Cell-autonomous Hedgehog signaling controls Th17 polarization and pathogenicity.

Th17 cells are key drivers of autoimmune disease. However, the signaling pathways regulating Th17 polarization are poorly understood. Hedgehog signaling regulates cell fate decisions during embryogenesis and adult tissue patterning. Here we find that cell-autonomous Hedgehog signaling, independent of exogenous ligands, selectively drives the polarization of Th17 cells but not other T helper cell subsets. We show that endogenous Hedgehog ligand, Ihh, signals to activate both canonical and non-canonical Hedgehog pathways through Gli3 and AMPK. We demonstrate that Hedgehog pathway inhibition with either the clinically-approved small molecule inhibitor vismodegib or genetic ablation of Ihh in CD4+ T cells greatly diminishes disease severity in two mouse models of intestinal inflammation. We confirm that Hedgehog pathway expression is upregulated in tissue from human ulcerative colitis patients and correlates with Th17 marker expression. This work implicates Hedgehog signaling in Th17 polarization and intestinal immunopathology and indicates the potential therapeutic use of Hedgehog inhibitors in the treatment of inflammatory bowel disease.

Autoinhibition of Cnn binding to γ-TuRCs prevents ectopic microtubule nucleation and cell division defects.

γ-Tubulin ring complexes (γ-TuRCs) nucleate microtubules. They are recruited to centrosomes in dividing cells via binding to N-terminal CM1 domains within γ-TuRC-tethering proteins, including Drosophila Centrosomin (Cnn). Binding promotes microtubule nucleation and is restricted to centrosomes in dividing cells, but the mechanism regulating binding remains unknown. Here, we identify an extreme N-terminal CM1 autoinhibition (CAI) domain found specifically within the centrosomal isoform of Cnn (Cnn-C) that inhibits γ-TuRC binding. Robust binding occurs after removal of the CAI domain or with the addition of phosphomimetic mutations, suggesting that phosphorylation helps relieve inhibition. We show that regulation of Cnn binding to γ-TuRCs is isoform specific and that misregulation of binding can result in ectopic cytosolic microtubules and major defects during cell division. We also find that human CDK5RAP2 is autoinhibited from binding γ-TuRCs, suggesting conservation across species. Overall, our results shed light on how and why CM1 domain binding to γ-TuRCs is regulated.

Engineering mono- and multi-valent inhibitors on a modular scaffold.

Here we exploit the simple, ultra-stable, modular architecture of consensus-designed tetratricopeptide repeat proteins (CTPRs) to create a platform capable of displaying both single as well as multiple functions and with diverse programmable geometrical arrangements by grafting non-helical short linear binding motifs (SLiMs) onto the loops between adjacent repeats. As proof of concept, we built synthetic CTPRs to bind and inhibit the human tankyrase proteins (hTNKS), which play a key role in Wnt signaling and are upregulated in cancer. A series of mono-valent and multi-valent hTNKS binders was assembled. To fully exploit the modular scaffold and to further diversify the multi-valent geometry, we engineered the binding modules with two different formats, one monomeric and the other trimeric. We show that the designed proteins are stable, correctly folded and capable of binding to and inhibiting the cellular activity of hTNKS leading to downregulation of the Wnt pathway. Multivalency in both the CTPR protein arrays and the hTNKS target results in the formation of large macromolecular assemblies, which can be visualized both in vitro and in the cell. When delivered into the cell by nanoparticle encapsulation, the multivalent CTPR proteins displayed exceptional activity. They are able to inhibit Wnt signaling where small molecule inhibitors have failed to date. Our results point to the tremendous potential of the CTPR platform to exploit a range of SLiMs and assemble synthetic binding molecules with built-in multivalent capabilities and precise, pre-programmed geometries.

Mutational inactivation of Apc in the intestinal epithelia compromises cellular organisation.

The adenomatous polyposis coli (Apc) protein regulates diverse effector pathways essential for tissue homeostasis. Truncating oncogenic mutations in Apc removing its Wnt pathway and microtubule regulatory domains drives intestinal epithelia tumorigenesis. Exuberant cell proliferation is one well-established consequence of oncogenic Wnt pathway activity; however, the contribution of other deregulated molecular circuits to tumorigenesis has not been fully examined. Using in vivo and organoid models of intestinal epithelial tumorigenesis we found that Wnt pathway activity controls intestinal epithelial villi and crypt structure, morphological features lost upon Apc inactivation. Although the Wnt pathway target gene c-Myc (also known as Myc) has critical roles in regulating cell proliferation and tumorigenesis, Apc specification of intestinal epithelial morphology is independent of the Wnt-responsive Myc-335 (also known as Rr21) regulatory element. We further demonstrate that Apc inactivation disrupts the microtubule cytoskeleton and consequently localisation of organelles without affecting the distribution of the actin cytoskeleton and associated components. Our data indicates the direct control over microtubule dynamics by Apc through an independent molecular circuit. Our study stratifies three independent Apc effector pathways in the intestinal epithelial controlling: (1) proliferation, (2) microtubule dynamics and (3) epithelial morphology.This article has an associated First Person interview with the first author of the paper.

Author Correction: Organoid culture media formulated with growth factors of defined cellular activity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Organoid culture media formulated with growth factors of defined cellular activity.

The media formulations necessary for deriving and sustaining organoids from epithelial tissues such as prostate, colon, gastric, liver, pancreas, and others have been established. Critical components of organoid media are a set of growth factors that include R-spondins and BMP signalling antagonists such as Noggin or Gremlin 1. Currently, the practical limitations for formulating organoid media of reproducible potency and larger-scale media production that have hampered further technological applications of organoid technology include: the cost of growth factors such as R-spondins and Gremlin 1/Noggin and their production as defined specific activities free of contaminants that may affect organoid growth. Here we report the production of highly pure recombinant Gremlin 1 and R-spondin 1 from bacterial expression for use in organoid media. We detail the workflow for Gremlin 1 and R-spondin 1 expression, purification, quantification of cellular activity, quality control and use in media formulated for culturing organoids derived from a number of tissues. The development of precisely formulated, cost-effective media of defined specific activity will engender the development of novel applications for organoid technology.

Overview of the oncogenic signaling pathways in colorectal cancer: Mechanistic insights.

Colorectal cancer is a multifaceted disease which is therapeutically challenging. Based on insights gleaned from almost a quarter century of research, it is obvious that deregulation of spatio-temporally controlled signaling pathways play instrumental role in development and progression of colorectal cancer. High-throughput technologies have helped to develop a sharper and broader understanding of the wide ranging signal transduction cascades which also contribute to development of drug resistance, loss of apoptosis and, ultimately, of metastasis. In this review, we have set the spotlight on role of JAK/STAT, TGF/SMAD, Notch, WNT/ß-Catenin, SHH/GLI and p53 pathways in the development and progression of colorectal cancer. We have also highlighted recent reports on TRAIL-mediated pathways and molecularly distinct voltage-gated sodium channels in colorectal cancer.

Macrocyclized Extended Peptides: Inhibiting the Substrate-Recognition Domain of Tankyrase.

We report a double-click macrocyclization approach for the design of constrained peptide inhibitors having non-helical or extended conformations. Our targets are the tankyrase proteins (TNKS), poly(ADP-ribose) polymerases (PARP) that regulate Wnt signaling by targeting Axin for degradation. TNKS are deregulated in many different cancer types, and inhibition of TNKS therefore represents an attractive therapeutic strategy. However, clinical development of TNKS-specific PARP catalytic inhibitors is challenging due to off-target effects and cellular toxicity. We instead targeted the substrate-recognition domain of TNKS, as it is unique among PARP family members. We employed a two-component strategy, allowing peptide and linker to be separately engineered and then assembled in a combinatorial fashion via click chemistry. Using the consensus substrate-peptide sequence as a starting point, we optimized the length and rigidity of the linker and its position along the peptide. Optimization was further guided by high-resolution crystal structures of two of the macrocyclized peptides in complex with TNKS. This approach led to macrocyclized peptides with submicromolar affinities for TNKS and high proteolytic stability that are able to disrupt the interaction between TNKS and Axin substrate and to inhibit Wnt signaling in a dose-dependent manner. The peptides therefore represent a promising starting point for a new class of substrate-competitive inhibitors of TNKS with potential for suppressing Wnt signaling in cancer. Moreover, by demonstrating the application of the double-click macrocyclization approach to non-helical, extended, or irregularly structured peptides, we greatly extend its potential and scope, especially given the frequency with which such motifs mediate protein-protein interactions.

LEF1 and B9L shield ß-catenin from inactivation by Axin, desensitizing colorectal cancer cells to tankyrase inhibitors.

Hyperactive ß-catenin drives colorectal cancer, yet inhibiting its activity remains a formidable challenge. Interest is mounting in tankyrase inhibitors (TNKSi), which destabilize ß-catenin through stabilizing Axin. Here, we confirm that TNKSi inhibit Wnt-induced transcription, similarly to carnosate, which reduces the transcriptional activity of ß-catenin by blocking its binding to BCL9, and attenuates intestinal tumors in Apc(Min) mice. By contrast, ß-catenin's activity is unresponsive to TNKSi in colorectal cancer cells and in cells after prolonged Wnt stimulation. This TNKSi insensitivity is conferred by ß-catenin's association with LEF1 and BCL9-2/B9L, which accumulate during Wnt stimulation, thereby providing a feed-forward loop that converts transient into chronic ß-catenin signaling. This limits the therapeutic value of TNKSi in colorectal carcinomas, most of which express high LEF1 levels. Our study provides proof-of-concept that the successful inhibition of oncogenic ß-catenin in colorectal cancer requires the targeting of its interaction with LEF1 and/or BCL9/B9L, as exemplified by carnosate.

PakB binds to the SH3 domain of Dictyostelium Abp1 and regulates its effects on cell polarity and early development.

Dictyostelium p21-activated kinase B (PakB) phosphorylates and activates class I myosins. PakB colocalizes with myosin I to actin-rich regions of the cell, including macropinocytic and phagocytic cups and the leading edge of migrating cells. Here we show that residues 1-180 mediate the cellular localization of PakB. Yeast two-hybrid and pull-down experiments identify two proline-rich motifs in PakB-1-180 that directly interact with the SH3 domain of Dictyostelium actin-binding protein 1 (dAbp1). dAbp1 colocalizes with PakB to actin-rich regions in the cell. The loss of dAbp1 does not affect the cellular distribution of PakB, whereas the loss of PakB causes dAbp1 to adopt a diffuse cytosolic distribution. Cosedimentation studies show that the N-terminal region of PakB (residues 1-70) binds directly to actin filaments, whereas dAbp1 exhibits only a low affinity for filamentous actin. PakB-1-180 significantly enhances the binding of dAbp1 to actin filaments. When overexpressed in PakB-null cells, dAbp1 completely blocks early development at the aggregation stage, prevents cell polarization, and significantly reduces chemotaxis rates. The inhibitory effects are abrogated by the introduction of a function-blocking mutation into the dAbp1 SH3 domain. We conclude that PakB plays a critical role in regulating the cellular functions of dAbp1, which are mediated largely by its SH3 domain.

An intrinsically labile α-helix abutting the BCL9-binding site of ß-catenin is required for its inhibition by carnosic acid.

Wnt/ß-catenin signalling controls development and tissue homeostasis. Moreover, activated ß-catenin can be oncogenic and, notably, drives colorectal cancer. Inhibiting oncogenic ß-catenin has proven a formidable challenge. Here we design a screen for small-molecule inhibitors of ß-catenin's binding to its cofactor BCL9, and discover five related natural compounds, including carnosic acid from rosemary, which attenuates transcriptional ß-catenin outputs in colorectal cancer cells. Evidence from NMR and analytical ultracentrifugation demonstrates that the carnosic acid response requires an intrinsically labile α-helix (H1) amino-terminally abutting the BCL9-binding site in ß-catenin. Similarly, in colorectal cancer cells with hyperactive ß-catenin signalling, carnosic acid targets predominantly the transcriptionally active ('oncogenic') form of ß-catenin for proteasomal degradation in an H1-dependent manner. Hence, H1 is an 'Achilles' Heel' of ß-catenin, which can be exploited for destabilization of oncogenic ß-catenin by small molecules, providing proof-of-principle for a new strategy for developing direct inhibitors of oncogenic ß-catenin.

Structural and functional properties of glycerol-3-phosphate dehydrogenase from a mammalian hibernator.

Glycerol-3-phosphate dehydrogenase (G3PDH; E.C.1.1.1.8) was purified from liver and skeletal muscle of black-tailed prairie dogs (Cynomys ludivicianus), a hibernating species. Native and subunit molecular masses of the dimeric enzyme were 77 and 40 kD, respectively, and both tissues contained a single isozyme with a pI of 6.4. Kinetic parameters of purified G3PDH from prairie dog liver and muscle were characterized at 22 and 5 °C and compared with rabbit muscle G3PDH. Substrate affinities for hibernator muscle G3PDH were stable (NAD) or increased significantly (K(m) G3P and DHAP decreased) at low temperature whereas K(m) NAD and DHAP of rabbit G3PDH increased. Prairie dog G3PDH showed greater conservation of K(m) G3P over a wide temperature range as well as greater thermal stability and resistance to chemical denaturation by guanidine hydrochloride than the rabbit enzyme. In addition, using the protein sequence of the hibernating thirteen-lined ground squirrel (Ictidomys tridecemlineatus) and bioinformatics tools, the deduced protein structure of G3PDH was compared between heterothermic and homeothermic mammals. Structural and functional characteristics of G3PDH from the hibernating species would support enzyme function over a wide range of core body temperatures over cycles of torpor and arousal.

Dvl2 promotes intestinal length and neoplasia in the ApcMin mouse model for colorectal cancer.

APC mutations cause activation of Wnt/beta-catenin signaling, which invariably leads to colorectal cancer. Similarly, overexpressed Dvl proteins are potent activators of beta-catenin signaling. Screening a large tissue microarray of different staged colorectal tumors by immunohistochemistry, we found that Dvl2 has a strong tendency to be overexpressed in colorectal adenomas and carcinomas, in parallel to nuclear beta-catenin and Axin2 (a universal transcriptional target of Wnt/beta-catenin signaling). Furthermore, deletion of Dvl2 reduced the intestinal tumor numbers in a dose-dependent way in the Apc(Min) model for colorectal cancer. Interestingly, the small intestines of Dvl2 mutants are shortened, reflecting in part a reduction of their crypt diameter and cell size. Consistent with this, mammalian target of rapamycin (mTOR) signaling is highly active in normal intestinal crypts in which Wnt/beta-catenin signaling is active, and activated mTOR signaling (as revealed by staining for phosphorylated 4E-BP1) serves as a diagnostic marker of Apc(Min) mutant adenomas. Inhibition of mTOR signaling in Apc(Min) mutant mice by RAD001 (everolimus) reduces their intestinal tumor load, similarly to Dvl2 deletion. mTOR signaling is also consistently active in human hyperplastic polyps and has a significant tendency for being active in adenomas and carcinomas. Our results implicate Dvl2 and mTOR in the progression of colorectal neoplasia and highlight their potential as therapeutic targets in colorectal cancer.

A role of Pygopus as an anti-repressor in facilitating Wnt-dependent transcription.

Wnt/beta-catenin signaling controls animal development and tissue homeostasis, and is also an important cancer pathway. Pygopus (Pygo) is a conserved nuclear Wnt signaling component that is essential for Wingless-induced transcription throughout Drosophila development. It associates with Armadillo/beta-catenin and T cell factor (TCF) through the Legless/BCL9 adaptor, but its molecular function in TCF-mediated transcription is unknown. Here, we use a groucho-null allele to show that Groucho represses Wingless target genes during Drosophila development. Interestingly, groucho pygo double-mutants revealed that Pygo is not obligatory for transcriptional and phenotypic Wingless signaling outputs if the interaction between Groucho and Drosophila TCF is compromised genetically. Pygo function is also non-essential for Wingless outputs in the absence of other transcriptional antagonists of Wingless signaling. This indicates an anti-repressor function of Pygo: we propose that Pygo predisposes Drosophila TCF target genes for rapid Wingless-induced transcription, or that it protects them against premature shut-down.

The function of BCL9 in Wnt/beta-catenin signaling and colorectal cancer cells.

BACKGROUND: Most cases of colorectal cancer are initiated by hyperactivation of the Wnt/beta-catenin pathway due to mutations in the APC tumour suppressor, or in beta-catenin itself. A recently discovered component of this pathway is Legless, which is essential for Wnt-induced transcription during Drosophila development. Limited functional information is available for its two mammalian relatives, BCL9 and B9L/BCL9-2: like Legless, these proteins bind to beta-catenin, and RNAi-mediated depletion of B9L/BCL9-2 has revealed that this protein is required for efficient beta-catenin-mediated transcription in mammalian cell lines. No loss-of-function data are available for BCL9. METHODS: We have used overexpression of dominant-negative forms of BCL9, and RNAi-mediated depletion, to study its function in human cell lines with elevated Wnt pathway activity, including colorectal cancer cells. RESULTS: We found that BCL9 is required for efficient beta-catenin-mediated transcription in Wnt-stimulated HEK 293 cells, and in the SW480 colorectal cancer cell line whose Wnt pathway is active due to APC mutation. Dominant-negative mutants of BCL9 indicated that its function depends not only on its beta-catenin ligand, but also on an unknown ligand of its C-terminus. Finally, we show that BCL9 and B9L are both Wnt-inducible genes, hyperexpressed in colorectal cancer cell lines, indicating that they are part of a positive feedback loop. CONCLUSION: BCL9 is required for efficient beta-catenin-mediated transcription in human cell lines whose Wnt pathway is active, including colorectal cancer cells, indicating its potential as a drug target in colorectal cancer.

Wingless-independent association of Pygopus with dTCF target genes.

The Wnt signaling pathway controls numerous cell fates during animal development. Its inappropriate activity can lead to cancer in many human tissues. A key effector of the canonical Wnt pathway is beta-catenin (or Drosophila Armadillo), a highly unstable phosphorylated protein that shuttles rapidly between nucleus and cytoplasm. Wnt signaling inhibits its phosphorylation and degradation; this allows it to associate with TCF/LEF factors bound to Wnt target genes and to stimulate their transcription by recruiting chromatin modifying and remodeling factors. The transcriptional activity of Armadillo/beta-catenin also depends on Pygopus (Pygo), a nuclear protein with which it associates through the Legless/BCL9 adaptor. It has been proposed that Pygo associates with TCF target genes during Wnt signaling through Armadillo and Legless to recruit a transcriptional coactivator through its Nbox motif. Here, we report that Pygo is associated constitutively with dTCF target genes in Drosophila salivary glands and tissue-culture cells. Our evidence indicates that this association depends on dTCF and on the Nbox motif of Pygo, but not on Legless. We thus propose an alternative model according to which Pygo functions at the onset of Wnt signaling, or at low signaling levels, to capture Armadillo at dTCF target genes, thus enabling the interaction between Armadillo and dTCF and, consequently, the Armadillo-mediated recruitment of transcriptional coactivators.

Identification and characterization of an 8-kDa light chain associated with Dictyostelium discoideum MyoB, a class I myosin.

Dictyostelium discoideum MyoB is a single-headed class I myosin. Analysis of purified MyoB by SDS-PAGE indicated the presence of an approximately 9-kDa light chain. A tryptic digest of MyoB yielded a partial sequence for the light chain that exactly matched a sequence in a 73-amino acid, 8,296-Da protein (dictyBase number DDB0188713). This protein, termed MlcB, contains two EF-hand motifs and shares approximately 30% sequence identity with the N- and C-terminal lobes of calmodulin. FLAG-MlcB expressed in Dictyostelium co-immunoprecipitated with MyoB but not with the related class myosins and MyoD. Recombinant MlcB bound Ca2+ with a Kd value of 0.2 microm and underwent a Ca2+-induced change in conformation that increased alpha-helical content and surface hydrophobicity. Mutational analysis showed that the first EF-hand was responsible for Ca2+ binding. In the presence and absence of Ca2+ MlcB was a monomer in solution and bound to a MyoB IQ motif peptide with a Kd value of approximately 0.5 microm. A MyoB head-neck construct with a Ser to Glu mutation at the TEDS site bound MlcB and displayed an actin-activated Mg2+ ATPase activity that was insensitive to Ca2+. We conclude that MlcB represents a novel type of small myosin light chain that binds to IQ motifs in a manner comparable with a single lobe of a typical four-EF-hand protein.

Cellular distribution and functions of wild-type and constitutively activated Dictyostelium PakB.

Dictyostelium PakB, previously termed myosin I heavy chain kinase, is a member of the p21-activated kinase (PAK) family. Two-hybrid assays showed that PakB interacts with Dictyostelium Rac1a/b/c, RacA (a RhoBTB protein), RacB, RacC, and RacF1. Wild-type PakB displayed a cytosolic distribution with a modest enrichment at the leading edge of migrating cells and at macropinocytic and phagocytic cups, sites consistent with a role in activating myosin I. PakB fused at the N terminus to green fluorescent protein was proteolyzed in cells, resulting in removal of the catalytic domain. C-terminal truncated PakB and activated PakB lacking the p21-binding domain strongly localized to the cell cortex, to macropinocytic cups, to the posterior of migrating cells, and to the cleavage furrow of dividing cells. These data indicate that in its open, active state, the N terminus of PakB forms a tight association with cortical actin filaments. PakB-null cells displayed no significant behavioral defects, but cells expressing activated PakB were unable to complete cytokinesis when grown in suspension and exhibited increased rates of phagocytosis and pinocytosis.

The Dictyostelium class I myosin, MyoD, contains a novel light chain that lacks high-affinity calcium-binding sites.

Dictyostelium discoideum MyoD, a long-tailed class I myosin, co-purified with two copies of a 16 kDa light chain. Sequence analysis of the MyoD light chain showed it to be a unique protein, termed MlcD, that shares 44% sequence identity with Dictyostelium calmodulin and 43% sequence identity with Acanthamoeba castellanii myosin IC light chain. MlcD comprises four EF-hands; however, EF-hands 2-4 contain mutations in key Ca2+-co-ordinating residues that would be predicted to impair Ca2+ binding. Electrospray ionization MS of MlcD in the presence of Ca2+ and La3+ showed the presence of one major and one minor metal-binding site. MlcD contains a single tryptophan residue (Trp39), the fluorescence intensity of which was quenched upon addition of Ca2+ or Mg2+, yielding apparent dissociation constants ( K'(d)) of 52 microM for Ca2+ and 450 microM for Mg2+. The low affinity of MlcD for Ca2+ indicates that it cannot function as a sensor of physiological Ca2+. Ca2+ did not affect the binding of MlcD to MyoD or to either of the two MyoD IQ (Ile-Gln) motifs. FLAG-MlcD expressed in Dictyostelium formed a complex with MyoD, but not with the two other long-tailed Dictyostelium myosin I isoenzymes, MyoB and MyoC. Through its specific association with the Ca2+-insensitive MlcD, MyoD may exhibit distinct regulatory properties that distinguish it from myosin I isoenzymes with calmodulin light chains.

Dictyostelium discoideum has a single diacylglycerol kinase gene with similarity to mammalian theta isoforms.

Diacylglycerol kinases (DGKs) phosphorylate the neutral lipid diacylglycerol (DG) to produce phosphatidic acid (PA). In mammalian systems DGKs are a complex family of at least nine isoforms that are thought to participate in down-regulation of DG-based signalling pathways and perhaps activation of PA-stimulated signalling events. We report here that the simple protozoan amoeba Dictyostelium discoideum appears to contain a single gene encoding a DGK enzyme. This gene, dgkA, encodes a deduced protein that contains three C1-type cysteine-rich repeats, a DGK catalytic domain most closely related to the theta subtype of mammalian DGKs and a C-terminal segment containing a proline/glutamine-rich region and a large aspargine-repeat region. This gene corresponds to a previously reported myosin II heavy chain kinase designated myosin heavy chain-protein kinase C (MHC-PKC), but our analysis clearly demonstrates that this protein does not, as suggested by earlier data, contain a protein kinase catalytic domain. A FLAG-tagged version of DgkA expressed in Dictyostelium displayed robust DGK activity. Earlier studies indicating that disruption of this locus alters myosin II assembly levels in Dictyostelium raise the intriguing possibility that DG and/or PA metabolism may play a role in controlling myosin II assembly in this system.