13 research outputs found
Comparative Genetic Screens in Human Cells Reveal New Regulatory Mechanisms in WNT Signaling
The comprehensive understanding of cellular signaling pathways remains a challenge due to multiple layers of regulation that may become evident only when the pathway is probed at different levels or critical nodes are eliminated. To discover regulatory mechanisms in canonical WNT signaling, we conducted a systematic forward genetic analysis through reporter-based screens in haploid human cells. Comparison of screens for negative, attenuating and positive regulators of WNT signaling, mediators of R-spondin-dependent signaling and suppressors of constitutive signaling induced by loss of the tumor suppressor adenomatous polyposis coli or casein kinase 1α uncovered new regulatory features at most levels of the pathway. These include a requirement for the transcription factor AP-4, a role for the DAX domain of AXIN2 in controlling β-catenin transcriptional activity, a contribution of glycophosphatidylinositol anchor biosynthesis and glypicans to R-spondin-potentiated WNT signaling, and two different mechanisms that regulate signaling when distinct components of the β-catenin destruction complex are lost. The conceptual and methodological framework we describe should enable the comprehensive understanding of other signaling systems
Hedgehog-Interacting Protein is a multimodal antagonist of Hedgehog signalling
Hedgehog (HH) morphogen signalling, crucial for cell growth and tissue patterning in animals, is initiated by the binding of dually lipidated HH ligands to cell surface receptors. Hedgehog-Interacting Protein (HHIP), the only reported secreted inhibitor of Sonic Hedgehog (SHH) signalling, binds directly to SHH with high nanomolar affinity, sequestering SHH. Here, we report the structure of the HHIP N-terminal domain (HHIP-N) in complex with a glycosaminoglycan (GAG). HHIP-N displays a unique bipartite fold with a GAG-binding domain alongside a Cysteine Rich Domain (CRD). We show that HHIP-N is required to convey full HHIP inhibitory function, likely by interacting with the cholesterol moiety covalently linked to HH ligands, thereby preventing this SHH-attached cholesterol from binding to the HH receptor Patched (PTCH1). We also present the structure of the HHIP C-terminal domain in complex with the GAG heparin. Heparin can bind to both HHIP-N and HHIP-C, thereby inducing clustering at the cell surface and generating a high-avidity platform for SHH sequestration and inhibition. Our data suggest a multimodal mechanism, in which HHIP can bind two specific sites on the SHH morphogen, alongside multiple GAG interactions, to inhibit SHH signalling
Correction to Inhibition of Hypoxia Inducible Factor 1–Transcription Coactivator Interaction by a Hydrogen Bond Surrogate α-Helix
Direct organocatalytic coupling of carboxylated piperazine-2,5-diones with indoles through conjugate addition of carbon nucleophiles to indolenine intermediates
Chromatin-Remodeling Complex SWI/SNF Controls Multidrug Resistance by Transcriptionally Regulating the Drug Efflux Pump ABCB1
R-spondins engage heparan sulfate proteoglycans to potentiate WNT signaling
R-spondins (RSPOs) amplify WNT signaling during development and regenerative responses. We previously demonstrated that RSPOs 2 and 3 potentiate WNT/β-catenin signaling in cells lacking leucine-rich repeat-containing G-protein coupled receptors (LGRs) 4, 5 and 6 (Lebensohn and Rohatgi, 2018). We now show that heparan sulfate proteoglycans (HSPGs) act as alternative co-receptors for RSPO3 using a combination of ligand mutagenesis and ligand engineering. Mutations in RSPO3 residues predicted to contact HSPGs impair its signaling capacity. Conversely, the HSPG-binding domains of RSPO3 can be entirely replaced with an antibody that recognizes heparan sulfate (HS) chains attached to multiple HSPGs without diminishing WNT-potentiating activity in cultured cells and intestinal organoids. A genome-wide screen for mediators of RSPO3 signaling in cells lacking LGRs 4, 5 and 6 failed to reveal other receptors. We conclude that HSPGs are RSPO co-receptors that potentiate WNT signaling in the presence and absence of LGRs
Suppression of Tumor Growth by Designed Dimeric Epidithiodiketopiperazine Targeting Hypoxia-Inducible Transcription Factor Complex
Hypoxia
is a hallmark of solid tumors, is associated with local
invasion, metastatic spread, resistance to chemo- and radiotherapy,
and is an independent, negative prognostic factor for a diverse range
of malignant neoplasms. The cellular response to hypoxia is primarily
mediated by a family of transcription factors, among which hypoxia-inducible
factor 1 (HIF1) plays a major role. Under normoxia, the oxygen-sensitive
α subunit of HIF1 is rapidly and constitutively degraded but
is stabilized and accumulates under hypoxia. Upon nuclear translocation,
HIF1 controls the expression of over 100 genes involved in angiogenesis,
altered energy metabolism, antiapoptotic, and pro-proliferative mechanisms
that promote tumor growth. A designed transcriptional antagonist,
dimeric epidithiodiketopiperazine (ETP <b>2</b>), selectively
disrupts the interaction of HIF1α with p300/CBP coactivators
and downregulates the expression of hypoxia-inducible genes. ETP <b>2</b> was synthesized via a novel homo-oxidative coupling of the
aliphatic primary carbons of the dithioacetal precursor. It effectively
inhibits HIF1-induced activation of <i>VEGFA</i>, <i>LOX</i>, <i>Glut1</i>, and <i>c-Met</i> genes
in a panel of cell lines representing breast and lung cancers. We
observed an outstanding antitumor efficacy of both (±)-ETP <b>2</b> and <i>meso</i>-ETP <b>2</b> in a fully
established breast carcinoma model by intravital microscopy. Treatment
with either form of ETP <b>2</b> (1 mg/kg) resulted in a rapid
regression of tumor growth that lasted for up to 14 days. These results
suggest that inhibition of HIF1 transcriptional activity by designed
dimeric ETPs could offer an innovative approach to cancer therapy
with the potential to overcome hypoxia-induced tumor growth and resistance