RICERCA ITALIANA     

Research program

Identification of folding and misfolding determinants by site-directed mutagenesis.

University Co-ordinator

Università degli Studi di GENOVA - FISICA - ()

Research Unit Leader

Annalisa Relini

Description

The research activity of the Genoa Unit will consist in the study of the aggregation of beta2-microglobulin (beta2-m) mutants by atomic force microscopy (AFM). Beta2-m mutants will be produced by the Pavia Unit and our AFM measurements will complement the results obtained in Pavia using thioflavine T fluorescence, Congo red staining and electron microscopy.
In particular, the tasks of the Genoa Unit are the following:
1 - to characterize aggregate morphology, both in liquid environment and in air, for the different mutants;
2 - to perform a kinetic analysis of fibril growth;
3 - to get insight into the effect of surface charge and hydrophobicity on the aggregation process.
These tasks will be developed according to the following items:
1. Structural characterization of prefibrillar and fibrillar aggregates: this task will span the entire duration of the project. Fibrillogenesis will be studied under the different aggregation conditions employed by the Pavia Unit:
i) incubation at 37°C in Na acetate 50 mM, pH 6.4 in the presence of collagen;
ii) incubation at 37°C in Na phosphate 50 mM, NaCl 100 mM, pH 7.4, in the presence of 20% trifluoroethanol and preformed seeds;
iii) incubation at 37°C in Na citrate 50 mM, NaCl 100 mM, pH 2.5 in the presence of preformed seeds.
Particular attention will be devoted to condition i), which is the closest to the physio-pathological state ever reported in the literature and therefore is particularly expected to provide significant contributions to the understanding of beta2-m amyloid deposition which occurs in dialysis-related amyloidosis. Aggregation of beta2-m mutants will be first studied in the presence of bovine fibrillar collagen of type I using the same procedures that we recently employed for wild-type beta2-m (Relini et al. 2006). Then experimental conditions will be extended to the presence of bovine collagen of type II and human collagen, with the aim of approaching physio-pathological conditions more closely. All collagen types will be prepared by the Pavia Unit.
Within this framework, the effect of heparin on the fibrillation process will also be tested. In fact, heparin is used as anticoagulant agent in haemodialysis; preliminary AFM measurements showed that in the presence of heparin concentrations comparable with those used in therapies, the fibrillation process of wild type beta2-m on collagen is faster.
For AFM imaging, sample aliquots will be extracted at different times during the aggregation process and will be deposited on a freshly cleaved mica surface. Tapping mode atomic force microscopy, which enables non-destructive imaging of soft samples, will be employed either in air or in solution. The deposition procedure will be optimized according to the aggregation condition employed. We recently found (Relini et al., 2006) that, in the presence of collagen, fibrillogenesis is strictly localized in close proximity to the collagen surface; therefore, in this case, care will be taken to deposit collagen with a drop of surrounding protein solution. When employing conditions ii) and iii), samples to be imaged in air will be strongly diluted before deposition on the substrate to avoid salt crystallization subsequent to the drying procedure. Aggregate height will be measured from the height in cross-section of topographic images. Fibril persistence length will also be evaluated.
2. Kinetic analysis of fibril growth: the length of a statistically significant fibril ensemble will be measured as function of the aggregation time. These experiments will allow us to determine the fibril elongation speed and will yield information about the kinetics of the aggregation process. The results obtained will be compared with those obtained by the Pavia Unit using other techniques such as ThT fluorescence and electron microscopy. Aggregation conditions and sample preparation will be the same described in item 1. This task will span the entire duration of the project.
3. Effect of surface charge and hydrophobicity on the aggregation process: the results obtained so far indicate that electrostatic interactions play an important role in beta2-m aggregation; in particular, positively charged surfaces (such as those present on collagen fibres) are required to obtain fibrillation of beta2-m at 37°C and pH 6.4. Zeta potential measurements will allow us to evaluate the aggregate surface charge at different times of the aggregation process for different beta2-m mutants.
Another interesting aspect concerns aggregate hydrophobicity. Relevant information can be obtained comparing the ability of different beta2-m mutants to interact with model membranes such as liposomes, planar membranes and phospholipid monolayers. Depending on the model system considered, these experiments will involve, respectively, fluorescence measurements of encapsulated calcein release, structural analysis of membrane morphology by AFM and monolayer surface pressure measurements. This task will be performed in the second project year and will involve a representative set of mutants, selected according to the results obtained in the first year by the research units involved in this project.

A. Relini, C. Canale, S. De Stefano, R. Rolandi, S. Giorgetti, M. Stoppini, A. Rossi, F. Fogolari, A. Corazza, G. Esposito, A. Gliozzi, V. Bellotti. (2006) Collagen plays an active role in the aggregation of beta2-microglobulin under physio-pathological conditions of dialysis-related amyloidosis. J. Biol. Chem., in press; doi 10.1074/jbc.M513827200.