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.
Naturally occurring and synthetic short arginine containing protein transduction domains (PTDs), including HIV1 TAT, poly-Arg and Antp, have been used to deliver a wide variety of macromolecular, ...biologically active therapeutic cargo into cells, including peptides, proteins, antisense oligonucleotides and liposomes, in vitro and to treat pre-clinical models of cancer and stroke. PTDs enter cells in a rapid, receptor-independent fashion. Recently, large TAT-fusion proteins (in excess of 30,000 Da) were shown to transduce into cells by fluid-phase macropinocytosis, a specialized form of endocytosis that is independent of caveloe, clathrin and dynamin. However, it remains controversial as to whether or not PTD peptides (1000–5000 Da) enter cells via macropinocytosis and/or through an unknown alternative mechanism. Due to strong ionic interactions with the cell surface, previous measurements of PTD peptide internalization were inaccurate. Cationic PTD peptides containing variable numbers of arginine residues and conditions entered cells exclusively through macropinocytosis. In addition, no PTD peptide was found to enter cells at 4 °C, a long held assumption of transduction. Taken together, these observations provide a solid scientific basis for the development of novel biologically active transducible anticancer PTD peptide therapeutics.
The TAT protein transduction domain (PTD) has been used to deliver a wide variety of biologically active cargo for the treatment of multiple preclinical disease models, including cancer and stroke. ...However, the mechanism of transduction remains unknown. Because of the TAT PTD's strong cell-surface binding, early assumptions regarding cellular uptake suggested a direct penetration mechanism across the lipid bilayer by a temperature- and energy-independent process. Here we show, using a transducible TAT-Cre recombinase reporter assay on live cells, that after an initial ionic cell-surface interaction, TAT-fusion proteins are rapidly internalized by lipid raft-dependent macropinocytosis. Transduction was independent of interleukin-2 receptor/raft-, caveolar- and clathrin-mediated endocytosis and phagocytosis. Using this information, we developed a transducible, pH-sensitive, fusogenic dTAT-HA2 peptide that markedly enhanced TAT-Cre escape from macropinosomes. Taken together, these observations provide a scientific basis for the development of new, biologically active, transducible therapeutic molecules.
The direct intracellular delivery of proteins, or active peptide domains, has, until recently, been difficult to achieve due primarily to the bioavailability barrier of the plasma membrane, which ...effectively prevents the uptake of macromolecules by limiting their passive entry. Traditional approaches to modulate protein function have largely relied on the serendipitous discovery of specific drugs and small molecules which could be delivered easily into the cell. However, the usefulness of these pharmacological agents is limited by their tissue distribution and unlike ‘information-rich’ macromolecules, they often suffer from poor target specificity, unwanted side-effects, and toxicity. Likewise, the development of molecular techniques, over the past several decades, for gene delivery and expression of proteins has provided for tremendous advances in our understanding of cellular processes but has been of surprisingly little benefit for the management of genetic disorders. Apart from these gains however, the transfer of genetic material into eukaryotic cells either using viral vectors or by non-viral mechanisms such as microinjection, electroporation, or chemical transfection remains problematic. Moreover, in vivo, gene therapy approaches relying on adenoviral vectors are associated with significant difficulties relating to a lack of target specificity and toxicity which have contributed to poor performance in several clinical trials. Remarkably, the recent identification of a particular group of proteins with enhanced ability to cross the plasma membrane in a receptor-independent fashion has led to the discovery of a class of protein domains with cell membrane penetrating properties. The fusion of these protein transduction domain peptide sequences with heterologous proteins is sufficient to cause their rapid transduction into a variety of different cells in a rapid, concentration-dependent manner. Moreover, this novel technique for protein and peptide delivery appears to circumvent many problems associated with DNA and drug based methods. This technique may represent the next paradigm in our ability to modulate cell function and offers a unique avenue for the treatment of disease.
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 (PrP(C)) to the infectious scrapie form (PrP(Sc)). However, the mechanism that exogenous PrP(Sc) infects cells and where pathologic conversion of PrP(C) to the PrP(Sc) 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 PrP(C) to the pathologic PrP(Sc) form in N2a cells exposed to strain RML PrP(Sc) infected brain homogenates, suggesting that a critical determinant of PrP(C) conversion occurs following macropinocytotic internalization and not through mere membrane association. Taken together, these observations provide a cellular mechanism that exogenous pathological PrP(Sc) infects cells by lipid raft dependent, macropinocytosis.
Protein transduction technology Wadia, Jehangir S; Dowdy, Steven F
Current Opinion in Biotechnology,
02/2002, Letnik:
13, Številka:
1
Book Review, Journal Article
Recenzirano
Intracellular delivery of macromolecules remains problematic because of the bioavailability restriction imposed by the cell membrane. Recent studies on protein transduction domains have circumvented ...this barrier, however, and have resulted in the delivery of peptides, full-length proteins, iron beads, liposomes, and radioactive isotopes into cells in culture and animal models
in vivo.
Recent studies on protein transduction domains have resulted in the delivery of peptides, full-length proteins, iron beads, liposomes, and radioactive isotopes into cells in culture and animal models in vivo.
Rare monogenic disorders often share molecular etiologies involved in the pathogenesis of common diseases. Congenital disorders of glycosylation (CDG) and deglycosylation (CDDG) are rare pediatric ...disorders with symptoms that range from mild to life threatening. A biological mechanism shared among CDG and CDDG as well as more common neurodegenerative diseases such as Alzheimer's disease and amyotrophic lateral sclerosis, is endoplasmic reticulum (ER) stress. We developed isogenic human cellular models of two types of CDG and the only known CDDG to discover drugs that can alleviate ER stress. Systematic phenotyping confirmed ER stress and identified elevated autophagy among other phenotypes in each model. We screened 1049 compounds and scored their ability to correct aberrant morphology in each model using an agnostic cell-painting assay based on >300 cellular features. This primary screen identified multiple compounds able to correct morphological phenotypes. Independent validation shows they also correct cellular phenotypes and alleviate each of the ER stress markers identified in each model. Many of the active compounds are associated with microtubule dynamics, which points to new therapeutic opportunities for both rare and more common disorders presenting with ER stress, such as Alzheimer's disease and amyotrophic lateral sclerosis.
Studies on amyloid beta (Aβ|), the peptide thought to play a crucial role in the pathogenesis of Alzheimer's disease, have implicated mitochondria in Aβ-mediated neurotoxicity. We used differentiated ...PC12 cells stably transfected with an inducible green fluorescent protein (GFP) fusion protein containing an N'-terminal mitochondrial targeting sequence (mtGFP), to examine the effects of sub-lethal Aβ on the import of nuclear-encoded proteins to mitochondria. Exposure to sub-lethal Aβ₂₅₋₃₅ (10 μmol/L) for 48 h inhibited mtGFP import to mitochondria; average rates decreased by 20 ± 4%. Concomitant with the decline in mtGFP, cytoplasmic mtGFP increased significantly while mtGFP expression and intramitochondrial mtGFP turnover were unchanged. Sub-lethal Aβ₁₋₄₂ inhibited mtGFP import and increased cytoplasmic mtGFP but only after 96 h. The import of two endogenous nuclear-encoded mitochondrial proteins, mortalin/mtHsp70 and Tom20 also declined. Prior to the decline in import, mitochondrial membrane potential (mmp), and reactive oxygen species levels were unchanged in Aβ-treated cells versus reverse phase controls. Sustained periods of decreased import were associated with decreased mmp, increased reactive oxygen species, increased vulnerability to oxygen-glucose deprivation and altered mitochondrial morphology. These findings suggest that an Aβ-mediated inhibition of mitochondrial protein import, and the consequent mitochondrial impairment, may contribute to Alzheimer's disease.
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.
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•A tau-specific antibody CBTAU-24.1 was isolated from human memory B cells•The CBTAU-24.1 crystal structure with a tau peptide identifies a unique tau epitope•CBTAU-24.1interacts with PHF-tau in neurofibrillary tangles and neuropil threads•CBTAU-24.1 recognizes tau irrespective of phosphorylation in its epitope
Antibodies targeting the tau protein can be used in immunotherapy or as diagnostic tools in Alzheimer disease. Zhang et al. report the biochemical, functional, and structural characterization of a human anti-tau antibody. Antibody CBTAU-24.1 recognizes a unique epitope in the proline-rich domain of tau and specifically detects pathological tau structures.
Several reports have described the presence of antibodies against Alzheimer’s disease-associated hyperphosphorylated forms of tau in serum of healthy individuals. To characterize the specificities ...that can be found, we interrogated peripheral IgG
+
memory B cells from asymptomatic blood donors for reactivity to a panel of phosphorylated tau peptides using a single-cell screening assay. Antibody sequences were recovered, cloned, and expressed as full-length IgGs. In total, 52 somatically mutated tau-binding antibodies were identified, corresponding to 35 unique clonal families. Forty-one of these antibodies recognize epitopes in the proline-rich and C-terminal domains, and binding of 26 of these antibodies is strictly phosphorylation dependent. Thirteen antibodies showed inhibitory activity in a P301S lysate seeded in vitro tau aggregation assay. Two such antibodies, CBTAU-7.1 and CBTAU-22.1, which bind to the proline-rich and C-terminal regions of tau, respectively, were characterized in more detail. CBTAU-7.1 recognizes an epitope that is similar to that of murine anti-PHF antibody AT8, but has different phospho requirements. Both CBTAU-7.1 and CBTAU-22.1 detect pathological tau deposits in post-mortem brain tissue. CBTAU-7.1 reveals a similar IHC distribution pattern as AT8, immunostaining (pre)tangles, threads, and neuritic plaques. CBTAU-22.1 shows selective detection of neurofibrillary changes by IHC. Taken together, these results suggest the presence of an ongoing antigen-driven immune response against tau in healthy individuals. The wide range of specificities to tau suggests that the human immune repertoire may contain antibodies that can serve as biomarkers or be exploited for therapy.
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