Caspases, a family of cysteine proteases, play a central role in apoptosis. During the last decade, major progress has been made to further understand caspase structure and function, providing a unique basis for drug design. This Review gives an overview of caspases and their classification, structure, and substrate specificity. We also describe the current knowledge of how interference with caspase signaling can be used to pharmacologically manipulate cell death.

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The dynamic modification of nuclear and cytoplasmic proteins with O-linked beta-N-acetylglucosamine (O-GlcNAc) by the O-linked N-acetylglucosaminyltransferase (OGT) is a regulatory post-translational modification that is responsive to various stimuli. Here, we demonstrate that OGT is a central factor for T- and B-lymphocytes activation. SiRNA-mediated knockdown of OGT in T cells leads to an impaired activation of the transcription factors NFAT and NFkappaB. This results in a reduction of IL-2 production consistent with prevention of T-cell activation. OGT is also required for the early activation of B cells mediated by stimulation of the B-cell receptor. Mechanistically, we demonstrate that NFkappaB as well as NFAT are glycosylated with O-GlcNAc after direct binding to OGT. Moreover, kinetic experiments show that O-GlcNAc modification prominently increased shortly after activation of lymphoid cells and it might be required for nuclear translocation of the transcription factors NFkappaB and NFAT.

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c-FLIP proteins (isoforms: c-FLIPL, c-FLIPS, and c-FLIPR) play an essential role in the regulation of death receptor–induced apoptosis. Here, we demonstrate that the cytoplasmic NH2-terminal procaspase-8 cleavage product of c-FLIP (p22-FLIP) found in nonapoptotic malignant cells, primary T and B cells, and mature dendritic cells (DCs) strongly induces nuclear factor κB (NF-κB) activity by interacting with the IκB kinase (IKK) complex via the IKKγ subunit. Thus, in addition to inhibiting apoptosis by binding to the death-inducing signaling complex, our data demonstrate a novel mechanism by which c-FLIP controls NF-κB activation and life/death decisions in lymphocytes and DCs.

Restimulation of the T-cell receptor (TCR) in activated T cells induces CD95 (Fas/Apo-1)-mediated activationinduced cell death (AICD). The TCR-proximal mechanisms leading to AICD are elusive. Here we characterize hematopoietic progenitor kinase 1 (HPK1) as a differentially regulated TCR-proximal signaling protein involved in AICD of primary T cells. We show that HPK1 is a functional component of the endogenous IjB kinase (IKK) complex and is crucial for TCR-mediated NFjB activation. While full-length HPK1 enhances IKKb phosphorylation, siRNAmediated knockdown of HPK1 blunts TCR-mediated NFjB activation and increases cell death. We also demonstrate proteolytic processing of HPK1 into HPK1-C, specifically in AICD-sensitive primary T cells. The cleavage product HPK1-C sequesters the inactive IKK complex and suppresses NFjB upon TCR restimulation by binding to IKKa and IKKb. T cells of HPK1-C transgenic mice are sensitized towards TCR-mediated AICD. Consequently, preventing HPK1-C generation in primary T cells by siRNA-mediated knockdown results in decreased AICD. Thus, these results show a novel mechanism of sensitization of T lymphocytes towards AICD by suppression of NFjB, and propose that HPK1 is a life/death switch in T lymphocytes.

Suramin is a polysulfonated derivative of urea and has been widely used both to treat infections and as a chemotherapeutic drug. Suramin has been shown to inhibit growth factor signaling pathways; however, its effect on apoptosis is unknown. Here we show that suramin inhibits apoptosis induced through death receptors in hepatoma and lymphoma cells. It also inhibits the proapoptotic effect of chemotherapeutic drugs. The antiapoptotic mechanism is specific to cell type and is caused by reduced activation, but not altered composition, of the death-inducing signaling complex (DISC), and by inhibition of the initiator caspases 8, 9 and 10. Suramin also shows similar effects in in vivo models: apoptotic liver damage induced by CD95 stimulation and endotoxic shock mediated by tumor-necrosis factor (TNF) are inhibited in mice, but necrotic liver damage is not inhibited in a rat model of liver transplantation. Thus, the antiapoptotic property of suramin in the liver may be therapeutically exploited.

Caspase-2 was reported to be involved in a number of apoptotic pathways triggered by various stimuli. However, the molecular mechanism of procaspase-2 activation in the course of apoptosis remains poorly defined. In this report, we demonstrate that procaspase-2 is recruited to the CD95 (Fas/APO-1) deathinducing signaling complex (DISC) in human T-and B-cell lines. We show that procaspase-2 is activated at the DISC on CD95 stimulation. Despite its presence at the DISC, caspase-2 does not initiate apoptosis on CD95 stimulation in caspase-8-deficient cell lines. Taken together, our data reveal that caspase-2 is activated at the DISC but does not play an initiating role in the CD95-induced apoptosis. Introduction CD95 (APO-1/Fas) is a member of the death receptor family, a subfamily of the TNF-R superfamily. [1][2][3][4][5] Crosslinking of CD95 with its natural ligand CD95L (FasL/CD178) 6 or with agonistic antibodies such as anti-APO-1 7 induces apoptosis in sensitive cells. The death-inducing signaling complex (DISC) is formed within seconds after CD95 stimulation. 8 The DISC consists of oligomerized, probably trimerized, CD95 receptors; the adaptor molecule FADD; 2 isoforms of procaspase-8 (procaspase-8/a and procaspase-8/b); procaspase-10; and c-FLIP L/S/R . 5,9 The interactions between molecules at the DISC are based on homotypic contacts. The death domain (DD) of the receptor interacts with the DD of FADD, whereas the death effector domain (DED) of FADD interacts with the N-terminal tandem DEDs of procaspase-8, -10, and c-FLIP L/S/R . The binding of procaspase-8 to the DISC results in processing of the zymogen. As a result the active caspase-8 heterotetramer p10 2 -p18 2 is released into the cytosol to propagate the apoptotic signal. 10 Two CD95 signaling pathways were established. 11 Type I cells are characterized by high levels of CD95 DISC formation and increased amounts of active caspase-8. Activated caspase-8 directly leads to the activation of downstream effector caspase-3 and -7. Type II cells are characterized by lower levels of CD95 DISC formation and, thus, lower levels of active caspase-8. 11 In this case, signaling requires an additional amplification loop that involves the cleavage by caspase-8 of the Bcl-2-family protein Bid to generate truncated (t) Bid and subsequent tBid-mediated release of Cytochrome C from mitochondria. The release of Cytochrome C from mitochondria results in apoptosome formation, followed by activation of procaspase-9, which in turn cleaves downstream, effector caspases. 12 Caspase-2 is referred to as an initiator caspase. [13][14][15] It is characterized by the presence of a caspase activation recruitment domain (CARD) and is structurally related to the initiator caspase-9. 16,17 The mechanism of procaspase-2 activation in apoptosis in contrast to other initiator caspases remains poorly defined. It was reported that caspase-2 is implicated in Cytochrome C release and is essential for drug-induced apoptosis in several human cell lines. [18][19][20] On the basis of these dat...