1. Cell Cycle and Apoptosis
September 21, 2017

2. Control of the Cell Cycle
-The proteins that control the cell cycle are different from the proteins that are involved in the process
-Series of checkpoints
-Each checkpoint serves as a biochemical switch

3. Control of the Cell Cycle
The control system:
1. Swithes are binary:
“On or Off” and once started the process continues to completion
2. Reliable due to back-up systems
3. Adaptable so it can be modified to different cell types or conditions

4. Key Control Components
1. Cyclin-dependent kinases (Cdk)
-Activities vary throughout the cell cycle
-The targets of phosphorylation then varies
2. Cyclins
-Control the activity of Cdk’s
-Undergo synthesis and degradation in
each cell cycle
Cycling of the cyclins creates the cycles of the cyclin/Cdk complex allowing for the varying activities resulting in progression through the cell cycle

5. Cyclin-Cdk Complexes 4 classes of cyclins
G1-cyclin
4 classes of cyclins
-All eucaryotes require 3 cyclins (G1/S, S, M)

6. Cyclin – activates the Cdk and helps direct it to the specific target
Yeast – 1 Cdk
Vertebrates – 4 Cdks
Cyclin – activates the Cdk and helps direct it to the specific target

7. Cdk Activation Cdk fullly active 1. Cyclin bound
2. Phosphorylated at the active site

8. Regulation of Cdk Activity
-Cdk activity can be fine tuned through 2 phosphorylations at the top of the active site
-Important in control of M-Cdk activity

9. Inhibition of Cyclin-Cdk Complexes
-Binding of inhibitors alters the structure of the active site
-Cdk inhibitors are utilized more early in the cell cycle

10. Cyclin-Cdk Complexes APC/C Anaphase-Promoting Complex, or Cyclosome
G1-cyclin
APC/C Anaphase-Promoting Complex, or Cyclosome
-Regulated by protein destruction

11. Marking Proteins by Ubiquitin

12. Ubiquitination Process I

13. Ubiquitination Process II
There are about 300 different E2-E3 each recognizing a different degradation signal. Therefore subsets of proteins can be regulated as a group.

14. Control of Proteolysis of APC/C
-Degrading S and M-cyclins stops the Cdk activity and the Cdk’s targets become dephosphorylated and inactive
or Cdh1
or S-cyclin
-APC/C is active in G1 keeping Cdks inactive

15. Control of Proteolysis by SCF
-Degrades Cdk Inhibitors of S-Cdks and allows S phase to occur
-F-box protein is constant through the cell cycle and is used to recognize the target

17. Cell-Cycle Control Overview
10% of yeast genes encode mRNAs which oscillate in the cell cycle

18. Creation of DNA Lesions

19. DNA Damage Signaling Pathways

20. DNA Damage Checkpoint Mdm2 – ubiquitin ligase
p21 – CKI (Cdk Inhibitor Protein)

21. Check1/2
Chk1/2 phosphorylate Cdc25 thereby inactivating the phosphatase activity resulting in inactive Cdk

22. DNA Damage -Cell cycle is arrested until the damage is repaired
-If it’s not repaired:
1. Unicellular organisms will resume their cycle taking a potential mutation over death
2. Multicellular organisms will sacrifice a cell over the health of the organism

23. Apoptosis

24. Apoptosis Apoptosis is one type of programmed Process of Apoptosis
-Cells shrink and condense
-Cytoskeleton collapses
-Nuclear envelope disassembles
-Nuclear chromatin fragments
-Cell surface belbs and may break up (apoptotic bodies)
-Cell surface is chemically altered
-Macrophage engulfs the cell
Process of Cell Necrosis
-Cell insult or injury
-Cells swell and burst
-Cell contents are released
-Inflammatory response

25. Examples of Apoptosis
1. Number of cells
-Nervous system

26. Examples of Apoptosis
2. Development
Paws of embryonic mouse

27. Examples of Apoptosis 3. Regulate cell numbers -Liver
4. Quality control
-Eliminates abnormal, misplaced, nonfunctional, or dangerous cells
-Developing T and B cells that do not produce useful antigen receptors or that are self-reactive
5. Supply of cells
-Large numbers of neutrophils are produced and stored awaiting infection

28. Biochemical Characteristics
DNA Cleavage
-Endonuclease cleaves
DNA into fragments
between nucleosomes

29. Biochemical Characteristics
TUNEL Assay - TdT-mediated
dUTP nick end labeling
Phosphatidylserine localization
-movement from the inner
to outer membrane
-marks the cell for
macrophages

30. Caspases -Enzymes responsible for apoptosis
-A family of proteases that cleave proteins at aspartic acid residues
-C for cysteine (in active site) and ASP for aspartic acid
-Synthesized as inactive precursors, procaspases
-Not all caspases are involved with apoptosis
-ICE, interleukin-1-converting enzyme

31. Procaspase Cleavage

32. Caspases -Initiator procaspase, start the proteolytic cascade
-Executioner procaspases, cleave and activate other executioner procaspases and other targets
-Targets include: nuclear lamins, endonuclease inhibitor, cytoskeleton components, cell-cell adhesion proteins
-The caspase cascade is:
-Very destructive
-Self-amplyfing
-Irreversible

33. Caspase Cascade

34. Caspases
-Initiator caspases have a caspase recruitment domain (CARD) that enables them to bind to adaptor proteins into activation complexes
-In the complex, the initiator caspases are close enough to activate each other starting the cascade
-2 Apoptotic pathways
1. Extrinsic Pathway
2. Intrinsic Pathway

35. Extrinsic Pathway
DISC – death-inducing signaling complex
Inhibitors such as decoy receptors and intracellular blocking proteins

36. Intrinsic Pathway Releases mitochondrial proteins into the cytoplasm
Cytochrome C release can trigger apoptosis through interaction with the adapter protein Apaf1

37. Intrinsic Pathway

38. Classes of Bcl2 Proteins
Bcl2 proteins –regulate apoptosis through controlling the release of cytochrome c

39. Pro-Apoptotic BH123

40. Regulation of Intrinsic Pathway
BH3-only proteins activate apoptosis through direct binding with anti-apoptotic proteins
-p53 activates BH3-only proteins (Puma and Noxa)
-Bid – extrinsic and intrinsic pathways

41. Model for IAPs

42. Inhibition of Apoptosis
(BH3-only
pro-apoptotic)
(Anti-apoptotic)