Molecular cloning and characterization of DEFCAP-L and -S, two isoforms of a novel member of the mammalian Ced-4 family of apoptosis proteins.

We report the deduced amino acid sequences of two alternately spliced isoforms, designated DEFCAP-L and -S, that differ in 44 amino acids and encode a novel member of the mammalian Ced-4 family of apoptosis proteins. Similar to the other mammalian Ced-4 proteins (Apaf-1 and Nod1), DEFCAP contains a caspase recruitment domain (CARD) and a putative nucleotide binding domain, signified by a consensus Walker's A box (P-loop) and B box (Mg(2+)-binding site). Like Nod1, but different from Apaf-1, DEFCAP contains a putative regulatory domain containing multiple leucine-rich repeats (LRR). However, a distinguishing feature of the primary sequence of DEFCAP is that DEFCAP contains at its NH(2) terminus a pyrin-like motif and a proline-rich sequence, possibly involved in protein-protein interactions with Src homology domain 3-containing proteins. By using in vitro coimmunoprecipitation experiments, both long and short isoforms were capable of strongly interacting with caspase-2 and exhibited a weaker interaction with caspase-9. Transient overexpression of full-length DEFCAP-L, but not DEFCAP-S, in breast adenocarcinoma cells MCF7 resulted in significant levels of apoptosis. In vitro death assays with transient overexpression of deletion constructs of both isoforms using beta-galactosidase as a reporter gene in MCF7 cells suggest the following: 1) the nucleotide binding domain may act as a negative regulator of the killing activity of DEFCAP; 2) the LRR/CARD represents a putative constitutively active inducer of apoptosis; 3) the killing activity of LRR/CARD is inhibitable by benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethyl ketone and to a lesser extent by Asp-Glu-Val-Asp (OMe)-fluoromethyl ketone; and 4) the CARD is critical for killing activity of DEFCAP. These results suggest that DEFCAP is a novel member of the mammalian Ced-4 family of proteins capable of inducing apoptosis, and understanding its regulation may elucidate the complex nature of the mammalian apoptosis-promoting machinery.

Negative regulation of selected bHLH proteins by eHAND.

The bHLH protein eHAND plays an important role in the development of extraembryonic, mesodermal, and cardiac cell lineages, presumably through heterodimerization with other HLH proteins and DNA binding. In this study, we have identified a novel transcriptional activity of eHAND. In transient transfection assays, eHAND is a potent inhibitor of activation by some but not all bHLH proteins. eHAND can prevent E-box DNA binding by these bHLH proteins. Interestingly, eHAND can also strongly inhibit transactivation activity by a MyoD approximately E47 tethered dimer, which suggests a distinct mechanism of action. eHAND also inhibits MyoD-dependent skeletal muscle cell differentiation and expression of the muscle-specific myosin heavy chain protein. In addition, we show that eHAND can repress activity of the natural p75LNGFR promoter, whose expression overlaps that of eHAND and dHAND. The inhibitory activity of eHAND may be attributed to multiple mechanisms, such as the ability to act as a corepressor, the presence of a repression domain, and its ability to sequester E proteins in an inactive complex. Based upon its inhibitory effect on bHLH proteins and cellular differentiation, we propose that eHAND may function by several mechanisms to promote placental giant cell proliferation by negatively regulating the activities of the bHLH protein MASH-2.