DQF - Artículos
https://hdl.handle.net/10481/14661
2024-03-28T10:21:12ZProtein-Protein Interactions in Crystals of the Human Receptor-Type Protein Tyrosine Phosphatase ICA512 Ectodomain
https://hdl.handle.net/10481/87304
Protein-Protein Interactions in Crystals of the Human Receptor-Type Protein Tyrosine Phosphatase ICA512 Ectodomain
Primo, María E.; Jakancic, Jean; Noguera, Martín E.; Risso, Valeria Alejandra; Sosa, Laura; Sica, Mauricio P.; Solimena, Michele; Poskus, Edgardo; Ermácora, Mario R.
ICA512 (or IA-2) is a transmembrane protein-tyrosine phosphatase located in secretory granules of neuroendocrine cells.
Initially, it was identified as one of the main antigens of autoimmune diabetes. Later, it was found that during insulin
secretion, the cytoplasmic domain of ICA512 is cleaved and relocated to the nucleus, where it stimulates the transcription of
the insulin gene. The role of the other parts of the receptor in insulin secretion is yet to be unveiled. The structures of the
intracellular pseudocatalytic and mature extracellular domains are known, but the transmembrane domain and several
intracellular and extracellular parts of the receptor are poorly characterized. Moreover the overall structure of the receptor
remains to be established. We started to address this issue studying by X-ray crystallography the structure of the mature
ectodomain of ICA512 (ME ICA512) and variants thereof. The variants and crystallization conditions were chosen with the
purpose of exploring putative association interfaces, metal binding sites and all other structural details that might help, in
subsequent works, to build a model of the entire receptor. Several structural features were clarified and three main different
association modes of ME ICA512 were identified. The results provide essential pieces of information for the design of new
experiments aimed to assess the structure in vivo.
Phenotypic comparisons of consensus variants versus laboratory resurrections of Precambrian proteins
https://hdl.handle.net/10481/87295
Phenotypic comparisons of consensus variants versus laboratory resurrections of Precambrian proteins
Risso, Valeria Alejandra; Gavira Gallardo, José Antonio; Gaucher, Eric A.; Sánchez Ruiz, José Manuel
Consensus-sequence engineering has generated protein variants with enhanced stability, and sometimes, with modulated
biological function. Consensus mutations are often interpreted as the introduction of ancestral amino acid residues. However,
the precise relationship between consensus engineering and ancestral protein resurrection is not fully understood.
Here, we report the properties of proteins encoded by consensus sequences derived from a multiple sequence alignment of
extant, class A b-lactamases, as compared with the properties of ancient Precambrian b-lactamases resurrected in the laboratory.
These comparisons considered primary sequence, secondary, and tertiary structure, as well as stability and catalysis
against different antibiotics. Out of the three consensus variants generated, one could not be expressed and purified (likely
due to misfolding and/or low stability) and only one displayed substantial stability having substrate promiscuity, although
to a lower extent than ancient b-lactamases. These results: (i) highlight the phenotypic differences between consensus variants
and laboratory resurrections of ancestral proteins; (ii) question interpretations of consensus proteins as phenotypic
proxies of ancestral proteins; and (iii) support the notion that ancient proteins provide a robust approach toward the preparation
of protein variants having large numbers of mutational changes while possessing unique biomolecular properties.
Thermostable and promiscuous Precambrian proteins
https://hdl.handle.net/10481/87187
Thermostable and promiscuous Precambrian proteins
Risso, Valeria Alejandra; Gavira Gallardo, José Antonio; Sánchez Ruiz, José Manuel
Work in the authors’ labs is supported by grants BIO2012– 34937, CSD2009-00088 (to J.M.S.-R.), BIO2010–16800, ‘Factoría Española de Cristalización’ Consolider-Ingenio 2010 (to J.A.G.) from the Spanish Ministry of Economy and Competitiveness, and Feder Funds (J.M.S.-R and J.A.G.).
Biotechnological and protein-engineering implications of ancestral protein resurrection
https://hdl.handle.net/10481/87099
Biotechnological and protein-engineering implications of ancestral protein resurrection
Risso, Valeria Alejandra; Sánchez Ruiz, José Manuel; Ozkan, S. Banu
Approximations to the sequences of ancestral proteins can be
derived from the sequences of their modern descendants.
Proteins encoded by such reconstructed sequences can be
prepared in the laboratory and subjected to experimental
scrutiny. These ‘resurrected’ ancestral proteins often display
remarkable properties, reflecting ancestral adaptations to
intra-cellular and extra-cellular environments that differed from
the environments hosting modern/extant proteins. Recent
experimental and computational work has specifically
discussed high stability, substrate and catalytic promiscuity,
conformational flexibility/diversity and altered patterns of
interaction with other sub-cellular components. In this review,
we discuss these remarkable properties as well as recent
attempts to explore their biotechnological and proteinengineering
potential.
Hyperstability and substrate promiscuity in laboratory resurrections of precambrian β-lactamases
https://hdl.handle.net/10481/87028
Hyperstability and substrate promiscuity in laboratory resurrections of precambrian β-lactamases
Risso, Valeria Alejandra; Gavira Gallardo, José Antonio; Gaucher, Eric A.; Mejia-Carmona, DF; Sánchez Ruiz, José Manuel
We report a sequence reconstruction analysis targeting several Precambrian nodes in the evolution of class-A β-lactamases and the preparation and experimental characterization of their encoded proteins. Despite extensive sequence differences with the modern enzymes (∼100 amino acid differences), the proteins resurrected in the laboratory properly fold into the canonical lactamase structure. The encoded proteins from 2–3 billion years (Gyr)-old β-lactamase sequences undergo cooperative two-state thermal denaturation and display very large denaturation temperature enhancements (∼35 °C) relative to modern β-lactamases. They degrade different antibiotics in vitro with catalytic efficiencies comparable to that of an average modern enzyme. This enhanced substrate promiscuity is not accompanied by significant changes in the active-site region as seen in static X-ray structures, suggesting a plausible role for dynamics in the evolution of function in these proteins. Laboratory resurrections of 2–3 Gyr-old β-lactamases also endowed modern microorganisms with significant levels of resistance toward a variety of antibiotics, opening up the possibility of performing laboratory replays of the molecular tape of lactamase evolution. Overall, these results support the notions that Precambrian life was thermophilic and that proteins can evolve from substrate-promiscuous generalists into specialists during the course of natural evolution. They also highlight the biotechnological potential of laboratory resurrection of Precambrian proteins, as both high stability and enhanced promiscuity (likely contributors to high evolvability) are advantageous features in protein scaffolds for molecular design and laboratory evolution.