Precision genetic cellular models identify therapies protective against ER stress.

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.

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.

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

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.

Immunological memory to hyperphosphorylated tau in asymptomatic individuals.

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.

DNA delivery into mammalian cells using bacteriophage λ displaying the TAT transduction domain.

The ability to modulate cellular function by the transfer and expression of novel genes or by affecting the levels of endogenous proteins by genetic means has been of tremendous benefit in studying cellular functions and offers great promise in the treatment of a variety of diseases. Consequently, the development of novel and efficient nonviral DNA delivery systems is an important goal. Small cationic peptides, termed peptide/protein transduction domains (PTDs), effectively deliver a wide variety of cargoes, including DNA, into all cells.

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 (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.

Chronic exposure to sub-lethal beta-amyloid (Abeta) inhibits the import of nuclear-encoded proteins to mitochondria in differentiated PC12 cells.

Studies on amyloid beta (Abeta|), the peptide thought to play a crucial role in the pathogenesis of Alzheimer's disease, have implicated mitochondria in Abeta-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 Abeta on the import of nuclear-encoded proteins to mitochondria. Exposure to sub-lethal Abeta(25-35) (10 mumol/L) for 48 h inhibited mtGFP import to mitochondria; average rates decreased by 20 +/- 4%.

Transmembrane delivery of protein and peptide drugs by TAT-mediated transduction in the treatment of cancer.

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.

Cationic TAT peptide transduction domain enters cells by macropinocytosis.

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 caveolae, clathrin and dynamin.

Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis.

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.

Flow cytometry and GFP: a novel assay for measuring the import and turnover of nuclear-encoded mitochondrial proteins in live PC12 cells.

Background: Mitochondrial protein import is typically measured by adding radiolabeled precursor proteins to isolated mitochondria. We have developed a novel, high-throughput method for measuring protein import in live differentiated PC12 cells using a tetracycline (Tet) regulated, nuclear encoded, mitochondrially-targeted GFP fusion protein and flow cytometry.

Methods: We generated a PC12 cell line stably transfected with an inducible GFP fusion protein (GFPmt) targeted to mitochondria.

Modulation of cellular function by TAT mediated transduction of full length proteins.

Due to the barrier imposed by the cell membrane, delivery of macromolecules in excess of 500 Daltons directly into cells remains problematic. However, proteins, which have been evolutionarily selected to perform specific functions, are therefore an attractive therapeutic agent to treat a variety of human diseases. In practice, the direct intracellular delivery of these proteins has, until recently, been difficult to achieve due primarily to the bioavailability barrier of the plasma membrane, which effectively prevents the uptake of the majority of peptides and proteins by limiting their passive entry.

Protein transduction technology.

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.