The tandem PDZ domains of syntenin promote cell invasion

Syntenin is a tandem PDZ protein that has recently been shown to be overexpressed in several cancer cells and tissues, and that might play an active role in tumor cell invasion and metastasis. Here we show that overexpression of the tandem PDZ domains of syntenin in non-invasive cells is necessary and sufficient to stimulate these cells to invade a collagen I matrix, and this effect can be regulated by ligand binding to the PDZ domains. Furthermore, we show that syntenin-induced invasion requires signaling through ras, rho and PI3K/MAPK signaling pathways and involves changes in cell-cell adhesion. Inversely, when we used RNA interference to inhibit syntenin expression in different invasive cancer cell lines, we observed a drastically decreased ability of these cells to migrate and invade into collagen type I or Matrigel. RNAi-treated cells also show increased cell aggregation, indicating that syntenin is important for cell-cell adhesion in epithelial cells. Together, these results suggest that downregulation of syntenin by RNA interference could provide a means of inhibiting tumor invasion and possibly metastasis in different cancers, and point to syntenin as a potential cancer biomarker and drug target.     

Deciphering the Kinetic Binding Mechanism of Dimeric Ligands Using a Potent Plasma-stable Dimeric Inhibitor of Postsynaptic Density Protein-95 as an Example

Dimeric ligands can be potent inhibitors of protein-protein or enzyme-substrate interactions. They have increased affinity and specificity toward their targets due to their ability to bind two binding sites simultaneously and are therefore attractive in drug design. However, few studies have addressed the kinetic mechanism of interaction of such bivalent ligands. We have investigated the binding interaction of a recently identified potent plasma-stable dimeric pentapeptide and PDZ1–2 of postsynaptic density protein-95 (PSD-95) using protein engineering in combination with fluorescence polarization, isothermal titration calorimetry, and stopped-flow fluorimetry. We demonstrate that binding occurs via a two-step process, where an initial binding to either one of the two PDZ domains is followed by an intramolecular step, which produces the bidentate complex. We have determined all rate constants involved in the binding reaction and found evidence for a conformational transition of the complex. Our data demonstrate the importance of a slow dissociation for a successful dimeric ligand but also highlight the possibility of optimizing the intramolecular association rate. The results may therefore aid the design of dimeric inhibitors in general.     

The PDZ-GEF Gef26 regulates synapse development and function via FasII and Rap1 at the Drosophila neuromuscular junction

Guanine nucleotide exchange factors (GEFs) are essential for small G proteins to activate their downstream signaling pathways, which are involved in morphogenesis, cell adhesion, and migration. Mutants of Gef26, a PDZ-GEF (PDZ domain-containing guanine nucleotide exchange factor) in Drosophila, exhibit strong defects in wings, eyes, and the reproductive and nervous systems. However, the precise roles of Gef26 in development remain unclear. In the present study, we analyzed the role of Gef26 in synaptic development and function. We identified significant decreases in bouton number and branch length at larval neuromuscular junctions (NMJs) in Gef26 mutants, and these defects were fully rescued by restoring Gef26 expression, indicating that Gef26 plays an important role in NMJ morphogenesis. In addition to the observed defects in NMJ morphology, electrophysiological analyses revealed functional defects at NMJs, and locomotor deficiency appeared in Gef26 mutant larvae. Furthermore, Gef26 regulated NMJ morphogenesis by regulating the level of synaptic Fasciclin II (FasII), a well-studied cell adhesion molecule that functions in NMJ development and remodeling. Finally, our data demonstrate that Gef26-specific small G protein Rap1 worked downstream of Gef26 to regulate the level of FasII at NMJs, possibly through a βPS integrin-mediated signaling pathway. Taken together, our findings define a novel role of Gef26 in regulating NMJ development and function.     

肿瘤转移相关基因syntenin的融合表达及纯化

肿瘤相关基因syntenin在乳腺癌的转移和浸润过程中发挥重要作用。syntenin基因编码蛋白的C端有2个串联的PDZ(PDZ1和PDZ2)结构域,它们与该蛋白的功能密切相关。PDZ结构域存在于多种蛋白质中。用PCR方法扩增了syntenin全长、N端（ΔPDZ)、串联重复的PDZ(2PDZ)、PDZ1和PDZ2结构域编码序列,并将其以正确相位与pGEX-2T载体中的GST序列编码融合,构建成重组质粒pGST-syntenin、pGST-ΔPDZ、pGST-2PDZ、pGST-PDZ1和pGST-PDZ2。将这些重组质粒分别转化E．coli DH50α后,分别表达了相应的GST融合蛋白。Westem blot检测结果表明,2种融合蛋白均能与GST抗体反应。表达的GST融合蛋白经谷胱甘肽-Sepharose 4B亲和层析获得了纯化的融合蛋白,为PDZ结构域及其相关蛋白功能研究提供了有用的材料。     

PDZ-domain-binding sites are common among cadherins

Cadherins are Ca²⁺-dependent cell adhesion molecules that play fundamental roles in animal development and homeostasis. A number of cadherins contain conserved binding sites for catenins in their cytoplasmic region that are important for the adhesive function of these cadherins by mediating their interaction to the cytoskeleton. However, most cadherins lack apparent binding sites for catenins and their cytoplasmic interacting partners are mostly unknown. In this paper, we show, using bioinformatics, that a number of insect and vertebrate cadherins lacking catenin-binding sites contain conserved consensus sequences for C-terminal PSD-95/Discs-large/ZO-1 (PDZ)-domain-binding sites. This suggests that PDZ-domain-containing proteins are common cytoplasmic interacting partners for cadherins lacking catenin-binding sites.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible to authorized users.     

Functional Modulation of the Glutamate Transporter Variant GLT1b by the PDZ Domain Protein PICK1

The dominant glutamate transporter isoform in the mammalian brain, GLT1, exists as at least three splice variants, GLT1a, GLT1b, and GLT1c. GLT1b interacts with the scaffold protein PICK1 (protein interacting with kinase C1), which is implicated in glutamatergic neurotransmission via its regulatory effect on trafficking of AMPA-type glutamate receptors. The 11 extreme C-terminal residues specific for the GLT1b variant are essential for its specific interaction with the PICK1 PDZ domain, but a functional consequence of this interaction has remained unresolved. To identify a functional effect of PICK1 on GLT1a or GLT1b separately, we employed the Xenopus laevis expression system. GLT1a and GLT1b displayed similar electrophysiological properties and EC₅₀ for glutamate. Co-expressed PICK1 localized efficiently to the plasma membrane and resulted in a 5-fold enhancement of the leak current in GLT1b-expressing oocytes with only a minor effect on [³H]glutamate uptake. Three different GLT1 substrates all caused a slow TBOA-sensitive decay in the membrane current upon prolonged application, which provides support for the leak current being mediated by GLT1b itself. Leak and glutamate-evoked currents in GLT1a-expressing oocytes were unaffected by PICK1 co-expression. PKC activation down-regulated GLT1a and GLT1b activity to a similar extent, which was not affected by co-expression of PICK1. In conclusion, PICK1 may not only affect glutamatergic neurotransmission by its regulatory effect on glutamate receptors but may also affect neuronal excitability via an increased GLT1b-mediated leak current. This may be particularly relevant in pathological conditions such as amyotrophic lateral sclerosis and cerebral hypoxia, which are associated with neuronal GLT1b up-regulation.     

Detection of a hidden folding intermediate of the third domain of PDZ

The folding pathway of the third domain of PDZ from the synaptic protein PSD-95 was characterized using kinetic and equilibrium methods by monitoring the fluorescence signal from a Trp residue that is incorporated at a near-surface position. Kinetic folding of this domain showed multiple exponential phases, whereas unfolding showed a single exponential phase. The slow kinetic phases were attributed to isomerization of proline residues, since there are five proline residues in this domain. We found that the logarithms of the rate constants for the fast phase of folding and unfolding are linearly dependent on the concentrations of denaturant. The unfolding free energy derived from these rate constants at zero denaturant was close to the value measured using the equilibrium method, suggesting the absence of detectable sub-millisecond folding intermediates. However, native-state hydrogen exchange experiments detected a partially unfolded intermediate under native conditions. It was further confirmed by a protein engineering study. These data suggest that a hidden intermediate exists after the rate-limiting step in the folding of the third domain of PDZ.