BIOLOGICAL OVERVIEW

Ced-3 is the product of a gene that is necessary for programmed cell death (PCD) in the nematode C. elegans. Using the sequence of Ced-3 in a Blast search, the Drosophila gene Dredd was identified, and found to be coded for by sequences at the 3' end of l(1)1Bi (accession No. U20542). Dredd (the name stands for "Death related ced-3/Nedd2-like") protein is a Drosophila member of the caspase gene family; it encodes a 128 kD nucleolar protein. To date, the mammalian caspase transcripts described are, under normal conditions, ubiquitously distributed in many, if not all, cell types. Similarly, constitutive embryonic expression has been reported for the two other Drosophila caspases, Dcp-1 and drICE (Fraser, 1997a and Song, 1997). In contrast to this, pronounced elevation of Dredd transcripts occurs in normal development and this unique regulation is tightly linked to apoptotic signaling by Reaper, Grim and Head involution defective (Hid). Expression of Reaper, Grim, and Hid triggers processing of Dredd protein precursors by means of a mechanism that is insensitive to, and upstream of, known caspase inhibitors (Chen, 1998b).

An example of stage specific expression of dredd is the expressing of dredd associated with PCD during oogenesis. During oogenesis, nurse cells synthesize essential cytoplasmic materials and transport these to the developing oocyte. Once this phase is accomplished (stage 12), the nurse cells degenerate, exhibiting apoptotic characteristics that include cellular condensation, DNA fragmentation, and changes in cytochrome c. An attractive feature of this system is that a stereotypical sequence of morphological changes permits the identification of doomed cells prior to any overt signs of apoptosis. For this reason, expression of DREDD mRNA was examined in developing egg chambers. DREDD mRNA first appears at stage 10 in both nurse cells and the developing oocute, suggesting that at least some DREDD mRNAS are maternally supplied to the egg. In later-stage egg chambers (stages 12-13), DREDD mRNA persists within nurse cells and accumulates to very high levels at a time coincident with nurse cell death (Chen, 1998b).

To discover whether expression of apoptosis activators reaper, grim and hid triggers the accumulation of DREDD mRNA, the three apoptosis activators were ectopically expressed in mesoderm, and the expression of DREDD mRNA examined. Expression of the apoptosis activators triggers excessive apoptosis in mesoderm. During stage 13 and beyond, DREDD mRNA is not widely expressed in the developing musculature in wild-type flies. However, when misexpression of each of the death activators is directed to these tissues, prominent levels of ectopic DREDD mRNA are detected. Expression of grim in the ectoderm also results in DREDD mRNA accumulation. DREDD mRNA accumulation has also been examined in embryos homozygous for crumbs (crb). In crb mutants, reaper is ectopically expressed in the disorganized epidermis. As anticipated, ectopic accumulation of DREDD mRNA is found scattered throughout the ectoderm in crb embryos, coincident with widespread patterns of rpr expression. Perhaps the most compelling evidence for a direct role for Dredd in apoptosis comes from an examination of accumulation of DREDD mRNA in embryos carrying a homozygous deletion of the entire reaper region (mutated for rpr, hid, and grim). No apoptosis occurs in these deletion mutants. The selective accumulation of DREDD mRNA fails to occur in these mutants. This is the first report of a molecular activity that is completely blocked by the absence of H99-associated signaling (Chen, 1998b).

Dredd has a long prodomain that contains significant sequence similarity to mammalian counterparts [caspase-8/FLICE/Mac5/MACH, and caspase-10(Mach4)]. This homology spans a region that is believed to promote homotypic interactions, which establish a regulatory interface between death signals and caspase function. In mammals, caspase-8 is the most upstream caspase activated by cell surface death receptors such as Fas and TNF. Dredd may serve a similar function related to the activation of other Drosophila caspases such as Dcp-1 and drICE. When apoptosis is independently triggered by expression of Rpr, Grim or Hid, processing of Dredd-gamma, -delta and -alpha (see below: Gene Structure) is readily observed. An N-terminal tagged version of Dredd was produced and tested for the production of the processed small subunit (p10) that represents the initial product of the caspase cleavage reaction. Using these N-terminal tagged versions of Dredd, intermediates were detected that anticipate the onset of apoptosis by at least two hours. Later, additional cleavage products were detected. Rpr- and Grim-induced apoptosis can be blocked by caspase peptide inhibitors, yet in the presence of inhibitors of apoptosis, initial processing intermediates of Dredd-gamma still appear. Therefore, proteolytic cleavage of Dredd is a direct consequence of signaling by death activators and is not inhibited by inhibitors of apoptosis, indicating that the processing of Dredd is a primary process and not a secondary consequence of apoptosis (Chen, 1998b).

Cells treated with caspase inhibitors accumulate Dredd proform and intermediates to levels that are notably higher than in the absence of inhibitors. This elevation might result from inhibition of downstream proteases responsible for further processing of intermediates. Thus despite the fact that classical caspase inhibitors and p35 completely prevent activator-induced apoptosis, the initial cleavage of Dredd is not prevented by these agents. These data raise the possibility that the initial step in the processing of Dredd may occur through proteolytic activities that are upstream of caspase action, suggesting that Dredd could function as an apical or initiator caspase for apoptosis in Drosophila. If signaling by Rpr, Grim and Hid engages Dredd at a direct level of activational processing, then these activators could propagate a feed-forward amplification loop of caspase activity. If such activity ultimately exceeds a threshold, the capacity of negative regulators, such as IAPs, may be overwhelmed and cell death may ensue (Chen, 1998b).

GENE STRUCTURE

Three alternate splice variants of Dredd have been identifed, two of which encode a relatively long prodomain and another that possibly utilizes an internal translation start truncating much of the prodomain. The Dredd-alpha and -gamma isoforms process the second intron at different splice donor sites and thus differ by six amino acids at the intron II position. However, this intron is retained in the beta variant and, as a result, the beta transcript includes a premature stop codon that separates the prodomain from the rest of the enzymatic open reading frame. In principle, the beta splice form could lead to an interrupted translation product producing only the prodomain; alternatively, translation starting at an internal AUC could produce a polypeptide including the large and small subunits but lacking much of the prodomain. Determinations of the translation initiation site suggests uniform utilization of a single 5' start, suggesting that the Dredd-alpha isoform is not formed. Uniformity of 3' ends of several alpha, beta and gamma cDNA clones suggests that most if not all the diversity associated with DREDD mRNAs arises from alternate processing of the second intron. PCR assays do not detect the gamma isoform, suggesting that this variant might be expressed at developmental stages that were not analyzed or that this isoform is expressed at levels below the limits of detection (Chen, 1998b).

PROTEIN STRUCTURE

Amino Acids - 488

Structural Domains

Drosophila Dredd shares extensive homology with all members of the caspase gene family. Dredd includes essential residues required for catalysis and stabilization of the P1 Asp found to be absolutely conserved among all caspases thus far identified. In addition, the catalytic site for this enzyme (QACQE) is unique among the caspases, bearing a glutamic acid in a position typically occupied by a glycine. Gapped Blast analysis identifies marked similarity to caspase-8 (Mch5/FLICE/MACH) and caspase 10 (Mch4) throughout the entire protein, including significant sequence similarities within the prodomain. These similarities span regions of the prodomain in caspase-8 and-10 referred to as death effector domains, which are believed to mediate critical protein interactions required for activation of some "initiator" caspases (Chen, 1998b).

date revised: 20 November 98

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