Transcript Slide 1
Apoptosis “Falling or dropping off” Ughhhh….. http://focosi.immunesig.org/apoptosis.htm Syndactlyly (1/2500 births) http://en.wikipedia.org/wiki/Webbed_toes http://www.indianahandcenter.com/hot_child.html Apoptosis is a balancing act Too much: -Alzheimers’ -Parkinson’s -Huntington’s -AIDS -stroke Too little: -cancer -autoimmunity -persistent infections http://research.yale.edu/ysm/article.jsp?articleID=357 Do all cells undergo apoptosis??? Plant Bacteria http://web.mala.bc.ca/simmsw/postergriffiths.htm http://www.cbs.dtu.dk/staff/dave/roanoke/bio101ch06.htm Yeast http://www.pkdiet.com/habits.htm •All undergo their own version of programmed cell death (PCD) and exhibit physiological changes similar to apoptosis in mammalian cells. Both plants and yeast have “caspaselike” proteins while to date, bacteria do not. Cytotoxic drugs Irradiation http://www.dkfz.de/en/phd-program/projects/immunogen.html Caspases..the central players: Asp175 Asp9 Asp28 Caspase-3 1 Pro Large Small 277 •The caspase family is highly conserved; at least 14 members (11 of these are found in mammals •We will only be talking about Caspase-8, Caspase-9, and Caspase-3 in this class •Classified as cysteine aspartate specific proteases due to the required cysteine in the active site and the fact that they cleave after an aspartate residue •In order to be active, the precursor caspase (a zymogen) must be processed by cleavage of the prodomain/large subunit and the large subunit/small subunit •Caspases can be classified as “initiator” or “effector” •Substrates of caspases include cell structural components, regulatory proteins, and inhibitors of apoptosis Caspase-3 Caspase-8 Caspase-9 http://people.bath.ac.uk/pr1cemb/Apoptosis.htm Cytotoxic drugs Irradiation http://www.dkfz.de/en/phd-program/projects/immunogen.html http://en.wikipedia.org/wiki/Mitochondria The Bcl2 family: Members are either anti- or pro- apoptotic Bid and Bad are examples of “BH3” only proteins Other proteins besides cytochrome c are also released from the mitochondria and Include antagonists of caspase inhibitors and endonuclease G http://www.sgul.ac.uk/depts/immunology/~dash/apoptosis/mito.htm Cytotoxic drugs Irradiation http://www.dkfz.de/en/phd-program/projects/immunogen.html So, what are we doing this week? A. Caspase assay: used to detect the enzymatic activity of Caspase-3 as an indicator of apoptosis You will be inducing apoptosis (w/Staurosporine) today, freezing them in LN2 and doing the assay on Day 2 B. TUNEL assay: used to measure DNA fragmentation characteristic of cells which have initiated apoptosis You will use cells that have already been induced with Staurosporine to perform the assay, but will not look at your slides until Day 2. A. + Jurkat Cells Apoptosis is induced Cells are frozen staurosporine B. Cells are lysed; caspase activity is measured +/- inhibitor. Don’t forget the standard Curve! Asp-Glu-Val-Asp DEVD-pNA Caspase-3 DEVD + pNA pNA detectable at λ405 nm Specificity of Caspase-3 for the substrate will be determined through use of the inhibitor DEVD-CHO Figure 2 Overview of PDK1PDK1-kinase domain bound to staurosporine Biochemical Journal www.biochemj.org Biochem. J. (2003) 375, 255255-262 Staurosporine binds to the ATP binding pocket of many kinases inhibiting their activity A. + Jurkat Cells Apoptosis is induced Cells are frozen staurosporine B. Cells are lysed; caspase activity is measured +/- inhibitor. Don’t forget the standard Curve! Asp-Glu-Val-Asp DEVD-pNA Caspase-3 DEVD + pNA pNA detectable at λ405 nm Specificity of Caspase-3 for the substrate will be determined through use of the inhibitor DEVD-CHO So, what are we doing this week? A. Caspase assay: used to detect the enzymatic activity of Caspase-3 as an indicator of apoptosis You will be inducing apoptosis (w/Staurosporine) today, freezing them in LN2 and doing the assay on Day 2 B. TUNEL assay: used to measure DNA fragmentation characteristic of cells which have initiated apoptosis You will use cells that have already been induced with Staurosporine to perform the assay, but will not look at your slides until Day 2. CAD=Caspase Activated Deoxyribonuclease An example of DNA laddering: Note: DNA fragmentation is a result of cleavage from multiple endonucleases http://www.actaps.com.cn/qikan/manage/htmlwenzhang/2006-3-08.htm TUNEL assay (TdT-mediated dUTP Nick-End Labeling) TdT=Terminal deoxynucleotidyl transferase Fluorescein-12-dUTP Visualized with fluorescence Microscope at ~520 nm http://www.emdbiosciences.com/html/cbc/Apoptosis_Resource_changes_in_nucleus.htm http://www.nearingzero.net/screen_res/nz253.jpg