Prion Protein- Torrent Et Al., Biochemistry 2003, 2004; Cordeiro Et Al., JBC2001; JBC 2004; Biophys J

A Química Inteligente a Serviço da Medicina

Biologia Estrutural de Macromoléculas Envolvidas em Doenças Neurodegenerativas e Câncer

Jerson Lima Silva Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem (INBEB) Centro Nacional de Ressonância Magnética Nuclear Jiri Jonas (CNRMN) Laboratório de Termodinâmica de Proteínas e Vírus GREGORIO WEBER (LTPV) 65 YEARS AGO: Do we need a new chemistry or physics to understand biological phenomena?

• ―...... from all we have learnt about the structure of living matter, we must be prepared to find it working in a manner that cannot be reduced to the ordinary laws of Physics. And that not on the ground that there is any ‗new force‘, directing the behaviour of the single atoms within a living organism, but because the construction is different from any thing we have yet tested in the physical laboratory.‖ E. Schrödinger (1944) In “What is Life” Física, Química e Bioquímica de Biologia Molecular Proteínas Biologia Molecular Estrutural Genética e Medicina Biologia Celular

Structural Molecular Biology: How did it start? Difração de Raios-X por Fibra de DNA (diagramas obtidos por Rosalind Franklin – King’s College)

DNA tipo “B” (hidratado e pouco cristalino)

O diagrama com um “X” é a assinatura de estruturas em hélices

As reflexões do “X” indicam que a periodicidade da hélice é ~34Ǻ

A reflexão axial indica diâmetro da hélice da ordem de 20 Ǻ (a) Ribosome (50S) (b) Rhodopsin (c) Reovirures core (d) HMG-CoA red (e) Yeast RNA pol (f) ATP synthase (g) Nucleosome (h) Mismath repair MutS (i) HIV gp120 complexed with CD4 (D1D2) and Ab

Ainda resta muito para se aprender a respeito da estrutura de proteínas National Institute for Science and Technology in Structure Biology and Bioimaging – INBEB Prion Amiloid b -synuclen p53

Transmissible Encefalopathies Alzheimer’s Disease Parkinson’s Disease Cancer

Silva et al., Acc. Chem. Res. 2010 43: 271-9. Why people get sick when protein folding goes wrong? Hypothesis: Hydration Controls Protein Conformation Diseases

H2O H2O

Increased

Volume

H2O

Increased Increased Volume

Foguel and Silva, Biochemistry 2004 Hydration and packing can be probed by high pressure studies

•Peng, Jonas, Silva (1993) PNAS 91: 8244 •Foguel and Silva (1994) PNAS 90: 1776

•Hummer et al., PNAS 1998 •Hillson N, Onuchic and Garcia, PNAS 1999 •McCarthy and Grigera, BBA 2006 •Oliveira et al., JMB 1994 Hydrophobic interaction is weaker Silva, Foguel, Royer, TiBS 2001 under pressure BAD MISFOLDING The volume approach to proteins that cause misfolding diseases: Trasnsthyretin- Ferrão-Gonzales et al., PNAS, 2000, JMB 2003; Dirix et al., JMB 2005 -synuclein- involved in Parkinson's disease. Foguel et al., PNAS 2003; Follmer et al., 2007 p53- Ishimaru et al., Biochemistry 2003; Ishimaru et al., JMB 2003; Biophys. J. 2004; Biochemistry 2009; Ano Bom et al., 2010

Prion Protein- Torrent et al., Biochemistry 2003, 2004; Cordeiro et al., JBC2001; JBC 2004; Biophys J. 2005; Silva et al., Acc Chem Res 2010

Disulfide deficient Hen lysozyme- Niraula, Akasaka et al., PNAS 2004

The volume approach to dissociate inclusion bodies: St John RJ, Carpenter JF, Randolph TW, PNAS Partially Folded 1999 Foguel D, Robinson C, Sousa PC, Silva JL, Robinson AS. Biotechnol. Bioeng. 1999; Foguel and Silva, Biochemistry 2004 Lefebre and Robinson, 2003

Parkinson´s disease • Involves the aggregation of -synuclein: wt and variants (A30P and A53T) • Parkinson's disease afflicts the substantia nigra in the brain. It is a dopaminergic region that controls movements soluble 1,0 20 WT fibril 1,0 after pressure 0 0,8 WT 0,8 -20

)

0 0,6 A53T ) (mdeg) CD -40 0 0,6 A30P -60 0,4 200 210 220 230 240 250 260 (LS/LS 0,4

(LS/LS Wavelength (nm) 0,2 0,2

0,0 A 0,0 B 0 500 1000 1500 2000 2500 0 50 100 150 200 250 Pressure (bar)

Time under 1,700 bar (min) Thioflavin T binding (green bars) (green binding T Thioflavin

WT A53T C 18 2,5 D 100 16 2,0 A53T 14 80 12 1,5 WT 10 60 8 1,0 40 6

O.D. (330 nm) 4 0,5 20

Congo Red (red bars) 2 0,0 0 0 0 20 40 60 80 100 fibrils fibrils soluble soluble Time (min)

after pressure after pressure

Foguel et al.(2003) PNAS 100: 9831 Follmer et al. (2007) Biochemistry 46: 472

Braga et al. (2011) J Mol Biol 405: 254 The intriguing prion diseases Transmissible Spongiform Encephalopathies Riek R, Hornemann S, Wider G, Billeter M, Glockshuber R, Wüthrich K (1996) Nature 382: 180-2

PrPSc (Scrapie) PrPC (cellular PrP) *Protease resistance; *Protease sensitive; *Higher b-sheet content; *-helical and random coil content; *Highly insoluble; *Soluble. *Formation of amorphous and fibrillar aggregates. DeMarco et al., 2005 (a) Section from frontal cortex showing a field with aggregates of plaques surrounded by spongiform degeneration. (b) Multiple plaques and amorphous deposits are PrP immunopositive. Prusiner, http://depts.washington.edu 1998. PROBLEMS WITH THE PROTEIN-ONLY HYPOTHESES

Many questions remain

unanswered: U

1) Why the low efficiency of I infection (ratio 1:100,000)? 2) Can recombinant PrPC

molecules cause disease in energy free Gibbs the absence of infectious Adjuvant prion? Conformation coordinate 3) Are there adjuvant factors? *Protein chaperones? (DebBurman etal., PNAS 1997); Another macromolecule *Sulfated Glycans? (Caughey et al., J. Virol 1994; Vieira et (provisionally designated al., JACS 2011; Silva et al., Methods protein X by Stanley Prusiner) 2011) may interact with the prion *Nucleic Acids? Micro RNAs, protein acting as an adjuvant iRNAS, DNA (Cordeiro et al., JBC factor. 2001; 2004; Nandi et al., JMB 2002; Deleault et al., Nature 2003; Silva et al;, TiBS 2008; Gomes et al., JBC 2008) Comparison of the stability against pressure between -rPrP and b-rPrP

 =(IRobs–IRinicial/IRfinal–IRinicial) = s  Q/(T p m V) Thermal expansion coefficient -rPrP  hydrated  ASA -rPrP b-rPrP b-rPrP  hydrated  ASA

Cordeiro et al. (2004), JBC 279: 32354 Volume (A) and Gibbs free-energy (B) diagrams of -rPrP and b-rPrP.

A The high pressure studies support the findings that other

molecules such as nucleic acid (ml/mol) may participate in the prion

29.2 conversion reaction by U 43.6 changing the solvent

Volume Volume U‘ accessible surface and the Conformation coordinate distribution of cavities B U The disordered segments of

-rPrP PrPC would fold either by 4

5. U‘ b-rPrP binding to other molecules,

such as an RNA or DNA, or (kcal/mol) Nucleic 2.8 by assembling into oligomers

acid or amyloid fibrils. Gibbs free energy free Gibbs Conformation coordinate Cordeiro et al. JBC 2004b Cordeiro et al. Biophys. J. 2005 -rPrP  hydrated  ASA Gomes et al., JBC 2008 b-rPrP  hydrated  ASA Silva et al., Acc. Chem Res. 2010 The prion protein is highly hydrated and malleable Nucleic acid acts as catalyst of the prion conversion

 Poli AT Oligo 28 Poli GC

E2DBS 5’-GTAACCGAAATCGGTTGA-3’R R3’-CATTGGCTTTAGCCAACT-5’ Nucleic acid acts as catalyst of the prion conversion

PrP (109-149) M

0,0 0,5 1,0 1,5 2,0 500 25 20 450

) 15 400 mP Factor X 10 350 5

300 0 -5 250 Polarization ( Polarization -10

Light scattering (320nm) Light

200 -15 

0 20 40 60 80 100 120 0,5 1,0 1,5 2,0 mPrP23-231 (nM) mPrP 23-231 (M)

Cordeiro et al., JBC 2001 The Prion Paradox: Protein Only Or Something Else

DNA? RNA? GAG?

Cordeiro et al., JBC 2001; Cordeiro et al., JBC 2004; Gomes et al., JBC 2008; Silva et al., TiBS 2008; Acc Chem Res 2010; Vieira et al., JACS 2011 Binding of nucleic acid to prion protein occurs mostly with interaction with the globular domain that is unusually hydrated Three-dimensional reconstruction of rPrP from SAXS measurements.

rPrP23-231 (light gray) rPrP32-121 (dark gray). rPrP23-231 (light gray) rPrP globular domain DNA 16pb (2BOP.pdb) (1AG2) D32-121 Lima et al., Biochemistry 2006 rPrP :DNA complex. NMR Illuminates Amino Acid-Specific information about he PrP-Oligonucleotide Complex.

Lima et al., Biochemistry 2006 Most amino acids in the globular domain that display significant changes in HSQC are directed to the same side, which is a typical design of helical DNA binding motifs

Lima et al., Biochemistry 2006 Predicted DNA-binding sites Electrostatic Potential Distribution

Human

Cattle

Mouse

Syrian hamster

Rabbit

Silva et al., Methods 2011 How about RNA? Prion Protein complexed to RNA through its N- terminal domain aggregates and is toxic to neuroblastoma cells

260 15 5

10 0 ) 240

3 -5 5

Raw Ellipticity (mdeg) Ellipticity Raw 210 240 270 300 220 Wavelength (nm) 0 200

Anisotropy (x10 Anisotropy Raw Ellipticity (mdeg) Ellipticity Raw -5

180 -10 0,1 1 10 100 200 220 240 260 280 300 320 N2aRNA [g/mL] Wavelength (nm) () FITC-PrP23-231

PrP 23-231 binds to N2aRNA with high affinity and its secondary structure changes upon this interaction .

Gomes et al., J. Biol. Chem. 2008 Prion Protein complexed to RNA through its N-terminal domain aggregates and is toxic to neuroblastoma cells

Gomes et al., J. Biol. Chem. 2008 Toxicity of PrP:N2aRNA complex

Gomes et al., JBC 2008 PrP:RNA Interaction – HSQC NMR

rPrP23-231 + SAF9343-59 Fosfato de sódio 20 mM NaCl 100 mM pH 7,4

Gomes et al., J. Biol. Chem. 2008 Binding of nucleic acids and GAGs to PrP protein

Leads to Dehydration Biological Relevance and Potential Antiscrapie Therapeutic Applications of Nucleic-acid Binding to PrP:

2008 Yin S, Fan X, Yu S, Li C, Sy MS. Binding of recombinant but not endogenous prion protein to DNA causes DNA internalization and expression in mammalian cells. J Biol Chem. 2008 Jul 11. [Epub ahead of print Where is the Nucleic Acid? The RNA revolution

Biology's Big Bang Jun 14th 2007

From The Economist print edition What physics was to the 20th century, biology will be to the 21st—and RNA will be a vital part of it Searching for More Specific RNAs in Neuroblastoma Cells

1) Padrão de peso; 2) Extrato N2aRNA; 3) RNA extraído pós agregação, sem tratamento; 4) RNA extraído pós agregação, tratado com RNase A por 1h a 37º; 5) RNA extraído pós agregação, lavado tampão contendo NaCl 250 mM. How about Glycosaminoglycans (GAGs)?

0.11

0.10

0.09

0.08

0.07

0.06

Anisotropy Anisotropy at 280nm pH 5.5 0.05 pH 7.4

0.04 0 2 4 6 8 10 12 14 16 [Heparin] uM Aggregation and binding kinetics of rPrP (23-231) with heparin, 1:1

50 12 0.35

pH 5.5 0.30 pH 7.4 pH 7.4 10

LS/LS 0.25 8

30 0.20

pH 5.5 pH

6 7.4 pH 0.15 20

anisotropy 0.10

LS/LS Δ

0.05 10 2 0.00

0 0 -0.05

0 2 4 6 8 10 0 2 4 6 8 10 2 t=h t=h pH 7.4 0

-2

-4

Raw Ellipticity Ellipticity Raw (mdeg) -6

200 210 220 230 240 250 Wavelength (nm) Vieira et al., J Am Chem Soc 133: 334-344, 2011 Vieira et al., J Am Chem Soc 133: 334-344, 2011 GAG traps PrP in the ―Dry State‖ but appears to prevent conversion

N2aRNA effect on PrP:Hep complex

(ml/mol) 29.2

U 43.6

Volume Volume U‘ Conformation coordinate B U

-rPrP 4

5. U‘ b-rPrP

(kcal/mol) 2.8

NA Gibbs free energy free Gibbs Conformation coordinate

Vieira et al., J Am Chem Soc 2011 - rPrP WT - GAG Aggregation Disaggregation (kobs LS) (k1obs LS, k2obs LS)

RNA Aggregation No aggregation RNA

Vieira et al., J Am Chem Soc 133: 334-344, 2011 PrP is a highly promiscuous protein

Glycosaminoglycans

STI1

2+ Cu Cu2+ Cu2+ Cu2+ Cu2+ Cu2+

RNA DNA Antiprion Compounds

(R)-6-(1-Hydroxybut-2- ylamino)phenanthridine

 rPrP23-231 (1 M) monitorada livre em solução;  rPrP23-231 (1 M) + N2aRNA (11,5 g/mL);  GA;  GAi;  6AP;  6APi. Teste t: *p<0,05; **p<0,001. a b Compound % Aggregation with LogBBB C50% 1M (1) NH2 4-amino-7- 8.4 % 0.08 ~ 0.2M chloroquinoline Cl N (2) CH3 HN 7-chloro-4- 38.6 % 0.29 > 0.9M methylaminoquinoline

Cl N OH (3) HN 2-(7-chloro-4- 97 % 0.06 > 1M quinolinyl)- aminoethanol Cl N (6) NH2 HN N-(7-chloro-4- 7.9 % 0.03 ~ 0.1M quinolinyl)-1,2- ethanediamine Cl N (7) CH3 N N2-(7-chloro-4- 82 % 0.20 > 1M HN CH 3 quinolinyl)-N1,N1- dimethyl-1,2- ethanediamine Cl N (9) CH3 N2-(7- 0.29 N CH3 HN trifluoromethylthio-4- 54 % 42> 1 M Macedo et al., Eur. J. Med. Chem., 2010 quinolinyl)-N1,N1- diethyl-1,2- ethanediamine F3CS N

(10)

HN 4-(cyclopentylamino)- 88 % 0.57 > 1M 7-chloro-quinoline

Cl N

Conclusions • Hydration change is the key event in the conversion between the cellular and the scrapie PrP isoforms;

• Nucleic acid molecules inhibit prion domains aggregation in vitro and its interaction with native rmPrP23-231 converts the more hydrated PrP species into the less hydrated conformation;

• We propose that an hypothetical NA molecule could act as a catalyst of PrPC into PrPSc conversion, by reducing the energetic barriers (involved in hydration changes) that prevents this conversion; • NMR and SAXS data show how the interaction between the globular domain and DNA/RNA occurs. NMR data reveal significant changes in the disordered domain, which demonstrate a contribution of this region for the formation of the consolidated complex. •The binding of NAs and GAGs by the prion protein occur with ordering of the N-domain as well as structural modification of the NA. Both GAGs and NA aptamers lead the prion protein to a less hydrated state and might prevent scrapie conversion.

Is the Prion Protein a Nucleic Acid Chaperone?

TARGETS FOR THERAPEUTIC INTERVENTION Cancer: The Big Picture

(e.g.src, ras, abl)

Proteins made by cancer genes are components of cell circuits controlling growth and development

(e.g. p53, Rb, BRCA) Adapted from Prof. Harold Varmus GLEEVEC (IMATINIB)

DISCOVERED AND DEVELOPED BY NOVARTIS AND DFCI IN THE 1990’S

INHIBITS THREE PROTEIN TYROSINE KINASES ENCODED BY PROTO-ONCOGENES

ABL (AND BCR-ABL)

KIT (RECEPTOR FOR SCF)

PDGF RECEPTORS Drug in active site of Abl kinase

NLS NLS NLS

Myr CAP SH3 SH2 SH1 DNABD FABD COOH

SH2L NSH3 NSH3SH2L

MGQQPGKVLGDQRR MGQQPGKVLGDQRRPSLPALHFIKGAG PVNSLEKHSWYHGP PSLPALHFIKGAGKKES KKESSRHGGPHCNVFVEHEALQRPVASD VSRNAAEYLLSSGING SRHGGPHCNVFVEHE FEPQGLSEAARWNSKENLLAGPSENDPN SFLVRESESSPGQRSIS ALQRPVASDFEPQGLS LFVALYDFVASGDNTLSITKGEKLRVLGYN LRYEGRVYHYRINTAS EAARWNSKENLLAGP HNGEWCEAQTKNGQGWVPSNYITPVN DGKLYVSSESRFNTLA SENDPNLFVALYDFVA SLEKHSWYHGPVSRNAAEYLLSSGINGSF ELVHHHSTVADGLITT SGDNTLSITKGEKLRVL LVRESESSPGQRSISLRYEGRVYHYRINTAS LHYPAPKRNKPTVYG GYNHNGEWCEAQTK DGKLYVSSESRFNTLAELVHHHSTVADGLI VSPNYDKWEMERTD NGQGWVPSNYITPV TTLHYPAPKRNKPTVYGVSPNYDKWEME RTD Shape Reconstruction of SH2-kinase linker domain and SH2-kinase linker domain alignment (created by 3D-JIGsaw)

~90°

1 2 3

1 - SH2-kinase linker domain alignment (created by 3D-JIGsaw)

3 - Superimposed model by SUPCOMB Cancer is a disease related to loss of control of cellular division The p53 gene is mutated in more than half of all human cancers ? 24 h Barra: 240 nm 310  10 nm / 220  20 nm 130  30 nm / 60  10 nm Inset: 340 x 260 nm

1 month 1 month Barra: 265 nm Barra: 240 nm > 1 mm / 260  60 nm / 20 ± 5 nm 60  3 nm Aggregation of proteins related to PFD (protein folding diseases) p53c aggregation behaves similar to amyloidogenic proteins p53 accumulates in the cytoplasm of neuroblastoma cells

p53c (fibrillar)

p53c (annular) Moll et al., Mol. Cell. Biol 1996 A “Prion-like” mechanism for the participation of amyloid aggregate of p53 in cancer Negative Dominance Effect Negative dominance phenomenon

Ishimaru et al., Biochemistry 2003 Aggregation of p53C at 37°C

R248Q R248Q

WT WT

Negative dominance phenomenon – ―Prion-like‖ effect. • Most p53 mutants (translated from a single mutant allele) are able to drive wild-type p53 protein (translated from the remaining wild-type p53 allele) into a mutant conformation, in a way that resembles the action of the prion protein (Milner, J. & Medcalf, Cell 1991) Structural Characterization of the Amyloid Structure by X-Ray Diffraction

~10 Å

~4,7 Å R248Q pH 7

wt pH 7

wt pH 5

R248Q pH 5 mutante p53 R248Q Co- localization of p53 and protein aggregates in breast cancer tissues

WT p53

Co-localization of p53 (green) and protein aggregates (red) in a tumor sample (T74) with WT p53

R237H p53

Co-localization of p53 (green) and protein aggregates (red) in a tumor sample (T21) with mutant p53 R273H

R175H p53

Co-localization of p53 (green) and protein aggregates (red) in a tumor sample (T18) with mutant p53 R175H Toxic Aggregates Formed by Amyloid Oligomers and Fibrils of p53 Is p53 Tumor-Related Mutant a Prionoid? Sequence-specific DNA binding stabilizes p53C Binding of small 24-bp nucleic acids

(a) 20 (b) 40 0

) ) 0

-1

-1 15 30 -300 -200 20 -600 10-400

-900 -600 5 10

Center of Mass (cm

Center of Mass (cm Mass of Center

Light Scattering Ratio -1200 Ratio Scattering Light

  -800 0 0 -1500 -1000 0 1 2 3 0 0 1 1 2 2 3 3 0 1 2 3 Pressure (kbar) PressurePressure (kbar) (kbar) Pressure (kbar) Low salt High salt Small aptamers are potential lead compouds to stabilize p53C and to rescue misfolded conformations Ishimaru et al., Biochemistry 2009 Aptameric cognate DNA induces recovery of the misfolded conformation

8000 7 (A) 7000 6 (B) 6000 WT p53 5 5000 - DNA (HP) 4 4000 +DNA + DNA (HP 3 3000 ) 2000 2

Light Scattering ratio 1000 1

Fluorescence intensityFluorescence (A.U.) 0 0 300 320 340 360 380 400 420 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Wavelength (nm) Pressure (kbar)

atmospheric pressure return in ausence of DNA presence of DNA return in the presence of DNA

Ishimaru et al., Biochemistry 2009 Silva et al., Acc Chem Res 2010 Thioaptamers to rescue misfolded p53 Consensus p53 DNA 5’ TTTC*CTAGAC*ATGCC*TAATTA 3’ 3’ AAAG*GATCTG*TACGG*ATTAAT 5’ Poly(GC) DNA 5’ ATAATTG*CGCGCG*CGCGCAGG*AAA 3’ 3’ TATTAAC*GCGCGC*GCGCGTCC*TTT 5’ * 200

)

-1 -200

-400 phosphodiester phosphorothioate

-600

Center of Mass (cm



-800

2,5e+6 -1000 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 p53wt 5 M + dna consenso 10 M Pressure (Kbar) p53wt 5 M 16 2,0e+6

12 1,5e+6

8 1,0e+6

Light Scattering (A.U.) 4

Light Scattering Ratio 5,0e+5 2

0 0,0 0 2000 4000 6000 8000 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 Pressure (kbar) Time (s) Conclusions • We conclude that aggregation of mutant p53 into a heterogeneous mixture of amyloid species, fibrillar and oligomers, is involved in cancer and accounts for the negative dominance. • Formation of the oligomers would occur in a fast equilibrium encompassing wild type and mutant forms, in a way similar to a prion conversion process, conferring a prionoid character for the misfolded conformation. Further aggregation into fibrils would act as a sink. • Our model explains how misfolding of both WT and mutant p53 lead to loss of function and to the prion-like negative dominance effect. •The inhibition of oligomeric species and aggregation appears to be a good target for therapeutic intervention in tumor diseases.

ACKNOWLEDGEMENTS CNRMN • Yraima Cordeiro Luzineide Tinoco • Débora Foguel Lenize F. Maia • Mariana P. B. Gomes IBCCF • Tuane Vieira •CNPq • Ana Paula Ano Bom Rafael Linden Sebastian Romano • Daniella Ishimaru •INCT and USP-IQ Millennium Institute • Luís Maurício T. R. Lima Sergio Verjovski-Almeida • Thiago A. Millen Murilo Sena Programs • Priscila S. F. Silva Universität Dortmund •FAPERJ • Marcius da Silva FT-IR and PPC Almeida •MS/DECIT • Adriana Marques Roland Winter Julia Kraineva • Narcisa Leal Cunha e •PRONEX

Silva (IBCCF/UFRJ) Ludwig •FINEP • Claudia Gallo (IBRAG, Vilma R. Martins UERJ) • Fabio Almeida • Ana Paula Valente Rocky Mountain Labs. NIH • Luciana P. Rangel Byron Caughey