The orphan nuclear receptor steroidogenic factor 1 (SF-1) regulates the differentiation and function of endocrine glands. Although SF-1 is constitutively active in cell-based assays, it is not known whether this transcriptional activity is modulated by ligands. Here, we describe the 1.5 angstroms crystal structure of the SF-1 ligand binding domain in complex with an LXXLL motif from a coregulator protein. The structure reveals the presence of a phospholipid ligand in a surprisingly large pocket (approximately 1600 angstroms3), with the receptor adopting the canonical active conformation. The bound phospholipid is readily exchanged and modulates SF-1 interactions with coactivators. Mutations designed to reduce the size of the SF-1 pocket or to disrupt hydrogen bonds with the phospholipid abolish SF-1/coactivator interactions and significantly reduce SF-1 transcriptional activity. These findings provide evidence that SF-1 is regulated by endogenous ligands and suggest an unexpected relationship between phospholipids and endocrine development and function.

Matrix metalloproteinases (MMP)-13 activity is necessary for normal skeletal development and plays a central role in cartilage degeneration associated with osteoarthritis (OA). The studies we described here examine the interactions of the hemopexin domain of MMP-13 with proteins secreted by human chondrocytes in culture. The hemopexin domain of the MMPs and many other proteins in which this structure is found mediates protein function by forming the primary site of interaction with other proteins. We have modified a tandem affinity expression tag (hTAP) to enable efficient expression of the tagged bait protein. In this case the MMP-13 C-terminal domain (CTD) comprises hinge and hemopexin domain, and we immobilized the fusion construct on a column of agarose bound immunoglobin G. The MMP-13 CTD affinity column so generated enabled the efficient and gentle isolation of interacting proteins from the culture medium of human articular chondrocytes. TIMP1 and alpha2-macroglobulin previously shown to interact with MMP-13 as well as several proteins, fibronectin, type VI collagen and xylosyltransferase 1 and several proteoglycans, decorin, syndecan 4 and serglycin not previously recognized as interacting with MMP-13 were identified by mass spectrometry. The interaction between isolated proteins and MMP-13 CTD was verified by yeast two hybrid analysis. We also demonstrated serglycin expression by chondrocytes for the first time and its co localization with MMP-13 in a cytoplasmic granular morphology. The consequence of these interactions remains to be demonstrated, however; binding to MMP-13 suggests a role in the regulation of cartilage degradation.

Emerging evidence indicates that Nanog is intimately involved in tumorigenesis in part through regulation of the cancer initiating cell population. However, the regulation and role of Nanog in tumorigenesis are still poorly understood. In this study, human Nanog was identified to be phosphorylated by human PKCε at multiple residues including T200 and T280. Our work indicated that phosphorylation at T200 and T280 modulates Nanog function through several regulatory mechanisms. Results with phosphorylation-insensitive and phosphorylation-mimetic mutant Nanog revealed that phosphorylation at T200 and T280 enhance Nanog protein stability. Moreover, phosphorylation-insensitive T200A and T280A mutant Nanog had a dominant-negative function to inhibit endogenous Nanog transcriptional activity. Inactivation of Nanog was due to impaired homodimerization, DNA binding, promoter occupancy, and p300, a transcriptional co-activator, recruitment resulting in a defect in target gene promoter activation. Ectopic expression of phosphorylation-insensitive T200A or T280A mutant Nanog reduced cell proliferation, colony formation, invasion, migration, and the cancer initiating cell population in head and neck squamous cell carcinoma (HNSCC) cells. The in vivo cancer initiating ability was severely compromised in HNSCC cells expressing phosphorylation-insensitive T200A or T280A mutant Nanog; 87.5% (14/16), 12.5% (1/8), and 0% (0/8) for control, T200A, and T280A, respectively. Nanog occupied the Bmi1 promoter to directly transactivate and regulate Bmi1. Genetic ablation and rescue experiments demonstrated that Bmi1 is a critical downstream signaling node for the pleiotropic, pro-oncogenic effects of Nanog. Taken together, our study revealed, for the first time, that post-translational phosphorylation of Nanog is essential to regulate Bmi1 and promote tumorigenesis.

Geldanamycin and its derivative 17AAG [17-(Allylamino)-17-demethoxygeldanamycin, telatinib] bind selectively to the Hsp90 chaperone protein and inhibit its function. We discovered that these drugs associate with mitochondria, specifically to the mitochondrial membrane voltage-dependent anion channel (VDAC) via a hydrophobic interaction that is independent of HSP90. In vitro, 17AAG functions as a Ca 2+ mitochondrial regulator similar to benzoquinone-ubiquinones like Ub0. All of these compounds increase intracellular Ca 2+ and diminish the plasma membrane cationic current, inhibiting urokinase activity and cell invasion. In contrast, the HSP90 inhibitor radicicol, lacking a bezoquinone moiety, has no measurable effect on cationic current and is less effective in influencing intercellular Ca 2+ concentration. We conclude that some of the effects of 17-AAG and other ansamycins are due to their effects on VDAC and that this may play a role in their clinical activity.