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Abstract

The presence of insoluble amyloid aggregates in various human tissues correlates with the development of
many human disorders (amyloidoses), including neurodegenerative diseases. Deposition of the aggregated
misfolded proteins in the brain, where aggregates can be localized extracellularly or intracellularly, is the
main characteristic of Alzheimer's, Parkinson's, Huntington's, prion diseases and many others. Since the
mechanisms behind the neurodegenerative diseases are far from understood, no efficient therapy is
available for their treatment.
Misfolded proteins frequently aggregate into ordered structures, named amyloid fibrils. Despite a lack
of sequence and structure similarities of the proteins involved in amyloid fibril formation, the amyloid
fibrils share common properties. They are very stable, elongated constructs, consisting of intertwined β
strands (β strands are running perpendicularly to the fibril axes) that are resistant to protease degradation.
Amyloid fibrils specifically bind certain dye molecules, such as ThT and Congo red.
The goal of our study was to contribute to understanding of the mechanism of amyloid fibril formation
by studying amyloid formation in in vitro as well as in vivo systems.
In the field of amyloid fibrils three leading hypotheses have been launched. Namely, the first hypothesis
states that the ability to form amyloid structures is a generic property of all polypeptides, second hypothesis
states that proteins not connected with any known disease share common features of amyloid-induced
toxicity compared to pathological proteins and third one argues that species influencing cell viability
(causing cytotoxicity) are not the fibrils themselves but rather the prefibrilar oligomers and that. According
to these hypotheses stefin B is very suitable model to study the phenomenon of amyloid fibril formation.
Human stefin B is a cysteine proteases inhibitor. Because of its broad distribution, a general protective
role was suggested. Deficiency of stefin B was found associated with development of the progressive
myoclonus epilepsy of type 1, EPM1. Stefin B forms amyloid fibrils rather easily and represents a well
studied system as its stability, folding and amyloid-fibril formation have been studied thoroughly.
In vitro studies included modelling the mechanism of amyloid fibril formation process of stefin B from
the kinetical, morphological and structural data and estimating the effect of microenvironment on stefin B
fibrillation, capturing copper influence and the interaction with amyloid beta peptide.
The model of stefin B amyloid fibril formation predicts formation of domain-swapped dimers that can
evolve to “closed” form, characteristic for off-pathway, or can proceed further in a propagated manner to
nucleate the fibrillation reaction. According to temperature and protein concentration dependence
measurements, the nucleation step was found to be a first-order reaction, followed by the fibril growth step
a second-order reaction. The prediction of the off-pathway from the oligomeric intermediates stems from
the observed anomaly of a reverse protein concentration dependence of the rates of fibrillation at higher
temperatures. Three energies of activation were derived from global fitting to the kinetic scheme. The
highest enthalpy of activation (95 ± 5 kcal mol-1) is characteristic for initial domain-swapping reaction,
nucleation with Ni irreversible transitions (global fitting insinuated Ni=64) corresponds to enthalpy of
activation 55 ± 4 kcal mol-1 and the enthalpy of activation 27 ± 5 kcal mol-1 obtained for the process of
fibril growth, is characteristic for conformational changes due to proline trans to cis isomerization.
It was shown that stefin B is, unlike its structural homolog stefin A, a high affinity copper binding
protein. Further, presence of copper inhibited amyloid fibril formation of stefin B. Since elevated
concentrations of metals, including Cu, Al, Zn, Fe have been located in the brain of patients with
neurodegenerative diseases, a role of copper binding could be related to specific function(s) of stefin B,
which remains to be confirmed by in vivo studies.
Stefins were found co-aggregated in senile plaques samples from patients with various
neurodegenerative disorders. Amyloid beta peptide is the main component of extracellular plaques specific
for Alzheimer's disease. To contribute to understanding of the role of cystatins in Alzheimer's disease, we
studied the interaction between different forms of stefin B and amyloid beta peptide. We were able to
demonstrate direct binding of the two monitored proteins that was oligomer specific, which is a new feature
differing from cystatin C. Hence, only tetramers of the wild type stefin B E31 and the variant stefin B Y31
(which is predominantly dimeric) showed concentration dependent interaction with amyloid beta peptide
and consequently inhibition of amyloid fibril growth of the latter. Thus, stefin B is another amyloid-binding
protein in vitro and, as preliminary experiments have shown, likely in cells. Possible »amateur« chaperon
activity of stefin B (similarly as was proposed for gelsolin, apolipoproteins and heparin-sulfateproteoglycans)
is discussed.
In the in vivo studies we decided to introduce stefin B into yeast cells. The amyloidogenic protein stefin
B was expressed in yeast Saccharomyces cerevisiae to acquire information on pathways affected in
eukaryotic cell by the presence of amyloid aggregates and amyloid-induced toxicity. We decided to
monitor gene expression pattern after inducing expression of stefin B in yeast cells using DNA microarray
technology. The construct of stefin B was also introduced into collection of all yeast strains from single
knock out gene library. Systematic construction of double mutants enabled a global analysis of synthetic
lethal genetic interactions, where mutants with significantly decreased growth rate were identified and thus
revealed genetic interactions of stefin B with the whole yeast genome. Unfortunately, the wild type stefin B
E31 was not aggressive enough to cause substantial effect on yeast strains, therefore, the next step of yeast
studies would be to introduce a highly aggregate-prone mutant of stefin B into S. cerevisiae cells.