Muerte celular

1. Apoptosis celular

2. Puntos de Regulación Durante la interfase hay dos puntos de regulación esenciales: uno durante G1 , antes de la entrada a la fase S y el otro en G2 , previo al inicio de la fase M . En la etapa de división se encuentra el punto M Ciclo Celular-UAM-I ALTO ALTO ALTO

6. A 、 Normal cell B 、 Apoptosis: Apoptotic bodies

8.  Características bioquímicas apoptosis Apoptosis inducida por Cyto C Lane 1 ． 0 h 2 ． 1 h 3 ． 2 h 4 ． 3 h 5 ． 4 h 6 ． Control 7 ． Marker 2.0kbp 1.0 0.5 0.2 DNA ladder: 180~200bp Acumulación de tTG , PS flip-flop

17. (1) Via extrinsica: Fas Signaling Fas (también llamada Apo-1 o CD95) receptor de la superfamilia del factor de necrosis tumoral ( TNFR). Apoptosis mediada por Receptor Bcl-2 Familia (factores citoplasmicos) : Bad,Bid y bax: promotores de apoptosis; Bcl-X, Bcl-w y Bcl-2: previenen apoptosis. Estímulos internos: Daño al DNA, Ca 2+ alto, estrés oxidativo.

19. External signals: Death receptor pathway

20. (2) Vía intrinseca: Mitocondrial pathway Apoptosis mediada por mitochondria Varous tipos de estrés celular La familia Bcl-2: Bad o Bax se insertan en OM de Mitocondría Liberación de citocrome C desde el espacio I-O de la mitocondria Se forma el complejo de multi-subunidades: cascada de Caspasas .…

21. Se requiere la activación de Caspasa -2 para permeabiulizar de la mitocondria, liberación de cytochrome C, y apoptosis

22. Internal signals: Mitochondrial pathway

23. Vías de muerte celular mamíferos

24. Apoptosis (Program cell death) Factors that induce apoptosis: Internal stimuli: abnormalities in DNA External stimuli: removal of growth factors,addition of cytokines (Tumor Necrosis Factor or TNF) Signal transduction pathways leading to apoptosis: There are two major pathways: intrinsic pathway ( mitochondria-dependent) and extrinsic pathway ( mitochondria-independent) . • •

25. Mitochondria The outer membrane and intermembrane space are involved in apoptosis signaling

26. Internal (Mitochondrial) Pathway

27. The bcl-2 family of proteins BH: bcl-2 homology 6 proteins 18 proteins

28. The bcl-2 family proteins homo- and hetero-dimerize The Bcl-2 family members can form homo- or hetero-dimers through the BH3 domains. The pro-apoptotic Bax homo-dimers promotes apoptosis. Bcl-2 forms hetero-dimers with Bax, leading to the inhibition of the apoptotic activity of Bax. How do these proteins regulate apoptosis? • • • Thus, the relative levels of pro-survival and pro-apoptosis Bcl-2 family proteins determine cell survival or apoptosis.

29. Bcl-2 family proteins control cytochrome c translocation Bax forms homo-dimers in the presence of apoptotic signals. This homodimer presumably promotes the opening of a channel that controls the translocation of cytochrome c from the intermembrane space to cytoplasm • Bcl-2 interferes with Bax function by forming a hetero dimer with Bax. This leads to the closing of the channel and inhibition of cytochrome c translocation • intermembrane space cytoplasm

30. Cytochrome c translocation initiates caspase cascade In the cytosol, cytochrome c binds to Apaf-1 to form apoptosome • Apaf-1 Apoptosome Caspase 9 Procaspase 9 Procaspase 3 Caspase 3 Cyt. c The apoptosome recruits procaspase 9 to generate the active caspase 9 Caspase 9 then activates the executioner caspase 3, 6, 7 • •

31. Extrinsic and intrinsic pathways converge at executioner caspases Caspase 9 ligands Death receptors FADD/DISC Caspase 8, 10 Caspase 3, 6, 7 Lamin, CAD,actin, Adhesion molecules, etc Pro-apoptotic Bcl-2 proteins Cytochrome c translocation Apoptosome (Apaf-1/cyt.c) Intrinsic apoptosis signals

32. Caspase assay-- measure caspase activation TUNEL assay-- measure DNA fragmentation Activation of caspase 3 results in the cleavage of a synthetic caspase 3 substrate called DEVD-pNA (aspartate-glutamate-valine-aspartate p-nitroanalide). Cleavage of DEVD-pNA releases pNA, which can then be detected by a change it its absorbance at 405 nm. Apoptosis lab: Induction of apoptosis of Jurkat human T-cells by staurosporine.

33. Methods to measure apoptosis TUNEL assay: measure DNA fragmentation TdT ( t erminal deoxynucleotidyl transferase): adds dNTPs to the 3’ end of DNA molecules in the absence of a template Bright field FITC 3’ 5’ 3’ 5’ TdT FITC-dUTP