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Riunione Scientifica di Istituto, Pozzuoli, 22-23 Ottobre 2014
Paolo Ruzza
Protein folding
The folded three-dimensional structure of
proteins is a compromise between
thermodynamic stability and conformational
flexibility required for their function
Consequently, proteins are often marginally
stable in their physiological environment.
Riunione Scientifica di Istituto, Pozzuoli, 22-23 Ottobre 2014
Paolo Ruzza
Protein misfolding diseases
Loss of function (LOF) diseases
cystic fibrosis, lysosomal storage diseases such as
Gaucher’s disease, α1-antitrypsin deficiency, and certain
cancers
Increasing
protein
misfolding
PROTEIN MISFOLDING DISEASES
Declining
proteostasis
capacity
Gain of function (GOF) diseases
Alzheimer’s and Parkinson’s diseases, ALS, and a wide
range of amyloidoses
Riunione Scientifica di Istituto, Pozzuoli, 22-23 Ottobre 2014
Paolo Ruzza
Protein misfolding diseases
Biochimica et Biophysica Acta 1830 (2013) 4860–4871
Riunione Scientifica di Istituto, Pozzuoli, 22-23 Ottobre 2014
Paolo Ruzza
Protein misfolding diseases
Riunione Scientifica di Istituto, Pozzuoli, 22-23 Ottobre 2014
Paolo Ruzza
Protein misfolding diseases
Biochimica et Biophysica Acta 1830 (2013) 4860–4871
Riunione Scientifica di Istituto, Pozzuoli, 22-23 Ottobre 2014
Paolo Ruzza
Protein misfolding diseases
Small molecules are attractive
because:
can be administered orally,
have more potential to access to
most cell types,
can cross the blood brain
barrier,
do not cause autoimmune
responses,
have lower manufacturing cost.
Riunione Scientifica di Istituto, Pozzuoli, 22-23 Ottobre 2014
Paolo Ruzza
Periventricular
white matter of
mice at 3
months of age
Riunione Scientifica di Istituto, Pozzuoli, 22-23 Ottobre 2014
Three dimensional visualization
modelled structure of GFAP. Annals
Research, 2011, 2, 40-50
of
the
of Biological
Paolo Ruzza
Ceftriaxone – GFAP – Alexander’s disease
Cef
GFAP alone
GFAP + Cef (2 eq.) det.
GFAP + Cef (2 eq.) calc.
12
Ellipticity (mdeg)
8
4
0
-4
-8
200
220
240
Wavelength (nm)
UK Diamond Light Source Synchrotron facility
where B23 SRCD beamline is located
Riunione Scientifica di Istituto, Pozzuoli, 22-23 Ottobre 2014
Far-UV SRCD spectra of GFAP alone or in presence of two
equivalents of ceftriaxone.
Paolo Ruzza
Ceftriaxone – GFAP – Alexander’s disease
4.9°C
20.2°C
12
Ellipticity (mdeg)
8
20.1°C
Far-UV CD spectra of GFAP alone (0.66 mg/ml) in
aqueous solution at different temperature.
4
0
-4
90.0°C
-8
1
200
220
8
240
The higher photon flux of B23 beamline can induce
protein denaturation in structured proteins with
significant content of α-helical or β-sheet
conformations.
Ellipticity (mdeg)
Wavelength (nm)
4
0
-4
20 Repeated CD scans of GFAP (0.66 mg/ml) in
aqueous solution measured with B23 module B.
20
-8
200
220
240
Wavelength (nm)
Riunione Scientifica di Istituto, Pozzuoli, 22-23 Ottobre 2014
Paolo Ruzza
Ceftriaxone – GFAP – Alexander’s disease
6
GFAP alone
GFAP + cef
GFAP + dph
8
GFAP alone
GFAP + cef
GFAP + dph
UV-protein
denaturation curves
∆CD (mdeg) @ 191 nm
∆CD (mdeg) @ 191 nm
10
6
4
2
4
2
CD melting curves
0
0
0
20
40
60
80
100
0
5
10
15
20
No of scans
Temperature (°C)
0.24
GFAP alone
GFAP + cef
GFAP + dph
0.20
0.20
% of α-helix content
% of α-helix content
0.16
0.16
0.12
0.08
0.12
0.08
GFAP alone
GFAP + cef
GFAP + dph
0.04
0.04
% of a-helix content
0.00
0.00
0
20
40
60
80
100
Temperature (°C)
Riunione Scientifica di Istituto, Pozzuoli, 22-23 Ottobre 2014
0
5
10
15
20
No of scans
Paolo Ruzza
Ceftriaxone – α-synuclein – Parkinson's disease
A – Lewi’ s body
B – normal brain pigmentation
C – nucleous of the cell
Riunione Scientifica di Istituto, Pozzuoli, 22-23 Ottobre 2014
Paolo Ruzza
Ceftriaxone – α-synuclein – Parkinson's disease
0
-3.2x10-4
A
B
-2
∆A=AL-AR (195nm)
-3.3x10-4
CD (mdeg)
-4
0.00 µM
1.25 µM
2.50 µM
3.75 µM
0.50 µM
7.50 µM
10.0 µM
12.5 µM
15.0 µM
17.5 µM
-6
-8
-10
-12
180
-3.4x10-4
-3.5x10-4
Kd = 0.53 ± 0.2 µM
0.0
190
200
210
220
230
240
250
260
Wavelength (nm)
Far-UV SRCD spectra of α-synuclein (5 µM) in 5 mM
Tris-HCl buffer, pH 6.8, in the presence of increasing
amount of ceftriaxone.
Riunione Scientifica di Istituto, Pozzuoli, 22-23 Ottobre 2014
5.0x10-6
1.0x10-5
1.5x10-5
2.0x10-
[Ceftriaxone] (M)
Determination of the Kd value by CD spectroscopy
analyses. The ΔA values at 195 nm, measured as a
function of different ligand/AS molar ratios, are
plotted versus the ceftriaxone concentration. Errors
are less than 1.0%.
Paolo Ruzza
Ceftriaxone – α-synuclein – Parkinson's disease
Alone
In presence of ceftriaxone (5.0 eq.)
0
-2
∆A = AL - AR 10-4
∆A = AL - AR 10-4
0
0
2
4
7
9
11
-4
-2
0
2
4
9
11
-4
200
220
240
200
Wavelength (nm)
240
Far-UV CD spectra of α-synuclein (6.31 µM) in 10 mM
phosphate buffer, pH 6.8, in 0.1 cm path length quartz
cells incubated at 37 C.
AS
AS-ceftriaxone
0.0000
220
Wavelength (nm)
∆A = AL- AR
-0.0001
-0.0002
Time-course of the intensity of CD band at 200 nm of αsynuclein alone or in presence of 5.0 equivalents of
ceftriaxone.
-0.0003
0
2
4
6
8
10
12
days
Riunione Scientifica di Istituto, Pozzuoli, 22-23 Ottobre 2014
Paolo Ruzza
Ceftriaxone – α-synuclein – Parkinson's disease
α-Synuclein- ceftriaxone docking
PDB: 1XQ8
Riunione Scientifica di Istituto, Pozzuoli, 22-23 Ottobre 2014
Paolo Ruzza
Ceftriaxone – α-synuclein – Parkinson's disease
MTT assay
In vitro effects of ceftriaxone (CTX)
on viability and α-synuclein
expression of PC12 cells exposed to
6-hydroxydopamine (6-OHDA).
α-synuclein immunoreactivity
Riunione Scientifica di Istituto, Pozzuoli, 22-23 Ottobre 2014
Paolo Ruzza
Future directions
The cross-reactivity of small molecules.
The use of chemical chaperones (osmolytes)
in reversing protein misfolding.
Riunione Scientifica di Istituto, Pozzuoli, 22-23 Ottobre 2014
Paolo Ruzza
Anna Marchiani
Mehmet Islami
Barbara Biondi
Andrea Calderan
Stefano Mammi
Paolo Ruzza
Giovanna Delogu
Davide Fabbri
Maria A. Dettori
Daniele Sanna
Giuliano Siligardi
Rohanah Hussain
Isabella Tessari
Luigi Bubacco
Mario Sechi
Nicolino Pala
Sonia Dedola
Ylenia Spissu
Rossana Migheli
Pier Andrea Serra
GianPietro Sechi
Riunione Scientifica di Istituto, Pozzuoli, 22-23 Ottobre 2014
Stefano Mammi
FIRB-MERIT Program "Molecular bases in
ageing-related
degenerative
syndromes"
(RBNE08HWLZ) and EU’s FP7 Programme
(FP7/2007-2013) under grant agreement nº
226716.
Paolo Ruzza