Diabetes reversal by inhibition of the low-molecular-weight tyrosine phosphatase.

Obesity-associated insulin resistance plays a central role in type 2 diabetes. As such, tyrosine phosphatases that dephosphorylate the insulin receptor (IR) are potential therapeutic targets. The low-molecular-weight protein tyrosine phosphatase (LMPTP) is a proposed IR phosphatase, yet its role in insulin signaling in vivo has not been defined. Here we show that global and liver-specific LMPTP deletion protects mice from high-fat diet-induced diabetes without affecting body weight. To examine the role of the catalytic activity of LMPTP, we developed a small-molecule inhibitor with a novel uncompetitive mechanism, a unique binding site at the opening of the catalytic pocket, and an exquisite selectivity over other phosphatases. This inhibitor is orally bioavailable, and it increases liver IR phosphorylation in vivo and reverses high-fat diet-induced diabetes. Our findings suggest that LMPTP is a key promoter of insulin resistance and that LMPTP inhibitors would be beneficial for treating type 2 diabetes.

αE-catenin is an autoinhibited molecule that coactivates vinculin.

αE-catenin, an essential component of the adherens junction, interacts with the classical cadherin-ß-catenin complex and with F-actin, but its precise role is unknown. αE-catenin also binds to the F-actin-binding protein vinculin, which also appears to be important in junction assembly. Vinculin and αE-catenin are homologs that contain a series of helical bundle domains, D1-D5. We mapped the vinculin-binding site to a sequence in D3a comprising the central two helices of a four-helix bundle. The crystal structure of this peptide motif bound to vinculin D1 shows that the two helices adopt a parallel, colinear arrangement suggesting that the αE-catenin D3a bundle must unfold in order to bind vinculin. We show that αE-catenin D3 binds strongly to vinculin, whereas larger fragments and full-length αE-catenin bind approximately 1,000-fold more weakly. Thus, intramolecular interactions within αE-catenin inhibit binding to vinculin. The actin-binding activity of vinculin is inhibited by an intramolecular interaction between the head (D1-D4) and the actin-binding D5 tail. In the absence of F-actin, there is no detectable binding of αE-catenin D3 to full-length vinculin; however, αE-catenin D3 promotes binding of vinculin to F-actin whereas full-length αE-catenin does not. These findings support the combinatorial or "coincidence" model of activation in which binding of high-affinity proteins to the vinculin head and tail is required to shift the conformational equilibrium of vinculin from a closed, autoinhibited state to an open, stable F-actin-binding state. The data also imply that αE-catenin must be activated in order to bind to vinculin.

Structural basis for the association of PLEKHA7 with membrane-embedded phosphatidylinositol lipids.

PLEKHA7 (pleckstrin homology domain containing family A member 7) plays key roles in intracellular signaling, cytoskeletal organization, and cell adhesion, and is associated with multiple human cancers. The interactions of its pleckstrin homology (PH) domain with membrane phosphatidyl-inositol-phosphate (PIP) lipids are critical for proper cellular localization and function, but little is known about how PLEKHA7 and other PH domains interact with membrane-embedded PIPs. Here we describe the structural basis for recognition of membrane-bound PIPs by PLEHA7. Using X-ray crystallography, nuclear magnetic resonance, molecular dynamics simulations, and isothermal titration calorimetry, we show that the interaction of PLEKHA7 with PIPs is multivalent, distinct from a discrete one-to-one interaction, and induces PIP clustering. Our findings reveal a central role of the membrane assembly in mediating protein-PIP association and provide a roadmap for understanding how the PH domain contributes to the signaling, adhesion, and nanoclustering functions of PLEKHA7.

Structural basis for vinculin activation at sites of cell adhesion.

Vinculin is a highly conserved intracellular protein with a crucial role in the maintenance and regulation of cell adhesion and migration. In the cytosol, vinculin adopts a default autoinhibited conformation. On recruitment to cell-cell and cell-matrix adherens-type junctions, vinculin becomes activated and mediates various protein-protein interactions that regulate the links between F-actin and the cadherin and integrin families of cell-adhesion molecules. Here we describe the crystal structure of the full-length vinculin molecule (1,066 amino acids), which shows a five-domain autoinhibited conformation in which the carboxy-terminal tail domain is held pincer-like by the vinculin head, and ligand binding is regulated both sterically and allosterically. We show that conformational changes in the head, tail and proline-rich domains are linked structurally and thermodynamically, and propose a combinatorial pathway to activation that ensures that vinculin is activated only at sites of cell adhesion when two or more of its binding partners are brought into apposition.

Identification and characterization of small molecule inhibitors of the ubiquitin ligases Siah1/2 in melanoma and prostate cancer cells.

Inhibition of ubiquitin ligases with small molecule remains a very challenging task, given the lack of catalytic activity of the target and the requirement of disruption of its interactions with other proteins. Siah1/2, which are E3 ubiquitin ligases, are implicated in melanoma and prostate cancer and represent high-value drug targets. We utilized three independent screening approaches in our efforts to identify small-molecule Siah1/2 inhibitors: Affinity Selection-Mass Spectrometry, a protein thermal shift-based assay and an in silico based screen. Inhibitors were assessed for their effect on viability of melanoma and prostate cancer cultures, colony formation, prolyl-hydroxylase-HIF1α signaling, expression of selected Siah2-related transcripts, and Siah2 ubiquitin ligase activity. Several analogs were further characterized, demonstrating improved efficacy. Combination of the top hits identified in the different assays demonstrated an additive effect, pointing to complementing mechanisms that underlie each of these Siah1/2 inhibitors.

Modulation of nongenomic activation of PI3K signalling by tetramerization of N-terminally-cleaved RXRα.

Retinoid X receptor-alpha (RXRα) binds to DNA either as homodimers or heterodimers, but it also forms homotetramers whose function is poorly defined. We previously discovered that an N-terminally-cleaved form of RXRα (tRXRα), produced in tumour cells, activates phosphoinositide 3-kinase (PI3K) signalling by binding to the p85α subunit of PI3K and that K-80003, an anti-cancer agent, inhibits this process. Here, we report through crystallographic and biochemical studies that K-80003 binds to and stabilizes tRXRα tetramers via a 'three-pronged' combination of canonical and non-canonical mechanisms. K-80003 binding has no effect on tetramerization of RXRα, owing to the head-tail interaction that is absent in tRXRα. We also identify an LxxLL motif in p85α, which binds to the coactivator-binding groove on tRXRα and dissociates from tRXRα upon tRXRα tetramerization. These results identify conformational selection as the mechanism for inhibiting the nongenomic action of tRXRα and provide molecular insights into the development of RXRα cancer therapeutics.

Sulindac-derived RXRα modulators inhibit cancer cell growth by binding to a novel site.

Retinoid X receptor-alpha (RXRα), an intriguing and unique drug target, can serve as an intracellular target mediating the anticancer effects of certain nonsteroidal anti-inflammatory drugs (NSAIDs), including sulindac. We report the synthesis and characterization of two sulindac analogs, K-8008 and K-8012, which exert improved anticancer activities over sulindac in a RXRα-dependent manner. The analogs inhibit the interaction of the N-terminally truncated RXRα (tRXRα) with the p85α subunit of PI3K, leading to suppression of AKT activation and induction of apoptosis. Crystal structures of the RXRα ligand-binding domain (LBD) with K-8008 or K-8012 reveal that both compounds bind to tetrameric RXRα LBD at a site different from the classical ligand-binding pocket. Thus, these results identify K-8008 and K-8012 as tRXRα modulators and define a binding mechanism for regulating the nongenomic action of tRXRα.

Selective and potent furin inhibitors protect cells from anthrax without significant toxicity.

Furin and related proprotein convertases cleave the multibasic motifs R-X-R/K/X-R in the precursor proteins and, as a result, transform the latent proproteins into biologically active proteins and peptides. Furin is present both in the intracellular secretory pathway and at the cell surface. Intracellular furin processes its multiple normal cellular targets in the Golgi and secretory vesicle compartments while cell-surface furin appears to be essential only for the processing of certain pathogenic proteins and, importantly, anthrax. To design potent, safe and selective inhibitors of furin, we evaluated the potency and selectivity of the derivatized peptidic inhibitors modeled from the extended furin cleavage sequence of avian influenza A H5N1. We determined that the N- and C-terminal modifications of the original RARRRKKRT inhibitory scaffold produced selective and potent, nanomolar range, inhibitors of furin. These inhibitors did not interfere with the normal cellular function of furin because of the likely functional redundancy existing between furin and other proprotein convertases. These furin inhibitors, however, were highly potent in blocking the furin-dependent cell-surface processing of anthrax protective antigen-83 both in vitro and cell-based assays and in vivo. We conclude that the inhibitors we have designed have a promising potential as selective anthrax inhibitors, without affecting major cell functions.