Thermoresponsive Controlled Association of Protein with a Dynamic Nanogel of Hydrophobized Polysaccharide and Cyclodextrin:  Heat Shock Protein-Like Activity of Artificial Molecular Chaperone

Nomura, Yuta; Sasaki, Yoshihiro; Takagi, M.; Narita, Tadashi; Aoyama, Yasuhiro; Akiyoshi, Kazunari

doi:10.1021/bm049501t

Cited by 95 publications

(80 citation statements)

References 33 publications

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“…[15][16][17][18] The hydrogel NPs can also be used as a drug carrier system in medicine 17,19,20 as well as artificial molecular chaperones in biotechnology. [21][22][23] On the basis of these, our group developed a new method to synthesize cholesterol-modified pullulan (CHSP) by directly grafting proper cholesterol residues onto pullulan using succinyl linkages, thus providing a new strategy for the effective and safe synthesis of CHSP. In vitro studies indicated that CHSP self-assembled NPs showed potential as a sustained-release carrier of mitoxantrone.…”

Section: Introductionmentioning

confidence: 99%

Cellular uptake mechanism and intracellular fate of hydrophobically modified pullulan nanoparticles

Jiang¹,

Li²,

Liu³

et al. 2013

IJN

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Abstract:The cellular uptake mechanism and intracellular fate of self-assembled nanoparticles (NPs) of cholesterol-modified pullulan (CHSP) by human hepatocellular carcinoma (HepG2) cells were investigated. Covalent conjugation with fluorescein isothiocyanate (FITC) yielded stably labeled CHSP (FITC-CHSP), which was successfully formulated into NPs (mean particle size 63.0 ± 1.9 nm) by dialysis. A cytotoxicity assay clearly indicated that the CHSP NPs did not show significant toxicity in HepG2 cells. The effects of NP concentration, incubation time, and temperature on the cellular uptake of the NPs were systematically evaluated by fluorometry, and the results suggested that cellular uptake of the NPs was concentration-, time-, and temperature-dependent. In vitro experiments with endocytic inhibitors revealed that clathrin-mediated endocytosis and macropinocytosis were involved in the internalization of CHSP NPs. The intracellular trafficking study demonstrated that CHSP NPs were entrapped in the lysosomes at 1 hour after incubation; colocalization of NPs with either the Golgi apparatus or the endoplasmic reticula was not observed during the entire course of the study. These results suggested that the CHSP NPs may serve as a versatile carrier for intracellular delivery of therapeutic agents.

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Section: Introductionmentioning

confidence: 99%

Cellular uptake mechanism and intracellular fate of hydrophobically modified pullulan nanoparticles

Jiang¹,

Li²,

Liu³

et al. 2013

IJN

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“…Our previous results indicate that CHP nanogels can selectively trap unfolded proteins or refolding intermediates [21][22][23] that expose relatively hydrophobic domains to the aqueous solution. Thus, one hypothesis to explain the inhibition of GFP folding is that the unfolded or partially folded GFP peptides become trapped in the hydrogel matrix of CHP nanogels.…”

Section: Nanogel Chaperone In Cell-free Protein Synthesis Y Sasaki Et Almentioning

confidence: 99%

“…20 For example, nanogels formed with cholesteryl group-bearing pullulan (CHP) can trap heatdenatured proteins 21 or chemically denatured proteins (that is, protein-refolding intermediates formed after chemical denaturation by urea or guanidinium hydrochloride). 22,23 The CHP nanogel consists of stable monodisperse nanoparticles (B30 nm) with B4-5 CHP polymers per nanogel and approximately eight non-covalent crosslinking regions that consist of cholesteryl groups. [24][25][26] The structure of the CHP nanogel is disrupted by the addition of cyclodextrins and the protein complexed with the nanogel is released and folds into its mature form, so that the CHP nanogels and cyclodextrins together function as an artificial chaperone.…”

Section: Introductionmentioning

confidence: 99%

Polysaccharide nanogel–cyclodextrin system as an artificial chaperone for in vitro protein synthesis of green fluorescent protein

Sasaki

Nomura

Sawada

et al. 2010

Polym J

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Polysaccharide nanogels have been demonstrated to aid the refolding processes of chemically or thermally denatured proteins, a function that is similar to that of natural molecular chaperones. In this study, we examined the possibilities of using the nanogel chaperone system to mediate protein folding in a cell-free (in vitro) protein synthesis system containing transcription/ translation factors. High-performance liquid chromatography showed that a polysaccharide nanogel comprising cholesteryl group-bearing pullulan (CHP) trapped unfolded or partially folded green fluorescent protein (GFP) expressed in the cell-free system. The protein release and refolding processes, which are induced by ATP in natural molecular chaperone systems, were also simulated by methyl-b-cyclodextrin (M-b-CD). The CHP nanogels dissociate on complexation with M-b-CD to yield dissociated CHP. Thus, the dissociation of the CHP nanogel-protein complex subsequently allows for the release and folding of GFP. The folding kinetics in the presence of the CHP nanogel and M-b-CD was comparable to that of spontaneous folding in the absence of CHP/M-b-CD, indicating that the CHP nanogels did not affect protein synthesis in the cell-free system, providing correctly folded active proteins.

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“…In this study, carbonic anhydrase (CA), lipase and aamylase were selected as target proteins, which are easy to assay their activity; they are often used as a model enzyme to examine the stability and improvement of the activity. 15,31,32 EXPERIMENTAL PROCEDURE Materials gPGAs (sodium salt form, molecular weight: 50 and 500 kDa) were commercial products of BioLeaders Corp. (Daejeon, Korea). CA from bovine erythrocytes, Amano lipase AK from Pseudomonas fluorescens, a-amylase type VI-B from porcine pancreas, p-nitrophenyl acetate (pNPA), fluorescein isothiocyanate (FITC) isomer I and phosphate-buffered saline (pH 7.4) were purchased from Sigma-Aldrich (St Louis, MO, USA).…”

Section: Introductionmentioning

confidence: 99%

Enhancement of enzyme activity and stability by poly(γ-glutamic acid)

Lee

Tsujimoto

Uyama

et al. 2010

Polym J

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The effects of poly(c-glutamic acid) (cPGA), a water-soluble poly(amino acid), on the enzyme activity and stability have been investigated. The activity of carbonic anhydrase (CA) was distinctly improved in the presence of 0.30-2.0% cPGA at pH 7. cPGA was also effective to enhance the activity of lipase and a-amylase. cPGA efficiently suppressed denaturation of the enzyme by the thermal treatment and repeated freeze-thaw process. The interaction of cPGA and CA was investigated by using fluorescence probe-labeled protein. The Michaelis-Menten kinetics on the CA-catalyzed hydrolysis of p-nitrophenyl acetate in the absence or presence of cPGA were performed, which demonstrated that the enhancement of the CA activity by cPGA is ascribed to the increase of the catalytic constant k cat .

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Thermoresponsive Controlled Association of Protein with a Dynamic Nanogel of Hydrophobized Polysaccharide and Cyclodextrin: Heat Shock Protein-Like Activity of Artificial Molecular Chaperone

Cited by 95 publications

References 33 publications

Cellular uptake mechanism and intracellular fate of hydrophobically modified pullulan nanoparticles

Cellular uptake mechanism and intracellular fate of hydrophobically modified pullulan nanoparticles

Polysaccharide nanogel–cyclodextrin system as an artificial chaperone for in vitro protein synthesis of green fluorescent protein

Enhancement of enzyme activity and stability by poly(γ-glutamic acid)

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