An in vitro model for vitamin A transport across the human blood-brain barrier.

Vitamin A, supplied by the diet, is critical for brain health, but little is known about its delivery across the blood-brain barrier (BBB). Brain microvascular endothelial-like cells (BMECs) differentiated from human-derived induced pluripotent stem cells (iPSCs) form a tight barrier that recapitulates many of the properties of the human BBB. We paired iPSC-derived BMECs with recombinant vitamin A serum transport proteins, retinol-binding protein (RBP), and transthyretin (TTR), to create an in vitro model for the study of vitamin A (retinol) delivery across the human BBB.

An in vitro model for vitamin A transport across the human blood-brain barrier.

Vitamin A, supplied by the diet, is critical for brain health, but little is known about its delivery across the blood-brain barrier (BBB). Brain microvascular endothelial-like cells (BMECs) differentiated from human-derived induced pluripotent stem cells (iPSC) form a tight barrier that recapitulates many of the properties of the human BBB. We paired iPSC-derived BMECs with recombinant vitamin A serum transport proteins, retinol binding protein (RBP) and transthyretin (TTR), to create an model for the study of vitamin A (retinol) delivery across the human BBB.

Necroptosis is associated with Rab27-independent expulsion of extracellular vesicles containing RIPK3 and MLKL.

Extracellular vesicle (EV) secretion is an important mechanism used by cells to release biomolecules. A common necroptosis effector-mixed lineage kinase domain like (MLKL)-was recently found to participate in the biogenesis of small and large EVs independent of its function in necroptosis. The objective of the current study is to gain mechanistic insights into EV biogenesis during necroptosis.

Nanoparticle tracking analysis and statistical mixture distribution analysis to quantify nanoparticle-vesicle binding.

Nanoparticle tracking analysis (NTA) is a single particle tracking technique that in principle provides a more direct measure of particle size distribution compared to dynamic light scattering (DLS). Here, we demonstrate how statistical mixture distribution analysis can be used in combination with NTA to quantitatively characterize the amount and extent of particle binding in a mixture of nanomaterials. The combined approach is used to study the binding of gold nanoparticles to two types of phospholipid vesicles, those containing and lacking the model ion channel peptide gramicidin A.

Membrane Remodeling and Stimulation of Aggregation Following α-Synuclein Adsorption to Phosphotidylserine Vesicles.

α-Synuclein is an intrinsically disordered protein abundant in presynaptic terminals in neurons and in synaptic vesicles. α-Synuclein's interaction with lipid bilayers is important not only for its normal physiological function but also in its pathological aggregation and deposition as Lewy bodies in Parkinson's disease. α-Synuclein binds preferentially to lipids with acidic head groups and to high-curvature vesicles and can modulate membrane curvature.

Retinol binding protein IV purified from Escherichia coli using intein-mediated cleavage as a suitable replacement for serum sources.

Retinol binding protein IV (RBP) functions as the principal carrier of retinol (Vitamin A) in the blood, where RBP circulates bound to another serum protein, transthyretin. Isolation of pure RBP from the transthyretin complex in human serum can be difficult, but expression of RBP in recombinant systems can circumvent these purification issues. Human recombinant RBP has previously been successfully expressed and purified from E.

ROSETTA-informed design of structurally stabilized cyclic anti-amyloid peptides.

β-amyloid oligomers are thought to be the most toxic species formed en route to fibril deposition in Alzheimer's disease. Transthyretin is a natural sequestering agent of β-amyloid oligomers: the binding site to β-amyloid has been traced to strands G/H of the inner β-sheet of transthyretin. A linear peptide, with the same primary sequence as the β-amyloid binding domain on transthyretin, was moderately effective at inhibiting β-amyloid fibril growth.

Evaluation of Nanoparticle Tracking Analysis for the Detection of Rod-Shaped Particles and Protein Aggregates.

Nanoparticle tracking analysis (NTA) is an important technique for measuring hydrodynamic size of globular biological particles including liposomes and viruses. Less attention has been paid to NTA of rod-like particles, despite their considerable interest. For example, amyloid fibrils and protofibrils are protein aggregates with rod-like morphology, diameters of 2-15 nm, and lengths from 50 nm to 1 μm, and linked to diseases including Alzheimer's and Parkinson's.

Retinol-Binding Protein Interferes with Transthyretin-Mediated β-Amyloid Aggregation Inhibition.

β-Amyloid (Aβ) aggregation is causally linked to Alzheimer's disease. On the basis of in vitro and transgenic animal studies, transthyretin (TTR) is hypothesized to provide neuroprotection against Aβ toxicity by binding to Aβ and inhibiting its aggregation. TTR is a homotetrameric protein that circulates in blood and cerebrospinal fluid; its normal physiological role is as a carrier for thyroxine and retinol-binding protein (RBP).

Nanoparticle Tracking for Protein Aggregation Research.

Protein aggregates are the pathological agents in several neurodegenerative disorders such as Alzheimer's and Huntington's disease. In the pharmaceutical industry, protein aggregation poses significant challenges to the manufacture of biologics. Nanoparticle tracking is an emerging technology that allows particle-by-particle measurement of aggregate size and concentration.

Transthyretin Mimetics as Anti-β-Amyloid Agents: A Comparison of Peptide and Protein Approaches.

β-Amyloid (Aβ) aggregation is causally linked to neuronal pathology in Alzheimer's disease; therefore, several small molecules, antibodies, and peptides have been tested as anti-Aβ agents. We developed two compounds based on the Aβ-binding domain of transthyretin (TTR): a cyclic peptide cG8 and an engineered protein mTTR, and compared them for therapeutically relevant properties. Both mTTR and cG8 inhibit fibrillogenesis of Aβ, with mTTR inhibiting at a lower concentration than cG8.

Cerebrospinal Fluid Proteins as Regulators of Beta-amyloid Aggregation and Toxicity.

Amyloid disorders, such as Alzheimer's, are almost invariably late-onset diseases. One defining diagnostic feature of Alzheimer's disease is the deposition of beta-amyloid as extracellular plaques, primarily in the hippocampus. This raises the question: are there natural protective agents that prevent beta-amyloid from depositing, and is it loss of this protection that leads to onset of disease? Proteins in cerebrospinal fluid (CSF) have been suggested to act as just such natural protective agents.

A mechanistic model to predict effects of cathepsin B and cystatin C on β-amyloid aggregation and degradation.

β-Amyloid (Aβ) aggregation is thought to initiate a cascade of neurodegenerative events in Alzheimer's disease (AD). Much effort is underway to develop strategies to reduce Aβ concentration or inhibit aggregation. Cathepsin B (CatB) proteolytically degrades Aβ into non-aggregating fragments but is potently inhibited by cystatin C (CysC).

Insights into the mechanism of cystatin C oligomer and amyloid formation and its interaction with β-amyloid.

Cystatin C (CysC) is a versatile and ubiquitously-expressed member of the cysteine protease inhibitor family that is present at notably high concentrations in cerebrospinal fluid. Under mildly denaturing conditions, CysC forms inactive domain-swapped dimers. A destabilizing mutation, L68Q, increases the rate of domain-swapping and causes a fatal amyloid disease, hereditary cystatin C amyloid angiopathy.

TANGO-Inspired Design of Anti-Amyloid Cyclic Peptides.

β-Amyloid peptide (Aβ) self-associates into oligomers and fibrils, in a process that is believed to directly lead to neuronal death in Alzheimer's disease. Compounds that bind to Aβ, and inhibit fibrillogenesis and neurotoxicity, are of interest as an anti-Alzheimer therapeutic strategy. Peptides are particularly attractive for this purpose, because they have advantages over small molecules in their ability to disrupt protein-protein interactions, yet they are amenable to tuning of their properties through chemical means, unlike antibodies.

Transthyretin variants with improved inhibition of β-amyloid aggregation.

Aggregation of β-amyloid (Aβ) is widely believed to cause neuronal dysfunction in Alzheimer's disease. Transthyretin (TTR) binds to Aβ and inhibits its aggregation and neurotoxicity. TTR is a homotetrameric protein, with each monomer containing a short α-helix and two anti-parallel β-sheets.

Expression, purification, and characterization of human cystatin C monomers and oligomers.

Human cystatin C (cysC) is a soluble basic protein belonging to the cysteine protease inhibitor family. CysC is a potent inhibitor of cathepsins--proteolytic enzymes that degrade intracellular and endocytosed proteins, remodel extracellular matrix, and trigger apoptosis. Inhibition is via tight reversible binding involving the N-terminus as well as two β-hairpin loops of cysC.

Asparagine Repeat Peptides: Aggregation Kinetics and Comparison with Glutamine Repeats.

Amino acid repeat runs are common occurrences in eukaryotic proteins, with glutamine (Q) and asparagine (N) as particularly frequent repeats. Abnormal expansion of Q-repeat domains causes at least nine neurodegenerative disorders, most likely because expansion leads to protein misfolding, aggregation, and toxicity. The linkage between Q-repeats and disease has motivated several investigations into the mechanism of aggregation and the role of Q-repeat length in aggregation.

Evaluation of Nanoparticle Tracking for Characterization of Fibrillar Protein Aggregates.

Amyloidogenesis is the process of formation of protein aggregates with fibrillar morphology. Because amyloidogenesis is linked to neurodegenerative disease, there is interest in understanding the mechanism of fibril growth. Kinetic models of amyloidogenesis require data on the number concentration and size distribution of aggregates, but this information is difficult to obtain using conventional methods.

A Cyclic Peptide Mimic of the β-Amyloid Binding Domain on Transthyretin.

Self-association of β-amyloid (Aβ) into oligomers and fibrils is associated with Alzheimer's disease (AD), motivating the search for compounds that bind to and inhibit Aβ oligomerization and/or neurotoxicity. Peptides are an attractive class of such compounds, with potential advantages over small molecules in affinity and specificity. Self-complementation and peptide library screening are two strategies that have been employed in the search for peptides that bind to Aβ.

Synthesis and disaggregation of asparagine repeat-containing peptides.

Of all amino acid repeats in eukaryotes, polyglutamine (polyQ) is the most frequent, followed by polyasparagine (polyN). Glutamine repeats are expanded in proteins associated with several neurodegenerative disorders. The expanded polyQ domain is known to induce aggregation, and it is hypothesized that aggregation is directly causative of pathology.

Transthyretin-derived peptides as β-amyloid inhibitors.

Self-association of β-amyloid (Aβ) into soluble oligomers and fibrillar aggregates is associated with Alzheimer's disease pathology, motivating the search for compounds that selectively bind to and inhibit Aβ oligomerization and/or neurotoxicity. Numerous small-molecule inhibitors of Aβ aggregation or toxicity have been reported in the literature. However, because of their greater size and complexity, peptides and peptidomimetics may afford improved specificity and affinity as Aβ aggregation modulators compared to small molecules.

Protein misfolding and aggregation research: some thoughts on improving quality and utility.

Once misfolded and aggregated proteins were as interesting as yesterday's trash, just a bothersome byproduct of productive activities. Today, they attract sustained interest from both basic researchers and practicing engineers. In the burgeoning biopharmaceutical industry, protein misfolding and aggregation pose significant challenges to the economic manufacture of safe and effective protein products.

Kinetic analysis of aggregation data.

Aggregation of repeat-containing proteins is associated with neurodegenerative disorders; a specific example is the established link between expansion of the polyglutamine domain in huntingtin and the appearance of nuclear inclusions in Huntington's disease. This connection between aggregation and pathology has motivated numerous investigations into the kinetics of aggregation. Quantitative analysis of kinetic data is needed both for comparative purposes (e.