La modellistica dei sistemi biologici a scala molecolare. Applicazioni a processi infiammatori e

malattie autoimmuni e segnalazione cellulare tramite Calcio

Enrico Pieroni1 & Amit Kumar2

CRS41 & University of Cagliari2 11 February 2015

A short history of the last 4 years

Molecular Modeling

Multiple Sclerosis

Fibromyalgic Syndrome

Ca++ buffering in CASQ1/2

Outline

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Protein structure based on similarity with known crystal structure

Protein : Molecule (Drug : Receptor in drug design) Protein : Peptide (cell signaling & regulation, immune system antigen recognition) Protein : Protein (pMHC:TCR, muscle, cell signaling & metabolism) Protein : Lipid (Receptor:Lipid, cell signaling, immune system, metabolic syndrome) Protein : Carbohydrate (inflammation, pathogen recognition, adhesion & cell signaling) Protein : Polymer (drug delivery)

Molecular Modelling: Applications

Molecular Dynamics

Multiple Sclerosis: Autoimmune disease

The most common early symptoms of MS include: • Tingling • Numbness • Loss of balance • Weakness in one or more limbs • Blurred or double vision

Age group : 25 -35 years

Female:Male ratio = 2:1http://www.discoverprobita.co.uk/hydrolysed-collagen/visi-multiple-sclerosis/

• Genetic Factors

• Monozygotic twins: 25-30 %

• Dizygotic twins: 3-4%

• Parents with MS- child: 2%

• Siblings of MS - 0.9 %

What causes MS?

• Environmental Factors

• Sunlight (Vitamin D)

• Epstein-Barr Virus

• Smoking

Cocco, E., Sardu, C., Pieroni, E., et al. 2012 PLoS One Kakalacheva, K., Lunemann, J.D. 2011 FEBS Lett

International Multiple Sclerosis Genetics, C., Wellcome Trust Case Control, Sawcer, S.,et al. 2011 Nature

Human Leukocyte Antigen (HLA) : main genetic determinant region linked to MS

MS Prevalance per 100.000

0

60

120

180

240 EuropeItalySardinia

http://www.drbriffa.com/2011/10/20/why-might-shift-workers-be-at-increased-risk-of-multiple-sclerosis/

Sunlight is a relatively precious commodity = Vitamin D

Cocco E, et al. 2013 Interaction between HLA-DRB1-DQB1 Haplotypes in Sardinian Multiple Sclerosis Population. PLoS ONE 8(4):

e59790.

Geographical distribution of MS

Antigen presentation

Molecular Mimicry Mechanism

Foreign antigen that shares sequence or structural similarities with self-antigens.

Gras S, et al. 2011 The structural bases of direct T-cell allorecognition: Implications for T-cell mediated transplant rejection.

Immunol. Cell. Biol. 89, 388-395.

Myelin proteins: MBP, MOG, PLP, … with regions having sufficient “identity” w/r to pathogenic agent proteins (e.g. EBV/EBNA-1 MAP). NT “Identity” can be defined at various, non equivalent, levels: Sequence (simple, BLAST), Structural (complex 3D structure), Functional (dynamics, Affinities: MD)

Genetic information identified

Identified alleles associated to MS

Cocco E, et al. 2012 HLA-DRB1-DQB1 Haplotypes confer susceptibility and resistance to MS in Sardinia. PLOS One 2012

Investigate plasticity of peptide-MHC (pMHC)

complex and correlate the obtained structural and

dynamical characteristics to disease susceptibility

employing molecular dynamics simulations.

OUR AIM

X-ray Structure (www.pdb.org)

Yes

Model system: hydrogen atoms + water box + minimization (VMD software)

F=-∇V(r); Vinteraction potential = V(Bonded terms) + V(Nonbonded terms)

Vbonded = V(bonds, angles,torsion) VNonbonded = V(Van der Waals, Electrostatics) NAMD software

Homology Modeling: high sequence similarity template X-ray structure

(www-salilab.org)

MD Simulation

Stability, local flexibility, Interaction network (h-bond, hydrophobic), Binding free Energy (∆G)

TRAJECTORY ANALYSIS

No

HOW DO WE DO?

MD simulation movie: 0.12 μsPeptide-HLA Free HLA

STEP ONE

Key Actors

MBP(85-9

EBNA-1(400-413

Kumar A, et al. 2013 Structural and Dynamical Insights on HLA-DR2 Complexes That Confer Susceptibility to Multiple Sclerosis

in Sardinia: A Molecular Dynamics Simulation Study. PLoS ONE 8(3): e59711.

1-Protective (*16:01),1-Predisposing (*15:01) alleles complexed: to 1-self (MBP) and 1-non-self (EBNA-1) peptides

MHC class II peptide binding groove

Kumar A, et al. 2013 Structural and Dynamical Insights on HLA-DR2 Complexes That Confer Susceptibility to

Multiple Sclerosis in Sardinia: A Molecular Dynamics Simulation Study. PLoS ONE 8(3): e59711.

Interaction Network: H-bond interactions

polymorphism at position 71

Differential H-bond interaction pattern for EBNA1 peptide complex between predisposing and protective alleles

Kumar A, et al. 2013 Structural and Dynamical Insights on HLA-DR2 Complexes That Confer Susceptibility to Multiple Sclerosis

in Sardinia: A Molecular Dynamics Simulation Study. PLoS ONE 8(3): e59711.

Aromatic Stacking interactions

Aromatic Stacking Interaction Hydrophobic Interaction

*15:01 allele

MBP αPhe54-Phe91MBP, βPhe26-Phe92MBP, βHis81-His90MBP

βAsn82-Val89MBP, βVal85-Val89MBP

EBNA-1 βTyr78-H407EBNA-1 βVal85-Pro404EBNA-1

*16:01 allele

MBP αPhe54-Phe91MBP, βPhe26-Phe92MBP, βArg71-Phe92MBP

βAsn82-Val89MBP, βVal85-Val89MBP

EBNA-1 Absent Absent

polymorphism at position 71 reflected in absence of stacking interaction

Kumar A, et al. 2013 Structural and Dynamical Insights on HLA-DR2 Complexes That Confer Susceptibility to

Multiple Sclerosis in Sardinia: A Molecular Dynamics Simulation Study. PLoS ONE 8(3): e59711.

Binding free energy estimation

Kumar A, et al. 2013 Structural and Dynamical Insights on HLA-DR2 Complexes That Confer Susceptibility to

Multiple Sclerosis in Sardinia: A Molecular Dynamics Simulation Study. PLoS ONE 8(3): e59711.

Preferential binding characteristic shown by *16:01 towards MBP.

Extended Investigation to DQB1 alleles

Kumar A, et al. 2014 Antigenic peptide molecular recognition by the DRB1–DQB1 haplotype modulates

multiple sclerosis susceptibility. Molecular. Biosystems RSC 10, 2043-2054.

STEP TWO

Predisposing Protective

Binding Free Energy estimates

Mutation A38V of DQB1*06:02 MBP complex and V86G of DRB1*15:01 EBNA-1 complex displayed favourable binding with respect to their WT counterparts.

Preferential binding to EBNA-1 peptide

WT cases

Mutant cases

Binding energy estimate: DRB1-DQB1 haplotype

Predisposing allele DRB1*15:01 and protective allele DQB1*05:02 displays degenerate binding characteristic towards MBP and EBNA-1 peptides.

Kumar A, et al. 2014 Antigenic peptide molecular recognition by the DRB1–DQB1 haplotype modulates

multiple sclerosis susceptibility. Mol. Biosyst. 10, 2043-2054.

A robust synergistic action and a differential role of DRB1 and DQB1 in tissues and in the time-steps towards autoimmunity (MS animal models)

Take home message

STEP 3: 2 Pred. & 2. Prot.

Sequence Analysis

Principal component analysis

Collective motion analysis

Water dynamics and Energy estimates

Higher number of total water in protective alleles.

Higher value of entropy not destructive for MAP binding to predisposing alleles.

Conclusions: DR2

DRB1 86 Gly/Val (prot/pred): fundamental role. Predisposing DR2 allele has similar binding configuration to both MBP and EBV peptides. Protective allele binds weaker to EBNA1 & w/distinct binding configs to the two peptides. Local flexibility confirms global results. All these findings coherently support the molecular mimicry hypothesis.

Experimental Support

MS patients EBV+ have similar B-mediated immune response for EBNA1 and MBP peptides.

Ab reactive against MBP peptide are cross-reactive against EBNA1 peptide.

EBNA1 and MBP peptides do not share sequence homology, thus may share conformational homology as presented by MHC proteins.

APC bearing protective haplotype: present stable and durable MBP complexes via both DRB1 & DQB1 ... and intermediate lifetime EBNA1 via DQB1 alone higher Ag presentation higher activation efficiency optimal and distinct TCR triggering pathways? Risk: higher cross-reactivity! Predisposing haplotype has partly lost discrimination ability MS predisposition DRB1-DQB1 loosing discrimination ability between self/nonself + DRB1/DQB1 complementarity in antigen molecular recognition process

Conclusions: DRB1-DQB1

Given that: i) DRB1 and DQB1 expression can be tissue dependent & ii) molecular mimicry requires prior peripheral T cell activation by nonself peptide, then central reactivation by self peptide ....

• DRB1 role could be more relevant in the peripheral activation of T lymphocytes, while ...

• DQB1 could be dominant in the reactivation of the clonally expanded T cell repertoire, leading to autoimmune attack in CNS.

more robust and flexible recognition system for the protective haplotype (?), tissue-specific (?), w/risk of crossreactivity (MS) & supporting molecular mimicry.

Conclusions: DRB1-DQB1

• Design of small peptides ...

• modulating MHC affinity toward self/nons. Ags,

• enhancing/suppressing HLA peptide loading,

• ... directly or indirectly (e.g. DM mediated).

• ... acting in a selective way w/r to

• HLA allele and pathogenic antigen,

• specific tissue.

• Phenotypic, genetic, clinical and modelling data integration into an individualised risk model and drug response prediction.

Therapeutic potentialities

• Disorder of pain processing due to abnormalities in how pain signals are processed in the central nervous system

• Exact cause unknown but believed to involve psychological, genetic, neurobiological and environmental factors

• The American College of Rheumatology classify fibromyalgia as a functional somatic syndrome.

• Defining symptoms: chronic widespread pain, fatigue, sleep disturbance and allodynia (heightened pain in response to tactile pressure). Others: tingling of the skin, prolonged muscle spasms, weakness in the limbs, nerve pain, muscle twitching, palpitations and functional bowel disturbances.

Fibromyalgia

cccc

Key Players: PAFr and LysoPC ligands

LysoPC ligands expressed in Patients

PAF-receptor

Molecular Movies..

Interaction Picture

• No FMS metabolomic study, although pathogenesis unclear & no biomarkers.

• Observed increase of lysoPC (14:0) and lysoPC (16:0) in FMS patients.

• MD: lysoPC(16) and lysoPC(14) are slightly more flexible than PAF, but overall similar binding configurations to PAFR.

• RMSD variations in key intra-membrane domains smaller than in another MD investigation, but show signals of a conformational change upon binding, which on turn could be linked to receptor activation.

• LysoPCs might i) induce the PAFR open state, ii) activate PAFR & iii) initiate the signal transduction cascade through G-protein coupling, …

• ... with PAF-like function, thus playing a role in the clinical manifestation of FMS.

Discussion

• less relevant lysoPCs should also be analyzed; • understand the source (diet?) and the body processing of the lipids; • design of molecules interfering with PAFR & modulating pain response; • bio-markers from metabolic profiles, leading to clinical tests; • personalized diets.

Perspectives

Final Conclusions