Using cDNA microarrays, we identified StarD4 as a gene whose expression decreased more than 2-fold in the livers of mice fed a high-cholesterol diet. StarD4 expression in cultured 3T3 cells was also sterol-regulated, and known sterol regulatory element binding protein (SREBP)-target genes showed coordinate regulation. The closest homologues to StarD4 were two other StAR-related lipid transfer (START) proteins named StarD5 and StarD6. StarD4, StarD5, and StarD6 are 205-to 233-aa proteins consisting almost entirely of START domains. These three constitute a subfamily among START proteins, sharing Ϸ30% amino acid identity with one another, Ϸ20% identity with the cholesterol-binding START domains of StAR and MLN64, and less than 15% identity with phosphatidylcholine transfer protein (PCTP) and other START domains. StarD4 and StarD5 were expressed in most tissues, with highest levels in liver and kidney, whereas StarD6 was expressed exclusively in the testis. In contrast to StarD4, expression of StarD5 and MLN64 was not sterol-regulated. StarD4, StarD5, and StarD6 may be involved in the intracellular transport of sterols or other lipids.

We present a common allosteric mechanism for control of inflammatory and apoptotic caspases. Highly specific thiol-containing inhibitors of the human inflammatory caspase-1 were identified by using disulfide trapping, a method for site-directed small-molecule discovery. These compounds became trapped by forming a disulfide bond with a cysteine residue in the cavity at the dimer interface Ϸ15 Å away from the active site. Mutational and structural analysis uncovered a linear circuit of functional residues that runs from one active site through the allosteric cavity and into the second active site. Kinetic analysis revealed robust positive cooperativity not seen in other endopeptidases. Recently, disulfide trapping identified a similar small-molecule site and allosteric transition in the apoptotic caspase-7 that shares only a 23% sequence identity with caspase-1. Together, these studies show a general small-molecule-binding site for functionally reversing the zymogen activation of caspases and suggest a common regulatory site for the allosteric control of inflammation and apoptosis.procaspase ͉ allosteric regulation ͉ apoptosis ͉ fragment-based discovery ͉ inflammation C aspases are dimeric thiol endopeptidases that cleave specific proteins after aspartic acid residues and drive apoptosis or inflammation (for review, see refs. 1 and 2). Although of considerable medical interest, the active sites of caspases have been very difficult to target with drug-like compounds owing to the enzyme's preference for negatively charged chemotypes with electrophilic warheads that engage the catalytic cysteine (3).In past work to develop alternative small-molecule inhibitors to the active site of the apoptotic caspase-7, we used a fragmentbased discovery approach called disulfide trapping. This is a site-directed small-molecule-capture approach in which natural or engineered cysteines on the surface of the protein are screened with a library of disulfide-containing small molecules in a redox buffer (4). This approach has been broadly applied in fragment-based drug discovery for both enzymes and challenging protein-protein interface targets (for review see ref. 5). Although the active-site cysteine of caspase-7 failed to yield strong hits from a library of Ϸ10,000 disulfide-containing compounds, several strong inhibitors were found for another cysteine in a cavity at the dimer interface.Procaspase-7, the inactive precursor or zymogen, is known to undergo a large structural transition after auto-or transproteolysis. This transition results in cleavage of the N-terminal 23 residues and an internal cut that yields two large and two small subunits of mature caspase-7. Structural studies of caspase-7 containing the disulfide-trapped inhibitors at the dimerinterface cavity showed that the compounds reversed this structural transition and induced a conformation virtually identical to the zymogen form (6).We wondered whether other caspases could be inhibited by a similar mechanism. The class of human inf lammator y caspases-1, -4, and -5 is...

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Bruton's tyrosine kinase (BTK), a member of the TEC family of kinases, plays a crucial role in B-cell maturation and mast cell activation. Although the structures of the unphosphorylated mouse BTK kinase domain and the unphosphorylated and phosphorylated kinase domains of human ITK are known, understanding the kinase selectivity profiles of BTK inhibitors has been hampered by the lack of availability of a high resolution, ligand-bound BTK structure. Here, we report the crystal structures of the human BTK kinase domain bound to either Dasatinib (BMS-354825) at 1.9 Å resolution or to 4-amino-5-(4-phenoxyphenyl)-7H-pyrrolospyrimidin-7-yl-cyclopentane at 1.6 Å resolution. This data provides information relevant to the development of small molecule inhibitors targeting BTK and the TEC family of nonreceptor tyrosine kinases. Analysis of the structural differences between the TEC and Src families of kinases near the Trp-Glu-Ile motif in the N-terminal region of the kinase domain suggests a mechanism of regulation of the TEC family members.