The 2.7 A X-ray crystal structure of the HNF4gamma ligand binding domain (LBD) revealed the presence of a fatty acid within the pocket, with the AF2 helix in a conformation characteristic of a transcriptionally active nuclear receptor. GC/MS and NMR analysis of chloroform/methanol extracts from purified HNF4alpha and HNF4gamma LBDs identified mixtures of saturated and cis-monounsaturated C14-18 fatty acids. The purified HNF4 LBDs interacted with nuclear receptor coactivators, and both HNF4 subtypes show high constitutive activity in transient transfection assays, which was reduced by mutations designed to interfere with fatty acid binding. The endogenous fatty acids did not readily exchange with radiolabeled palmitic acid, and all attempts to displace them without denaturing the protein failed. Our results suggest that the HNF4s may be transcription factors that are constitutively bound to fatty acids.

The peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that regulate glucose and lipid homeostasis. The PPAR␥ subtype plays a central role in the regulation of adipogenesis and is the molecular target for the 2,4-thiazolidinedione class of antidiabetic drugs. Structural studies have revealed that agonist ligands activate the PPARs through direct interactions with the C-terminal region of the ligand-binding domain, which includes the activation function 2 helix. GW0072 was identified as a high-affinity PPAR␥ ligand that was a weak partial agonist of PPAR␥ transactivation. X-ray crystallography revealed that GW0072 occupied the ligand-binding pocket by using different epitopes than the known PPAR agonists and did not interact with the activation function 2 helix. In cell culture, GW0072 was a potent antagonist of adipocyte differentiation. These results establish an approach to the design of PPAR ligands with modified biological activities.The nuclear hormone receptors are ligand-activated transcription factors that regulate target genes essential for mammalian physiology and development (1). The peroxisome proliferatoractivated receptors (PPARs) are nuclear receptors activated by fatty acids and their eicosanoids metabolites, which regulate genes involved in the biosynthesis, storage, and metabolism of these ligands (2). The pharmacology of synthetic PPAR ligands demonstrated the role of these receptors in regulating glucose and lipid homeostasis and established their utility as molecular targets for the development of drugs for the treatment of diabetes and cardiovascular disease (3).Biochemical and structural studies with several nuclear receptors revealed that hormone binding induces allosteric changes in the conformation of the ligand-binding domain, which promote recruitment of transcriptional coactivator proteins such as steroid receptor coactivator 1 (SRC1) (4) and CREB binding protein (CBP) (5). We recently reported x-ray crystallographic analysis of the ternary complex of PPAR␥ with the 2,4-thiazolidinedione (TZD) rosiglitazone (Fig. 1A) and the coactivator SRC1 (6), as well as the complexes of PPAR␦ with either the fibrate GW2433 or the essential fatty acid eicosapentaenoic acid (7). Despite differences in their gross chemical structure, all of these small molecule PPAR agonists share a common binding mode, in which the acidic head groups form a network of hydrogen bonds with Y473, H449, and H323 within the ligand-binding pocket. These interactions stabilize a charge clamp (6) between the Cterminal activation function 2 (AF-2) helix and a conserved lysine residue on the surface of the receptor, through which coactivator proteins are recruited to the receptor.

Unlike mammalian and yeast cells, little is known about how plants regulate G 1 progression and entry into the S phase of the cell cycle. In mammalian cells, a key regulator of this process is the retinoblastoma tumor suppressor protein (RB). In contrast, G 1 control in Saccharomyces cerevisiae does not utilize an RB-like protein.We report here the cloning of cDNAs from two Zea mays genes, RRB1 and RRB2, that encode RB-related proteins. Further, RRB2 transcripts are alternatively spliced to yield two proteins with different C termini. At least one RRB gene is expressed in all the tissues examined, with the highest levels seen in the shoot apex. RRB1 is a 96-kDa nuclear protein that can physically interact with two mammalian DNA tumor virus oncoproteins, simian virus 40 large-T antigen and adenovirus E1A, and with a plant D-type cyclin. These associations are abolished by mutation of a conserved cysteine residue in RRB1 that is also essential for RB function. RRB1 binding potential is also sensitive to deletions in the conserved A and B domains, although differences exist in these effects compared to those of human RB. RRB1 can also bind to the AL1 protein from tomato golden mosaic virus (TGMV), a protein which is essential for TGMV DNA replication. These results suggest that G 1 regulation in plant cells is controlled by a mechanism which is much more similar to that found in mammalian cells than that in yeast.Progression through the G 1 phase of the eukaryotic cell cycle is tightly regulated, allowing cells to integrate internal and external cues before initiating DNA replication and committing to a round of cell division. This process is governed by both positive-and negative-acting regulatory factors. Although substantial progress has been made in understanding the mechanisms that govern these events in yeast and mammals (reviewed in reference 61), relatively little is known about G 1 regulation in plants. The existence of cyclin-dependent kinases (Cdks) and their associated cyclin subunits in plants (reviewed in reference 14) suggests that at least some of the basic mechanisms which regulate the cell cycle have been conserved throughout eukaryotic evolution. However, identification of additional regulatory components of the plant cell cycle is clearly essential for understanding plant growth and development.In the yeast Saccharomyces cerevisiae, progression through the G 1 phase is regulated by the Cdk Cdc28 (50), which in conjunction with G 1 cyclins activates the heterodimeric Swi4/ Swi6 transcription factor (40), resulting in the transcriptional activation of genes necessary for G 1 progression and S-phase entry (12, 61). In mammalian cells, G 1 progression also depends upon a Cdk-cyclin-activated transcriptional control pathway. However, a major regulatory protein in this pathway, the retinoblastoma protein (RB), has not been found in yeast. RB is the 110-kDa product of the retinoblastoma susceptibility tumor suppressor gene and plays key roles in regulating both cell cycle progression through the G 1 ph...

The tumor necrosis factor-␣-converting enzyme (TACE) is a membrane-anchored zinc metalloprotease involved in precursor tumor necrosis factor-␣ secretion. We designed a series of constructs containing full-length human TACE and several truncate forms for overexpression in insect cells. Here, we demonstrate that fulllength TACE is expressed in insect cells inefficiently: only minor amounts of this enzyme are converted from an inactive precursor to the mature, functional form. Removal of the cytoplasmic and transmembrane domains resulted in the efficient secretion of mature, active TACE. Further removal of the cysteine-rich domain located between the catalytic and transmembrane domains resulted in the secretion of mature catalytic domain in association with the precursor (pro) domain. This complex was inactive and function was only restored after dissociation of the complex by dilution or treatment with 4-aminophenylmercuric acetate. Therefore, the pro domain of TACE is an inhibitor of the catalytic domain, and the cysteine-rich domain appears to play a role in the release of the pro domain. Insect cells failed to secrete a deletion mutant encoding the catalytic domain but lacking the inhibitory pro domain. This truncate was inactive and extensively degraded intracellularly, suggesting that the pro domain is required for the secretion of functional TACE. TNF␣1 is a potent cytokine that is secreted by activated monocytes and macrophages in a tightly regulated manner (1). Upon release, TNF␣ mediates the recruitment and activation of inflammatory cells to injured or infected tissues (2). Elevated levels of circulating TNF␣ have been demonstrated in several acute and chronic pathological states, such as lipopolysaccharide-induced septic shock, arthritis, pleurisy, Crohn's disease, and inflammatory bowel disease (3). TNF␣ is synthesized as a pro, membrane-anchored form facing the lumenal/extracellular side of the secretory pathway. Our group and others have shown that proTNF␣ is released from cells after endoproteolytic cleavage at positions Ala 76 -Val 77 , mediated by a zinc metalloprotease sensitive to hydroxamic acid inhibitors (4 -6). Because neutralization of TNF␣ activity has been demonstrated in the clinic, this enzyme constitutes a potential target for drug discovery.The TNF␣-converting enzyme (TACE) was purified to homogeneity and cloned (7,8). Analysis of its amino acid sequence demonstrates a multidomain protein closely resembling members of the disintegrin family of metalloproteases, also commonly referred to as ADAMs or metalloprotease and disintegrin-containing proteins (9). Starting at the N terminus, TACE exhibits a classical signal peptide followed by a ϳ200-residue pro domain that includes a consensus cysteine switch motif (PKVCGY 186 ), which can act as an inhibitor by ligating the zinc ion in the catalytic site (10, 32). The catalytic domain starts downstream from a consensus furin cleavage site (RVKRR 215 ) and contains a canonical zinc binding site and a MYP loop involved in formation of the P1Ј p...