Unraveling the APP/HB-EGF interaction

• Autor(a) principal: Bastos, Luísa Filipe Henriques Martins de
• Data de Publicação: 2013
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/12503
• topic: Biomedicina; Doença de Alzheimer; Marcadores bioquímicos; Proteínas

Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder worldwide and the leading cause of dementia in the elderly. Abnormal processing of Alzheimer’s amyloid precursor protein (APP) and increased generation of its amyloid beta (Aβ) fragment are central events in the AD pathogenesis, propelling major studies on APP biology. APP is thought to be involved in important processes such as cell adhesion, survival, migration and differentiation. Therefore, because of the imperative need to study APP biological functions, the search for APP-binding partners has stand out. Recently in our laboratory, a plethora of putative APP-binding proteins was unraveled through the use of the Yeast Two Hybrid (YTH) system. Among them, the heparin-binding epidermal growth factor-like growth factor (HB-EGF) has emerged as an interesting target of study. HB-EGF is a heparin-binding member of the EGF family of growth factors that stimulates growth and differentiation. It has been purposed has an important trophic factor in the developing and adult central nervous system (CNS), being expressed at much higher levels than EGF in the CNS, which indicates that HB-EGF may serve as a major ligand for EGFR in neurons. HB-EGF is synthesized as a pre-pro-form of 208 amino acids in length and is expressed at the cell surface as a 20-30kDa type I transmembrane precursor, named proHB-EGF. This larger membrane-anchored precursor is then proteolytically processed, generating the mature soluble HB-EGF (sHB-EGF) that is released to the extracellular medium. Very interestingly, HB-EGF presents autocrine, paracrine and juxtacrine biological activities, with proHB-EGF evidencing unique biological characteristics distinct from sHB-EGF. In the work here described, we first validated the APP/HB-EGF interaction by yeast co-transformation, and unraveled that the interaction is not mediated by the APP intracellular domain (AICD). We further confirmed it as an in vivo interaction by GFP-Trap pull-down assays and accessed the physiological relevance of this novel interaction through co-localization and signaling studies in HeLa cells transfected with the APP-GFP and HB-EGF cDNAs. Although the functional role of the APP/HB-EGF complex was not determined, results suggest that these proteins physically and functionally interact, having potential value in regulating each other signal pathways, with a role for APP in inducing the activation of the MAPK signaling being evidenced. In addition, a putative novel interaction with a proEGF species was also detected and APP was shown to act synergistically with EGF to activate the MAPK signaling. These results deepen our understanding of the APP biology, a crucial protein in cerebral physiology and AD pathophysiology.