Pathologic Prion Protein Infects Cells by Lipid-Raft Dependent Macropinocytosis

Transmissible spongiform encephalopathies, including variant-Creutzfeldt-Jakob disease (vCJD) in humans and bovine spongiform encephalopathies in cattle, are fatal neurodegenerative disorders characterized by protein misfolding of the host cellular prion protein (PrPC) to the infectious scrapie form (PrPSc). However, the mechanism that exogenous PrPSc infects cells and where pathologic conversion of PrPC to the PrPSc form occurs remains uncertain. Here we report that similar to the mechanism of HIV-1 TAT-mediated peptide transduction, processed mature, full length PrP contains a conserved N-terminal cationic domain that stimulates cellular uptake by lipid raft-dependent, macropinocytosis. Inhibition of macropinocytosis by three independent means prevented cellular uptake of recombinant PrP; however, it did not affect recombinant PrP cell surface association. In addition, fusion of the cationic N-terminal PrP domain to a Cre recombinase reporter protein was sufficient to promote both cellular uptake and escape from the macropinosomes into the cytoplasm. Inhibition of macropinocytosis was sufficient to prevent conversion of PrPC to the pathologic PrPSc form in N2a cells exposed to strain RML PrPSc infected brain homogenates, suggesting that a critical determinant of PrPC conversion occurs following macropinocytotic internalization and not through mere membrane association. Taken together, these observations provide a cellular mechanism that exogenous pathological PrPSc infects cells by lipid raft dependent, macropinocytosis

Cellular uptake of rPrP occurs by macropinocytosis.

<p>(A) N2a cells were pretreated with either 100 µM EIPA or 5 mM cytochalasin D for 30 min prior the addition of 2.0 µM fluorescently labeled recombinant rPrP-Alexa546. After 2 hr, cells were washed and analyzed by live cell confocal microscopy. Scale bar = 10 µm. (B) N2a cells were treated with 0.5 mg/mL 70 kDa neutral dextran-FITC for 30 min in the presence of increasing concentrations of rPrP (0, 0.25, 0.5, 1.0 or 2.0 µM). Fluid phase uptake of dextran was measured by flow cytometry (values±SD, Student's T-test: ** = p<0.02, *** = p<0.002). (C) N2a cells were treated with 0.5 mg/mL 70 kDa neutral dextran-FITC for 30 min in the presence of 1.0 µM TAT-Cre, rPrP (23–231), rPrP (30–321) or rPrP (100–231). Fluid phase uptake of dextran was measured by flow cytometry.</p

Cellular uptake of rPrP occurs by endocytosis.

<p>(A) Reporter cells containing <i>lox</i>P-STOP-<i>lox</i>P GFP reporter gene were treated with indicated proteins for 1 hr, incubated overnight, then assayed for GFP expression by flow cytometry (±SD). (B) Reporter cells were treated with rPrP (23–90)-Cre in the presence of heparin or chondroitin sulfate B, washed, incubated overnight, then assayed for GFP-positive cells by flow cytometry (±SD). (C) N2a cells were transfected with Cav1α-GFP expression plasmid, followed 48 hr later by treatment with rPrP-546 (red) and live cell confocal microscopy. Scale bar = 5 µm. (D) N2a cells co-transfected with pDyn^K44A-HA dominant-negative and pZ/EG <i>lox</i>P-stop-<i>lox</i>P GFP reporter plasmids were treated with rPrP (23–90)-Cre protein. Scale bar = 25 µm. (E) N2a cells co-transfected with pDyn^K44A-HA and pEGFP (constitutive GFP expression) plasmids (10∶1) were incubated with fluorescent transferrin-TMR (red) as a positive control for Dyn^K44A activity. Arrows indicate Dyn^K44A/EGFP transfected cells. Scale bar = 20 µm.</p

Pathological PrP^Sc conversion of PrP^C requires macropinocytosis.

<p>(A) N2aPK1 cells were exposed to 10⁻⁵ dilution of strain RML scrapie PrP^Sc-infected murine brain homogenate and increasing concentrations of the macropinocytosis inhibitor EIPA (25, 50 and 100 µM) for 48 hr. Infected cells were then grown on cover slips, blotted, digested with proteinase K (PK) and probed with anti-PrP antibodies (A) and Ponceau S staining (B).</p

Exogenous rPrP is endocytosed into cells and co-localizes with TAT-Cre.

<p>(A) Alignment of putative PrP^C transduction domain with the HIV-1 TAT PTD and schematic of rPrP-Cre recombinase fusion proteins. (B) Co-localization of rPrP (residues 23–231) and TAT-Cre. N2a cells were treated with rPrP-Alexa546 (red) and TAT-Cre-Alexa488 (green) for 1 hr, then assayed by live cell confocal microscopy. Yellow fluorescence indicates areas of co-localization. Scale bar = 5 µm. (C) N2a cells pre-incubated for 30 min with 50 µg/mL heparin or 5 mM nystatin followed by rPrP-Alexa546 protein incubation for 2 hr prevented surface binding and internalization, respectively. Scale bar = 10 µm.</p

Structural Basis for Recognition of a Unique Epitope by a Human Anti-tau Antibody

Aggregation of the hyperphosphorylated protein tau into neurofibrillary tangles and neuropil threads is a hallmark of Alzheimer disease (AD). Identification and characterization of the epitopes recognized by anti-tau antibodies might shed light on the molecular mechanisms of AD pathogenesis. Here we report on the biochemical and structural characterization of a tau-specific monoclonal antibody CBTAU-24.1, which was isolated from the human memory B cell repertoire. Immunohistochemical staining with CBTAU-24.1 specifically detects pathological tau structures in AD brain samples. The crystal structure of CBTAU-24.1 Fab with a phosphorylated tau peptide revealed recognition of a unique epitope (Ser235-Leu243) in the tau proline-rich domain. Interestingly, the antibody can bind tau regardless of phosphorylation state of its epitope region and also recognizes both monomeric and paired helical filament tau irrespective of phosphorylation status. This human anti-tau antibody and its unique epitope may aid in development of diagnostics and/or therapeutic AD strategies

Structural basis for recognition of the central conserved region of RSV G by neutralizing human antibodies

<div><p>Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in infants and the elderly, and yet there remains no effective treatment or vaccine. The surface of the virion is decorated with the fusion glycoprotein (RSV F) and the attachment glycoprotein (RSV G), which binds to CX3CR1 on human airway epithelial cells to mediate viral attachment and subsequent infection. RSV G is a major target of the humoral immune response, and antibodies that target the central conserved region of G have been shown to neutralize both subtypes of RSV and to protect against severe RSV disease in animal models. However, the molecular underpinnings for antibody recognition of this region have remained unknown. Therefore, we isolated two human antibodies directed against the central conserved region of RSV G and demonstrated that they neutralize RSV infection of human bronchial epithelial cell cultures in the absence of complement. Moreover, the antibodies protected cotton rats from severe RSV disease. Both antibodies bound with high affinity to a secreted form of RSV G as well as to a peptide corresponding to the unglycosylated central conserved region. High-resolution crystal structures of each antibody in complex with the G peptide revealed two distinct conformational epitopes that require proper folding of the cystine noose located in the C-terminal part of the central conserved region. Comparison of these structures with the structure of fractalkine (CX3CL1) alone or in complex with a viral homolog of CX3CR1 (US28) suggests that RSV G would bind to CX3CR1 in a mode that is distinct from that of fractalkine. Collectively, these results build on recent studies demonstrating the importance of RSV G in antibody-mediated protection from severe RSV disease, and the structural information presented here should guide the development of new vaccines and antibody-based therapies for RSV.</p></div

Crystal structure of Fab CB017.5 bound to an RSV G peptide.

<p>(A) Ribbon diagram of the Fab CB017.5 variable domain (Fv) in complex with the subtype A RSV G peptide. (B) Ribbon-and-stick representation of Fab CB017.5 in complex with the G peptide. Black dotted lines indicate hydrogen bonds and the red dotted line indicates a salt bridge. (C) Ribbon-and-stick representation of the G peptide and a molecular surface representation of Fab CB017.5, rotated 45° from the view in (B). In panels (A–C) the heavy chain is colored green, the light chain is colored white, and the G peptide is colored on a spectrum from yellow to red, N- to C-terminus, respectively. For stick models, oxygen atoms are colored red, nitrogen are blue, and sulfur are yellow.</p

Fabs CB017.5 and CB002.5 bind with high affinity to RSV G and a G peptide.

<p>Surface plasmon resonance (SPR) response curves of Fab CB002.5 (top) and Fab CB017.5 (bottom) binding to wild-type RSV sG from strain A2 (A) and a subtype A RSV G peptide encompassing the central conserved region (B). The raw data are plotted in black, and the calculated best fit to a 1:1 binding model is plotted in red. The equilibrium dissociation constant (<i>K</i>_D) for each interaction is displayed above the respective SPR curve. (C) Sequence alignment of the 45-residue G peptide and the corresponding region of RSV G from strains A2 and B1. The strictly conserved residues, the cystine noose, and CX3C motif are labeled.</p