Protein refolding by pH-triggered chaperone binding and release.
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ABSTRACT:
SUBMITTER: Tapley TL
PROVIDER: S-EPMC2803140 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Heat shock protein 47 (HSP47) is an endoplasmic reticulum (ER)-resident collagen-specific chaperone and essential for proper formation of the characteristic collagen triple helix. It preferentially binds to the folded conformation of its clients and accompanies them from the ER to the Golgi compartment, where it releases them and is recycled back to the ER. Unlike other chaperones, the binding and release cycles are not governed by nucleotide exchange and hydrolysis, but presumably the dissociation of the HSP47-procollagen complex is triggered by the lower pH in the Golgi (pH 6.3) compared with the ER (pH 7.4). Histidine residues have been suggested as triggers due to their approximate textbook pKa value of 6.1 for their side chains. We present here an extensive theoretical and experimental study of the 14 histidine residues present in canine HSP47, where we have mutated all histidine residues in the collagen binding interface and additionally all of those that were predicted to undergo a significant change in protonation state between pH 7 and 6. These mutants were characterized by biolayer interferometry for their pH-dependent binding to a collagen model. One mutant (H238N) loses binding, which can be explained by a rearrangement of the Arg(222) and Asp(385) residues, which are crucial for specific collagen recognition. Most of the other mutants were remarkably silent, but a double mutant with His(273) and His(274) exchanged for asparagines exhibits a much less pronounced pH dependence of collagen binding. This effect is mainly caused by a lower koff at the low pH values.
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Project description:Liposomes are representative lipid nanoparticles widely used for delivering anticancer drugs, DNA fragments, or siRNA to cancer cells. Upon targeting, various internal and external triggers have been used to increase the rate for contents release from the liposomes. Among the internal triggers, decreased pH within the cellular lysosomes has been successfully used to enhance the rate for releasing contents. However, imparting pH sensitivity to liposomes requires the synthesis of specialized lipids with structures that are substantially modified at a reduced pH. Herein, we report an alternative strategy to render liposomes pH sensitive by encapsulating a precursor which generates gas bubbles in situ in response to acidic pH. The disturbance created by the escaping gas bubbles leads to the rapid release of the encapsulated contents from the liposomes. Atomic force microscopic studies indicate that the liposomal structure is destroyed at a reduced pH. The gas bubbles also render the liposomes echogenic, allowing ultrasound imaging. To demonstrate the applicability of this strategy, we have successfully targeted doxorubicin-encapsulated liposomes to the pancreatic ductal carcinoma cells that overexpress the folate receptor on the surface. In response to the decreased pH in the lysosomes, the encapsulated anticancer drug is efficiently released. Contents released from these liposomes are further enhanced by the application of continuous wave ultrasound (1 MHz), resulting in substantially reduced viability for the pancreatic cancer cells (14%).
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Project description:Cinnamaldehyde is a natural product with antibacterial, antifungal, and anti-inflammatory properties, poorly stable in environmental conditions. Systems for the controlled release of cinnamaldehyde are of great interest to the food and pharmaceutical industries. Here, a new oxide-based construct for the release of cinnamaldehyde catalyzed by acidic pH was obtained by a facile grafting method based on amino-silane linkers and imine chemistry. The grafting procedure led to a loading of ca. 5 molecules/nm2, determined on oxide powders with CHN and TGA measurements. The covalent grafting of cinnamaldehyde, demonstrated by FTIR analyses, preserved the molecule stability, simplifying storage. Release tests were performed at different pH values (between 5.0 and 7.4). Thanks to imine chemistry, a fast cinnamaldehyde (CIN) release was observed in a pH 5.0 environment. Using 1 mg/mL suspensions, CIN concentrations within the range adopted in the food industry were obtained (12.4 ppm). The grafting procedure was also performed on a porous film based on a photocatalytic oxide, demonstrating the versatility of this method, adaptable to both powders and macroscopic materials. By taking advantage of the photoactivity of the oxide, regeneration of the fouled film was achieved upon UV irradiation for 1 h, opening the door to reusable devices for the controlled release of cinnamaldehyde.
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Project description:Magnetic nanocapsules were synthesized for controlled drug release, magnetically assisted delivery, and MRI imaging. These magnetic nanocapsules, consisting of a stable iron nanocore and a mesoporous silica shell, were synthesized by controlled encapsulation of ellipsoidal hematite in silica, partial etching of the hematite core in acid, and reduction of the core by hydrogen. The iron core provided a high saturation magnetization and was stable against oxidation for at least 6 months in air and 1 month in aqueous solution. The hollow space between the iron core and mesoporous silica shell was used to load anticancer drug and a T1-weighted MRI contrast agent (Gd-DTPA). These multifunctional monodispersed magnetic "nanoeyes" were coated by multiple polyelectrolyte layers of biocompatible poly-l-lysine and sodium alginate to control the drug release as a function of pH. We studied pH-controlled release, magnetic hysteresis curves, and T1/T2 MRI contrast of the magnetic nanoeyes. They also served as MRI contrast agents with relaxivities of 8.6 mM-1 s-1 (r1) and 285 mM-1 s-1 (r2).
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Project description:RIP2, one of the RIP kinases, interacts with p75 neurotrophin receptor, regulating the neuron survival, and with NOD1 and NOD2 proteins, causing the innate immune response against gram-negative and gram-positive bacteria via its caspase recruitment domain (CARD). This makes RIP2 a prospective target for novel therapies, aimed to modulate the inflammatory diseases and neurogenesis/neurodegeneration. Several studies report the problems with the stability of human RIP2 CARD and its production in bacterial hosts, which is a prerequisite for the structural investigation with solution NMR spectroscopy. In the present work, we report the high yield production and refolding protocols and resolve the structure of rat RIP2 CARD. The structure reveals the important differences to the previously published conformation of the homologous human protein. Using solution NMR, we characterized the intramolecular mobility and pH-dependent behavior of RIP2 CARD, and found the propensity of the protein to form high-order oligomers at physiological pH while being monomeric under acidic conditions. The oligomerization of protein may be explained, based on the electrostatic properties of its surface. Analysis of the structure and sequences of homologous proteins reveals the residues which are significant for the unusual fold of RIP2 CARD domains from different species. The high-throughput protein production/refolding protocols and proposed explanation for the protein oligomerization, provide an opportunity to design the stabilized variants of RIP2 CARD, which could be used to study the structural details of RIP2/NOD1/NOD2 interaction and perform the rational drug design.
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Project description:AlphaA-crystallin is a molecular chaperone; it prevents aggregation of denaturing proteins. We have previously demonstrated that upon modification by a metabolic alpha-dicarbonyl compound, methylglyoxal (MGO), alphaA-crystallin becomes a better chaperone. AlphaA-crystallin also assists in refolding of denatured proteins. Here, we have investigated the effect of mild modification of alphaA-crystallin by MGO (with 20-500 microM) on the chaperone function and its ability to refold denatured proteins. Under the conditions used, mildly modified protein contained mostly hydroimidazolone modifications. The modified protein exhibited an increase in chaperone function against thermal aggregation of beta(L)- and gamma-crystallins, citrate synthase (CS), malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) and chemical aggregation of insulin. The ability of the protein to assist in refolding of chemically denatured beta(L)- and gamma-crystallins, MDH and LDH, and to prevent thermal inactivation of CS were unchanged after mild modification by MGO. Prior binding of catalytically inactive, thermally denatured MDH or the hydrophobic probe, 2-p-toluidonaphthalene-6-sulfonate (TNS) abolished the ability of alphaA-crystallin to assist in the refolding of denatured MDH. However, MGO modification of chaperone-null TNS-bound alphaA-crystallin resulted in partial regain of the chaperone function. Taken together, these results demonstrate that: 1) hydroimidazolone modifications are sufficient to enhance the chaperone function of alphaA-crystallin but such modifications do not change its ability to assist in refolding of denatured proteins, 2) the sites on the alphaA-crystallin responsible for the chaperone function and refolding are the same in the native alphaA-crystallin and 3) additional hydrophobic sites exposed upon MGO modification, which are responsible for the enhanced chaperone function, do not enhance alphaA-crystallin's ability to refold denatured proteins.
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Project description:One of the greatest challenges in cancer therapy is to develop methods to deliver chemotherapy agents to tumor cells while reducing systemic toxicity to noncancerous cells. A promising approach to localizing drug release is to employ drug-loaded nanoparticles with coatings that release the drugs only in the presence of specific triggers found in the target cells such as pH, enzymes, or light. However, many parameters affect the nanoparticle distribution and drug release rate, and it is difficult to quantify drug release in situ. In this work, we show proof-of-principle for a "smart" radioluminescent nanocapsule with an X-ray excited optical luminescence (XEOL) spectrum that changes during release of the optically absorbing chemotherapy drug, doxorubicin. XEOL provides an almost background-free luminescent signal for measuring drug release from particles irradiated by a narrow X-ray beam. We study in vitro pH-triggered release rates of doxorubicin from nanocapsules coated with a pH-responsive polyelectrolyte multilayer using HPLC and XEOL spectroscopy. The doxorubicin was loaded to over 5% by weight and released from the capsule with a time constant in vitro of ∼36 days at pH 7.4 and 21 h at pH 5.0, respectively. The Gd₂O₂S:Eu nanocapsules are also paramagnetic at room temperature with similar magnetic susceptibility and similarly good MRI T₂ relaxivities to Gd₂O₃, but the sulfur increases the radioluminescence intensity and shifts the spectrum. Empty nanocapsules did not affect cell viability up to concentrations of at least 250 μg/mL. These empty nanocapsules accumulated in a mouse liver and spleen following tail vein injection and could be observed in vivo using XEOL. The particles are synthesized with a versatile template synthesis technique which allows for control of particle size and shape. The XEOL analysis technique opens the door to noninvasive quantification of drug release as a function of nanoparticle size, shape, surface chemistry, and tissue type.
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