Proteases i inhibidors com a models per a estudis de relació estructura-funció i agregació

by Pallarès i Goitiz, Irantzu

Abstract (Summary)
The present thesis reflects the two main research lines of our group. The first part is related with the study of the structure-function relationship in metallocarboxypeptidases and their inhibitors. While the second part, is focused on the study of folding and protein aggregation and more specifically on the study of protein models in which the formation of amyloid structures can be induced and in the development of protein aggregation prediction methods from their primary sequence. Part I: Carboxypeptidase A1 is the prototype of the A/B carboxypeptidase subfamily. In this thesis has been obtained the three dimensional structure of human CPA1 at 1.6 Å resolution. This structure at high resolution provides a template for modeling the structure of physiologically relevant carboxypeptidases, as the ones implied in hormone and neuropeptide processing, as well as for the specific design of inhibitors. In the second chapter the three dimensional structure of human latexin in complex with human CPA4 was been solved. The functional characterization of latexin by inhibition assays describes it as a non-competitive inhibitor of vertebrate A/B metallocarboxypeptidases. In the complex, the inhibitor sits on the top of the funnel rim occluding the entrance to the active site covering a higher area than in other typical protease-inhibitor interfaces. In contrast to other protein inhibitors of metalloproteases, inhibition by latexin does not involve any of its termini. Our molecular studies show that structural determinants simultaneously confer vertebrate-linked specificity to latexins and sufficient variability to strongly inhibit all vertebrate forms tested. Latexins could represent the family of endogenous MCP inhibitors in vertebrates. Part II: The third chapter, which opens the second part, indicates that latexin could be a good model for the study of the prefibrillar states originate from an unfolded state. In contrast to latexin, which can be used as an intrinsically unstructured protein model, in the fourth chapter of the thesis a work developed with ?chymotripsin is presented. This is a protein which in the native state presents a ?barrel fold, one of the most abundant in nature. In this case, the amyloid fibril formation requires the previous formation of a partially folded intermediate state rich in ?-sheet structure. The first and second works of the second part of the thesis summarizes the two proteins models that are involved in the development of amyloidogenic diseases. In the case of latexin, the amyloid structure formation requires the completely unfolding of the protein, so it can be a good model for the study of the diseases where the protein that originates the aggregation event is completely unfolded. While, the ?chymotripsin model fits with the globular proteins, where the aggregation process requires the formation of a partially unfolded state rich in ?-sheet structure. The obtained results indicate that the ability to form these structures is general for all the polypeptide chains, although the propensity changes between different sequences. In the last work, a method to predict the aggregation prone regions of a sequence has been developed. Prediction of such sequence stretches is important since they are potential therapeutic targets. The main difference between the few prediction methods available in the literature and the one presented in the thesis is that the origin of the data used to generate the aggregation profile; while in the other methods mathematical formulas with theoretical values are assigned to specific region of the sequence, in this work, we have used experimental data obtained in an in vivo system using ?-amyloid peptide has been used as a model to derive the relative aggregation propensities of each individual natural amino acid when placed in the central position of the CHC of A?.
This document abstract is also available in Catalan.
Bibliographical Information:

Advisor:Vendrell i Roca, Josep; Ventura i Zamora, Salvador

School:Universitat Autónoma de Barcelona

School Location:Spain

Source Type:Master's Thesis

Keywords:406 departament de bioquimica i biologia molecular


Date of Publication:07/18/2006

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