Fundamental structural and biochemical features for the obestatin/gpr39 system mitogenic action

  1. Otero Alén, Begoña
Dirixida por:
  1. Tomás García-Caballero Parada Director
  2. Yolanda Pazos Randulfe Co-director
  3. Felipe Casanueva Freijó Co-director

Universidade de defensa: Universidade de Santiago de Compostela

Fecha de defensa: 16 de setembro de 2016

Tribunal:
  1. Jesús Jiménez Barbero Presidente/a
  2. J. Enrique Domínguez Muñoz Secretario
  3. María Dolores Mayán Santos Vogal
Departamento:
  1. Departamento de Ciencias Morfolóxicas

Tipo: Tese

Resumo

In 2005, using a bioinformatics approach, a new peptide derived from the ghrelin peptide precursor called obestatin, was discovered. It was described as a physiological opponent of ghrelin, but, ruling out the role of obestatin in food intake, numerous biological functions have been described for it. One of the most important is its mitogenic ability. Furthermore, obestatin was initially discovered as the cognate ligand for GPR39, however, several groups published their results challenging these data. All this information became obestatin a controversial peptide. In recent years, some issues have been clarified but many of them are still unrevealed. Obestatin and the GPR39 receptor were reported to be involved in the control of mitogenesis of gastric cancer cell lines; the controversy generated by the obestatin/GPR39 system regarding its mode of interaction, led us to investigate obestatin/GPR39 signalling and the relationship between the system and gastric cancer progression and explored their potential functional roles. Moreover, the quest for therapeutic applications of obestatin involves, as a first step, the determination of its 3D solution structure and the relationship between this structure and the biological activity of obestatin. On this basis, we have employed a combination of circular dichroism, nuclear magnetic resonance spectroscopy, and modelling techniques to understand the structural requirements for this hormone bioactivity and help the future design of new drugs related to these pathologies. Therefore, the main objective of this thesis project was to establish the relationship between obestatin and GPR39 receptor in both normal and tumour environments, from the structural to tissue level. Conformational studies reveal the structural features of human obestatin required for the interaction with its receptor. Amidation at the C-terminus is essential to adopt an α-helix structure and stabilize the GPR39 conformations necessary for the full range of receptor activities. Furthermore, human (11-23)-obestatin is able to induce selective coupling to the β-arrestin-dependent signalling, representing the first example of an endogenous biased ligand for GPR39. Mouse and human obestatin exhibit clear conformational differences beyond their primary structure. This evidence supports the species-specific activity of obestatin. Finally, NMR in living cells indicates that obestatin-GPR39 interaction might involve an E/Z isomerization of the peptide and the posibility that GPR39 could be acting as a prolyl cis-trans isomerase. Regarding the activation/regulation mechanism of GPR39 receptor signalling triggered by obestatin, our results show that obestatin increases GPR39 phosphorylation and induces receptor endocytosis. The RTKs and proteases expression profiles confirm the implication of EGFR and MMPs in the obestatin signalling pathway, and introduce other proteases and RTKs in the process of GPR39 transactivation. In human tissues we observe that the obestatin/GPR39 system regulates pepsinogen secretion. This result provide the first biological function for the obestatin/GPR39 system in healthy stomach. This system also regulates proliferation, motility, EMT, and invasion of gastric cancer cells. More importantly, the GPR39 expresion levels found in human gastric adenocarcinomas provide the rationale for including GPR39 as a prognostic marker of these tumours. The ubiquitous expression of GPR39 causes that in pathological conditions as cancer, it becomes overexpressed and, together with obestatin, provokes the proliferation and cell motillity of diverse human cancer cell lines. Moreover, these effects depend not only on GPR39, but also on the expression of key components of its signalling pathway.