PROJECT ADMINISTRATION AGREEMENT KN2122/EN

Agreement to invite PJA

Role of Smac in cephalostatin-induced cell death.

Cephalostatin 1 is a natural compound isolated from a marine worm that induces apoptosis in tumor cells via an apoptosome-independent but caspase-9-dependent pathway and through an endoplasmic reticulum stress response that is accompanied by caspase-4 activation. Here, we show that cephalostatin evokes mitochondrial Smac (second mitochondria-derived activator of caspases) but not cytochrome c release in various carcinoma cell lines. We also show that Smac is critically involved in caspase-9 activation as evidenced by gene silencing experiments. Remarkably, caspase-2 appears to be a major target for cephalostatin-induced cytosolic Smac. Using biochemical and genetic inhibition experiments, we demonstrate that caspase-2 participates in the apoptotic machinery induced by cephalostatin. Cephalostatin-activated caspase-2 appears to act as initiator caspase and is not involved in the activation of caspase-9. Importantly, experiments immunoprecipitating PIDD (p53-induced protein with a DD), RAIDD (RIP-associated ICH-1/CED-3-homologous protein with DD) and caspase-2 identify cephalostatin as an experimental drug that induces the formation of the PIDDosome. The bis-steroid cephalostatin proves to be both a helpful tool to investigate apoptotic signaling and a promising chemotherapeutic agent.Cell Death and Differentiation advance online publication, 19 September 2008; doi:10.1038/cdd.2008.125

The marine product cephalostatin 1 activates an endoplasmic reticulum stress-specific and apoptosome-independent apoptotic signaling pathway

Cephalostatin 1, a bis-steroidal marine natural product, has been reported to induce apoptosis without the requirement of an active caspase-8 or mitochondrial cytochrome c release and apoptosome formation. Here we show that despite the absence of these events, caspase-9 activation is essential for cephalostatin 1-induced apoptosis. Cephalostatin 1 initiates a rapid endoplasmic reticulum stress response characterized by phosphorylation of eukaryotic initiation factor-2 alpha-subunit and increased expression of the chaperone immunoglobulin heavy chain-binding protein GRP78 as well as the transcription factor C/EBP homologous protein (CHOP)/GADD153. Cephalostatin 1 activates apoptosis signal-regulating kinase 1 and c-Jun N-terminal kinase (JNK). However, this pathway does not play a major role in cephalostatin 1-induced apoptosis, as assessed by stable expression of a dominant negative apoptosis signal-regulating kinase 1. Importantly, the endoplasmic reticulum-associated caspase-4 is required and as shown by biochemical and genetic inhibition experiments, acts upstream of caspase-9 in cephalostatin-induced apoptosis

5' Trans-Splicing Repair of the PLEC1 Gene

The efficient treatment of hereditary disorders, especially of those caused by dominant-negative mutations still remains an obstacle to be overcome. Allele specificity is a critical aspect that must be addressed by silencing therapies such as small interfering RNA, which has the potential risk of also reducing expression of the normal allele. To overcome this hurdle, we used spliceosome-mediated RNA trans-splicing (SMaRT) to replace mRNA exon segments in an in vitro disease model. In this model, a heterozygous insertion of a leucine codon into exon 9 of the plectin gene (PLEC1) leads to aggregation of plectin peptide chains and subsequent protein degradation recapitulating, together with a nonsense mutation on the other allele, the blistering skin disease epidermolysis bullosa simplex with muscular dystrophy (EBS-MD). Transient transfection of EBS-MD fibroblasts with a 5' pre-trans-splicing molecule encoding wild-type exons 2–9 led to specific replacement of the mutated 5' portion of the endogenous PLEC1 transcript through trans-splicing. This treatment reduced the levels of mutant mRNA and restored a wild-type pattern of plectin expression as revealed by immunofluorescence microscopy. When EBS-MD fibroblasts were transfected with retroviral constructs, the level of full-length plectin protein in the corrected fibroblasts increased by 58.7%. Thus, SMaRT may be a promising new tool for treatment of autosomal-dominant genetic diseases

Divergent functions of two ancient Hydra Brachyury paralogues suggest specific roles for their C-terminal domains in tissue fate induction

Homologues of the T-box gene Brachyury play important roles in mesoderm differentiation and other aspects of early development in all bilaterians. In the cliploblast Hydra, the Brachyury homologue HyBra1 acts early in the formation of the hypostome, the location of the organiser in adult Hydra. We now report the isolation and characterisation of a second Brachyury gene, HyBra2. Sequence analysis suggests that HyBra1 and HyBra2 are paralogues, resulting from an ancient lineage-specific gene duplication. We show that both paralogues acquired novel functions, both at the level of their cis-regulation as well as through significant divergence of the coding sequence. Both genes are expressed in the hypostome, but HyBra1 is predominantly endodermal, whereas HyBra2 transcripts are found primarily in the ectoderm. During bud formation, both genes are activated before any sign of evagination, suggesting an early role in head formation. During regeneration, HyBra1 is an immediate-early response gene and is insensitive to protein synthesis inhibition, whereas the onset of expression of HyBra2 is delayed and requires protein synthesis. The functional consequence of HyBra1/2 protein divergence on cell fate decisions was tested in Xenopus. HyBra1 induces mesoderm, like vertebrate Brachyury proteins. By contrast, HyBra2 shows a strong cement-gland and neural-inducing activity. Domain-swapping experiments show that the C-terminal domain of HyBra2 is responsible for this specific phenotype. Our data support the concept of sub- and neofunctionalisation upon gene duplication and show that divergence of cis-regulation and coding sequence in paralogues can lead to dramatic changes in structure and function