No abstract

The Ets family of transcription factors, of which there are now about 35 members regulate gene expression during growth and development. They share a conserved domain of around 85 amino acids which binds as a monomer to the DNA sequence 5'-C/AGGAA/T-3'. We have determined the crystal structure of an ETS domain complexed with DNA, at 2.3-A resolution. The domain is similar to alpha + beta (winged) 'helix-turn-helix' proteins and interacts with a ten-base-pair region of duplex DNA which takes up a uniform curve of 8 degrees. The domain contacts the DNA by a novel loop-helix-loop architecture. Four of amino acids that directly interact with the DNA are highly conserved: two arginines from the recognition helix lying in the major groove, one lysine from the 'wing' that binds upstream of the core GGAA sequence, and another lysine, from the 'turn' of the 'helix-turn-helix' motif, which binds downstream and on the opposite strand.

Diverse guanine-rich RNAs and DNAs that fold to form guanine quadruplexes are known to form tight complexes with Fe(III) heme. We show here that a wide variety of such complexes robustly catalyze two-electron oxidations, transferring oxygen from hydrogen peroxide to thioanisole, indole, and styrene substrates. Use of (18)O-labeled hydrogen peroxide reveals the source of the oxygen transferred to form thioanisole sulfoxide and styrene oxide to be the activated ferryl moiety within these systems. Hammett analysis of the kinetics of thioanisole sulfoxide formation is unable to distinguish between a one-step, direct oxygen transfer and a two-step, oxygen rebound mechanism for this catalysis. Oxygen transfer to indole produces a range of products, including indigo and related dyes. Docking of heme onto a high-resolution structure of the G-quadruplex fold of Bcl-2 promoter DNA, which both binds heme and transfers oxygen, suggests a relatively open active site for this class of ribozymes and deoxyribozymes. That heme-dependent catalysis of oxygen transfer is a property of many RNAs and DNAs has ramifications for primordial evolution, enzyme design, cellular oxidative disease, and anticancer therapeutics.

Apaf1/CED4 family members play central roles in apoptosis regulation as activators of caspase family cell death proteases. These proteins contain a nucleotidebinding (NB) self-oligomerization domain and a caspase recruitment domain (CARD). A novel human protein was identified, NAC, that contains an NB domain and CARD. The CARD of NAC interacts selectively with the CARD domain of Apaf1, a caspase-activating protein that couples mitochondria-released cytochrome c (cyt-c) to activation of cytosolic caspases. Cyt-c-mediated activation of caspases in cytosolic extracts and in cells is enhanced by overexpressing NAC and inhibited by reducing NAC using antisense/DNAzymes. Furthermore, association of NAC with Apaf1 is cyt c-inducible, resulting in a mega-complex (>1 MDa) containing both NAC and Apaf1 and correlating with enhanced recruitment and proteolytic processing of pro-caspase-9. NAC also collaborates with Apaf1 in inducing caspase activation and apoptosis in intact cells, whereas fragments of NAC representing only the CARD or NB domain suppress Apaf1-dependent apoptosis induction. NAC expression in vivo is associated with terminal differentiation of short lived cells in epithelia and some other tissues. The ability of NAC to enhance Apaf1-apoptosome function reveals a novel paradigm for apoptosis regulation.CED4 family proteins constitute a unique family of caspaseactivating molecules. The founding member of this family, CED4, was discovered in the nematode Caenorhabditis elegans in screens for genes that are essential for developmental programmed cell death (1). CED4 contains an N-terminal CARD 1 followed by an NB domain, the later containing classical Walker A and B box motifs recognized as important in binding nucleotide triphosphates. CED4 functions as an activator of the caspase, CED3, in vitro and in vivo (2, 3). The NB domain of CED4 oligomerizes in an ATP-dependent manner (4, 5), whereas the CARD binds a complementary N-terminal CARD found in the zymogen proform of CED3 (6). Protease activation is thought to result from the induced proximity of CED3 zymogens bound to oligomerized CED4, where the weak intrinsic protease activity of the proenzymes is sufficient for trans-proteolysis of closely juxtaposed pro-caspases (4, 7). Proteolytic cleavage of pro-CED3 then produces the large and small subunits of the heterotetrameric, autonomously active enzyme.The closest homologue of CED4 identified in humans and other mammals thus far is Apaf1 (apoptosis protease-activating factor-1) (8). Similar to CED4, the Apaf1 protein contains a CARD, followed by an NB domain that shares significant amino acid sequence identity with the NB domains of CED4 and a family of ATPases associated with pathogen resistance (R genes) in plants (3, 5, 9), thus constituting the NB-ARC (Apaf-1/R gene/CED4) domain family (also known as NACHT domain). Unlike CED4, however, the NB-ARC domain of Apaf1 is followed by multiple WD repeats. These WD domains participate in auto-repression of Apaf1, locking it into an inactive, unoligomerized state ...