Procarboxipeptidases humanes: estudis funcionals i estructurals de formes pancreàtiques i de noves formes reguladores
This doctoral thesis focuses on the structure/function relationship of human metallocarboxypeptidases, also known as carboxypeptidases (CPs). This work has been devided into two different parts: the first part is about functional studies and the second comprises structural studies.
Some CPs are synthesized as inactive precursosrs named procarboxypeptidases (PCPs), which contain long activation segments (91-96 residues) that are cleaved as a result of limited proteolysis through the action of a protease, such as trypsin. In the first part of this work, optimization of an efficient recombinant expression system and purification for human PCPA1 and PCPB allowed a detailed characterization of their tryptic activation processes.
Previous detailed studies on the tryptic activation mechanism of the porcine proenzymes showed that the proteolytic processing of PCPB to a mature enzyme is a much faster process than that of PCPA1, the former following a monotonic curve and the latter a biphasic curve, as a result of the activation segment of porcine PCPA1 being a powerful competitive inhibitor of the enzyme moiety. The study reported here shows that, while human PCPB follows the general behaviour described above, this is not the case for human PCPA1 whose proteolytic processing, in terms of activation conditions, velocity of the process and shape of the activation curve, ressembles that of PCPB, as happens in the activation process of PCPA2.
A reversed-specificity mutant of human PCPB, named D255K, was expressed and purified in the same manner as the wild-type form in order to study the effect of this point mutation on the tryptic activation process of PCPB. These studies demonstrate the involvement of the generated CPB in the trimming of the severed pro-segment.
The second part is concerned with structural studies. On the one hand, the three-dimensional structure of human pancreatic PCPB has been solved, which is similar to previously reported PCPs structures, also in that the most variable region is the connecting segment that links both globular moieties. However, the empty active site shows subtle differences in some of the key residues for substrate binding. On the basis of this structure and others previously reported, a three-dimensional model of human TAFI, a protein of biomedical interest, has been built in order to generate a structure that may help us to understand its behaviour and also as a basis for the design of drugs to modulate its biological activity. The analysis of this model and its pseudo-potential energy profile allows us to propose an explanation for the intrinsic conformational instability of its active enzyme.
On the other hand, amino acid homology searches of the human genome were performed and revealed three members of the metallocarboxypeptidase family. According to their predicted specificities these enzymes were named CPA5, CPA6 and CPO. Modelling analysis shows the overall structure of these proteins to be very similar to that of other members of the family of metallocarboxypeptidases. The active sites of CPA5 and CPA6 are predicted to cleave substrates with C-terminal hydrophobic residues, as do CPA1 and CPA2. CPO, is predicted to cleave substrates with C-terminal acidic residues, a unique activity among human CPs.
Advisor:Avilés Puigvert, Francesc Xavier; Vendrell Roca, Josep
School:Universitat Autónoma de Barcelona
Source Type:Master's Thesis
Keywords:406 departament de bioquimica i biologia molecular
Date of Publication:12/13/2002