Anti-apolipoprotein A-I antibodies in the pathogenesis of Alzheimer disease : project plan

RESUMO: A doença de Alzheimer (AD) é a forma mais comum de demência em todo o mundo e sua prevalência deverá duplicar até 2050. Os mecanismos precisos responsáveis pela AD são desconhecidas mas as características histopatológicas estão bem caracterizadas. A hipótese mais importante para a perda neuronal e declínio cognitivo na AD é a cascata amilóide que indica que AD é o resultado da sobreprodução de beta amilóide (Aβ) e / ou remoção ineficaz; a acumulação do BA no cérebro seria o passo crítico na patogénese da AD. Actualmente, a identificação de proteínas que se ligam ao Aβ e modulam a sua agregação e neurotoxicidade pode proporcionar a base para novas abordagens terapêuticas. A apolipoproteína AI (ApoA-I), o principal componente das HDL humanas, interage com o domínio extracelular da proteína precursora de amilóide (APP), bem como com o Aβ. Estudos epidemiológicos têm mostrado uma diminuição acentuada da ApoA-I plasmática em doentes com AD, com uma correlação inversa entre o nível de ApoA-I e o risco de AD. Este trabalho pretende apresentar um projecto que tem como objectivo investigar se os anticorpos anti-apo AI podem impedir a formação de complexos Aβ / ApoA-I, bloqueando o efeito protector da ApoA-I. A hipótese baseia-se na possibilidade dos doentes com AD terem anticorpos anti-ApoA-I plasmáticos e de estes poderem interferir com a formação do complexo no LCR.------- ABSTRACT:Alzheimer’s disease (AD) is the most common form of dementia world-wide and its prevalence is expected to double by the year 2050. The precise mechanisms responsible for AD are unknown but the histopathologic features are well-characterised. The most compelling hypothesis for neuronal loss and cognitive decline in AD is the amyloid cascade hypothesis which states that AD is the result of amyloid beta (Aβ) overproduction and/or ineffective clearance and its accumulation in the brain would be the critical step in AD pathogenesis. Currently, identification of proteins that bind Aβ and modulate its aggregation and neurotoxicity could provide the basis for novel treatment approaches. Apolipoprotein A-I (ApoA-I), the main constituent of human HDL, ApoA-I interacts with the extracellular domain of amyloid precursor protein (APP), as well as with Aβ itself. Epidemiological studies have shown a marked decrease of plasma ApoA-I levels in AD patients, with an inverse correlation between the ApoA-I level and the risk of AD. This work intends to present a project that aims to investigate if anti-ApoA-I antibodies may prevent the formation of the Aβ /ApoA-I complex and by doing so blocking the protective effect of ApoA-I in AD. We base the hypothesis on the possibility that patients with AD might have anti-ApoA-I antibodies in plasma and that these can interfere with the complex formation in the cerebrospinal fluid (CSF).

Assessment of Notch1 ligands production - key protein targets in breast cancer

Cell-to-cell communication is required for many biological processes in development and adult life. One of the most common systems utilized by a wide range of eukaryotes is the Notch signalling pathway. Four Notch receptors and five ligands have been identified in mammals that interact via their extracellular domains leading to transcription activation. Studies have shown that the Notch ligands expression is undetectable in normal breast tissues, but moderate to high expression has been detected in breast cancer. Thus, any of the Notch1 ligands can be studied as possible therapeutic targets for breast cancer. To study Notch pathway proteins there is the need to obtain stable protein solutions. E. coli is the host of excellence for recombinant proteins for the ease of use, fast growth and high cell densities. However, the expression of mammalian proteins in such systems may overwhelm the bacterial cellular machinery, which does not possess the ability for post-translational modifications, or dedicated compartments for protein synthesis. Mammalian cells are therefore preferred, despite their technical and financial increased demands. We aim to determine the best expression and purification conditions for the different ligand protein constructs, to develop specific function-blocking antibodies using the Phage Display technology. Moreover, we propose to crystallize the Notch1 ligands alone and in complex with the phage display selected antibodies, unveiling molecular details. hJag2DE3 and hDll1DE6 proteins were purified from refolded inclusion bodies or mammalian cell culture supernatants, respectively, and purity was confirmed by SDS-PAGE (>95%). Protein produced in mammalian cells showed to be more stable, apparently with the physiological disulfide pattern, contrary to what was observed in the refolded protein. Several nano-scale crystallization experiments were set up in 96-well plates, but no positive result was obtained. We will continue to pursue for the best expression for the Notch ligand constructs in both expression systems.