Mechanistic Insights in to the
p53-CYPD interactions
Under the guidance of
Dr. Rajanikant G.K.
Associate Professor
School ...
BACKGROUND
• p53 is a central stress sensor responding to multiple insults, including
oxidative stress

• In response to o...
INTRODUCTION:

p53

• p53 (also known as protein 53 or tumor protein 53), is a tumor
suppressor protein that in humans is ...
P53 regulations
Activated by
•
•
•
•

DNA damage,
Oxidative stress,
Osmotic stress ,
Ribonucleotide depletion.

Activation...
Cyclophilin D (CYPD)
• Located on the matrix of mitochondria,
• It modulates opening of mitochondrial permeability transit...
OBJECTIVES
• Studying the binding modes and interactions of p53 and CYPD
• Analyzing the effect of CYPD inhibitor, Cyclosp...
Protein structure prediction
• Protein structure prediction is the prediction of the threedimensional structure of a prote...
Comparative protein modeling
• Comparative protein modeling uses previously solved structures
as starting points, or templ...
1)Homology modeling
•

It is based on the reasonable assumption that
two homologous proteins will share very similar struc...
2)Protein threading
• Protein threading, also known as fold recognition, is a method of
protein modeling (i.e. computation...
•. Homology modeling is for those targets which have homologous
proteins with known structure(usually/maybe of same family...
Macromolecular docking
• Macromolecular docking is the computational modeling of
the quaternary structure of complexes for...
METHODOLOGY
a) Modeling of CYPD and p53
• No data is available regarding the p53-CYPD interactions and binding
conformatio...
METHODOLOGY
b) Docking of CYPD and p53
• Protein-Protein docking of CYPD and p53 modeled structures was carried
out using ...
RESULTS

3/25/2013
RESULTS

3/25/2013
RESULTS
Binding Site

CYPD-p53 Complex after Docking
3/25/2013
DISCUSSION
• p53 binds close to the binding site of CYPD,
• CYPD-p53 interaction may be inhibited by CYPD inhibitors,
• Cs...
RESULTS

CsA bound to CYPD (PDB Id 2Z6W) was modeled in to the complete CYPD protein
3/25/2013
RESULTS

p53 docked to CYPD-CsA complex
3/25/2013
RESULTS

CsA inhibits the interaction between CYPD and p53

3/25/2013
CONCLUSIONS
• p53 binds close to the active site of CYPD

• Due to this, CsA is able to inhibit CYPD-p53 interaction
• The...
REFERENCES
•

Vaseva AV, Marchenko ND, Ji K, Tsirka SE, Holzmann S, Moll UM. “p53 opens the mitochondrial
permeability tra...
THANK YOU

3/25/2013
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B.tech mini project computational biology NITC

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B.tech mini project computational biology NITC

  1. 1. Mechanistic Insights in to the p53-CYPD interactions Under the guidance of Dr. Rajanikant G.K. Associate Professor School of Biotechnology, NITC Mini Project Report by YANAMALA VIJAY RAJ MATHEW ALEXANDER CHERIAN CHRISTINE JAMES MOHAN 3/25/2013 NATIONAL INSTITUTE OF TECHNOLOGY, CALICUT.
  2. 2. BACKGROUND • p53 is a central stress sensor responding to multiple insults, including oxidative stress • In response to oxidative stress, p53 accumulates in the mitochondrial matrix and triggers mitochondrial permeability transition pore (MPTP) opening by physical interaction with the PTP regulator cyclophilin D (CypD) (Angelina et al, 2012 ) • CypD is a component of mitochondrial permeability transition (MPT) and mediates cell death (Schinzel et al, 2005) • Intriguingly, a robust p53-CypD complex forms during cell death processes like apoptosis and necrosis (Mihara et al, 2003) 3/25/2013
  3. 3. INTRODUCTION: p53 • p53 (also known as protein 53 or tumor protein 53), is a tumor suppressor protein that in humans is encoded by the TP53 gene located on the short arm of chromosome 17 • Human p53 is 393 amino acids long and has seven domains. FUNCTIONS: p53 has many mechanisms of anticancer function, and plays a role in apoptosis, genomic stability, and inhibition of angiogenesis. In its anticancer role, p53 works through several mechanisms: • It can activate DNA repair proteins when DNA has sustained damage. • It can induce growth arrest by holding the cell cycle at the G1/S regulation point on DNA damage recognition (if it holds the cell here for long enough, the DNA repair proteins will have time to fix the damage and the cell will be allowed to continue the cell cycle). • It can initiate apoptosis, the programmed cell death, if DNA damage 3/25/2013 proves to be irreparable.
  4. 4. P53 regulations Activated by • • • • DNA damage, Oxidative stress, Osmotic stress , Ribonucleotide depletion. Activation marked by two events • Half life of p53 increased drastically, leading to quick accumulation ofp53, • Conformational change of p53 to transcriptional regulation . 3/25/2013
  5. 5. Cyclophilin D (CYPD) • Located on the matrix of mitochondria, • It modulates opening of mitochondrial permeability transition pore (MPTP), • Opening of mitochondrial permeability transition pore involved in regulating cell death by inducing a sustained and irreversible loss of inner mitochondrial potential, • Proteins of MPTP located in between in between inner and outer mitochondrial membranes , • Cyclophilin stands as the only genetically verified component of MPTP. 3/25/2013
  6. 6. OBJECTIVES • Studying the binding modes and interactions of p53 and CYPD • Analyzing the effect of CYPD inhibitor, Cyclosporin A (CsA) on p53-CYPD interactions 3/25/2013
  7. 7. Protein structure prediction • Protein structure prediction is the prediction of the threedimensional structure of a protein from its amino acid sequence that is, the prediction of its secondary, tertiary, and quaternary structure from its primary structure. • Structure prediction is fundamentally different from the inverse problem of protein design. • Protein structure prediction is one of the most important goals pursued by bioinformatics and theoretical chemistry 3/25/2013
  8. 8. Comparative protein modeling • Comparative protein modeling uses previously solved structures as starting points, or templates. This is effective because it appears that although the number of actual proteins is vast, there is a limited set of tertiary structural motifs to which most proteins belong. It has been suggested that there are only around 2,000 distinct protein folds in nature, though there are many millions of different proteins. • These methods may also be split into two groups 1) Homology modeling 2) Protein threading 3/25/2013
  9. 9. 1)Homology modeling • It is based on the reasonable assumption that two homologous proteins will share very similar structures. • Because a protein's fold is more evolutionarily conserved than its amino acid sequence, a target sequence can be modeled with reasonable accuracy on a very distantly related template, provided that the relationship between target and template can be discerned through sequence alignment. • Unsurprisingly, homology modeling is most accurate when the target and template have similar sequences. 3/25/2013
  10. 10. 2)Protein threading • Protein threading, also known as fold recognition, is a method of protein modeling (i.e. computational protein structure prediction) which is used to model those proteins which have the same fold as proteins of known structures, but do not have homologous proteins with known structure. • It differs from the homology modeling method of structure prediction as it (protein threading) is used for proteins which do not have their homologous protein structures deposited in the Protein Data Bank (PDB), whereas homology modeling is used for those proteins which do. 3/25/2013
  11. 11. •. Homology modeling is for those targets which have homologous proteins with known structure(usually/maybe of same family), while protein threading is for those targets with only fold-level homology found • Threading works by using statistical knowledge of the relationship between the structures deposited in the PDB and the sequence of the protein which one wishes to model. 3/25/2013
  12. 12. Macromolecular docking • Macromolecular docking is the computational modeling of the quaternary structure of complexes formed by two or more interacting biological macromolecule • Protein–ligand docking is a molecular modeling technique. The goal of protein–ligand docking is to predict the position and orientation of a ligand (a small molecule) when it is bound to a protein receptor or enzyme • Computational capacity has increased dramatically over the last decade making possible the use of more sophisticated and computationally intensive methods in computer-assisted drug design 3/25/2013
  13. 13. METHODOLOGY a) Modeling of CYPD and p53 • No data is available regarding the p53-CYPD interactions and binding conformations • To study the interactions, complete structures of p53 and CYPD are required • CYPD structure is predicted through Homology Modeling using bovine CYPD (PDB Id 1IHG) as template (94% identical) • p53 doesn’t contain templates with 100% sequence coverage. So, structure is predicted through Threading using I-Tasser • The modeled proteins were verified using PROCHECK and Ramachandran plot 3/25/2013
  14. 14. METHODOLOGY b) Docking of CYPD and p53 • Protein-Protein docking of CYPD and p53 modeled structures was carried out using Hex 6.3 software • The proteins were docked based on the correlation of shape and electrostatics 3/25/2013
  15. 15. RESULTS 3/25/2013
  16. 16. RESULTS 3/25/2013
  17. 17. RESULTS Binding Site CYPD-p53 Complex after Docking 3/25/2013
  18. 18. DISCUSSION • p53 binds close to the binding site of CYPD, • CYPD-p53 interaction may be inhibited by CYPD inhibitors, • CsA has been reported to inhibit the CYPD-p53 interaction (Liu J et al, 2008), • CsA binds at the binding site of CYPD. 3/25/2013
  19. 19. RESULTS CsA bound to CYPD (PDB Id 2Z6W) was modeled in to the complete CYPD protein 3/25/2013
  20. 20. RESULTS p53 docked to CYPD-CsA complex 3/25/2013
  21. 21. RESULTS CsA inhibits the interaction between CYPD and p53 3/25/2013
  22. 22. CONCLUSIONS • p53 binds close to the active site of CYPD • Due to this, CsA is able to inhibit CYPD-p53 interaction • Therefore, CYPD inhibitors may act in a similar way and inhibit CYPD-p53 interactions • There are no inhibitors of CYPD apart from its natural inhibitor, CsA • Thus, design & discovery of novel inhibitors targeting the CYPD binding site holds great therapeutic promise • Similar studies can be carried out to check whether the novel CYPD inhibitors inhibit CYPD-p53 interaction 3/25/2013
  23. 23. REFERENCES • Vaseva AV, Marchenko ND, Ji K, Tsirka SE, Holzmann S, Moll UM. “p53 opens the mitochondrial permeability transition pore to trigger necrosis.” Cell, 2012, 149, 1536-1548. • Schinzel AC, Takeuchi O, Huang Z, Fisher JK. “Cyclophilin D is a component of mitochondrial permeability transition and mediates neuronal cell death after focal cerebral ischemia.” Proc Natl Acad Sci U S A. 2005, 102, 12005-12010. • Mihara M, Erster S, Zaika A, Petrenko O. “p53 has a direct apoptogenic role at the mitochondria.” Mol Cell. 2003, 11, 577-590. • Jacobson MP, Friesner RA, Xiang Z, Honig B. "On the Role of Crystal Packing Forces in Determining Protein Sidechain Conformations." J. Mol. Biol., 2002, 320, 597-608. • Roy A, Kucukural A, Zhang Y. “I-TASSER: a unified platform for automated protein structure and function prediction.” Nat Protoc, 2010 , 5, 725-738. • Ritchie DW, Kozakov D, Vajda S. “Accelerating and focusing protein-protein docking correlations using multi-dimensional rotational FFT generating functions.” Bioinformatics. 2008, 24, 1865-1873. • Liu J, St Clair DK, Gu X, Zhao Y. “Blocking mitochondrial permeability transition prevents p53 mitochondrial translocation during skin tumor promotion.” FEBS Lett. 2008, 582, 1319-1324. 3/25/2013
  24. 24. THANK YOU 3/25/2013

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