cleavage at the appropriate sites. These results suggest the involvement of PACE in the processing of HPC in vivo and represent an example of the engineering of animal organs into bioreactors with enhanced protein processing capacity.

miRNAs as therapeutic agents in neurodegeneration, a pilot study

L’échec des différents essais cliniques souligne la nécessité de développer des nouvelles thérapies pour la maladie d’Alzheimer (MA), la cause la plus commune de démence. Les microARNs (miARNs) sont les ARNs non-codants les plus étudiés et ils jouent un rôle important dans la modulation de l’expression des gènes et de multiples voies de signalisation. Des études antérieures, dont celles de mon laboratoire d’accueil, ont permis de développer l’hypothèse que certains membres de la famille miR-15/107 (c.-à-d. miR-15ab, miR-16, miR-195, miR-424, and miR-497) pourraient être utilisés comme agents thérapeutiques dans MA. En effet, cette famille avait le potentiel de réguler de multiples gènes associés à MA, tels que la protéine précurseur de l’amyloïde (APP), la β-secrétase (BACE1), et la protéine Tau. Tel que démontré dans ce projet de thèse, j’ai choisi miR-16 comme cible thérapeutique potentielle pour MA parmi tous les membres de la famille. L’essai luciférase dans ce projet confirme que miR-16 peut réguler simultanément APP et BACE1, directement par une interaction avec la région non-codante en 3’ de l’ARNm). Notamment, nous observons aussi une réduction de la production des peptides amyloïdes et de la phosphorylation de Tau après une augmentation de miR-16 en cellule. J’ai ensuite validé mes résultats in vivo dans la souris en utilisant une méthode de livraison de miR-16 via une pompe osmotique implanté dans le cerveau. Dans ce cas, l’expression des protéines d’intérêts (APP, BACE1, Tau) a été mesurée par immunobuvardage et PCR à temps réel. Après validation, ces résultats ont été complémentés par une étude protéomique (iTRAQ) du tronc cérébral et de l’hippocampe, deux régions associées à la maladie. Ces données m’ont permis d’identifier d’autres protéines régulées par miR-16 in vivo, incluant α-Synucléine, Transferrine receptor1, et SRm300. Une autre observation intéressante : les voies régulées par miR-16 in vivo sont directement en lien avec le stress oxydatif et la neurodégénération. En résumé, ce travail démontre l’efficacité et la faisabilité d’utiliser un miARN comme outil thérapeutique pour la maladie d’Alzheimer. Ces résultats rentrent dans un cadre plus vaste de découvrir de nouvelles cibles pour MA, et en particulier la forme sporadique de la maladie qui représente plus de 95% de tous les cas. Évidemment, la découverte d’une molécule pouvant cibler simultanément les deux pathologies de la maladie (plaques amyloïdes et hyper phosphorylation de tau) est nouvelle et intéressante, et ce domaine de recherche ouvre la porte aux autres petits ARNs non-codants dans MA et les maladies neurodégénératives connexes.