Natural Chaperones Research Articles

Temperature-Responsive Self-Assembly Nanochaperone Protects Green Fluorescent Proteins from Thermal Denaturation

Protein products perform important roles in the biochemistry field, but the thermal inactivation of proteins will increase the difficulty of their transport, storage and application. Therefore, improving the thermal stability of proteins has become a thorny challenge. Natural molecular chaperones can efficiently improve the resistance of proteins to environmental stimuli by reversible supramolecular assembly with proteins. Inspired by this machine, herein we designed a nanochaperone (nChap) with thermo-responsive amphiphilic surfaces that can prevent thermal denaturation and facilitate refolding of green fluorescent proteins (GFPs). By mimicking the hydrophobic microregion of natural chaperones, this nChap can effectively capture free GFPs and hide them into surface confined spaces, thereby shielding exposed hydrophobic sites of GFPs and preventing their irreversible thermal aggregation. When the heat stimulation disappeared, the thermosensitive segments of the nChaps underwent the hydrophilic transition, which provided suitable microenvironments for GFPs refolding. More importantly, nChaps could also actively adsorb to the surface of immobilized GFPs at high temperatures and realize the satisfactory dissociation of the nChap-protein complex upon cooling, which exhibited excellent chaperone-like activity. This work provides significant insights for understanding and developing strategies to improve protein stability.

Arabinogalactan-Proteins as Boron-Acting Enzymes, Cross-Linking the Rhamnogalacturonan-II Domains of Pectin

Most pectic rhamnogalacturonan-II (RG-II) domains in plant cell walls are borate-bridged dimers. However, the sub-cellular locations, pH dependence, reversibility and biocatalyst involvement in borate bridging remain uncertain. Experiments discussed here explored these questions, utilising suspension-cultured plant cells. In-vivo pulse radiolabelling showed that most RG-II domains dimerise extremely quickly (<4 min after biosynthesis, thus while still intraprotoplasmic). This tallies with the finding that boron withdrawal causes cell wall weakening within 10–20 min, and supports a previously proposed biological role for boron/RG-II complexes specifically at the wall/membrane interface. We also discuss RG-II monomer ↔ dimer interconversion as monitored in vitro using gel electrophoresis and a novel thin-layer chromatography method to resolve monomers and dimers. Physiologically relevant acidity did not monomerise dimers, thus boron bridge breaking cannot be a wall-loosening mechanism in ‘acid growth’; nevertheless, recently discovered RG-II trimers and tetramers are unstable and may thus underpin reversible wall loosening. Dimerising monomers in vitro by B(OH)3 required the simultaneous presence of RG-II-binding ‘chaperones’: co-ordinately binding metals and/or ionically binding cationic peptides. Natural chaperones of the latter type include highly basic arabinogalactan protein fragments, e.g., KHKRKHKHKRHHH, which catalyse a reaction [2 RG-II + B(OH)3 → RG-II–B–RG-II], suggesting that plants can ‘enzymically’ metabolise boron.

Proteolytically Stable Diaza-Peptide Foldamers Mimic Helical Hot Spots of Protein-Protein Interactions and Act as Natural Chaperones.

A novel class of peptidomimetic foldamers based on diaza-peptide units are reported. Circular dichroism, attenuated total reflection -Fourier transform infrared, NMR, and molecular dynamics studies demonstrate that unlike the natural parent nonapeptide, the specific incorporation of one diaza-peptide unit at the N-terminus allows helical folding in water, which is further reinforced by the introduction of a second unit at the C-terminus. The ability of these foldamers to resist proteolysis, to mimic the small helical hot spot of transthyretin-amyloid β (Aβ) cross-interaction, and to decrease pathological Aβ aggregation demonstrates that the introduction of diaza-peptide units is a valid approach for designing mimics or inhibitors of protein-protein interaction and other therapeutic peptidomimetics. This study also reveals that small peptide foldamers can play the same role as physiological chaperone proteins and opens a new way to design inhibitors of amyloid protein aggregation, a hallmark of more than 20 serious human diseases such as Alzheimer's disease.

An Arabidopsis thaliana arabinogalactan-protein (AGP31) and several cationic AGP fragments catalyse the boron bridging of rhamnogalacturonan-II

Rhamnogalacturonan-II (RG-II) is a complex pectic domain in plant primary cell walls. In vivo, most RG-II domains are covalently dimerised via borate diester bridges, essential for correct cell-wall assembly, but the dimerisation of pure RG-II monomers by boric acid in vitro is extremely slow. Cationic ‘chaperones’ can promote dimerisation, probably by overcoming the mutual repulsion between neighbouring anionic RG-II molecules. Highly effective artificial chaperones include Pb2+ and polyhistidine, but the proposed natural chaperones remained elusive. We have now tested cationic peptide fragments of several Arabidopsis thaliana arabinogalactan-proteins (AGPs) as candidates. Fragments of AGP17, 18, 19 and 31 were effective, typically at ∼25 µg/ml (9–19 µM), promoting the boron bridging of 16–20 µM monomeric RG-II at pH 4.8 in vitro. Native AGP31 glycoprotein was also effective, and hexahistidine was moderately so. All chaperones tested interacted reversibly with RG-II and were not consumed during the reaction; thus they acted catalytically, and may constitute the first reported boron-acting enzyme activity, an RG-II borate diesterase. Many of the peptide chaperones became less effective catalysts at higher concentration, which we interpret as due to the formation of RG-II–peptide complexes with a net positive charge, as mutually repulsive as negatively charged pure RG-II molecules. The four unique AGPs studied here may serve an enzymic role in the living plant cell, acting on RG-II within Golgi cisternae and/or in the apoplast after secretion. In this way, RG-II and specific AGPs may contribute to cell-wall assembly and hence plant cell expansion and development.

Structural basis for the inhibition of IAPP fibril formation by the co-chaperonin prefoldin

Chaperones, as modulators of protein conformational states, are key cellular actors to prevent the accumulation of fibrillar aggregates. Here, we integrated kinetic investigations with structural studies to elucidate how the ubiquitous co-chaperonin prefoldin inhibits diabetes associated islet amyloid polypeptide (IAPP) fibril formation. We demonstrated that both human and archaeal prefoldin interfere similarly with the IAPP fibril elongation and secondary nucleation pathways. Using archaeal prefoldin model, we combined nuclear magnetic resonance spectroscopy with electron microscopy to establish that the inhibition of fibril formation is mediated by the binding of prefoldin’s coiled-coil helices to the flexible IAPP N-terminal segment accessible on the fibril surface and fibril ends. Atomic force microscopy demonstrates that binding of prefoldin to IAPP leads to the formation of lower amounts of aggregates, composed of shorter fibrils, clustered together. Linking structural models with observed fibrillation inhibition processes opens perspectives for understanding the interference between natural chaperones and formation of disease-associated amyloids.

Artificial Molecular Chaperone Systems for Proteins, Nucleic Acids, and Synthetic Molecules.

Molecular chaperones play critical roles in biological functions. They are closely involved in the maintenance of cell homeostasis, proper folding of proteins and nucleic acids, and inhibition of irreversible aggregation in denatured proteins. In addition to protein production, molecular chaperone function is widely recognized as important for peptide and protein drug delivery systems. Therefore, much effort has been made in recent decades to develop chaperone-mimetic molecules that have similar structures and biological functions to natural chaperones. These artificial molecular chaperone systems have been demonstrated to facilitate proper protein and nucleic acid folding, in addition to the formation of higher-order structures of synthetic molecules. Furthermore, the functions of these artificial systems show promising clinical applications in drug delivery and biomolecule detection. This topical review focuses on recent advances in the design, construction, characterization, and potential applications of different artificial molecular systems with distinct functional roles, such as the folding of water-soluble and membrane proteins, nucleic acids, and the self-assembly of synthetic molecules. Strategies used in the construction of some artificial molecule chaperone systems for proteins (such as pairs of amphiphilic molecules or self-assembled nanogels) and their applications as biomaterials are described. Specific examples from each design strategy are also highlighted to demonstrate the mechanisms, challenges, and limitations of the different artificial molecular systems. By highlighting the many new developments that have expanded the applications of the artificial chaperones beyond protein folding, this review aims to stimulate further studies on their design and applications.

Nanochaperones Mediated Delivery of Insulin

Insulin would undergo unfolding and fibrillation under stressed conditions, which may cause serious biotechnological and medical problems. Herein, by mimicking the structure and functions of natural chaperones HSP70s, self-assembled polymeric micelles are used as nanochaperones for the delivery of insulin. The confined hydrophobic domains on the surface of nanochaperones adsorb partially unfolded insulin, inhibiting the aggregation and fibrillation and enhancing the stability of insulin. The bioactivity of insulin is well-reserved after incubation with the nanochaperones at 37 °C for 7 d or heating at 70 °C for 1 h. The stealthy poly(ethylene glycol) chains around the confined domains protect the adsorbed insulin from enzymatic degradation and prolong the circulation time. More importantly, the excellent glucose sensitivity of the hydrophobic domains enables the nanochaperones to release and refold insulin in native form in response to hyperglycemia. This kind of nanochaperone may offer a hopeful strategy for the protection and delivery of insulin.

Artificial chaperones based on thermoresponsive polymers recognize the unfolded state of the protein

Stabilization of the enzymes under stress conditions is of special interest for modern biochemistry, bioengineering, as well as for formulation and target delivery of protein-based drugs. Aiming to achieve an efficient stabilization at elevated temperature with no influence on the enzyme under normal conditions, we studied chaperone-like activity of thermoresponsive polymers based on poly(dimethylaminoethyl methacrylate) (PDMAEMA) toward two different proteins, glyceraldehyde-3-phosphate dehydrogenase and chicken egg lysozyme. The polymers has been shown to do not interact with the folded protein at room temperature but form a complex upon heating to either protein unfolding or polymer phase transition temperature. A PDMAEMA-PEO block copolymer with a dodecyl end-group (d-PDMAEMA-PEO) as well as PDMAEMA-PEO without the dodecyl groups protected the denatured protein against aggregation in contrast to PDMAEMA homopolymer. No effect of the polymers on the enzymatic activity of the client protein was observed at room temperature. The polymers also partially protected the enzyme against inactivation at high temperature. The results provide a platform for creation of artificial chaperones with unfolded protein recognition which is a major feature of natural chaperones.

Chaperone-like activity of synthetic polyanions can be higher than the activity of natural chaperones at elevated temperature

Polyelectrolytes are a prospective tool for protection of proteins against aggregation. We compared synthetic polyanion, poly(styrene sulfonate), and natural chaperones of different types, namely, GroEL-like chaperonin from Pseudomonas aeruginosa phage EL and human small heat shock protein HspB5 (αB-crystallin), in their ability to prevent aggregation of client proteins. At 45 °C, all three agents efficiently suppressed thermal aggregation of phage endolysin. At higher temperatures, HspB5 and poly(styrene sulfonate) also inhibited endolysin aggregation, though polyanion became less efficient than HspB5 at 55 °C and 60 °C. However, the polyanion completely protected another protein, glyceraldehyde-3-phosphate dehydrogenase, even at 60 °C, in contrast to both natural chaperones whose effect disappeared at 50–55 °C. These results provide a platform for the development of artificial chaperones based on synthetic polyelectrolytes.

Rational Design of a Synthetic Peg-Like Polymer for Protein Stabilization

Organophosphorus hydrolase (OPH) is an enzyme that can deteriorate acutely toxic chemicals known as organophosphates, which are ubiquitously employed as pesticides and are thus considerable environmental hazards. However, the proclivity of OPH to aggregate in aqueous media severely hinders its biological activity. Inspired by natural chaperones, we have designed and synthesized heteropolymers to encapsulate and stabilize OPH. When OPH is complexed with the designed heteropolymer, it exhibits a drastic increase in biological activity in a buffer solution - approximately 15 times greater than the native biological activity of pure OPH in the same environment. We have confirmed via dynamic light scattering that while pure OPH aggregates in the buffer solution, the heteropolymer prevents aggregation from occurring.Two model proteins, bovine serum albumin and lysozyme, have also been studied with the heteropolymer to aid our understanding of the protein-polymer interactions that occur in our system. The structure and particle size of the different protein-polymer mixtures at several temperatures have been probed via dynamic light scattering, circular dichroism, and Fourier transform infrared spectroscopy. These characterization techniques have elucidated the mechanisms of interactions between the polymer and proteins.

Modulation of Amyloid-β1-40 Transport by ApoA1 and ApoJ Across an in vitro Model of the Blood-Brain Barrier.

Amyloid-β (Aβ) accumulation in Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA) is likely caused by the impairment of its brain clearance that partly occurs through the blood-brain barrier (BBB). In this context, an in vitro BBB model is a valuable tool for studying the molecular mechanisms that regulate this process. This study assessed brain Aβ elimination across the BBB and its modulation by the natural chaperones Apolipoprotein A1 (ApoA1) and Apolipoprotein J/Clusterin (ApoJ). The model was based on primary cerebral endothelial cells that were cultured on Matrigel-coated Transwells and treated with fluorescently labeled-Aβ1-40 to track its efflux across the BBB, which corresponds to trafficking from the basolateral (brain) to apical (blood) compartments. We observed that the transport of basolateral Aβ1-40 was enhanced when it was complexed to rApoJ, whereas the complex formed with rApoA1 did not influence Aβ1-40 efflux. However, the presence of rApoA1 in the apical compartment was able to mobilize Aβ1-40 from the basolateral side. We also observed that both rApoA1 and rApoJ moderately crossed the monolayer (from blood to brain) through a mechanism involving the LDL receptor-related protein family. In contrast to the increased rApoJ efflux when complexed to Aβ1-40, rApoA1 trafficking was restricted when it was bound to the Aβ peptide. In summary, the present study highlights the role of ApoJ and ApoA1 in the in vitro modulation of Aβ elimination across the BBB.

Taurine-Conjugated Bile Acids Protect Expanding Hematopoietic Stem/Progenitor Cells from Unfolded Protein Stress As Natural Chaperones

Hematopoietic stem cells (HSCs) give rise to all lineages of hematopoietic cells in the body for entire life span and are thus protected from risk factors by multiple defense systems. We have recently discovered that HSCs are highly susceptible to stress caused by accumulation of mis-/un-folded proteins, so called endoplasmic reticulum (ER) stress upon enhanced growth conditions, and addition of a specific type of bile acid (BA), Tauroursodeoxycholic acid (TUDCA), known as a chemical chaperone can maintain functional murine HSCs for 2 weeks in vitro, by reducing ER stress (Miharada et al., Cell Rep. 2014). This work depicts the importance of proper protein quality control in HSC maintenance, particularly during the expansion.HSCs are kept in dormant state in the adult body, but actively expanding in the fetal liver. BAs are synthesized from cholesterol in the liver. Interestingly, bile acid synthesis is highly up-regulated in the fetal liver during embryogenesis and the composition of fetal BAs gradually reduces after birth. In addition, composition of bile acids in the fetus is different from adult liver, with the vast majority of fetal BAs are of Taurine-conjugated form that is more stable and non-toxic. Of note, hematopoietic cells and hepatocytes producing BAs are in close contact in the fetal liver and HSCs are therefore exposed to BAs, whereas the adult liver has anatomically isolated bile duct structures that separate blood flow and bile flow. However the role for these fetal BAs has been unknown.Here we report that bile acids support expansion of hematopoietic stem and progenitor cells (HSPCs) in the fetal liver and ex vivo. Since TUDCA is a rare component in human and mouse BAs, even in the fetal liver, we sought analogue(s) that similarly function as ER stress inhibitors. We identified that Taurocholic acid (TCA), one of the main components of fetal BA, and Tauro-alpha-muricholic acid (TαMCA) that is a rodent specific BA have a potential to reduce ER stress, similar to TUDCA. Mouse HSCs cultured with TCA or TαMCA in vitro for 2 weeks showed a robust increase in the reconstitution level compared to non-treated cells (14-fold, n=14, p<0.001 and 13-fold, n=9, p<0.05, respectively), which has comparable or even better potential to support HSC function than TUDCA.To study physiological roles of BA in ER stress reduction and HSC expansion in the fetal liver, an inhibitor of BA synthesis, GW4064 (an agonist of a nuclear receptor FxR that negatively regulates key enzymes in the BA biosynthesis, CYP7A1 and CYP8B1), was intraperitoneally injected into pregnant mice. E16.5 fetuses derived from GW4064-injected pregnant mice showed severe decrease in the number of HSPCs (0.40-fold, n=28-33, p<0.001) in the fetal liver, due to increased apoptosis triggered by elevated ER stress levels. Importantly, co-injection of TCA or Salubrinal (inhibitor of the ER stress-induced apoptosis signal) rescued the effects of GW4064 on cellularity of the fetal liver and levels of ER stress, confirming that the phenotype seen here is due to increased ER stress resulting from lowered levels of BA.Analyses of CYP27A1 knockout (KO) mice that have reduced BA synthesis observed decreased HSC number and increased ER stress in the fetal liver, whereas CYP8B1 KO mice that have increased Tα/βMCA synthesis instead of lack of TCA didn’t show any difference.These findings strongly suggest that fetal BA, particularly TCA and TαMCA, supports the expansion of HSPCs in the fetal liver and the ex vivo culture as chemical chaperones by lowering ER stress levels. Our findings propose a new role of bile acids in hematopoiesis as natural chaperones and provide a novel connection between hematopoiesis and fetal liver. DisclosuresNo relevant conflicts of interest to declare.

Inhibiting effect of α s1-casein on Aβ 1–40 fibrillogenesis

Background α s1-Casein is one of the four types of caseins, the largest protein component of bovine milk. The lack of a compact folded conformation and the capability to form micelles suggest a relationship of α s1-casein with the class of the intrinsically disordered (or natively unfolded) proteins. These proteins are known to exert a stabilizing activity on biomolecules through specific interaction with hydrophobic surfaces. In the present work we focused on the effect of α s1-casein on the fibrillogenesis of 1–40 β-amyloid peptide, involved in Alzheimer's disease. Methods The aggregation kinetics of β-peptide in presence and absence of α s1-casein was followed under shear at 37 °C by recording the Thioflavine fluorescence, usually taken as an indicator of fibers formation. Measurements of Static and Dynamic Light Scattering, Circular Dichroism, and AFM imaging were done to reveal the details of α s1-casein–Aβ 1–40 interaction. Results and discussions α s1-Casein addition sizably increases the lag-time of the nucleation phase and slows down the entire fibrillization process. α s1-Casein sequesters the amyloid peptide on its surface thus exerting a chaperone-like activity by means a colloidal inhibition mechanism. General significance Insights on the working mechanism of natural chaperones in preventing or controlling the amyloid aggregation.