Structural and functional studies on escherichia coli ¿2-macroglobulin: a snap-trap peptidase inhibitor
- Autores: Irene García Ferrer
- Directores de la Tesis: Francesc Xavier Gomis-Rüth (dir. tes.), Theodoros Goulas (codir. tes.)
- Lectura: En la Universitat de Barcelona ( España ) en 2015
- Idioma: español
- Materias:
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- Resumen
The balance between proteolytic and antiproteolytic activity is crucial in many biological processes such as nutrition, immune defence, virulence and tissue remodelling. Therefore, it is controlled by several mechanisms, among which by peptidic peptidase inhibitors that are encoded in the genomes of many organisms, representing up to 1% of genes in metazoa but being scarce and sketchy in bacteria. However, among bacterial peptidase inhibitors, some proteins with homology to the highly abundant metazoan ¿2- macroglobulins (¿2Ms) have been described, which may have been acquired by bacteria from metazoa by horizontal gene transfer. Although ¿2Ms have been extensively characterised in metazoa, where they play important roles in innate immunity, the biological role, mechanism of action and molecular structure of bacterial ¿2Ms (b¿2Ms) remained largely unknown.
In this thesis, the characterisation of the Escherichia coli ¿2M, ECAM, was undertaken in order to elucidate its role in the bacterial cell. The results unveiled a novel mechanism of peptidase inhibition, called the snap-trap mechanism, that is probably shared by other monomeric ¿2Ms, both from bacteria and metazoa. In this, attacking endopeptidases cleave the native inhibitor in an accessible bait region, thus causing a major conformational rearrangement and producing induced species that covalently trap peptidases through a highly reactive thioester bond. The rearrangement, involving most of the 13 domains of ECAM, and key structural elements of the mechanism, were described by producing the first atomic models of ECAM in both conformations by using X-ray crystallography and cryo-electron microscopy. Through a covalent trap, ECAM prevents peptidases from cleaving large substrates, such as components of the bacterial cell wall, thus protecting E. coli cells against potentially damaging proteolytic activity. Therefore, it seems that ECAM participates in defence mechanisms in bacteria that thrive in the presence of peptidases, for which this thesis provides the first experimental evidence through in vivo functional assays.
In summary, through a multifaceted approach that combined structural, biochemical and functional characterisation, this thesis yielded a mechanistic model for ECAM, significantly enriching our understanding of b¿2Ms and providing new insights into bacterial defence mechanisms
