P53-MDM2 & APOPTOSIS

P53-MDM2 & APOPTOSIS : 

P53-MDM2 & APOPTOSIS

P53: 

P53 Apoptosis ( also called programmed cell death ) :- involves several morphological and biochemical events . The physiological purpose of apoptosis is to eliminate unwanted cells without harming neighboring cells . Apoptosis is tightly regulated by many protein . The most important protein controlling apoptosis is P53 .

Structure of P53 : 

Structure of P53 The tumor suppressor protein p53 was first described in 1979 (Lane & Crawford 1979, DeLeo et al. 1979, Linzer & Levine 1979 ). the TP53 gene that contains 11 exons is located in chromosome 17p13.1, the coded protein is approximately 53 kDa in size, human protein containing 393 amino acids. The p53 protein consists of an acidic N-terminus with a transactivation domain, a hydrophobic central DNA-binding core and a basic C-terminus with regulatory and oligomerisation domains

PowerPoint Presentation: 

Human p53 protein (Hp53) can be divided into five domains, each corresponding to specific functions: The amino-terminus part 1-42 contains the acidic transactivation domain and the mdm2 protein binding site. It also contains the Highly Conserved Domain I (HCD I) II) Region 40-92 contains series repeated proline residues that are conserved in the majority of p53. it also contains a second transactivation domain. III) The central region (101-306) contains the DNA binding domain. It is the target of 90% of p53 mutations found in human cancers. It contains HCD II to V. IV) The oligomerization domain (307-355, 4D) consists of a beta-strand, followed by an alpha-helix necessary for dimerization , as p53 is composed of a dimer of two dimers . A nuclear export signal (NES) is localized in this oligomerization domain. V) The carboxy -terminus of p53 (356-393) contains 3 nuclear localization signals (NLS) and a non-specific DNA binding domain that binds to damaged DNA. This region is also involved in downregulation of DNA binding of the central domain.

PowerPoint Presentation: 

The amino-terminus of p53 AD1: activation domain 1 AD2: activation domain 2 PRD: proline rich domain NES: nuclear exclusion domain HCD I: Highly Conserved Domain I

PowerPoint Presentation: 

The carboxy -terminus of p53 Tetra (4D): oligomerization domain AD2: activation domain 2 NEG: negative regulation domain NES: nuclear exclusion domain NLS: nuclear localization domain

p53 isoforms: Recent studies using more sensitive methodology has uncovered at least 8 isoforms, the function of which remains unknown. : 

p53 isoforms : Recent studies using more sensitive methodology has uncovered at least 8 isoforms , the function of which remains unknown.

PowerPoint Presentation: 

P53 is : 1-Guardian of the cell 2- Transcription factor ( p53 is a sequence-specific nuclear transcription factor that binds to defined consensus sites within DNA as a tetramer and affects the transcription of its target genes ) 3- Tumor suppressor gene ( Controlling proto oncogenes )

Post transitional modifications of P53 affecting its function : 

Post transitional modifications of P53 affecting its function

P53 Pathway : 

P53 Pathway - in normal unstressed cells, the level of p53 protein is low .  After genotoxic or non- genotoxic stresses, activation of p53 is a two steps process: --> First p53 protein level is increased via the inibition of its interaction with mdm2 and the other negative regulators. Overtranslation of p53 RNA is a complementary that will also ensure p53 accumulation. --> Second , a series of modulator ( kinases , acetylases ) will activates p53 transcriptional activity.

PowerPoint Presentation: 

p53 pathway: Upstream pathway i )The stress signals that activate the pathway ii) The upstream mediators that detect and interpret the upstream signals. iii) the core regulation of p53 through its interacion with several proteins that modulate its stability

PowerPoint Presentation: 

p53 pathways: downstream pathway iii) the core regulation of p53 through its interacion with several proteins that modulate its stability iv) the downstream events, mainly transcriptional activation or protein protein interactions v) The final outcome, growth arrest, apoptosis or DNA repair.

PowerPoint Presentation: 

- Regardless of the type of stress , the final outcome of p53 activation is either cell cycle arrest and DNA repair or apoptosis, but the mechanism leading to the choice between these fates has not yet been elucidated . -Extensive studies on this topic have been made, and several factors influencing the response of the cell have been suggested: cell type, oncogenic cell composition, intensity of stress, level of TP53 expression, interaction of p53 with specific proteins and affinity of p53 for promoters

PowerPoint Presentation: 

P53 - Intiates apoptosis - long term effect (cell arrest)  N.B: P53 is involved in DNA repair via an intirnsic 3’-5’ exonuclease activity . 1- Cell arrest : -activation of p21\Waf1 which binds to and inhibits cyclin -dependent kinases cuasing hypophosphorylation of Rb thus preventing release of E2F & causing cycle arrest

PowerPoint Presentation: 

2- Apoptosis : Cell death can be intiated by two different mechanisms referred as , the extrinsic & intrinsic pathways . The key player to both are aspartate specific cystein protease ( CASPASES ) . Extrinsic pathway : Consists of several protein members including death receptor , membrane bound FasL & FasDD and caspases 8,10 which activate the rest of effector caspases & in some cells activation of caspase8 is enough to initiate cell death .

PowerPoint Presentation: 

In other cells caspase8 interact with intrinsic pathways to activate mitochondrial release of CytC . N.B : initiation of extrinsic pathway done up on binding of Fas to Death receptors . Intrinsic pathway : -Is triggered by external signals ( UV , gamma rad , drugs ,…..) -Tightly regulated by Bcl2 family of proteins. -pro-apoptotic proteins such as Bad, Bax , Bcl-xs , Bim act as activators for CytC release . - Ratio bet Bax and Bcl2 control mitochondrial permeability .

PowerPoint Presentation: 

-When proapototic proteins are exceed ,outer mitochondrial membrane becomes permeable leading to cascade of events . ( attacking valuable proteins required by cell ) CytC release ---> caspase-3 activation ---> Ca+2 efflux ---> release of Caspase12 ---> caspase 9 & caspase 3 ---- finally lead to ---> APOPTOSIS

Regulation of P53 : 

Regulation of P53 -In normal unstressed cells, the level of p53 protein is downregulated via the binding of proteins such as MDM2, COP1, PIRH2 or JNK that promote p53 degradation via the ubiquitin / proteasome pathway. -it is generally believed that the principal mechanisms governing the activity of p53 occur at the protein level . These include post-translational modifications, regulation of the stability of p53 protein, and control of its sub-cellular localization .  The human p53 protein has been shown to be modified at least at 17 different sites.

PowerPoint Presentation: 

-P53 regulates its own activity by inducing expression of MDM2 this phenomena forms a vital negative feed back loop .  Mech.; N-terminus of MDM2 binds to P53 masks its transactivationg domain. The E3 ubiquitin ligase activity of MDM2 ubiquitinates p53 targeting its degradation by exporting p53 from nucleus to cytoplamic proteosome .