Apoptosis“Falling or dropping off”

Ughhhh…..

http://focosi.immunesig.org/apoptosis.htm

http://en.wikipedia.org/wiki/Webbed_toes

Syndactlyly (1/2500 births)

http://www.indianahandcenter.com/hot_child.html

Too much: -Alzheimers’ -Parkinson’s -Huntington’s -AIDS -stroke

Too little: -cancer -autoimmunity -persistent infections

Apoptosis is a balancing act

http://research.yale.edu/ysm/article.jsp?articleID=357

Do all cells undergo apoptosis???

http://web.mala.bc.ca/simmsw/postergriffiths.htm http://www.pkdiet.com/habits.htm

BacteriaYeast

•All undergo their own version of programmed cell death (PCD) and exhibit physiologicalchanges similar to apoptosis in mammalian cells. Both plants and yeast have “caspase-like” proteins while to date, bacteria do not.

http://www.cbs.dtu.dk/staff/dave/roanoke/bio101ch06.htm

Plant

http://www.dkfz.de/en/phd-program/projects/immunogen.html

Cytotoxic drugsIrradiation

Pro Large Small

Asp9 Asp28 Asp175

1 277Caspase-3

Caspases..the central players:

•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

http://people.bath.ac.uk/pr1cemb/Apoptosis.htm

Caspase-3

Caspase-8

Caspase-9

http://www.dkfz.de/en/phd-program/projects/immunogen.html

Cytotoxic drugsIrradiation

http://en.wikipedia.org/wiki/Mitochondria

The Bcl2 family: Members are either anti- or pro- apoptotic

Other proteins besides cytochrome c are also released from the mitochondria andInclude antagonists of caspase inhibitors and endonuclease G

Bid and Bad are examples of “BH3” only proteins

http://www.sgul.ac.uk/depts/immunology/~dash/apoptosis/mito.htm

http://www.dkfz.de/en/phd-program/projects/immunogen.html

Cytotoxic drugsIrradiation

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.

staurosporine

+ Jurkat Cells

DEVD-pNA DEVD + pNApNA detectable at λ405 nm

Caspase-3

Apoptosis is inducedCells are frozen

A.

B. Cells are lysed; caspase activity is measured +/- inhibitor. Don’t forget the standardCurve!

Asp-Glu-Val-Asp

Specificity of Caspase-3 for the substrate will be determined through use ofthe inhibitor DEVD-CHO

Figure 2 Overview of PDK1Figure 2 Overview of PDK1--kinase domain bound to staurosporine kinase domain bound to staurosporine

Biochemical Journal www.biochemj.org Biochem.Biochemical Journal www.biochemj.org Biochem. J. (2003) 375, 255J. (2003) 375, 255--262 262

Staurosporine binds to the ATP binding pocket of many kinases inhibiting their activity

staurosporine

+ Jurkat Cells

DEVD-pNA DEVD + pNApNA detectable at λ405 nm

Caspase-3

Apoptosis is inducedCells are frozen

A.

B. Cells are lysed; caspase activity is measured +/- inhibitor. Don’t forget the standardCurve!

Asp-Glu-Val-Asp

Specificity of Caspase-3 for the substrate will be determined through use ofthe 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

http://www.actaps.com.cn/qikan/manage/htmlwenzhang/2006-3-08.htm

An example of DNA laddering:

Note: DNA fragmentation isa result of cleavage from multiple endonucleases

TUNEL assay

Fluorescein-12-dUTP

http://www.emdbiosciences.com/html/cbc/Apoptosis_Resource_changes_in_nucleus.htm

Visualized with fluorescence Microscope at ~520 nm

(TdT-mediated dUTP Nick-End Labeling)TdT=Terminal deoxynucleotidyl transferase

http://www.nearingzero.net/screen_res/nz253.jpg