Caspase-3 is necessary and sufficient for cleavage of protein synthesis eukaryotic initiation factor 4G during apoptosis.

Induction of apoptosis BJAB cells is accompanied by the rapid cleavage of protein synthesis eukaryotic initiation factor 4G and the appearance of a fragment of approximately 76 kDa. Inhibition of apoptotic proteases (caspases) has previously been shown to prevent the cleavage of eukaryotic initiation factor 4G. In MCF-7 breast carcinoma cells, which are deficient in caspase-3, eukaryotic initiation factor 4G is not cleaved but in vivo expression of caspase-3 restores eukaryotic initiation factor 4G cleavage following induction of apoptosis. Recombinant caspase-3 can also cleave eukaryotic initiation factor 4G to yield the 76 kDa fragment both in cell extracts and when the eukaryotic initiation factor 4G is presented in a purified eukaryotic initiation factor 4F complex. These results indicate that caspase-3 activity is necessary and sufficient for eukaryotic initiation factor 4G degradation.     

The adenomatous polyposis coli protein and retinoblastoma protein are cleaved early in apoptosis and are potential substrates for caspases

Apoptosis in human monocytic THP.1 tumour cells, induced by diverse stimuli, was accompanied by proteolytic cleavage of the adenomatous polyposis coli gene product (APC) and by sequential cleavage of the retinoblastoma susceptibility gene product (Rb). Cleavage of poly(ADP-ribose) polymerase (PARP), APC and the initial cleavage of Rb at the carboxy terminal region all occurred at a similar time, early in the apoptotic process. Subsequently, Rb underwent a secondary cleavage to 43 kDa and 30 kDa protein fragments. Two caspase inhibitors, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD.FMK) and acetyl-Tyr-Val-Ala-Asp chloromethyl ketone (YVAD.CMK), had markedly different effects on the induction of apoptosis. Z-VAD.FMK inhibited the primary and secondary cleavage of Rb, cleavage of APC and PARP, and apoptosis assessed by flow cytometry. In marked contrast, YVAD.CMK inhibited cleavage of APC and the secondary cleavage of Rb to the 43 kDa and 30 kDa protein fragments but did not inhibit the primary carboxy terminal cleavage of Rb, PARP proteolysis or apoptosis assessed by flow cytometry. These results suggest that different caspases are responsible for the cleavage of different substrates at different stages during the apoptotic process and that a caspase may either cleave APC directly or may be involved in the pathway leading to APC proteolysis. This is the first report suggesting that a cytoplasmic tumour suppressor gene (APC) may be cleaved by a caspase during apoptosis.     

Prostate apoptosis response 4 (Par-4), a novel substrate of caspase-3 during apoptosis activation

Prostate apoptosis response 4 (Par-4) is a ubiquitously expressed proapoptotic tumor suppressor protein. Here, we show for the first time, that Par-4 is a novel substrate of caspase-3 during apoptosis. We found that Par-4 is cleaved during cisplatin-induced apoptosis in human normal and cancer cell lines. Par-4 cleavage generates a C-terminal fragment of ~25 kDa, and the cleavage of Par-4 is completely inhibited by a caspase-3 inhibitor, suggesting that caspase-3 is directly involved in the cleavage of Par-4. Caspase-3-deficient MCF-7 cells do not show Par-4 cleavage in response to cisplatin treatment, and restoration of caspase-3 in MCF-7 cells produces a decrease in Par-4 levels, with the appearance of a cleaved fragment. Additionally, knockdown of Par-4 reduces caspase-3 activation and apoptosis induction. Site-directed mutagenesis reveals that Par-4 cleavage by caspase-3 occurs at an unconventional site, EEPD(131)↓G. Interestingly, overexpression of wild-type Par-4 but not the Par-4 D131A mutant sensitizes cells to cisplatin-induced apoptosis. Upon caspase-3 cleavage, the cleaved fragment of Par-4 accumulates in the nucleus and displays increased apoptotic activity. Overexpression of the cleaved fragment of Par-4 inhibits IκBα phosphorylation and blocks NF-κB nuclear translocation. We have identified a novel specific caspase-3 cleavage site in Par-4, and the cleaved fragment of Par-4 retains proapoptotic activity.     

N‐terminal cleavage of Bax by calpain generates a potent proapoptotic 18‐kDa fragment that promotes Bcl‐2‐independent cytochrome C release and apoptotic cell death

Upon apoptosis induction, the proapoptotic protein Bax is translocated from the cytosol to mitochondria, where it promotes release of cytochrome c, a caspase‐activating protein. However, the molecular mechanisms by which Bax triggers cytochrome c release are unknown. Here we report that before the initiation of apoptotic execution by etoposide or staurosporin, an active calpain activity cleaves Bax at its N‐terminus, generating a potent proapoptotic 18‐kDa fragment (Bax/p18). Both the calpain‐mediated Bax cleavage activity and the Bax/p18 fragment were found in the mitochondrial membrane‐enriched fraction. Cleavage of Bax was followed by release of mitochondrial cytochrome c, activation of caspase‐3, cleavage of poly(ADP‐ribose) polymerase, and fragmentation of DNA. Unlike the full‐length Bax, Bax/p18 did not interact with the antiapoptotic Bcl‐2 protein in the mitochondrial fraction of drug‐treated cells. Pretreatment with a specific calpain inhibitor calpeptin inhibited etoposide‐induced calpain activation, Bax cleavage, cytochrome c release, and caspase‐3 activation. In contrast, transfection of a cloned Bax/p18 cDNA into multiple human cancer cell lines targeted Bax/p18 to mitochondria, which was accompanied by release of cytochrome c and induction of caspase‐3‐mediated apoptosis that was not blocked by overexpression of Bcl‐2 protein. Therefore, Bax/p18 has a cytochrome c–releasing activity that promotes cell death independent of Bcl‐2. Finally, Bcl‐2 overexpression inhibited etoposide‐induced calpain activation, Bax cleavage, cytochrome c release, and apoptosis. Our results suggest that the mitochondrial calpain plays an essential role in apoptotic commitment by cleaving Bax and generating the Bax/p18 fragment, which in turn mediates cytochrome c release and initiates the apoptotic execution. J. Cell. Biochem. 80:53–72, 2000. © 2000 Wiley‐Liss, Inc.     

Apoptosis in sulfur mustard treated A549 cell cultures

The chemical warfare agent sulfur mustard (SM) is a strong alkylating agent that leads to erythema and ulceration of the human skin several hours after exposure. Although SM has been intensively investigated, the cellular mechanisms leading to cell damage remain unclear. Apoptosis, necrosis and direct cell damage are discussed. In this study we investigated apoptotic cell death in pulmonary A549 cells exposed to SM (30-1000 microM, 30 min). 24 h after SM exposure DNA breaks were stained with the TUNEL method. Additionally, A549 cells were lysed and cellular protein was transferred to SDS page and blotted. Whole PARP as well as PARP cleavage into the p89 fragment, an indicator of apoptosis, were detected by specific antibodies. SM concentration dependent increase in TUNEL positive cells and PARP cleavage showed that SM is an inducer of apoptosis. It has been previously suggested that AChE is activated during apoptotic processes and may be involved in apoptosis regulation. Therefore, we examined AChE activity in A549 cells upon induction of apoptosis by SM (100-500 microM). Increased AChE activity was found in SM treated A549 cell cultures examined as determined by the Ellman's assay and by western blot. AChE activity showed a strong correlation with TUNEL positive cells. However, the broad caspase inhibitor zVAD and the PARP-inhibitor 3-aminobenzamide had no protective effect on A459 cells measured with AChE activity and frequency of TUNEL positive cells. In summary, our studies demonstrate that AChE activity may be a potential marker of apoptosis in A549 cells after SM injury. To what extent AChE is involved in apoptosis regulation during SM poisoning has to be further investigated.     

Regulation of lens connexin 45.6 by apoptotic protease,caspase-3

Gap junctions are important in maintaining lens homeostasis. Here we report that connexin 45.6 (Cx45.6) was partially truncated to a 46 kDa fragment during chicken lens development. This specific truncation initiated during embryonic days and the truncated fragment accumulated towards the later developmental stages. When membranes of the embryonic lens were subjected to caspase-3 treatment, the 46 kDa fragment of Cx45.6 was reproduced, suggesting apoptotic protease caspase-3 is a potential protease involved. The COOH-terminus of Cx45.6 in GST-fusion protein was also cleaved by caspase-3, confirming that Cx45.6 is a direct substrate of caspase-3. Induction of apoptosis in lens primary cultures regenerated the 46 kDa fragment and this cleavage was blocked by a caspase-3 inhibitor. Alteration of amino acid residue Asp364 or Glu367 to Ala prevented Cx45.6 from cleavage by caspase-3, suggesting that the cleavage site of Cx45.6 is likely to be between Glu367 and Gly361. Phosphorylation of Ser363, a known substrate for casein kinase II (CKII) in vivo, inhibited the cleavage of Cx45.6 by caspase-3. Thus, this study demonstrates that a lens connexin can be a direct target of caspase-3 and the cleavage by caspase-3 leads to the development-associated truncation of Cx45.6. Finally, caspase-3 mediated truncation can be modulated by the specific connexin phosphorylation.     

The fate of newly synthesized V-ATPase accessory subunit Ac45 in the secretory pathway.

The vacuolar H+-ATPase (V-ATPase) is a multimeric enzyme complex that acidifies organelles of the vacuolar system in eukaryotic cells. Proteins that interact with the V-ATPase may play an important role in controlling the intracellular localization and activity of the proton pump. The neuroendocrine-enriched V-ATPase accessory subunit Ac45 may represent such a protein as it has been shown to interact with the membrane sector of the V-ATPase in only a subset of organelles. Here, we examined the fate of newly synthesized Ac45 in the secretory pathway of a neuroendocrine cell. A major portion of intact approximately 46-kDa Ac45 was found to be N-linked glycosylated to approximately 62 kDa and a minor fraction to approximately 64 kDa. Trimming of the N-linked glycans gave rise to glycosylated Ac45-forms of approximately 61 and approximately 63 kDa that are cleaved to a C-terminal fragment of 42-44 kDa (the deglycosylated form is approximately 23 kDa), and a previously not detected approximately 22-kDa N-terminal cleavage fragment (the deglycosylated form is approximately 20 kDa). Degradation of the N-terminal fragment is rapid, does not occur in lysosomes and is inhibited by brefeldin A. Both the N- and C-terminal fragment pass the medial Golgi, as they become partially endoglycosidase H resistant. The Ac45 cleavage event is a relatively slow process (half-life of intact Ac45 is 4-6 h) and takes place in the early secretory pathway, as it is not affected by brefeldin A and monensin. Tunicamycin inhibited N-linked glycosylation of Ac45 and interfered with the cleavage process, suggesting that Ac45 needs proper folding for the cleavage to occur. Together, our results indicate that Ac45 folding and cleavage occur slowly and early in the secretory pathway, and that the cleavage event may be linked to V-ATPase activation.     

Caspase-mediated cleavage of the U snRNP-associated Sm-F protein during apoptosis

Recent studies have implicated the dying cell as a potential reservoir of modified autoantigens that might initiate and drive systemic autoimmunity in susceptible hosts. The spliceosomal Sm proteins are recognized by the so-called anti-Sm autoantibodies, an antibody population found exclusively in patients suffering from systemic lupus erythematosus. We have studied the effects of apoptosis on the Sm proteins and demonstrate that one of the Sm proteins, the Sm-F protein, is proteolytically cleaved in apoptotic cells. Cleavage of the Sm-F protein generates a 9-kDa apoptotic fragment, which remains associated with the U snRNP complexes in apoptotic cells. Sm-F cleavage is dependent on caspase activation and the cleavage site has been located near the C-terminus, EEED(81) downward arrow G. Use of different caspase inhibitors suggests that besides caspase-8 other caspases are implicated in Sm-F cleavage. A C-terminally truncated mutant of the Sm-F protein, representing the modified form of the protein, is capable of forming an Sm E-F-G complex in vitro that is recognized by many anti-Sm patient sera.     

MEK Kinase 1, a Substrate for DEVD-Directed Caspases, Is Involved in Genotoxin-Induced Apoptosis

MEK kinase 1 (MEKK1) is a 196-kDa protein that, in response to genotoxic agents, was found to undergo phosphorylation-dependent activation. The expression of kinase-inactive MEKK1 inhibited genotoxin-induced apoptosis. Following activation by genotoxins, MEKK1 was cleaved in a caspase-dependent manner into an active 91-kDa kinase fragment. Expression of MEKK1 stimulated DEVD-directed caspase activity and induced apoptosis. MEKK1 is itself a substrate for CPP32 (caspase-3). A mutant MEKK1 that is resistant to caspase cleavage was impaired in its ability to induce apoptosis. These findings demonstrate that MEKK1 contributes to the apoptotic response to genotoxins. The regulation of MEKK1 by genotoxins involves its activation, which may be part of survival pathways, followed by its cleavage, which generates a proapoptotic kinase fragment able to activate caspases. MEKK1 and caspases are predicted to be part of an amplification loop to increase caspase activity during apoptosis.     

Apoptosis-associated cleavage of beta-catenin in human colon cancer and rat hepatoma cells

Proteases belonging to the caspase family play a crucial role in apoptotic processes. Identification of protein cleavage specific to apoptosis may therefore provide further information about the mechanisms of apoptosis. In this study, apoptosis and necrosis were induced in cells of the human colon cancer cell lines, WiDr and DLD-1, and the resulting protein cleavage patterns investigated for beta-catenin. beta-Catenin was detected as a 92 kDa protein in control viable cells, while 65-72 kDa beta-catenin cleavage fragments were characteristically observed in apoptotic cells. These fragments were not observed in necrotic cell death. Similar apoptosis-specific beta-catenin cleavage was also demonstrated in the rat hepatoma cell line McA-RH7777, suggesting that the beta-catenin cleavage is a common event in apoptosis in various cell types. The formation of 65-72 kDa beta-catenin cleavage fragments was completely prevented by a caspase-1 inhibitor Z-VAD-CH2F and a caspase-3 inhibitor Z-DEVD-CH2F, indicating that the cleavage is associated with caspase-dependent process. Since beta-catenin is implicated in cell adhesion and signal transduction, these findings may suggest various possible roles of beta-catenin degradation in the dramatic cytoskeletal and morphological changes, as well as signaling events that accompany apoptosis.     

N-terminal cleavage of bax by calpain generates a potent proapoptotic 18-kDa fragment that promotes bcl-2-independent cytochrome C release and apoptotic cell death

Upon apoptosis induction, the proapoptotic protein Bax is translocated from the cytosol to mitochondria, where it promotes release of cytochrome c, a caspase-activating protein. However, the molecular mechanisms by which Bax triggers cytochrome c release are unknown. Here we report that before the initiation of apoptotic execution by etoposide or staurosporin, an active calpain activity cleaves Bax at its N-terminus, generating a potent proapoptotic 18-kDa fragment (Bax/p18). Both the calpain-mediated Bax cleavage activity and the Bax/p18 fragment were found in the mitochondrial membrane-enriched fraction. Cleavage of Bax was followed by release of mitochondrial cytochrome c, activation of caspase-3, cleavage of poly(ADP-ribose) polymerase, and fragmentation of DNA. Unlike the full-length Bax, Bax/p18 did not interact with the antiapoptotic Bcl-2 protein in the mitochondrial fraction of drug-treated cells. Pretreatment with a specific calpain inhibitor calpeptin inhibited etoposide-induced calpain activation, Bax cleavage, cytochrome c release, and caspase-3 activation. In contrast, transfection of a cloned Bax/p18 cDNA into multiple human cancer cell lines targeted Bax/p18 to mitochondria, which was accompanied by release of cytochrome c and induction of caspase-3-mediated apoptosis that was not blocked by overexpression of Bcl-2 protein. Therefore, Bax/p18 has a cytochrome c-releasing activity that promotes cell death independent of Bcl-2. Finally, Bcl-2 overexpression inhibited etoposide-induced calpain activation, Bax cleavage, cytochrome c release, and apoptosis. Our results suggest that the mitochondrial calpain plays an essential role in apoptotic commitment by cleaving Bax and generating the Bax/p18 fragment, which in turn mediates cytochrome c release and initiates the apoptotic execution.     

Regulation of Lens Connexin 45.6 by Apoptotic Protease,Caspase-3

Gap junctions are important in maintaining lens homeostasis. Here we report that connexin 45.6 (Cx45.6) was partially truncated to a 46 kDa fragment during chicken lens development. This specific truncation initiated during embryonic days and the truncated fragment accumulated towards the later developmental stages. When membranes of the embryonic lens were subjected to caspase-3 treatment, the 46 kDa fragment of Cx45.6 was reproduced, suggesting apoptotic protease caspase-3 is a potential protease involved. The COOH-terminus of Cx45.6 in GST-fusion protein was also cleaved by caspase-3, confirming that Cx45.6 is a direct substrate of caspase-3. Induction of apoptosis in lens primary cultures regenerated the 46 kDa fragment and this cleavage was blocked by a caspase-3 inhibitor. Alteration of amino acid residue Asp³⁶⁴or Glu³⁶⁷ to Ala prevented Cx45.6 from cleavage by caspase-3, suggesting that the cleavage site of Cx45.6 is likely to be between Glu³⁶⁷ and Gly³⁶⁸. Phosphorylation of Ser³⁶³, a known substrate for casein kinase II (CKII) in vivo, inhibited the cleavage of Cx45.6 by caspase-3. Thus, this study demonstrates that a lens connexin can be a direct target of caspase-3 and the cleavage by caspase-3 leads to the development-associated truncation of Cx45.6. Finally, caspase-3 mediated truncation can be modulated by the specific connexin phosphorylation.     

Cleavage of polypeptide chain initiation factor eIF4GI during apoptosis in lymphoma cells: characterisation of an internal fragment generated by caspase-3-mediated cleavage

Polypeptide chain initiation factor eIF4GI undergoes caspase-mediated degradation during apoptosis to give characteristic fragments. The most prominent of these has an estimated mass of approximately 76 kDa (Middle-Fragment of Apoptotic cleavage of eIF4G; M-FAG). Subcellular fractionation of the BJAB lymphoma cell line after induction of apoptosis indicates that M-FAG occurs in both ribosome-bound and soluble forms. Affinity chromatography on m7GTP-Sepharose shows that M-FAG retains the ability of eIF4GI to associate with both the mRNA cap-binding protein eIF4E and initiation factor eIF4A and that the ribosome-bound form of M-FAG is also present as a complex with eIF4E and eIF4A. These data suggest that the binding sites for eIF4E, eIF4A and eIF3 on eIF4GI are retained in the caspase-generated fragment. M-FAG is also a substrate for cleavage by the Foot-and-Mouth-Disease Virus-encoded L protease. These properties, together with the pattern of recognition by a panel of antibodies, define the origin of the apoptotic cleavage fragment. N-terminal sequencing of the products of caspase-3-mediated eIF4GI cleavage has identified the major cleavage sites. The pattern of eIF4GI degradation and the possible roles of the individual cleavage products in cells undergoing apoptosis are discussed.     

A presumed human nuclear autoantigen that translocates to plasma membrane blebs during apoptosis

The structure and subcellular localization of a number of molecules change during apoptosis. These molecules are recognized by the immune system, leading to the development of autoimmunity when apoptotic cells fail to be effectively cleared by phagocytosis. We searched for such molecules by analyzing sera from 12 individuals who suffered from autoimmune diseases and from 3 patients with amyotrophic lateral sclerosis. One serum sample, designated 681, detected an antigen that fulfilled the above criteria. In Western blotting of lysates of human Jurkat T cells, the 681 antigen appeared as a distinct signal with a molecular mass of 60 kDa in normal cells, and 2 additional signals with faster mobilities were detected in apoptotic cells. The results of subcellular fractionation and immunofluorescence experiments revealed this antigen to be strictly localized in the nucleus of normal cells, but to be translocated to a region near the plasma membrane, to membrane blebs in particular, after the induction of apoptosis. Under conditions in which membrane blebbing was inhibited in apoptotic cells, the antigen still moved away from the nucleus, but its accumulation at the periplasmic region was completely abolished. The apparent partial cleavage and intracellular redistribution of the 681 antigen in apoptotic cells mimics changes previously reported for the nuclear autoantigen La, but the 681 antigen was clearly distinct from La. These results suggest that cleavage-dependent exit from the nucleus during apoptosis is a phenomenon common to nuclear autoantigens.     

Proteome analysis of apoptosis signaling by S-trityl-L-cysteine, a potent reversible inhibitor of human mitotic kinesin Eg5

Mitotic kinesins represent potential drug targets for anticancer chemotherapy. Inhibitors of different chemical classes have been identified that target human Eg5, a kinesin responsible for the establishment of the bipolar spindle. One potent Eg5 inhibitor is S-trityl-L-cysteine (STLC), which arrests cells in mitosis and exhibits tumor growth inhibition activity. However, the underlying mechanism of STLC action on the molecular level is unknown. Here, cells treated with STLC were blocked in mitosis through activation of the spindle assembly checkpoint as shown by the phosphorylated state of BubR1 and the accumulation of mitosis specific phosphorylation on histone H3 and aurora A kinase. Using live cell imaging, we observed prolonged mitotic arrest and subsequent cell death after incubation of GFP-alpha-tubulin HeLa cells with STLC. Activated caspase-9 occurred before cleavage of caspase-8 leading to the accumulation of the activated executioner caspase-3 suggesting that STLC induces apoptosis through the intrinsic apoptotic pathway. Proteome analysis following STLC treatment revealed 33 differentially regulated proteins of various cellular processes, 31 of which can be linked to apoptotic cell death. Interestingly, four identified proteins, chromobox protein homolog, RNA-binding Src associated in mitosis 68 kDa protein, stathmin, and translationally controlled tumor protein can be linked to mitotic and apoptotic processes.     

Protein kinase B inhibits endostatin-induced apoptosis in HUVECs

Endostatin is a tumor-derived angiogenesis inhibitor, and the endogenous 20 kDa carboxyl-terminal fragment of collagen XVIII. In addition to inhibiting angiogenesis,endostatin inhibits tumor growth and the induction of apoptosis in several endothelial cell types. However, the mechanisms that regulate endostatin-induced apoptotic cell death are unclear. Here, we investigated apoptotic cell death and the underlying regulatory mechanisms elicited of endostatin in human umbilical vein endothelial cells (HUVECs). Endostatin was found to induce typical apoptotic features, such as, chromatin condensation and DNA fragmentation in these cells. Thus, as the phosphoinositide 3-OH kinase (PI3K)/protein kinase B (PKB) signaling pathway has been shown to prevent apoptosis in various cell types, we investigated whether this pathway could protect cells against endostatin induced apoptosis. It was found that the inhibition of PI3K/PKB significantly increased endostatin-induced apoptosis, and that endostatininduced cell death is physiologically linked to PKB-mediated cell survival through caspase-8.