1. Disease specific databases for personalized medicine: Fabry disease as a case of study
Maria Vittoria Cubellis
Dipartimento di Biologia ,
Università Federico II, Napoli, Italia
7th International Conference on Proteomics & Bioinformatics 
Rome, October 24-26, 2016.

2. Lysosomal alpha-galactosidase
429 aa
31 aa signal peptide
5 disulphide bridges
3 glycosylation sites
homodimer
17 X-ray structures

5. Endoplasmic reticulum
Autophagy
Golgi apparatus
Autophagosome
Phagocytosis
Primary
lysosomes
exocytosis
Secondary
lysosomes
Exocytosis
Late
endosome
Residual
bodies
endocytosis
Early
endosome

6. Endoplasmic reticulum
Autophagy
Golgi apparatus
Autophagosome
Phagocytosis
Primary
lysosomes
exocytosis
Secondary
lysosomes
Exocytosis
Late
endosome
Residual
bodies
endocytosis
Early
endosome

7. GLA is located on X chromosome

8. wide spectrum of heterogeneously clinical phenotypes Progressive
FABRY DISEASE
wide spectrum of heterogeneously clinical phenotypes
Progressive
Multiple organ systems
Morbidity
Cardiac complications
Stroke
Renal failure
Decreased lifespan

11. Enzymatic replacement therapy
ERT

12. ERT versus PC Enzyme replacement therapy ERT
High cost
Intravenous Infusion
Does not reach CNS
Immune reaction
Therapy with pharmacological chaperones(PC)
Low cost
Oral administration
Reaches CNS

13. ΔG (PROTEIN+CHAPERONE)= ΔG(PROTEIN) +RTln(1+CHAPERONE/Kd)
CHAPERONES ARE LIGANDS WHICH PREFERENTIALLY BIND THE FOLDED STATE OF PROTEINS
CHAPERONE
UNFOLDED WT PROTEIN
FOLDED WT PROTEIN
ΔG (PROTEIN+CHAPERONE)= ΔG(PROTEIN) +RTln(1+CHAPERONE/Kd)

14. DESTABILIZING MUTATIONS LOWER THE FREE ENERGY DIFFERENCE BETWEEN NATIVE AND UNFOLDED MUTANTS
ΔGN→D=1 D
A288P mildly
destabilizing
ΔGN→D=8
ΔGN→D=10
F50C severely
destabilizing N
wild

15. ΔG (PROTEIN+CHAPERONE)= ΔG(PROTEIN) +RTln(1+CHAPERONE/Kd)
CHAPERONES ARE LIGANDS WHICH PREFERETIALLY BIND THE FOLDED STATE OF SOME MUTANT PROTEINS
CHAPERONE
UNFOLDED MUTANT
FOLDED MUTANT
ΔG (PROTEIN+CHAPERONE)= ΔG(PROTEIN) +RTln(1+CHAPERONE/Kd)

16. UNFOLDING
FOLDING
PROTEASOME

17. Total amount of mutant A-Gal is lower than wild type in cells, but is increased by PC
WITHOUT CHAPERONE -
WITH CHAPERONE +
FIBROBLASTS FROM PATIENTS OR TRASFECTED MUTANTS
WESTERN BLOT
ALPHA GALACTOSIDASE

18. WESTERN BLOT ANALYSIS

19. ACTIVITY ASSAY
-DGJ
+DGJ 20 MICROMOLAR

21. One subunit of lysosomal alpha-galactosidase

22. Almost all amino-acids can be mutated

23. Some mutations retain residual activity
No residual activity
Some residual activity

24. Some mutations retain enzymatic activity
More than 500 missense mutations*, mostly private, are known for Fabry diseases
Some mutations retain enzymatic activity *

25. Some mutations respond to pharmacological chaperones
Responsive
Non responsive

27. Mutations affecting disulphide bridges do not retain residual activity in vivo and do not respond to chaperones

28. Mutations affecting the active site do not retain residual activity in vivo and do not respond to chaperones

29. Severely destabilizing mutations do not retain residual activity in vivo and do not respond to chaperones
ΔGN→D=5
F50C severely
destabilizing

32. FLEXIBILITY AND RESIDUAL ACTIVITY CORRELATE

33. OUTLIERS ARE MOSTLY GLYCINES

34. Mutations affecting the active site or disulphide bridges as well as severely destabilizing mutations, which prevent folding, do not retain residual activity
Mutations occurring at very rigid sites do not retain residual activity.
Residual activity correlates with flexibility

36. Assignment of PSSM scores to A-gal mutants
Let’s consider two cases: N122D and N139D

37. P S S M
O P U A
S E B T
I C S R
T I T I
I F I X
O I T
N C U T I O N

38. PSSM SCORES CORRELATE WITH RESPONSIVENESS
% Responsive (GALAFOLD)

41. Mutations occurring at non conserved sites retain residual activity and are responsive to chaperones

43. Residual activity in vitro of putative non pathological a-gal
mut
N hemiz
-DGJ
+DGJ
PSSM
humdiv
E66Q 3
47.6
53.66 -2
Probably damaging
R118C 8
24.5
27.8
S126G 18
51.3
67.4
benign
A143T 19
39.7
63.7 -1
D313Y
129
75.5
100.3
R363H 28
65.7
T385A 36 45
48.9
Possibly damaging
W399S 5 53
51.5 -4

44. Mutations retaining >25% residual activity might be considered non pathological variants

45. I would like to thank:
Dr G.Andreotti who is responsible for the experiments in vitro
Dr V.Citro who is responsible for the experiments with cells
M.Cammisa for develping Fabry_CEP
Telethon for funding us

46. References
Andreotti G, Guarracino M.R., Marco Cammisa M., Antonella Correra A., Cubellis MV.
Prediction of the responsiveness to pharmacological chaperones: lysosomal human alpha-galactosidase, a case of study.
Orphanet Journal of Rare Diseases, 2010, 5:36
Andreotti G, Citro V, De Crescenzo A, Orlando P, Cammisa M, Correra A, Cubellis MV.
Therapy of Fabry disease with pharmacological chaperones: from in silico predictions to in vitro tests.
Orphanet J Rare Dis :66
   
Cammisa M, Correra A, Andreotti G, Cubellis MV.
Identification and analysis of conserved pockets on protein surfaces.
BMC Bioinformatics. 2013; 14 Suppl 7:S9   
Fabry_CEP: a tool to identify Fabry mutations responsive to pharmacological chaperones.
Orphanet J Rare Dis :111
Andreotti G, Citro V, Correra A, Cubellis MV.
A thermodynamic assay to test pharmacological chaperones for Fabry disease.
Biochim Biophys Acta ;1840:
 Cubellis MV, Baaden M, Andreotti G.
Taming molecular flexibility to tackle rare diseases.
Biochimie Apr 2. pii: S (15) doi: /j.biochi [Epub ahead of print]
Andreotti G, Monticelli M, Cubellis MV.
Looking for protein stabilizing drugs with thermal shift assay.
Drug Test Anal Apr 5. doi: /dta [Epub ahead of print]
HayMele B, Citro V, Andreotti G., Cubellis MV
Drug repositioning can accelerate discovery of pharmacological chaperones.
Orphanet J Rare Dis May 7;10(1):55. doi: /s .

47. Palazzo Donn’Anna, Posillipo, Napoli
Thank you for your attention
Palazzo Donn’Anna, Posillipo, Napoli