Discovery of a PDZ Domain Inhibitor Targeting the Syndecan/Syntenin Protein-Protein Interaction: A Semi-Automated "Hit Identification-to-Optimization" Approach.

The rapid identification of early hits by fragment-based approaches and subsequent hit-to-lead optimization represents a challenge for drug discovery. To address this challenge, we created a strategy called "DOTS" that combines molecular dynamic simulations, computer-based library design (chemoDOTS) with encoded medicinal chemistry reactions, constrained docking, and automated compound evaluation. To validate its utility, we applied our DOTS strategy to the challenging target syntenin, a PDZ domain containing protein and oncology target.

The serine/threonine kinase MINK1 directly regulates the function of promigratory proteins.

Upregulation of the developmental Wnt planar cell polarity (Wnt/PCP) pathway is observed in many cancers and is associated with cancer development. We have recently shown that PRICKLE1, a core Wnt/PCP pathway component, is a marker of poor prognosis in triple-negative breast cancer (TNBC). PRICKLE1 is phosphorylated by the serine/threonine kinase MINK1 and contributes to TNBC cell motility and invasiveness.

Identification of PDZ Interactions by Affinity Purification and Mass Spectrometry Analysis.

Identification of protein networks becomes indispensable for determining the function of a given protein of interest. Some proteins harbor a PDZ binding motif (PDZBM) located at the carboxy-terminus end. This motif is necessary to recruit PDZ domain proteins which are involved in signaling, trafficking, and maintenance of cell architecture.

The Tumor Suppressor SCRIB is a Negative Modulator of the Wnt/β-Catenin Signaling Pathway.

SCRIB is a scaffold protein containing leucine-rich repeats (LRR) and PSD-95/Dlg-A/ZO-1 domains (PDZ) that localizes at the basolateral membranes of polarized epithelial cells. Deregulation of its expression or localization leads to epithelial defects and tumorigenesis in part as a consequence of its repressive role on several signaling pathways including AKT, ERK, and HIPPO. In the present work, a proteomic approach is used to characterize the protein complexes associated to SCRIB and its paralogue LANO.

ECT2 associated to PRICKLE1 are poor-prognosis markers in triple-negative breast cancer.

Background: Triple-negative breast cancers (TNBC) are poor-prognosis tumours candidate to chemotherapy as only systemic treatment. We previously found that PRICKLE1, a prometastatic protein involved in planar cell polarity, is upregulated in TNBC. We investigated the protein complex associated with PRICKLE1 in TNBC to identify proteins possibly involved in metastatic dissemination, which might provide new prognostic and/or therapeutic targets.

Mapping Cellular Polarity Networks Using Mass Spectrometry-based Strategies.

Cell polarity is a vital biological process involved in the building, maintenance and normal functioning of tissues in invertebrates and vertebrates. Unsurprisingly, molecular defects affecting polarity organization and functions have a strong impact on tissue homeostasis, embryonic development and adult life, and may directly or indirectly lead to diseases. Genetic studies have demonstrated the causative effect of several polarity genes in diseases; however, much remains to be clarified before a comprehensive view of the molecular organization and regulation of the protein networks associated with polarity proteins is obtained.

Wnt/Planar Cell Polarity Signaling: New Opportunities for Cancer Treatment.

Cancer cells are addicted to a large spectrum of extracellular cues implicated in initiation, stem cell renewal, tumor growth, dissemination in the body, and resistance to treatment. Wingless/Int-1 (Wnt) ligands and their associated signaling cascades contribute to most of these processes, paving the way for opportunities in therapeutic development. The developmental Wnt/planar cell polarity (PCP) pathway is the most recently described branch of Wnt signaling strongly implicated in cancer development at early and late stages.

PRICKLE1 Contributes to Cancer Cell Dissemination through Its Interaction with mTORC2.

Components of the evolutionarily conserved developmental planar cell polarity (PCP) pathway were recently described to play a prominent role in cancer cell dissemination. However, the molecular mechanisms by which PCP molecules drive the spread of cancer cells remain largely unknown. PRICKLE1 encodes a PCP protein bound to the promigratory serine/threonine kinase MINK1.

SAPCD2 Controls Spindle Orientation and Asymmetric Divisions by Negatively Regulating the Gαi-LGN-NuMA Ternary Complex.

Control of cell-division orientation is integral to epithelial morphogenesis and asymmetric cell division. Proper spatiotemporal localization of the evolutionarily conserved Gαi-LGN-NuMA protein complex is critical for mitotic spindle orientation, but how this is achieved remains unclear. Here we identify Suppressor APC domain containing 2 (SAPCD2) as a previously unreported LGN-interacting protein.

The PTK7 and ROR2 Protein Receptors Interact in the Vertebrate WNT/Planar Cell Polarity (PCP) Pathway.

The non-canonical WNT/planar cell polarity (WNT/PCP) pathway plays important roles in morphogenetic processes in vertebrates. Among WNT/PCP components, protein tyrosine kinase 7 (PTK7) is a tyrosine kinase receptor with poorly defined functions lacking catalytic activity. Here we show that PTK7 associates with receptor tyrosine kinase-like orphan receptor 2 (ROR2) to form a heterodimeric complex in mammalian cells.

Convergence of melatonin and serotonin (5-HT) signaling at MT2/5-HT2C receptor heteromers.

Inasmuch as the neurohormone melatonin is synthetically derived from serotonin (5-HT), a close interrelationship between both has long been suspected. The present study reveals a hitherto unrecognized cross-talk mediated via physical association of melatonin MT2 and 5-HT2C receptors into functional heteromers. This is of particular interest in light of the "synergistic" melatonin agonist/5-HT2C antagonist profile of the novel antidepressant agomelatine.

Asymmetry of VANGL2 in migrating lymphocytes as a tool to monitor activity of the mammalian WNT/planar cell polarity pathway.

Background: The WNT/planar-cell-polarity (PCP) pathway is a key regulator of cell polarity and directional cell movements. Core PCP proteins such as Van Gogh-like2 (VANGL2) are evolutionarily highly conserved; however, the mammalian PCP machinery is still poorly understood mainly due to lack of suitable models and quantitative methodology. WNT/PCP has been implicated in many human diseases with the most distinguished positive role in the metastatic process, which accounts for more than 90% of cancer related deaths, and presents therefore an attractive target for pharmacological interventions.

The human PDZome: a gateway to PSD95-Disc large-zonula occludens (PDZ)-mediated functions.

Protein-protein interactions organize the localization, clustering, signal transduction, and degradation of cellular proteins and are therefore implicated in numerous biological functions. These interactions are mediated by specialized domains able to bind to modified or unmodified peptides present in binding partners. Among the most broadly distributed protein interaction domains, PSD95-disc large-zonula occludens (PDZ) domains are usually able to bind carboxy-terminal sequences of their partners.

Mink1 regulates β-catenin-independent Wnt signaling via Prickle phosphorylation.

β-Catenin-independent Wnt signaling pathways have been implicated in the regulation of planar cell polarity (PCP) and convergent extension (CE) cell movements. Prickle, one of the core proteins of these pathways, is known to asymmetrically localize proximally at the adherens junction of Drosophila melanogaster wing cells and to locally accumulate within plasma membrane subdomains in cells undergoing CE movements during vertebrate development. Using mass spectrometry, we have identified the Ste20 kinase Mink1 as a Prickle-associated protein and found that they genetically interact during the establishment of PCP in the Drosophila eye and CE in Xenopus laevis embryos.

Calcium-sensing receptor modulates cell adhesion and migration via integrins.

The calcium-sensing receptor (CaSR) is a family C G protein-coupled receptor that is activated by elevated levels of extracellular divalent cations. The CaSR couples to members of the G(q) family of G proteins, and in the endocrine system this receptor is instrumental in regulating the release of parathyroid hormone from the parathyroid gland and calcitonin from thyroid cells. Here, we demonstrate that in medullary thyroid carcinoma cells, the CaSR promotes cellular adhesion and migration via coupling to members of the integrin family of extracellular matrix-binding proteins.

GPCR-interacting proteins, major players of GPCR function.

G protein-coupled receptors (GPCRs) are, with approximately 800 members, among the most abundant membrane proteins in humans. They are responding to a plethora of ligands and are involved in the transmission of extracellular signals inside the cell. GPCRs are synthesized in the endoplasmatic reticulum and are then transported to the cell surface where they are typically activated.

Tandem affinity purification and identification of heterotrimeric g protein-associated proteins.

Heterotrimeric G proteins are the main signal-transducing molecules activated by G protein-coupled receptors. Their GTP-dependent activation leads to the regulation of different effectors such as adenylyl cyclases, phospholipases, and RhoGEFs. To understand the full biological consequences of GPCR signalling and to further understand the cross-talk with other signalling pathways, the complement of proteins associating with heterotrimeric G proteins needs to be identified.

Tandem affinity purification and identification of GPCR-associated protein complexes.

The first tandem affinity purification (TAP) protocol was described in 1999. Originally designed for the purification of protein complexes in yeast RNA splicing, its application rapidly expanded towards whole proteome analysis in yeast and mammalian cells. More recently, TAP has been applied to the purification of G protein-coupled receptor (GPCR)-associated protein complexes (GAPCs).

Peptide affinity purification for the isolation and identification of GPCR-associated protein complexes.

Protein networks and their dynamic regulation play a fundamental role in biological systems. Seven transmembrane-spanning G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors controlling the flow of information from the extracellular environment into cells by inducing intracellular signaling pathways. Several GPCR-associated protein complexes (GAPCs), particularly those binding to the intracellular carboxyl-terminus (C-terminus), have been identified over the last 20 years.

The ubiquitin-specific protease USP34 regulates axin stability and Wnt/β-catenin signaling.

Wnt proteins control multiple cell behaviors during development and tissue homeostasis. However, pathological activation of Wnt signaling is the underlying cause of various human diseases. The ubiquitin-proteasome system plays important regulatory functions within the Wnt pathway by regulating the activity of several of its core components.

Molecular organization and dynamics of the melatonin MT₁ receptor/RGS20/G(i) protein complex reveal asymmetry of receptor dimers for RGS and G(i) coupling.

Functional asymmetry of G-protein-coupled receptor (GPCR) dimers has been reported for an increasing number of cases, but the molecular architecture of signalling units associated to these dimers remains unclear. Here, we characterized the molecular complex of the melatonin MT₁ receptor, which directly and constitutively couples to G(i) proteins and the regulator of G-protein signalling (RGS) 20. The molecular organization of the ternary MT₁/G(i)/RGS20 complex was monitored in its basal and activated state by bioluminescence resonance energy transfer between probes inserted at multiple sites of the complex.

G protein betagamma subunits regulate cell adhesion through Rap1a and its effector Radil.

The activation of several G protein-coupled receptors is known to regulate the adhesive properties of cells in different contexts. Here, we reveal that Gbetagamma subunits of heterotrimeric G proteins regulate cell-matrix adhesiveness by activating Rap1a-dependent inside-out signals and integrin activation. We show that Gbetagamma subunits enter in a protein complex with activated Rap1a and its effector Radil and establish that this complex is required downstream of receptor stimulation for the activation of integrins and the positive modulation of cell-matrix adhesiveness.

Recent methodological advances in the discovery of GPCR-associated protein complexes.

Protein-interaction networks have important roles in cellular homeostasis and the generation of complexity in biological systems. G-protein-coupled receptors (GPCRs), the largest family of membrane receptors and important drug targets, are integral parts of these networks. Ligand stimulation and the dynamic interaction with GPCR-associated protein complexes (GAPCs) constitute two important regulatory mechanisms of GPCR function.