Caspases Parp And Cancer

PARP, pro-Caspase-6

Lamins A, B1/B2, C, PARP PARP, pro-Caspases PARP

Pro-Caspases B, 7

subunit, which contains the active site cysteine, and a small subunit (Figure 3.2.1.A). Activation involves the proteolytic processing of the domains, followed by association between the large and small subunits in a heterodimer. This NH2-terminal prodomain is separated from the central large Caspase subunit (about 17 or 20 kD, called P17 or P20 subunit) by one or two asparagine cleavage sites. The large Caspase subunit itself is separated from the COOH-terminal small subunit (about 10 kD, called P10 subunit) by one asparagine cleavage site or a linker peptide (two asparagine cleavage sites). Like other proteases, their activity is also tightly controlled by protease inhibitors.

Caspases can activate themselves by autoproteol-ysis, or can be processed by other active Caspases. The proteolytic cleavage leads to the formation of the active Caspases, which consist of the P10 and P20 subunits. The active proteins are tetramers of 2 P20 subunits surrounding two adjacent P10 sub-units. Both, the P20 and P10 subunits are essential for catalytic activity.

The catalytic activity of Caspases depends on a critical cysteine residue within a highly conserved active site pentapeptide QAC(R,G,Q)G. This motif is located in the large subunit (P20 or P17) and forms the primary recognition pocket for the asparagine residue; however, several residues in the P10 and P20 (P17) subunits contribute to the specific binding of the substrate and form secondary recognition pockets. The central cysteine residue in the active site pentapeptide and a histidine residue form a pro-domain large small pro-domain large small

Figure 3.2.I.A. Basic Caspase structure. The scheme depicts the activation of pro-Caspase. The proenzyme is cleaved at two Caspase cleavage sequences (AspX, Asp = asparagine). Two large subunits and two small subunits combine to form the active tetrameric enzyme. The green structures indicate the catalytic domains.

Figure 3.2.I.A. Basic Caspase structure. The scheme depicts the activation of pro-Caspase. The proenzyme is cleaved at two Caspase cleavage sequences (AspX, Asp = asparagine). Two large subunits and two small subunits combine to form the active tetrameric enzyme. The green structures indicate the catalytic domains.

catalytic diad. They directly contribute to the formation of the tetrahedral intermediate state that is formed during the hydrolytic cleavage of substrate peptides by Caspases. While the active site pen-tapeptide is common to all Caspases, they differ in their recognition sites and their substrate specificity.

The Caspases specifically cleave their substrates after asparagine residues. The Caspases -3, -7, and -9 recognize the tetrapeptide sequences DEXD, which is present in Poly(ADP-ribose)Polymerase. The motif WEHD is cleaved by Caspases -1, -4, and -5. Caspase-6 is the only Caspase known to cleave Lamin A with the recognition sequence VEID. This cleavage contributes to chromatin condensation and nuclear shrinkage. Caspase-1 [Yuan et al. 1993] cleaves the cytokine pro-IL-1P. In general, the most significant differences in Caspase specificities are found in substrate position P4. In contrast, P3 specificities are similar among Caspases, and in P2 a wide range of amino acids is tolerated. The P4 preferences can be categorized as hydrophobic (Caspases -1, -4, and -6) or aspartate (Caspases -2, -3, and -7).

Several caspase genes are expressed as multiple forms by alternative splicing of the primary transcript, including caspase-1, -2, -3, -6, -7 and -8. Among those forms are enzymatically inactive variants, which are expressed as modified mRNAs or truncated proteins that may play a crucial role in the negative or positive regulation of Caspase activity. Caspase-8 is expressed in at least seven variants that differ by deletions or sequence variations in the NH2-terminal prodomain, containing the death effector domains (DEDs), or by loss of the COOH-terminal part that normally encodes the P10 and P20 Caspase subunits. The variant MACH a-3 has a dominant negative effect on the activity of the processed Caspase-8 enzyme and provides effective protection against CD95-mediated apoptosis [Boldin etal. 1996].

The inducer Caspase-8 is associated with apoptosis triggered through death receptors (extrinsic pathway). Upon their ligation, Caspase-8 oligomer-ization drives its own activation through autocat-alytic cleavage. Association with the adaptor protein FADD (FAS-Associated Via Death Domain, MORT-1) through the DED is also required. The Caspase-8/FADD complex is referred to as DISC (death inducing signaling complex). Caspase-8 then activates downstream effector Caspases. It also cleaves and activates BID, the truncated form of which (tBID) triggers the mitochondrial activation of Caspase-9 by inducing the homooligomerization and allosteric activation of BAK or BAX. Furthermore, Caspase-8 cleaves Poly-(ADP ribose) Polymerase (PARP) [Hopkins-Donaldson et al. 2000]. CD95 mediates the extrinsic, Caspase-8-dependent pathway to apoptosis. FLIP (FADD-Like ICE Inhibitory Protein) is a competitive inhibitor of Caspase-8 that may block death receptor-induced apoptosis by being incorporated into the DISC, but lacking proteolytic enzymatic activity.

The inducer Caspase-9 (APAF-3) is involved in death brought about by cytotoxic agents (intrinsic pathway). Many apoptotic stimuli induce the release of the Caspase activator Cytochrome c and the Caspase coactivator SMAC (DIABLO) from mitochondria into the cytosol, where it binds to APAF-1 and induces the interaction with pro-Caspase-9. The Caspase-9/APAF-1 complex is referred to as apopto-some. Binding of pro-Caspase-9 to APAF-1 through the CARD leads to its proteolytic activation by cleavage of the residues 316 through 330 from the Caspase-9 small subunit. The resulting Caspase-9 can no longer be inhibited by XIAP. The activated Caspase-9 cleaves and activates Caspase-3. ARC (Apoptosis Repressor with CARD) is a competitive inhibitor of Caspase-9. Survivin selectively inhibits the intrinsic, Caspase-9-dependent apoptotic pathway.

The inducer Caspase-2 (NEDD-2, ICH-1) of the intrinsic pathway is ubiquitously expressed. It is required for apoptosis in response to genotoxic stress. It induces the cleavage of BID, the translocation of BAX to the mitochondria, and the release of Cytochrome c from the mitochondria. Upon activation, Caspase-2 is recruited into a large protein complex, which includes the death domain-containing protein PIDD and the adaptor protein RAIDD [Tinel and Tschopp 2004]. Caspase-2 is required for apoptosis by some cancer cells [Lassus et al. 2002].

The accumulation of unfolded or malfolded proteins causes endoplasmic stress, a process that can lead to apoptosis independently of mitochondria or APAF-1. The relevant inducer Caspase located in the endoplasmic reticulum is Caspase-12. There, it mediates apoptotic responses to stress that affects the endo-plasmic reticulum (endoplasmic pathway, unfolded protein response) [Nakagawa et al. 2000]. TRAF2 interacts with pro-Caspase-12 and promotes its clustering with ensuing activation by cleavage. BAX and

BAK can also localize to the endoplasmic reticulum. In stress situations, they undergo conformational changes and oligomerization, which leads to Caspase-12 cleavage. Downstream, Caspase-7 is activated and the transcription factor eIF2a (eukaryotic Translation Initiation Factor 2a) is dephosphorylated and inactivated.

Caspase-3 is a prominent effector Caspase that mediates DNA fragmentation, cell rounding, and the formation of apoptotic bodies.

- Caspase-3 cuts the chaperone ICAD (Inhibitor of the Caspase-Activated Deoxyribonuclease). This releases the ICAD partner CAD (Caspase-Activated Deoxyribonuclease), which translocates to the nucleus and degrades DNA. This may account for the DNA laddering in apoptosis.

- Caspase-3 cuts Gelsolin, a protein that normally binds to the Actin filaments that help give a cell its shape. Cells with degraded Gelsolin round up.

-Caspase-3 cuts and activates PAK2 (P21-Activated Kinase-2) that regulates the cytoskele-ton and may contribute to the formation of apoptotic bodies.

- Apoptosis is regulated, in part, by phosphoryla-tion of serine 14 in the tail of Histone H2B. This event may trigger the chromatin condensation that is followed by DNA fragmentation. The active kinase in this process is MST1, which is induced by Caspase-3.

RGD containing peptides may enter cells and directly induce autoprocessing and enzymatic activity of pro-Caspase-3 through an interaction with its RGD-binding motif DMM. This mechanism may be activated after the cleavage and internalization of extracellular matrix proteins [Buckley et al. 1999].

Upon induction of apoptosis, pro-Caspase-6 is processed at aspartate residues to yield a large (18 kD) and a small (11 kD) subunit, which associate to form the active enzyme. Caspase-6 catalyzes the pro-teolysis of Poly-(ADP-ribose)Polymerase (PARP), an enzyme that is involved in DNA repair and genomic maintenance. Caspase-6 is the main effector Caspase in glucocorticosteroid-induced apoptosis.

The effector Caspase-7 (Mch3, ICE-LAP3, CMH-1) cleaves substrates responsible for producing the morphological and biochemical changes associated with apoptosis. Its a form of 303 amino acids is processed upon activation to a 20 and 12 kD subunit. A 253 amino acid P form results from alternative splicing and lacks the cysteine protease active site (QACRG). The P form may function as a dominant negative regulator of apoptosis. The active Caspase-7 is involved in the proteolysis of Poly(ADP-ribose)Polymerase (PARP).

• Methylation of caspase-8 occurs in some childhood tumors and in neuroendocrine lung tumors. caspase-8 expression is silenced by gene methylation in malignant neuroblastoma, and this correlates with resistance to cell death [Hopkins-Donaldson et al. 2000].

• eIF4E (Eukaryotic Translation Initiation Factor 4E) is an mRNA cap-binding protein required for the translation of cellular mRNA. eIF4E is a major target for the regulation of translation by growth factors and hormones. When overexpressed, eIF4E profoundly suppresses proto-oncogene-dependent apoptosis, causing malignant transformation. eIF4E rescues cells from endoplasmic reticulum stressors and functions as a pleiotropic regulator of cell viability. This is accomplished, in part, by preventing the release of Caspase-12 from the endoplasmic reticulum [Li et al. 2004].

• The tumor suppressor gene product DRS (Domain Rich in Serine, Pinin, PNN) activates Caspase-12 and ensuing programmed cell death. The release of Cytochrome c from the mitochondria into the cytoplasm is not associated with this form of apoptosis [Tambe et al. 2004]. The expression of drs {14q13} is markedly down-regulated in renal cell carcinoma, transitional cell carcinoma, and in cancers of the colon and the prostate. This may be caused by promoter methylation.

Endonucleases. The digestion of the genomic DNA within an apoptotic cell is accomplished by endonu-cleases. They cut the DNA into fragments of about 180 bp in size, reflecting the length of 1 wrap of DNA around Histone proteins (internucleosomal DNA cleavage).

DFF (DNA Fragmentation Factor) is composed of a heterodimer of a catalytic endonuclease subunit DFF40 (DFFB, CAD, Caspase-Activated Deo-xyribonuclease) {1p36.3} and a chaperone/inhibitor subunit DFF45 (45 kD DNA Fragmentation Factor, DFFA, DFF1, ICAD-L) {1p36.2-36.3} or DFF35 (35 kD DNA Fragmentation Factor, ICAD-S). Cleavage of the inhibitor DFF45 causes the release of the DNAse DFF40, which travels to the nucleus to fragment DNA. The cleavage of DFF45 or DFF35 by Caspase-3 is accompanied by DFF40 homooligomer formation, with a tetramer being the smallest unit. Intact DFF45 can inhibit the nuclease activity by associating with these homooligomers, without mediating their disassembly.

CIDE-A and CIDE-B (Cell Death-Inducing DFFA-Like Effectors A and B) [Inohara et al. 1998] activate apoptosis in a manner that is inhibitable by DFF45, but is independent of Caspases. Another family member, CIDE-3, is also competent to induce DNA fragmentation [Liang et al. 2003]. The COOH-terminal region of CIDE-A is necessary and sufficient for killing, whereas a region with homology to DFF45 located in the NH2-terminus is required for DFF45 to inhibit CIDE-A-induced apoptosis. The expression pattern of CIDE-3 is different from that of CIDE-B, with expression of CIDE-3 mainly in small intestine, heart, colon, and stomach, while CIDE-B is strongly expressed in the liver and small intestine, and at a lower level in colon, kidney, and spleen. CD95-mediated apoptosis can be enhanced by CIDEs.

ENDO-G (Endonuclease G) [Ruiz-Carrillo and Renaud 1987] is an Mg2+-dependent DNA endonu-clease that has a strong preference to nick within long tracts of guanine residues. It is located in the mitochondria. In response to apoptotic stimuli, ENDO-G is released simultaneously with Cyto-chrome c into the cytoplasm and travels to the nucleus. Once released from the mitochondria, ENDO-G cleaves chromatin DNA into nucleosomal fragments independently of Caspases [Li et al. 2001].

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