Classification of protein disulphide-bridge topologies.

The preferential occurrence of certain disulphide-bridge topologies in proteins has prompted us to design a method and a program, KNOT-MATCH, for their classification. The program has been applied to a database of proteins with less than 65% homology and more than two disulphide bridges. We have investigated whether there are topological preferences that can be used to group proteins and if these can be applied to gain insight into the structural or functional relationships among them. The classification has been performed by Density Search and Hierarchical Clustering Techniques, yielding thirteen main protein classes from the superimposition and clustering process. It is noteworthy that besides the disulphide bridges, regular secondary structures and loops frequently become correctly aligned. Although the lack of significant sequence similarity among some clustered proteins precludes the easy establishment of evolutionary relationships, the program permits us to find out important structural or functional residues upon the superimposition of two protein structures apparently unrelated. The derived classification can be very useful for finding relationships among proteins which would escape detection by current sequence or topology-based analytical algorithms.

Refinement of modelled structures by knowledge-based energy profiles and secondary structure prediction: application to the human procarboxypeptidase A2.

Knowledge-based energy profiles combined with secondary structure prediction have been applied to molecular modelling refinement. To check the procedure, three different models of human procarboxypeptidase A2 (hPCPA2) have been built using the 3D structures of procarboxypeptidase A1 (pPCPA1) and bovine procarboxypeptidase A (bPCPA) as templates. The results of the refinement can be tested against the X-ray structure of hPCPA2 which has been recently determined. Regions miss-modelled in the activation segment of hPCPA2 were detected by means of pseudo-energies using Prosa II and modified afterwards according to the secondary structure prediction. Moreover, models obtained by automated methods as COMPOSER, MODELLER and distance restraints have also been compared, where it was found possible to find out the best model by means of pseudo-energies. Two general conclusions can be elicited from this work: (1) on a given set of putative models it is possible to distinguish among them the one closest to the crystallographic structure, and (2) within a given structure it is possible to find by means of pseudo-energies those regions that have been defectively modelled.

Detection of molecular interactions by using a new peptide-displaying bacteriophage biosensor.

Foreign peptides fused to the carboxy terminus of P22 tailspike protein are solvent-exposed and highly antigenic when displayed on the surface of infectious virus particles. Binding of an anti-peptide specific Fab antibody fragment enhances the infectivity of chimeric bacteriophage particles in a titre-dependent fashion. Although the precise molecular basis of this enhanced infectivity remains unclear, experimental data and modelling approaches suggest that the antibody binding might restore conformational impairments in the assembled tail protein affecting its activity and performance during infection. These results suggest that in addition to free enzymes, peptide-displaying bacteriophages could be engineered as new biosensors to detect molecular interactions by using natural viral enzymes critical for cell infection.

Effects of counter-ions and volume on the simulated dynamics of solvated proteins. Application to the activation domain of procarboxypeptidase B.

Molecular dynamics (MD) simulations of the globular activation domain of porcine procarboxypeptidase B (ADBp) and its isolated alpha-helix 1 were performed in order to understand the effects of adding salts and using periodic boundary conditions (this being reflected in the box size) along the simulations. alpha-Helix 1 was chosen because it is the most charged element of the secondary structure within ADBp. Different types of MD simulations with the GROMOS package were performed, studying either the whole activation domain or the isolated alpha-helix 1 with different water box sizes and counter-ionic shells. The analyses of the trajectories show that simulations of solvated proteins are highly sensitive to the presence of counter-ions and less sensitive to the volume of the water box. The differences in protein potential energies, r.m.s. deviations and radius of gyration between the simulations with and without counter-ions demonstrate that during such studies secondary structures of proteins are more stable when their charges are carefully neutralized. This stresses the need for such a procedure when analysing significantly charged proteins. The results also showed that the enlargement of the water box helps in the stabilization of the system.

Protein similarities beyond disulphide bridge topology.

Structural superimposition is an important procedure to analyse the relationships between proteins. A new approach and program, KNOT-MATCH, has been developed for automated structural superimposition of proteins by means of their disulphide bridge topology. As a result of the superimposition, regular secondary structures, loops and clusters of residues become correctly aligned. This fact allows us to find out important structural overlaps of residues, sometimes with functional significance, not only among proteins belonging to the same family but also between apparently non-related proteins. Different disulphide-rich protein families, such as EGF-like, defensin-like and plant protease inhibitors, have been self or cross analysed with this approach. Some amino acids that have been experimentally determined to be structural and/or functional key residues for these proteins are conserved in the three-dimensional space after superimposition by KNOT-MATCH. The program can be very useful for finding relationships among proteins that would be hidden to the current alignment methods based on sequence and on main-chain topology.

Potato carboxypeptidase inhibitor, a T-knot protein, is an epidermal growth factor antagonist that inhibits tumor cell growth.

Epidermal growth factor (EGF) and its receptor (EGFR) are involved in many aspects of the development of carcinomas, including tumor cell growth, vascularization, invasiveness, and metastasis. Because EGFR has been found to be overexpressed in many tumors of epithelial origin, it is a potential target for antitumor therapy. Here we report that potato carboxypeptidase inhibitor (PCI), a 39-amino acid protease inhibitor with three disulfide bridges, is an antagonist of human EGF. It competed with EGF for binding to EGFR and inhibited EGFR activation and cell proliferation induced by this growth factor. PCI suppressed the growth of several human pancreatic adenocarcinoma cell lines, both in vitro and in nude mice. PCI has a special disulfide scaffold called a T-knot that is also present in several growth factors including EGF and transforming growth factor alpha. PCI shows structural similarities with these factors, a fact that can explain the antagonistic effect of the former. This is the first reported example of an antagonistic analogue of human EGF.

[Aspects of cognitive functioning in the aged. A transversal study]. / Aspecten van het cognitief functioneren van bejaarden. Een transversaal leeftijdsonderzoek.

In the context of a WHO study on psychosocial needs of the elderly, a sample of 519 subjects from the region of Louvain was psychometrically tested. This sample consisted of men and woman between 55 and 94 years of age, most of them living at home. The test battery was composed of ten verbal and nonverbal tests and tasks currently used in psychogeriatric clinical practice but lacking validation and normative data for those aged populations. This study (psychometric testing took place in 1979-1980) was intended as a first step in a follow up program to gather more definite information about mental deterioration. Up to now it can be considered however as a cross-sectional study, providing normative data for the assessment of cognitive functioning in aged individuals up to 94 years. The decline in performances, found on all these tests, reflect generation as well as aging effects; the increasing proportions of aged subjects failing on the tests however, seem to indicate a high occurrence of partial or global cognitive deficits in elderly people beyond the eighth decade.

[Idiopathic arterial calcification in infancy. A literature review and presentation of one case (author's transl)]. / Enfermedad con calcificaciones arteriales idiopáticas en la infancia. Revisión de la literatura y presentación de un caso.

A 26 days old male newborn, developing cardiac insufficiency followed by death, is reported. Necropsy revealed broadly spread calcifications with severe affectation to the coronary arteries. No justifying cause to those calcifications was found. Literature is reviewed, and the current state of knowledge of this uncommon disease's etiology and pathogeny is questioned.