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.

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

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

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

GLA is located on X chromosome

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

Enzymatic replacement therapy ERT

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

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)

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

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)

UNFOLDING FOLDING PROTEASOME

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

WESTERN BLOT ANALYSIS

ACTIVITY ASSAY -DGJ +DGJ 20 MICROMOLAR

One subunit of lysosomal alpha-galactosidase

Almost all amino-acids can be mutated

Some mutations retain residual activity No residual activity Some residual activity

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

Some mutations respond to pharmacological chaperones Responsive Non responsive

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

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

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

FLEXIBILITY AND RESIDUAL ACTIVITY CORRELATE

OUTLIERS ARE MOSTLY GLYCINES

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

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

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

PSSM SCORES CORRELATE WITH RESPONSIVENESS % Responsive (GALAFOLD)

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

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

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

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

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. 2011 6:66     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. 2013 8:111 Andreotti G, Citro V, Correra A, Cubellis MV. A thermodynamic assay to test pharmacological chaperones for Fabry disease. Biochim Biophys Acta. 2014 ;1840:1214-24  Cubellis MV, Baaden M, Andreotti G. Taming molecular flexibility to tackle rare diseases. Biochimie. 2015 Apr 2. pii: S0300-9084(15)00087-5. doi: 10.1016/j.biochi.2015.03.018. [Epub ahead of print] Andreotti G, Monticelli M, Cubellis MV. Looking for protein stabilizing drugs with thermal shift assay. Drug Test Anal. 2015 Apr 5. doi: 10.1002/dta.1798. [Epub ahead of print] HayMele B, Citro V, Andreotti G., Cubellis MV Drug repositioning can accelerate discovery of pharmacological chaperones. Orphanet J Rare Dis.. 2015 May 7;10(1):55. doi: 10.1186/s13023-015-0273-2. .

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