The structure of FADD has been solved in solution, revealing that the death effector domain (DED) and death domain (DD) are aligned with one another in an orthogonal, tail-to-tail fashion. Mutagenesis of FADD and functional reconstitution with its binding partners define the interaction with the intracellular domain of CD95 and the prodomain of procaspase-8 and reveal a self-association surface necessary to form a productive complex with an activated "death receptor." The identification of a procaspase-specific binding surface on the FADD DED suggests a preferential interaction with one, but not both, of the DEDs of procaspase-8 in a perpendicular arrangement. FADD self-association is mediated by a "hydrophobic patch" in the vicinity of F25 in the DED. The structure of FADD and its functional characterization, therefore, illustrate the architecture of key components in the death-inducing signaling complex.

Receptor-mediated programmed cell death proceeds through an activated receptor to which the death adaptor FADD and the initiator procaspases 8 and/or 10 are recruited following receptor stimulation. The adaptor FADD is responsible for both receptor binding and recruitment of the procaspases into the death-inducing signaling complex. Biochemical dissection of the FADD death effector domain and functional replacement with a coiled-coil motif demonstrates that there is an obligatory FADD self-association via the DED during assembly of the death-inducing signaling complex. Using engineered oligomerization motifs with defined stoichiometries, the requirement for FADD self-association through the DED can be separated from the caspase-recruitment function of the domain. Disruption of FADD self-association precludes formation of a competent signaling complex. On this basis, we propose an alternative architecture for the FADD signaling complex in which FADD acts as a molecular bridge to stitch together an array of activated death receptors.

The initiation of programmed cell death at CD95 (Fas, Apo-1) is achieved by forming a death-inducing signaling complex (DISC) at the cytoplasmic membrane surface. Assembly of the DISC has been proposed to occur via homotypic interactions between the death domain (DD) of FADD and the cytoplasmic domain of CD95. Previous analysis of the FADD/CD95 interaction led to the identification of a putative CD95 binding surface within FADD DD formed by ␣ helices 2 and 3. More detailed analysis of the CD95/FADD DD interaction now demonstrates that a bimodal surface exists in the FADD DD for interaction with CD95. An expansive surface on one side of the domain is composed of elements in ␣ helices 1, 2, 3, 5, and 6. This major surface is common to many proteins harboring this motif, whether or not they are associated with programmed cell death. A secondary surface resides on the opposite face of the domain and involves residues in helices 3 and 4. The major surface is topologically similar to the protein interaction surface identified in Drosophila Tube DD and the death effector domain of hamster PEA-15, two physiologically unrelated proteins which interact with structurally unrelated binding partners. These results demonstrate the presence of a structurally conserved surface within the DD which can mediate protein recognition with homoand heterotypic binding partners, whereas a second surface may be responsible for stabilizing the higher order complex in the DISC. The subgroup of tumor necrosis factor receptor (TNFR)1 superfamily members that contain a death domain (DD) within their cytoplasmic region have been termed "death receptors" (1). The receptor DD is the nucleus of an intracellular deathinducing cellular signaling complex (DISC), which forms in response to ligand binding by the receptor. The DISC of CD95 (Fas, Apo-1), one of six TNFRs that initiate cell death, is composed of the activated receptor (2, 3), the adaptor protein FADD (4, 5), and the initiator caspases, caspase-8 (6 -8) and caspase-10 (9, 10). A key molecule in this assembly is FADD, which links the death receptor to the caspases, a function attributed to FADD at nearly all death receptors identified to date (1). FADD contains two ϳ90-amino acid protein interaction modules, an N-terminal death effector domain (DED), and a C-terminal DD, each of which adopts a structurally similar six ␣-helical bundle (11-13). It has been suggested that the two domains of FADD function independently of each other (4, 5), with the DD responsible for interaction with CD95 and the DED responsible for the subsequent recruitment of caspases into the DISC (11-13).Despite knowledge of the three-dimensional structures of the DD (12-17) and DED (11, 18) motifs from a variety of proteins, the mechanism of protein recognition by these structurally homologous motifs has not been clearly defined. The crystal structure of Drosophila Tube DD bound to the DD from the serine/threonine kinase Pelle demonstrated that the DD motif possessed two distinct and opposing protein interaction surfac...

Patient-derived tumor xenograft models represent a promising preclinical cancer model that better replicates disease, compared with traditional cell culture; however, their use is low-throughput and costly. To overcome this limitation, patient-derived tumor organoids (PDOs) were established from human lung, ovarian and uterine tumor tissues, among others, to accurately and efficiently recapitulate the tissue architecture and function. PDOs were able to be cultured for >6 months, and formed cell clusters with similar morphologies to their source tumors. Comparative histological and comprehensive gene expression analyses proved that the characteristics of PDOs were similar to those of their source tumors, even following long-term expansion in culture. At present, 53 PDOs have been established by the Fukushima Translational Research Project, and were designated as Fukushima PDOs (F-PDOs). In addition, the in vivo tumorigenesis of certain F-PDOs was confirmed using a xenograft model. The present study represents a detailed analysis of three F-PDOs (termed REME9, 11 and 16) established from endometrial cancer tissues. These were used for cell growth inhibition experiments using anticancer agents. A suitable high-throughput assay system, with 96- or 384-well plates, was designed for each F-PDO, and the efficacy of the anticancer agents was subsequently evaluated. REME9 and 11 exhibited distinct responses and increased resistance to the drugs, as compared with conventional cancer cell lines (AN3 CA and RL95-2). REME9 and 11, which were established from tumors that originated in patients who did not respond to paclitaxel and carboplatin (the standard chemotherapy for endometrial cancer), exhibited high resistance (half-maximal inhibitory concentration >10 µM) to the two agents. Therefore, assay systems using F-PDOs may be utilized to evaluate anticancer agents using conditions that better reflect clinical conditions, compared with conventional methods using cancer cell lines, and to discover markers that identify the pharmacological effects of anticancer agents.

Interactions between the nuclear matrix and special regions of chromosomal DNA called matrix attachment regions (MARs) have been implicated in various nuclear functions. We have identified a novel protein from wheat, AT hookcontaining MAR binding protein1 (AHM1), that binds preferentially to MARs. A multidomain protein, AHM1 has the special combination of a J domain-homologous region and a Zn finger-like motif (a J-Z array) and an AT hook. For MAR binding, the AT hook at the C terminus was essential, and an internal portion containing the Zn finger-like motif was additionally required in vivo. AHM1 was found in the nuclear matrix fraction and was localized in the nucleoplasm. AHM1 fused to green fluorescent protein had a speckled distribution pattern inside the nucleus. AHM1 is most likely a nuclear matrix component that functions between intranuclear framework and MARs. J-Z arrays can be found in a group of (hypothetical) proteins in plants, which may share some functions, presumably to recruit specific Hsp70 partners as co-chaperones. INTRODUCTIONThe nuclear matrix, operationally defined, is the dynamic fibrogranular structure forming the skeletal framework that surrounds and penetrates the interphase nucleus; it has been implicated in most nuclear functions, including replication, repair, transcription, RNA processing, and RNA transport (Berezney and Jeon, 1995). The chromosomal DNAs are known to be associated with the nuclear matrix at specific regions called matrix attachment regions (MARs) and are thereby thought to be organized into topologically constrained loops, each of which represents a sort of functional or structural domain (or both) (Laemmli et al., 1992;Bode et al., 1996). In animals, interactions between the nuclear matrix and MARs have also been shown to be involved in DNA replication and repair and in various aspects of gene regulation, thus playing a key role in the essential functions of the nucleus (Boulikas, 1995;Bode et al., 1996).MARs consist of AT-rich sequences extending over at least a few hundred base pairs and containing various ATrich motifs as well as structural motifs such as base-unpairing regions and intrinsically curved portions (Boulikas, 1995). In animals, MAR binding activity has been found in a wide range of structurally and functionally diverse proteins (listed in Boulikas, 1995). These include topoisomerase II (Adachi et al., 1989); filament proteins such as lamins and NuMA (Ludérus et al., 1992(Ludérus et al., , 1994; ARBP, identical to a methylated CpG binding protein (von Kries et al., 1991;Weitzel et al., 1997); hnRNP-U/SAF1, an RNA binding protein involved in RNA processing (Fackelmayer et al., 1994;von Kries et al., 1994); SATB1, a tissue-specific transcription factor with high affinity for base-unpairing regions (Dickinson et al., 1992; de Belle et al., 1998); and architectural chromatin proteins such as histone H1 and HMG-I/Y (Zhao et al., 1993).In plants, MARs have been found in intergenic regions, often in or close to the regulatory regions of genes, and clo...