Learnings about Aβ from human brain recommend the use of a live-neuron bioassay for the discovery of next generation Alzheimer's disease immunotherapeutics.

Despite ongoing debate, the amyloid β-protein (Aβ) remains the prime therapeutic target for the treatment of Alzheimer's disease (AD). However, rational drug design has been hampered by a lack of knowledge about neuroactive Aβ. To help address this deficit, we developed live-cell imaging of iPSC-derived human neurons (iNs) to study the effects of the most disease relevant form of Aβ-oligomeric assemblies (oAβ) extracted from AD brain.

A calcium-sensitive antibody isolates soluble amyloid-β aggregates and fibrils from Alzheimer's disease brain.

Aqueously soluble oligomers of amyloid-β peptide may be the principal neurotoxic forms of amyloid-β in Alzheimer's disease, initiating downstream events that include tau hyperphosphorylation, neuritic/synaptic injury, microgliosis and neuron loss. Synthetic oligomeric amyloid-β has been studied extensively, but little is known about the biochemistry of natural oligomeric amyloid-β in human brain, even though it is more potent than simple synthetic peptides and comprises truncated and modified amyloid-β monomers. We hypothesized that monoclonal antibodies specific to neurotoxic oligomeric amyloid-β could be used to isolate it for further study.

biAb Mediated Restoration of the Linkage between Dystroglycan and Laminin-211 as a Therapeutic Approach for α-Dystroglycanopathies.

Patients with α-dystroglycanopathies, a subgroup of rare congenital muscular dystrophies, present with a spectrum of clinical manifestations that includes muscular dystrophy as well as CNS and ocular abnormalities. Although patients with α-dystroglycanopathies are genetically heterogeneous, they share a common defect of aberrant post-translational glycosylation modification of the dystroglycan alpha-subunit, which renders it defective in binding to several extracellular ligands such as laminin-211 in skeletal muscles, agrin in neuromuscular junctions, neurexin in the CNS, and pikachurin in the eye, leading to various symptoms. The genetic heterogeneity associated with the development of α-dystroglycanopathies poses significant challenges to developing a generalized treatment to address the spectrum of genetic defects.

SAR228810: an antibody for protofibrillar amyloid β peptide designed to reduce the risk of amyloid-related imaging abnormalities (ARIA).

Background: Anti-amyloid β (Aβ) immunotherapy represents a major area of drug development for Alzheimer's disease (AD). However, Aβ peptide adopts multiple conformations and the pathological forms to be specifically targeted have not been identified. Aβ immunotherapy-related vasogenic edema has also been severely dose limiting for antibodies with effector functions binding vascular amyloid such as bapineuzumab.

Reduction of inflammation and preservation of neurological function by anti-CD52 therapy in murine experimental autoimmune encephalomyelitis.

Alemtuzumab, a monoclonal antibody directed against human CD52, is used in the treatment of MS. To characterize the impact of anti-CD52 administration, a monoclonal antibody to mouse CD52 (anti-muCD52) was generated and evaluated in EAE mouse models of MS. A single course of anti-muCD52 provided a therapeutic benefit accompanied by a reduction in the frequency of autoreactive T lymphocytes and production of pro-inflammatory cytokines.

Capture-stabilize approach for membrane protein SPR assays.

Measuring the binding kinetics of antibodies to intact membrane proteins by surface plasmon resonance has been challenging largely because of the inherent difficulties in capturing membrane proteins on chip surfaces while retaining their native conformation. Here we describe a method in which His-tagged CXCR5, a GPCR, was purified and captured on a Biacore chip surface via the affinity tag. The captured receptor protein was then stabilized on the chip surface by limited cross-linking.

Erythrocytes encapsulated with phenylalanine hydroxylase exhibit improved pharmacokinetics and lowered plasma phenylalanine levels in normal mice.

Enzyme replacement therapy is often hampered by the rapid clearance and degradation of the administered enzyme, limiting its efficacy and requiring frequent dosing. Encapsulation of therapeutic molecules into red blood cells (RBCs) is a clinically proven approach to improve the pharmacokinetics and efficacy of biologics and small molecule drugs. Here we evaluated the ability of RBCs encapsulated with phenylalanine hydroxylase (PAH) to metabolize phenylalanine (Phe) from the blood and confer sustained enzymatic activity in the circulation.

LIMP-2 is a receptor for lysosomal mannose-6-phosphate-independent targeting of beta-glucocerebrosidase.

beta-glucocerebrosidase, the enzyme defective in Gaucher disease, is targeted to the lysosome independently of the mannose-6-phosphate receptor. Affinity-chromatography experiments revealed that the lysosomal integral membrane protein LIMP-2 is a specific binding partner of beta-glucocerebrosidase. This interaction involves a coiled-coil domain within the lumenal domain.

Effect of mannose chain length on targeting of glucocerebrosidase for enzyme replacement therapy of Gaucher disease.

Recombinant human glucocerebrosidase (imiglucerase, Cerezyme) is used in enzyme replacement therapy for Gaucher disease. Complex oligosaccharides present on Chinese hamster ovary cell-expressed glucocerebrosidase (GCase) are enzymatically remodeled into a mannose core, facilitating mannose receptor-mediated uptake into macrophages. Alternative expression systems could be used to produce GCase containing larger oligomannose structures, offering the possibility of an improvement in targeting to macrophages.

Chemical chaperones and permissive temperatures alter localization of Gaucher disease associated glucocerebrosidase variants.

Point mutations in the lysosomal hydrolase, glucocerebrosidase (GC), can cause Gaucher disease, a common lysosomal storage disease. Several clinically important GC mutations impede folding in the endoplasmic reticulum (ER) and target these enzymes for ER-associated degradation (ERAD). The removal of these misfolded proteins decreases the lysosomal concentration of GC, which results in glucosylceramide accumulation.

EPI64 regulates microvillar subdomains and structure.

EPI64 is a TBC domain-containing protein that binds the PDZ domains of EBP50, which binds ezrin, a major actin-binding protein of microvilli. High-resolution light microscopy revealed that ezrin and EBP50 localize exclusively to the membrane-surrounded region of microvilli, whereas EPI64 localizes to variable regions in the structures. Overexpressing EPI64 results in its and EBP50's relocalization to the base of microvilli, including to the actin rootlet devoid of ezrin or plasma membrane.

Protein tyrosine phosphatase PRL-3 in malignant cells and endothelial cells: expression and function.

Protein tyrosine phosphatase PRL-3 mRNA was found highly expressed in colon cancer endothelium and metastases. We sought to associate a function with PRL-3 expression in both endothelial cells and malignant cells using in vitro models. PRL-3 mRNA levels were determined in several normal human endothelial cells exposed or unexposed to the phorbol ester phorbol 12-myristate 13-acetate (PMA) and in 27 human tumor cell lines.

Secreted frizzled-related protein 4 is a potent tumor-derived phosphaturic agent.

Tumors associated with osteomalacia elaborate the novel factor(s), phosphatonin(s), which causes phosphaturia and hypophosphatemia by cAMP-independent pathways. We show that secreted frizzled-related protein-4 (sFRP-4), a protein highly expressed in such tumors, is a circulating phosphaturic factor that antagonizes renal Wnt-signaling. In cultured opossum renal epithelial cells, sFRP-4 specifically inhibited sodium-dependent phosphate transport.

Distinct cell type-specific expression of scaffolding proteins EBP50 and E3KARP: EBP50 is generally expressed with ezrin in specific epithelia, whereas E3KARP is not.

The ezrin/radixin/moesin (ERM) proteins are regulated microfilament membrane linking proteins. Previous tissue localization studies have revealed that the three related proteins show distinct tissue distributions, with ezrin being found predominantly in polarized epithelial cells, whereas moesin is enriched in endothelial cells and lymphocytes. EBP50 and E3KARP are two related scaffolding proteins that bind to the activated form of ERM proteins in vitro, and through their PDZ domains to the cytoplasmic domains of specific membrane proteins, including the Na+/H+ exchanger isoform (NHE3) present in kidney proximal tubules and the beta2-adrenergic receptor.

Identification of two distinct structural motifs that, when added to the C-terminal tail of the rat LH receptor, redirect the internalized hormone-receptor complex from a degradation to a recycling pathway.

We show that most of the internalized rat LH receptor is routed to a lysosomal degradation pathway whereas a substantial portion of the human LH receptor is routed to a recycling pathway. Chimeras of these two receptors identified a linear amino acid sequence (GTALL) present near the C terminus of the human LH receptor that, when grafted onto the rat LH receptor, redirects most of the rat LH receptor to a recycling pathway. Removal of the GTALL sequence from the human LH receptor failed to affect its routing, however.

Identification of EPI64, a TBC/rabGAP domain-containing microvillar protein that binds to the first PDZ domain of EBP50 and E3KARP.

The cortical scaffolding proteins EBP50 (ERM-binding phosphoprotein-50) and E3KARP (NHE3 kinase A regulatory protein) contain two PDZ (PSD-95/DlgA/ZO-1-like) domains followed by a COOH-terminal sequence that binds to active ERM family members. Using affinity chromatography, we identified polypeptides from placental microvilli that bind the PDZ domains of EBP50. Among these are 64- and/or 65-kD differentially phosphorylated polypeptides that bind preferentially to the first PDZ domain of EBP50, as well as to E3KARP, and that we call EPI64 (EBP50-PDZ interactor of 64 kD).

Hierarchy of merlin and ezrin N- and C-terminal domain interactions in homo- and heterotypic associations and their relationship to binding of scaffolding proteins EBP50 and E3KARP.

The neurofibromatosis 2 tumor suppressor gene product merlin has strong sequence identity to the ezrin-radixin-moesin (ERM) family over its approximately 300-residue N-terminal domain. ERM proteins are membrane cytoskeletal linkers that are negatively regulated by an intramolecular association between domains known as NH(2)- and COOH-ERM association domains (N- and C-ERMADs) that mask sites for binding membrane-associated proteins, such as EBP50 and E3KARP, and F-actin. Here we show that merlin has self-association regions analogous to the N- and C-ERMADs.

ERM-Merlin and EBP50 protein families in plasma membrane organization and function.

The ezrin-radixin-moesin (ERM) family of proteins have emerged as key regulatory molecules in linking F-actin to specific membrane proteins, especially in cell surface structures. Merlin, the product of the NF2 tumor suppressor gene, has sequence similarity to ERM proteins and binds to some of the same membrane proteins, but lacks a C-terminal F-actin binding site. In this review we discuss how ERM proteins and merlin are negatively regulated by an intramolecular association between their N- and C-terminal domains.

Structure of the ERM protein moesin reveals the FERM domain fold masked by an extended actin binding tail domain.

The ezrin-radixin-moesin (ERM) protein family link actin filaments of cell surface structures to the plasma membrane, using a C-terminal F-actin binding segment and an N-terminal FERM domain, a common membrane binding module. ERM proteins are regulated by an intramolecular association of the FERM and C-terminal tail domains that masks their binding sites. The crystal structure of a dormant moesin FERM/tail complex reveals that the FERM domain has three compact lobes including an integrated PTB/PH/ EVH1 fold, with the C-terminal segment bound as an extended peptide masking a large surface of the FERM domain.

A kinase-regulated PDZ-domain interaction controls endocytic sorting of the beta2-adrenergic receptor.

A fundamental question in cell biology is how membrane proteins are sorted in the endocytic pathway. The sorting of internalized beta2-adrenergic receptors between recycling endosomes and lysosomes is responsible for opposite effects on signal transduction and is regulated by physiological stimuli. Here we describe a mechanism that controls this sorting operation, which is mediated by a family of conserved protein-interaction modules called PDZ domains.

C-terminal threonine phosphorylation activates ERM proteins to link the cell's cortical lipid bilayer to the cytoskeleton.

The plasma membrane consists of a lipid bilayer with integral membrane proteins stabilized by regulated linkages to the cortical actin cytoskeleton. The regulation is necessary for cells to change shape ormigrate. The ERM (ezrin-radixin-moesin) proteins are believed to provide such links, with the N-terminal halves associating with integral membrane proteins, either directly or indirectly through adapter molecules like EBP50 (ERM binding phosphoprotein, 50 kDa), and their C-terminal halves associating with F-actin.

An apical PDZ protein anchors the cystic fibrosis transmembrane conductance regulator to the cytoskeleton.

The function of the cystic fibrosis transmembrane conductance regulator (CFTR) as a Cl- channel in the apical membrane of epithelial cells is extensively documented. However, less is known about the molecular determinants of CFTR residence in the apical membrane, basal regulation of its Cl- channel activity, and its reported effects on the function of other transporters. These aspects of CFTR function likely require specific interactions between CFTR and unknown proteins in the apical compartment of epithelial cells.

The carboxyl-terminal region of EBP50 binds to a site in the amino-terminal domain of ezrin that is masked in the dormant molecule.

EBP50 (ezrin-radixin-moesin-binding phosphoprotein 50) was recently identified by affinity chromatography on the immobilized NH2-terminal domain of ezrin. Here we map and characterize the regions in EBP50 and ezrin necessary for this association. Using blot overlays and in solution binding assays, the COOH-terminal 30 residues of EBP50 were found to be sufficient for an association with residues 1-286 of ezrin.

Ezrin: a protein requiring conformational activation to link microfilaments to the plasma membrane in the assembly of cell surface structures.

The cortical cytoskeleton of eucaryotic cells provides structural support to the plasma membrane and also contributes to dynamic processes such as endocytosis, exocytosis, and transmembrane signaling pathways. The ERM (ezrin-radixin-moesin) family of proteins, of which ezrin is the best studied member, play structural and regulatory roles in the assembly and stabilization of specialized plasma membrane domains. Ezrin and related molecules are concentrated in surface projections such as microvilli and membrane ruffles where they link the microfilaments to the membrane.

Identification of EBP50: A PDZ-containing phosphoprotein that associates with members of the ezrin-radixin-moesin family.

Members of the ezrin-radixin-moesin (ERM) family of membrane-cytoskeletal linking proteins have NH2- and COOH-terminal domains that associate with the plasma membrane and the actin cytoskeleton, respectively. To search for ERM binding partners potentially involved in membrane association, tissue lysates were subjected to affinity chromatography on the immobilized NH2-terminal domains of ezrin and moesin, which comprise the ezrin-radixin-moesin-association domain (N-ERMAD). A collection of polypeptides at 50-53 kD from human placenta and at 58-59 kD from bovine brain bound directly to both N-ERMADs.