Traffic of prion protein between different compartments on the neuronal surface, and the propagation of prion disease

The key mechanism in prion disease is the conversion of cellular prion protein into an altered, pathogenic conformation, in which cellular mechanisms play a poorly understood role. Both forms of prion protein are lipid-anchored and reside in rafts that appear to protect the native conformation against conversion. Neurons rapidly traffic their cellular prion protein out of its lipid rafts to be endocytosed via coated pits before recycling back to the cell surface. It is argued in this review that understanding the mechanism of this trafficking holds the key to understanding the cellular role in the conformational conversion of prion protein.     

The evolution of putative starch-binding domains

The present bioinformatics analysis was focused on the starch-binding domains (SBDs) and SBD-like motifs sequentially related to carbohydrate-binding module (CBM) families CBM20 and CBM21. Originally, these SBDs were known from microbial amylases only. At present homologous starch- and glycogen-binding domains (or putative SBD sequences) have been recognised in various plant and animal proteins. The sequence comparison clearly showed that the SBD-like sequences in genethonin-1, starch synthase III and glucan branching enzyme should possess the real SBD function since the two tryptophans (or at least two aromatics) of the typical starch-binding site 1 are conserved in their sequences. The same should apply also for the sequences corresponding with the so-called KIS-domain of plant AKINbetagamma protein that is a homologue of the animal AMP-activated protein kinase (AMPK). The evolutionary tree classified the compared SBDs into three distinct groups: (i) the family CBM20 (the motifs from genethonins, laforins, starch excess 4 protein, beta-subunits of the animal AMPK and all plant and yeast homologues, and eventually from amylopullulanases); (ii) the family CBM21 (the motifs from regulatory subunits of protein phosphatase 1 together with those from starch synthase III); and (iii) the (CBM20+CBM21)-related group (the motifs from the pullulanase subfamily consisting of pullulanase, branching enzyme, isoamylase and maltooligosyl trehalohydrolase).     

Substrate recognition and transport by multidrug resistance protein 1 (ABCC1)

Multidrug resistance protein (MRP) 1 belongs to the 'C' branch of the ABC transporter superfamily. MRP1 is a high-affinity transporter of the cysteinyl leukotriene C(4) and is responsible for the systemic release of this cytokine in response to an inflammatory stimulus. However, the substrate specificity of MRP1 is extremely broad and includes many organic anion conjugates of structurally unrelated endo- and xenobiotics. In addition, MRP1 transports unmodified hydrophobic compounds, such as natural product type chemotherapeutic agents and mutagens, such as aflatoxin B(1). Transport of several of these compounds has been shown to be dependent on the presence of reduced glutathione (GSH). More recently, GSH has also been shown to stimulate the transport of some conjugated compounds, including sulfates and glucuronides. Here, we summarize current knowledge of the substrate specificity and modes of transport of MRP1 and discuss how the protein may recognize its structurally diverse substrates.     

Construction of a fully active truncated alternansucrase partially deleted of its carboxy-terminal domain

Recombinant expression of the large alternansucrase (2057 amino acids) was hindered in E. coli due to poor enzyme solubility and protein degradation. The effects of deletions of the alternansucrase C-terminal CW-like and APY repeated motifs on enzyme solubility and specificity were investigated. A truncated variant deleted of the APY repeats but harboring four C-terminal CW-like repeats displayed a high specific activity and the same specificity of product synthesis as the native enzyme. It is more soluble and suffers less degradation than full length alternansucrase. Hence this truncated variant is a promising tool for the further structural and kinetic study of this interesting enzyme.     

Protein tyrosine phosphatase receptor type Z is inactivated by ligand-induced oligomerization

Receptor-type protein tyrosine phosphatases (RPTPs) are considered to transduce extracellular signals across the membrane through changes in their PTP activity, however, our understanding of the regulatory mechanism is still limited. Here, we show that pleiotrophin (PTN), a natural ligand for protein tyrosine phosphatase receptor type Z (Ptprz) (also called PTPzeta/RPTPbeta), inactivates Ptprz through oligomerization and increases the tyrosine phosphorylation of substrates for Ptprz, G protein-coupled receptor kinase-interactor 1 (Git1) and membrane associated guanylate kinase, WW and PDZ domain containing 1 (Magi1). Oligomerization of Ptprz by an artificial dimerizer or polyclonal antibodies against its extracellular region also leads to inactivation, indicating that Ptprz is active in the monomeric form and inactivated by ligand-induced oligomerization.     

Caspase-8 acts as a key upstream executor of mitochondria during justicidin A-induced apoptosis in human hepatoma cells

Justicia procumbens is a traditional Taiwanese herbal remedy used to treat fever, pain, and cancer. Justicidin A, isolated from Justicia procumbens, has been reported to suppress in vitro growth of several tumor cell lines as well as hepatoma cells. In this study, justicidin A activated caspase-8 to increase tBid, disrupted mitochondrial membrane potential (Delta psi(m)), and caused the release of cytochrome c and Smac/DIABLO in Hep 3B and Hep G2 cells. Justicidin A also reduced Bcl-x(L) and increased Bax and Bak in mitochondria. Caspase-8 inhibitor (Z-IETD) attenuated the justicidin A-induced disruption of Delta psi(m). Growth of Hep 3B implanted in NOD-SCID mice was suppressed significantly by oral justicidin A (20 mg/kg/day). These results indicate that justicidin A-induced apoptosis in these cells proceeds via caspase-8 and is followed by mitochondrial disruption.     

In vivo tracing of canonical Wnt signaling in Xenopus tadpoles by means of an inducible transgenic reporter tool

The canonical Wnt pathway is recurrently used during embryogenesis and adult life. To track the cellular output of Wnt signaling in a living organism, we designed a hormone-inducible Wnt responsive system, capable to dynamically and specifically report Wnt pathway activities through eGFP expression. In contrast to previous in vivo reporters, our system essentially avoids interference of consecutive signals by remaining dormant until addition of hormone, which makes it a valuable tool to map canonical Wnt signaling in post-embryonic stages. Transgenic Xenopus laevis embryos were analyzed revealing at tadpole stage in specific tissues and organs cell populations with high Wnt pathway activity.     

Characterization of osteoprotegerin binding to glycosaminoglycans by surface plasmon resonance: role in the interactions with receptor activator of nuclear factor kappaB ligand (RANKL) and RANK

Osteoprotegerin (OPG) is a decoy receptor for receptor activator of nuclear factor kappaB ligand (RANKL), a key inducer of osteoclastogenesis via its receptor RANK. We previously showed that RANK, RANKL, and OPG are able to form a tertiary complex and that OPG must be also considered as a direct effector of osteoclast functions. As OPG contains a heparin-binding domain, the present study investigated the interactions between OPG and glycosaminoglycans (GAGs) by surface plasmon resonance and their involvement in the OPG functions. Kinetic data demonstrated that OPG binds to heparin with a high-affinity (KD: 0.28 nM) and that the pre-incubation of OPG with heparin inhibits in a dose-dependent manner the OPG binding to the complex RANK-RANKL. GAGs from different structure/origin (heparan sulfate, dermatan sulfate, and chondroitin sulfate) exert similar activity on OPG binding. The contribution of the sulfation pattern and the size of the oligosaccharide were determined in this inhibitory mechanism. The results demonstrated that sulfation is essential in the OPG-blocking function of GAGs since a totally desulfated heparin loses its capacity to bind and to block OPG binding to RANKL. Moreover, a decasaccharide is the minimal structure that totally inhibits the OPG binding to the complex RANK-RANKL. Western blot analysis performed in 293 cells surexpressing RANKL revealed that the pre-incubation of OPG with these GAGs strongly inhibits the OPG-induced decrease of membrane RANKL half-life. These data support an essential function of the related glycosaminoglycans heparin and heparan sulfate in the activity of the triad RANK-RANKL-OPG.     

The Homer-1 protein Ania-3 interacts with the plasma membrane calcium pump

The Homer family of scaffold proteins couples NMDA receptors to metabotropic glutamate receptors and links extracellular signals to calcium release from intracellular stores. Ania-3 is a member of the Homer family and is rapidly inducible in brain in response to diverse stimuli. Here, we report the identification of the plasma membrane Ca2+ ATPase (PMCA) as a novel Ania-3/Homer-associated protein. Ania-3/Homer interacts with the b-splice forms of all PMCAs (PMCA1b, 2b, 3b, and 4b) via their PDZ domain-binding COOH-terminal tail. Ectopically expressed Ania-3 colocalized with the PMCA at the plasma membrane of polarized MDCK epithelial cells, and endogenous Ania-3/Homer and PMCA2 are co-expressed in the soma and dendrites of primary rat hippocampal neurons. The interaction between Ania-3/Homer and PMCAs may represent a novel mechanism by which local calcium signaling and hence synaptic function can be modulated in neurons.