PERSONAL DE APOYO
PRIETO Eduardo Daniel
congresos y reuniones científicas
Título:
TWO AMILOIDOGENIC APOLIPOPROTEIN A-I MUTANS: A FLUOROMETRIC APROACH
Autor/es:
RAMELLA, NAHUEL; PRIETO , EDUARDO DANIEL; RIMOLDI, OMAR; TRICERRI, MARIA ALEJANDRA
Lugar:
Búzios- Rio de Janeiro- Brasil
Reunión:
Congreso; VII Iberoamarican Congress of Biophysics 2009. (3-9-2009/3-10-2009) Búzios- Rio de Janeiro- Brasil; 2009
Resumen:
Amyloidosis is characterized by extra cellular
deposits of anomalous fibrillar proteins. Human apolipoprotein A-I (ApoA-I) does
not normally misfold, but certain single natural mutations favor its pathologic
aggregation, as Lys107-0 and Gly26Arg (Iowa).
Here we studied the folding of both mutants,
as compared with Wt apoA-I. Analysis of chemical denaturation shows that Iowa and Lys107-0 are
more unstable at both acidic and physiological pH respect to the Wt. Under
acidic conditions, mutant for Lys107-0.
denaturation is less cooperative, suggesting
intermediate folding states.
Thioflavin-T (ThT) fluorescence intensity test
showed high yield of amyloid-like aggregation at pH 4 and 5 of the three tested
proteins, minimal at pH 6 and almost none under neutral conditions. These
ThT-binding products sediment under mild centrifugation. Protein misfolding is
concentration-dependent, and can occur under diluted solutions. Fluorescence
intensity was always higher for Lys107-0, but no significant differences were
detected between Wt and Iowa.
Proteins show different types of aggregates as observed by electron microscopy.
It is well-known that heparin favors the
aggregation of different amyloidogenic proteins. At acidic pH heparin binding
induced a significant increase in scattering respect to proteins at neutral pH,
suggesting an increment in particles size, which is higher for Lys107-0.
Altogether these data
suggest that apoA-I is sensitive to acidic conditions, but Lys107-0 is more
sensitive to the pH environment. A strong conformational shift occurs between
pH 5 and 6, thus pI plays an important role in determining protein aggregation;
nevertheless, this is not the only factor, as the Iowa variant, having and extra positive
charge, behaves similar to Wt under this experimental set up, but induces
neuroamiloidosis in patients.