Cell Cycle and Apoptosis September 21, 2017

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

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

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

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

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

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

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

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

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

Marking Proteins by Ubiquitin

Ubiquitination Process I

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.

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

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

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

Creation of DNA Lesions

DNA Damage Signaling Pathways

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

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

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

Apoptosis

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

Examples of Apoptosis 1. Number of cells -Nervous system

Examples of Apoptosis 2. Development Paws of embryonic mouse

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

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

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

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

Procaspase Cleavage

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

Caspase Cascade

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

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

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

Intrinsic Pathway

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

Pro-Apoptotic BH123

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

Model for IAPs

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