Molecular Biology EU
Exercise 12/ Seminar 12
Molecular aspects of cell death
Types of cell death: autophagy, necrotic death (necrosis) of the cells, mitotic catastrophe, programmed cell death (apoptosis).
Caspases: types, structure - domain structure, homoactivation and heteroactivation, caspase activation cascade.
Pathways for inducing programmed cell death and their molecular regulation:extrinsic pathway, perforin/granzyme pathway, intrinsic pathway, mitochondrial pathway, sphingomyelin-ceramide pathway, apoptosis without caspases;activation of Caspian 2 via RIP and RAIDD/CRADD, megacanals- ANT, VDAC, BRP; hexokinase, creatine kinase, cyclophilin D, apoptosom, sphingomyelin, ceramide, phospholipase A2, MAPKs kinase, stress kinase SAPK/JNK, CAPK = ceramide activated protein kinase, CAPP = ceramede-activated protein phosphatase, Acinus (apoptosis chromatin condensation inducer in the nucleus), AIF (apoptosis inducing factor), Apaf-1/APAF-1 (apoptosis protease activating factor -1), ANT(adenine nucleotide translocator), Bad (Bcl-2 antagonist of cell death), Bax (Bcl-2-associated protein), Bcl-2 (B-cell leukemia/lymphoma 2), BH 1-4 ( Bcl-2 homology 1-4), Bid (BH3 interacting-domain death agonist), BPR (benzodiazepine peripheral receptor), CAD caspase –activated Dbase), CANP (Ca2+-activated neutral proteinase), CPAN (caspase-activated nuclease), DD (death domain), DED (death effector domain), DFF (defragmentation factor), DISC (death inducing signalling complex), FADD (Fas-associated death domain), Fas-L (Fas-ligand),
GrB (granzyme caspases (cysteine-dependent aspartate-directed proteases), NS (nuclear scaffold), PIGs (p53 induced genes), p53/TP53 (protein 53/tumor protein 53), PUMA (p53 upregulated modulator of apoptosis), Smac/DIABLO (second mitochondrial activator of caspase/direct IAP binding protein with low pI), tBid (truncated Bid), TRADD (TNF-R1 associated death domain), TRAIL (TNF-related apoptosis-indicing ligand), VDAC (voltage-dependent anion channel).
Mutations in p53 gene - Li-Fraumeni syndrome, Mdm2, ASPP = apoptotic specific regulator of p53, ARF = alternative reading frame, JMY = junction mediating and protein regulator, pRb, E2F and other passwords set forth below (apoptosis).
Recommended reading:
- “Molecular Biology of the Cell”, Alberts B. (e.g. Chapter 17. The Cell Cycle and Programmed Cell Death).
Available online:
- Elmore S. Apoptosis: A Review of Programmed Cell Death. Toxicologic pathology. 2007;35(4):495-516. doi:10.1080/01926230701320337.
Available online:
- SunY. andPengZ. Programmed cell death and cancer. Postgraduate Medical Journal.2009;85:134-140.
- Madame Curie Bioscience Database [Internet]. (e.g. Apoptosis)
Available online:
- Holland-Frei Cancer Medicine, 6th edition (e.g. Chapter 4. Apoptosis and Cancer)
Available online:
Genes/proteins which are involved in apoptosis regulation:
Acinus –apoptosis chromatin condensation inducer in the nucleus; nucleic protein inducing apoptotic chromatin condensation.
AIF –apoptosis inducing factor; protein involved in the non-caspase induced apoptosis pathway, after proteolytic release from the inner mitochondrial membrane, reaches the cytoplasm, whereby the NLS (nuclear localization sequence) can be translocated to the nucleus.
Apaf-1/APAF-1 –apoptosis protease activating factor -1; cytoplasmic binding protein released from mitochondria cytochrome C and ATP in the oligomeric apoptosome complex necessary for activation of caspase 9.
ANT –adenine nucleotide translocator;an element of mitochondrial megacanales present in the inner mitochondrial membrane.
Bad –Bcl-2 antagonist of cell death;one of the proapoptotic proteins.
Bax –the Bcl-2-associated protein; a cytoplasmic protein with proapoptotic activity, after a change of conformation, is incorporated into the mitochondrial outer membrane, where, by contacting with VDAC, positively regulates the opening megacanals at the interface of the two mitochondrial membranes (PTP- permeability transition pores). Opening of megacanals in the mitochondrial pathway of induction of apoptosis leads: to reduction of transmembrane potential, reduction in ATP production, decrease in intra-mitochondrial potency of thiol compounds, increase of Ca2+ concentration in the mitochondrial matrix, accompanied by the outflow from the intermembrane space into the cytosol proteins that promote apoptosis(cytochrome c, procaspase -2, -3, -9, Smac/DIABLO, AIF);
Bcl-2 –B-cell leukemia/lymphoma 2; Bcl-2 protooncogene product with antiapoptotic activity.
BH 1-4 –Bcl-2 homology 1-4;homology domain family of proteins Bcl-2 has the ability to determine the dimerization of the protein by facilitating the formation of homo- and heterodimers, as well as interaction with other proteins regulating apoptosis. Based on the presence of homology domains, the proteins of Bcl-2 family can be divided into 3 classes (subfamilies): I (subfamily Bcl-2) - anti-apoptotic proteins (including Bcl-2 protein) with BH 1-4 domain; II (Bax subfamily) - proapoptotic proteins without BH4 domain, or containing some of its motifs (including Bax and Bakproteins); III (BH3 subfamily) - proapoptotic proteins containing only the BH3 homology domain (including Bad and Bid proteins).
Bid –the BH3 interacting-domain death agonist; Bcl-2 family protein with proapoptotic activity, containing only the homologous BH3 homology domain, interacting with the Bax protein, facilitates its incorporation into the mitochondrial outer membrane.
BPR –benzodiazepine peripheral receptor:the mitochondrial megacanal element present in the mitochondrial outer membrane, builds a complex with VDAC protein, enzyme proteins (creatine kinase, cardiolipin synthase, hexokinase, glycerol kinase, glutathione peroxidase, dehydrogenase/3-beta hydroxy steroid dehydrogenase) and some matrix proteins (cyclophilin D).
CAD –caspase –activated DNase; Mg2+ dependent deoxyribonuclease, DFF homologue described in mouse lymphoid cells .
CANP –Ca2+-activated neutral proteinase; Ca2+dependent neutral endopeptidase, calpase, proteolytic enzyme involved in apoptosis; is involved in the degradation of cytoskeletal proteins, cytokines, enzymes of cell signaling pathways, but also in the proteolytic activation of caspases (3, 7, 9).
CPAN –caspase-activated nuclease;Mg2+dependent endonuclease activated by caspase 3.
DD –death domain.
DED –death effector domain.
DFF –defragmentation factor; Mg2+dependent deoxyribonuclease, responsible for effective fragmentation of chromatin DNA in apoptotic cells, cuts DNA between nucleosomes; the enzyme described in HeLa cells consists of two catalytic subunits (DFF40/CAD = 40 kDa/caspase activated DNase) and a DFF45/ICAD regulator = 45 kDa defibrillation factor/caspase activated DNase) acts as a care protein, providing adequate folding and inhibitor; The correct functioning of the DFF / CAD catalytic subunit is possible following the proteolytic inactivation of the enzyme subunit of this enzyme, involving caspases (or GrB).
DISC –death inducing signalling complex; the equivalent of the DISC heterocomplex in the mitochondrial pathway is apoptosome.
FADD –Fas-associated death domain.
Fas-L –Fas-ligand.
GrB –granzyme B; serine-protease involved in the pseudoreceptor pathway for induction of apoptosis, directed to the killed cell by T cells and NK cells, released simultaneously with a porinwhich allows the enzyme to enter. In the target cell granzyme B cuts the substrates between the aspartic acid residue and the rest of the next amino acid so that it can activate the caspase cascade: inactivate the DFF (DFF45/ICAD) nuclear endonuclease regulatory (inhibitor) subunit, or make a proteolytic cleavage of the Bid protein to form its active tBid derivative.
Kaspazy –caspases = cysteine-dependent aspartate-directed proteases;plays a key role in the pathways of inducing programmed cell death (but also in the regulation of the maturation of many cells, including erythrocytes and myoblasts, or development of inflammation).Caspases are divided into 3 functional groups: 1) inflammatory cytokine activators (caspase-1, -4, -5, -11, -12, -13, -14) 2) initiators (caspase-2, -8, -9, -10, [-12]), 3) effectors of the executive phase of apoptosis (caspase-3, -6, -7).
NS –nuclear scaffold; Ca2+dependent peptidase, responsible in apoptosis for the degradation of the nuclear lamins (proteins- the main components of the nuclear plate).
PIGs –p53 induced genes;coding for enzymatic proteins (oxidoreductases), whose products (reactive oxygen species) belong to the mitochondrial triggers of programmed cell death.
p53 –protein 53 but also tumor protein 53 = TP53 (Human p53 gene product, 17p13.1), a protein with a molecular weight of 53 kDa estimated by its isolation in polyacrylamide gel, the actual weight, based on the contents of amino acid residues is slightly smaller (43.7 kD). The transcription factor known as the tumor suppressor involved in cell cycle regulation (genome guard), in response to DNA damage, activates the proteins involved in its repair, while inhibiting the cell cycle at G1/S when DNA damage cannot be repaired, initiates apoptosis; Positively regulating the expression of genes encoding proteins with proapoptotic activity.
PUMA –the p53 upregulated modulator of apoptosis;Bcl-2 protein family with proapoptotic activity, binding proteins of this family with anti-apoptotic effects (Bcl-xL, Bcl-2, Mcl-1, Bcl-w, A1), thus unable to inhibit the activity of Bax proteins or Bak, in regulating the mitochondrial pathway of apoptosis induction.
Smac/DIABLO –second mitochondrial activator of caspase/direct IAPbinding protein with low pI;moitochondrial proapoptotic protein released upon opening of megacanals to the cytoplasm where it binds and inactivates protein caspase inhibitors (IAPs –inhibitor ofapoptosis proteins).
tBid –truncated Bid;shorter, active form of Bid protein.
TRADD TNF-R1 associated death domain.
TRAIL –TNF-related apoptosis-indicing ligand.
VDAC –voltage-dependent anion channel;porins present in mitochondrial outer membrane, entering in a complex with proteins of the Bcl-2 modifies the opening megacanals.
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