molecular cooperation to reinforce immune response during ... · system, the endocrine and immune...

Definition

Biological Intro: nucleic acids and protein structures

Data bank and modeling tools

Computational tool: Molecular Dynamics

Research project: a docking analysis

Teresa Rutigliano

STRUCTURAL BIOINFORMATICS

Structural bioinformatics: prediction and analysis of the three-dimensional structure of biological macromolecules

Generalizations about 3D : molecular folding, evolution, binding interactions, and structure/function relationships

Working both from experimentally solved structures (obtained e.g., by NMR or CD or X-ray) and from computational models

Definition

DNA structure

Deoxyribonucleic acid = DNA

Wikipedia

DNA stores biological information

RNA structureRibonucleic acid = RNA

Wikipedia

RNA catalyzes biological reactions (controlling gene expression, or sensing and communicating responses to cellular signals)

Protein structure

Lysine

Wikipedia

Proteins are essential parts of organisms and participate in every process within cells

From DNA to Protein Synthesis

YouTube

Just an easy explanation !!

Data bank and tools

The reference site for protein structures, crystallographic and NMR, is The Protein Data Bank http://www.wwpdb.org/

(NCBI for FASTA! https://www.ncbi.nlm.nih.gov/)

Download Tool: Structures, Sequences, and LigandsRCSB PDB - Download Files

Have a look at the file format!

• Download the PDB:

http://www.ebi.ac.uk/pdbe/

• Visualization and modeling:

DeepView - Swiss-PdbViewer C

Ascalaph Designer

Databases and tools

Structural bioinformatics: prediction and analysis of the three-dimensional structure of biological macromolecules

Generalizations about 3D : molecular folding, evolution, binding interactions, and structure/function relationships

Working both from experimentally solved structures (obtained e.g., by NMR or CD or X-ray) and from computational models

Definition

Computational tools

• Quantum Mechanics (QM) electronic structure based on Schrödinger Equationaccurate, expensive

• Classical Molecular Mechanics (MM)(MD)Empirical forces based on Newton’s Laws less accurate, fast

Molecular Dynamics

• Molecules interact with others (protein or nucleic acid or ligands..), their binding induce conformational changes that influence the activity or shape or accessibility ..

• MD studies the Evolution In Time of a System

INPUT:

-Topological properties (connectivity)

-Structural properties (conformation)

-Energetical properties (force Field)

-Thermodynamical properties

Molecular Dynamics

• Extend exploration of interactions• Study of system properties• Analysis of the conformational changes• Study of mutants in silico• Analysis of the structural flexibility• Drug discovery• Refinement of the 3D structure models• Useful guide to validate or explain experimental data

Aims of Molecular Dynamics

Research project: a docking analysis

8th International Conference on APPLIED MATHEMATICS, SIMULATION, MODELLING. Florence, 2014

Nervous system

Endocrine system

neurotransmitters

Hormones

(melatonin)

Immune system

White Blood Cell, cytokines (IL-2)

A complex network between the nervous system, the endocrine and immune system, a

bidirectional interrelationship, may affect their functions

e.g. cytokines produced by activated immune cells can affect, positively or negatively, the secretion of hormones

and vice versa

To validate if the immune system is simultaneously enhanced by the elements of the

neuroendocrine system and other stimulating agents of the immune system itself

OUR AIM

We used structural bioinformatics approach to analyse such interactions at a membrane receptor-ligand level

OUR AIM

Examples of membrane receptors:1. Ligands2. Receptors

Material and methodsThe Biological model

we emulated a malignant epithelial tissue where cells contain villin, ezrin and fimbrin proteins forming finger-like structures (microvilli)

POPE membrane with 340 lipids http://people.ucalgary.ca/~tieleman/download.html

On a bilayer membrane

We docked:

1) Ligands:

• One of the neuroendocrine system: Melatonin

• One of the immune system: IL-2

• One from external environment able to activate immunesystem: Lipopolysaccharide

2) Related receptors

Material and methodsThe biological model

Melatonin (MLT)

• It is a hormone secreted by the pineal gland

• Its secretion increases in darkness anddecreases during exposure to light

• It is implicated in the regulation of mood,learning and memory, dreaming, fertilityand reproduction

• Melatonin is an effective antioxidant

MLT exerts both direct and indirect anti-cancer effects insynergy with other molecules

Studies carried out an immunotherapy by administering high dosages of Melatonin (MLT), and low dosages of

Interleukin 2 (IL-2)

20% tumor regression

Melatonin in chemotherapy is useful to reduce toxicity

Interleukin-2 (IL-2)

• It is necessary for immune response

• It is a signalling molecule

• It activates certain White BloodCells, which fight diseases andinfections

IL-2 has been used in clinical trials for:

• The treatment of chronic viralinfections

• As a booster (adjuvant) for vaccines

• Anti cancer therapies

Bacterial lipopolysaccharide (LPS):

• It is the major outer surface membrane componentspresent in almost all Gram-negative bacteria

• It is an endotoxin, it is also an exogenous pyrogen

• It is cause of infection andinflammation

• In presence of neoplastic cellsand bacterial infection theimmune system isoverstimulated

• Biological functions are mediated by interactions betweenmolecules

• Interactions are determined by geometric, chemical-physicaland structural complementarity

• Interactions induce conformational changes

We want to analyze interactions between these molecules andknown receptors

Simulation techniques allow this exploration

Material and methodsSimulation method

• The docking tools allow to study in-silico conformationalchanges and the involved attractive forces, consideringmolecules both individually or in combination.

• This method is extremely useful when structural information(obtained e.g., by NMR or CD or X-ray) are not available, andfor analysis of intermolecular complexes

• Docked conformations and interaction energies wereachieved using the docking package HEX

Material and methodsSimulation method

• It is an interactive program for calculating and displayingfeasible docking of proteins and DNA molecules

• It can also calculate protein-ligand docking, assuming theligand is rigid, and it can superpose pairs of molecules usingknowledge of their 3D shapes

• It uses spherical polar Fourier (SPF) correlations (reached by

means of rotations and translations)

Source: Dave Ritchie-Team Orpailleur, INRIA ,France

http://hex.loria.fr/

Material and methodsDocking tool

HEX Rotational Search:

• A Cartesian grid is used to sample the molecular surfacesnumerically

• Molecules are oriented on the basis of their spherical polarcoordinates

HEX Docking Search:

1) Rotating the receptor andligand around their centroidson the basis of theirintermolecular distances

2) The receptor and ligand areeach assigned Euler rotationalangles

3) The final rotation is defined asa twist of the ligand about theintermolecular axis

4) Perform a full six-dimensionalsearch

Illustration of spherical polar docking with respect to the intermolecular axis

HEX Molecular Mechanics computation:

• Molecular mechanics energies are evaluated using a Newton-like model and energy minimization is applied

• These energies are calculated using hydrogen bond potentialsalong with an explicit charge-charge electrostatic contribution

HEX Clustering Docking Results:

• Docking calculations never give a unique solution. The bestorientations (lowest energy) are retained for viewing (1-500).The first solution is chosen for our analysis.

• It uses a clustering algorithm to group spatially similar dockingorientations:

– Each docking solution is first ordered by energy, and thelowest energy solution is taken for the first cluster.

– The process is then repeated on the basis of the main-chain alpha-carbon RMS , until all solutions have beenassigned to a cluster .

IL-2, MLT and LPS, and their known receptors were anchored to a lipid membrane with typical features of adenocarcinoma

Proteins

forming

microvilli

Villin Ezrin Fimbrin

Receptors MT-1 IL-2R TLR4

Ligands MLT IL-2 LPS

ResultsIL-2

IL-2 alone. In absence of other ligand IL-2(water green) is far from its receptor

(yellow)

Two ligands: IL-2 plus MLT. In presence of Melatonin (fuchsia) IL-2 ligand (water

green) moves toward IL-2 receptor (yellow)

ResultsMLT

One ligand: Melatonin alone is far from its receptor (blue)

Two ligands: IL-2 plus MLT. In presence of Melatonin (fuchsia) IL-2 ligand (water

green) moves toward IL-2 receptor (yellow). MLT is again far from its receptor

ResultsMLT

Two ligands: MLT plus LPS. MLT (fuchsia) seems to have an inner position in presence

of LPS (purple)

Three ligands: IL-2 plus MLT plus LPS

ResultsLPS

One ligand: LPS alone

Two ligands: IL-2 plus LPS

Three ligands: IL-2 plus MLT plus LPS

Conclusions

We were able to verify and confirm that IL-2 and LPS bindtheir known receptors (internal control)

Some ligands, and their receptors, behave in a differentway when other structures are present, in fact:

LPS, such as IL-2, is affected by the presence of othermolecules and is attracted by them where there are atleast two.

• The presence of LPS or IL-2 appears to promote immersion ofMelatonin in the membrane

• Melatonin is never located in its receptor

• IL-2 receptor appears to be of fundamental importance for allligands present in the study. Probably the surface of thisreceptor presents numerous “hot-spots”

Conclusions

• In summary, this study allows to observe that thesimultaneous presence of Melatonin, IL-2 and LPS resulted inin docking position changes, and this may explain resultsobtained in oncology field.

• It can therefore be concluded that, as hypothesized, themodel shows the existence of a synergetic interactionbetween neuroendocrine agents, immune substances andmediators of the inflammatory

• Further studies and in-vitro experiments may shed more lighton the effective action of a LPS- MLT- based therapy.

Conclusions

• M. Szczepanik, Melatonin and its influence on immune system, J Physiol Pharmacol, vol. 58 Suppl 6, 2007, pp. 115-124.

• Y.Yamaguchi, A. Ohshita A., Y. Kawabuchi , J. Hihar, E. Miyahara, K. Noma, T. Toge, Locoregionalimmunotherapy of malignant ascites by intraperitoneal administration of OK-432 plus IL-2 in gastric cancer patients, Japan Anticancer Research, vol. 15,1995, pp. 2201-2206.

• L. Nespoli, F. Uggeri, A. Nespoli, F. Brivo, L. Fumagalli, M. Sargenti, L. Gianotti, Modulation of systemic and intestinal immune response by Interleukin-2 therapy in gastrointestinal surgical oncology. Personal experience in the context of current knowledge and future perspectives, Anticancer Res., vol. 32, 2012,pp. 989-96

• P. Bongrand, Ligand-Receptor Interactions, Reports on Progress in Physics , vol. 62, 1999, pp. 921-968.

• D. V. Ritchie, V. Venkatraman, Ultra-fast FFT protein docking on graphics processors, Bioinformatics, vol. 26, 2010, pp. 2398–405

• David W Ritchie, Vishwesh Venkatraman, Lazaros Mavridis, Using graphics processors to accelerate protein docking calculations, Stud Health Technol Inform 2010 ;159:146-55

THANKS for the attention

molecular cooperation to reinforce immune response during ... · system, the endocrine and immune...

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