Protein refolding assisted by self-assembled nanogels as novel artificial molecular chaperone

Molecular chaperone-like activity for protein refolding was investigated using nanogels of self-assembly of cholesterol-bearing pullulan. Nanogels effectively prevented protein aggregation (i.e. carbonic anhydrase and citrate synthase) during protein refolding from GdmCl denaturation. Enzyme activity recovered in high yields upon dissociation of the gel structure in which the proteins were trapped, by the addition of cyclodextrins. The nanogels assisted protein refolding in a manner similar to the mechanism of molecular chaperones, namely by catching and releasing proteins. The nanogels acted as a host for the trapping of refolded intermediate proteins. Cyclodextrin is an effector molecule that controls the binding ability of these host nanogels to proteins. The present nanogel system was also effective at the renaturation of inclusion body of a recombinant protein of the serine protease family.     

Control of aggregation in protein refolding: cooperative effects of artificial chaperone and cold temperature

Refolding of GuHCl-denatured recombinant-human growth hormone (r-hGH) was investigated in both dilution additive and artificial chaperone assisted modes. In both techniques, it was found that CTAB is a better additive (in dilution mode) or a capturing agent (in artificial chaperone method). Neither of the two techniques was capable of complete inhibition of aggregates formed during refolding process. In dilution, using CTAB or alpha-cyclodextrin (alpha-CD) as two different additives, the aggregation was inhibited by almost 55%. However, the extent of inhibition raised to almost 82% in artificial chaperone assisted mode using CTAB as the capturing and alpha-CD as the stripping agents. Maximum inhibition of aggregation (up to 97%) was obtained when the entire process of refolding was done at 4 degrees C. However, under this temperature program, the far-UV CD and intrinsic fluorescence spectra of the refolded samples were not superimposable on their respective native spectra. The spectra superimposibilities were obtained when the refolding process was achieved under a well worked out temperature program: incubation of the sample for 3 min at 4 degrees C after initiation of the stripping step followed by overnight incubation at 22 degrees C. Based on these data, it is expected that higher activity recovery yields of recombinant proteins, particularly at relatively higher protein concentrations, could be achieved by getting a better molecular understanding of major factors responsive for aggregation and refolding pathways.     

Inhibitor-assisted refolding of protease: a protease inhibitor as an intramolecular chaperone

Pleurotus ostrearus proteinase A inhibitor 1 (POIA1), which was discovered as a protease inhibitor, is unique in that it shows sequence homology to the propeptide of subtilisin, which functions as an intramolecular of a cognate protease. In this study, we demonstrate that POIA1 can function as an intramolecular chaperone of subtilisin by in vitro and in vivo experiments. The specific cleavage between POIA1 and the mature region of subtilisin BPN' occurred in a refolding process of a chimera protein, and Bacillus cells transformed with a chimera gene formed a halo on a skim milk plate. The mutational analyses of POIA1 in the chimera protein suggested that the tertiary structure of POIA1 is required for such a function, and that an increase in its ability to bind to subtilisin BPN' makes POIA1 a more effective intramolecular chaperone.     

Evaluation of artificial chaperoning behavior of an insoluble cyclodextrin-rich copolymer: solid-phase assisted refolding of carbonic anhydrase

Insoluble beta-cyclodextrin (beta-CD) copolymers have been used for the refolding of thermally and/or chemically denatured carbonic anhydrase with refolding yield of 40% using 300 mg of the copolymer/ml refolding solution containing 0.042 mg/ml protein. In an attempt to enhance the refolding yield with lower quantities of the copolymer, a new beta-CD-rich copolymer with higher beta-CD content was synthesized. Regarding the need for rapid stripping of the detergent molecules from the detergent-protein complexes formed in the capture step of the technique (artificial chaperone-assisted refolding), experimental variables (e.g. copolymer and the protein contents) were optimized to improve the refolding yields along with depressing the aggregate formation. In addition, comparative studies using different ionic detergents and the copolymer were conducted to get a more comprehensive understanding of the detergent's tail length in the stripping step of the process. Our results indicated that under the optimal developed refolding environment, the denatured CA was refolded with a yield of 75% using only 5mg of the copolymer/1.2 ml refolding solution containing 0.0286 mg/ml protein. Taking into account the recycling potential of the copolymer, the new resin, with significant cost-cutting capability, is a suitable candidate for industrial applications.     

Construction of a new artificial biomineralization system

Hydroxyapatite (HAP) was mineralized in poly(vinyl alcohol) (PVA)/poly(acrylic acid) (PAA) complex hydrogel immersed in a salt solution containing PAA. The transparent HAP/polymer composite swelled in water depending on the HAP content; high HAP content gave small swelling and vice versa. The HAP content reached about 80 wt % at most. Observation of the cross section of the composite by energy-dispersive analysis of X-ray (EDAX) revealed that the composite consisted of two phases, i.e., a hard HAP-rich phase and a soft polymer-rich phase. In the HAP-rich phase, the space inside the hydrogel was occupied by HAP, while HAP was not mineralized in the polymer-rich phase. The nucleation seemed to take place, at first, at the middle depth of the hydrogel where the HAP-rich phase was formed. The HAP-rich phase grew its size toward the surface of the hydrogel at the cost of the polymer-rich phase. The presence of phosphorus, P, in the polymer-rich phase indicated the adsorption of HPO(4)(2-) on the polymer chain of the hydrogel via hydrogen bonding, accompanied with Ca(2+) because of electrostatic constraints. This adsorption of ions in addition to Donnan distribution of ions leads to the formation of a hypercomplex that can be regarded as a precursor of the HAP-rich phase. The change of the hypercomplex into the HAP-rich phase is discontinuous and hence concluded as a phase transition. By comparison of our mineralization system with the biomineralization system of HAP and CaCO(3), the physicochemical mechanism of the mineralization process in the present system was found to be similar to that in biological systems. In this sense, we termed the present system an artificial biomineralization system.     

Functions of the Hsp90 chaperone system: lifting client proteins to new heights

The molecular chaperone Hsp90 is an essential protein in eukaryotic organisms and is highly conserved throughout all kingdoms of life. It serves as a platform for the folding and maturation of many client proteins including protein kinases and steroid hormone receptors. To fulfill this task Hsp90 performs conformational changes driven by the hydrolysis of ATP. Further, it can resort to a broad set of co-chaperones, which fit the Hsp90 machinery to the needs of specific client proteins. During the last years the number of identified co-chaperones has been consistently rising, implying that the client spectrum of Hsp90 may be much more diverse and larger than currently known. Many cofactors contain a TPR-domain for interactions at the C-terminus of Hsp90 and in many cases their functions and client sets remain to be uncovered. Hsp90 is also a putative target to interfere with cancerous and infectious diseases. Thus the knowledge on more of its cellular functions would provide also more therapeutic options for the future. In this review we compile the current knowledge on the Hsp90 ATPase mechanism, cofactor regulation and prospects of Hsp90 inhibition.     

Structure of glycerol dehydratase reactivase: a new type of molecular chaperone

The function of glycerol dehydratase (GDH) reactivase is to remove damaged coenzyme B(12) from GDH that has suffered mechanism-based inactivation. The structure of GDH reactivase from Klebsiella pneumoniae was determined at 2.4 A resolution by the single isomorphous replacement with anomalous signal (SIR/AS) method. Each tetramer contains two elongated 63 kDa alpha subunits and two globular 14 kDa beta subunits. The alpha subunit contains structural features resembling both GroEL and Hsp70 groups of chaperones, and it appears chaperone like in its interactions with ATP. The fold of the beta subunit resembles that of the beta subunit of glycerol dehydratase, except that it lacks some coenzyme B(12) binding elements. A hypothesis for the reactivation mechanism of reactivase is proposed based on these structural features.     

Alkaline phosphatase from rat osseous plates: purification and biochemical characterization of a soluble form

A soluble form of an alkaline phosphatase obtained from rat osseous plates was purified 204-fold with a yield of 24.3%. The purified enzyme showed a single protein band of Mr 80,000 on SDS-PAGE and an apparent molecular weight of 163,000 by gel filtration on Sephacryl S-300 suggesting a dimeric structure for the soluble enzyme. The specific activity of the enzyme at pH 9.4 in the presence of 2 mM MgCl2 was 19,027 U/mg and the hydrolysis of p-nitrophenyl phosphate (K0.5 = 92 microM) showed positive cooperativity (n = 1.5). The purified enzyme showed a broad substrate specificity, however, ATP, bis(p-nitrophenyl) phosphate and pyrophosphate were among the less hydrolyzed substrates assayed. Surprisingly the enzyme was not stimulated by cobalt and manganese ions, in contrast with a 20-25% stimulation observed for magnesium and calcium ions. Zinc ions exerted a strong inhibition on p-nitrophenylphosphatase activity of the enzyme. This paper provides a simple experimental procedure for the isolation of a soluble form of alkaline phosphatase which is induced by demineralized bone matrix during endochondral ossification.     

Dechlorination of polychlorinated methanes by a sequential methanogenic-denitrifying bioreactor system

A two-stage bioreactor has been developed to link dechlorination of halogenated methane compounds to the anaerobic processes of methanogenesis and denitrification. A digester methanogenic consortium was shown to dechlorinate chloroform (CF) and carbon tetrachloride (CT) to dichloromethane (DCM), and DCM was then mineralized by an acclimated denitrifying biological activated carbon consortium. Combining these two processes, a sequential methanogenic-denitrifying bioreactor (SMDB) system that completely degraded polychlorinated methanes including CT, CF, and DCM was developed. More than 95% of the added CT and CF was dechlorinated in the methanogenic bioreactor with methanol as the primary substrate, and the resultant DCM was biodegraded in the denitrifying bioreactor with nitrate as the electron acceptor. In the denitrifying bioreactor, the residual CF was completely removed, and the DCM removal efficiency was more than 95%. This novel bioreactor system eliminates the need for aeration and so avoids the air contamination associated with aerobic biotreatment of volatile chlorinated pollutants. This SMDB system provides an alternative to conventional biotreatment of wastewaters and other matrices contaminated with polychlorinated methanes and is, to our knowledge, the first report on such a sequential anoxic system. Received: 26 May 1999 / Accepted: 1 November 1999     

Erythrocyte ghost cell-alkaline phosphatase: construction and characterization of a vesicular system for use in biomineralization studies

Alkaline phosphatase is required for the mineralization of bone and cartilage. This enzyme is localized in the matrix vesicle, which plays a role key in calcifying cartilage. In this paper we standardize a method to construction a resealed ghost cell-alkaline phosphatase system to mimic matrix vesicles and examine the kinetic behavior of the incorporated enzyme. Polidocanol-solubilized alkaline phosphatase, free of detergent, was incorporated into resealed ghost cells. This process was time-dependent and practically 50% of the enzyme was incorporated into the vesicles in 40 h of incubation, at 25 degrees C. Alkaline phosphatase-ghost cell systems were relatively homogeneous with diameters of about 300 nm and were more stable when stored at -20 degrees C. Alkaline phosphatase was completely released from the resealed ghost cell-system using only phospholipase C. These experiments confirm that the interaction between alkaline phosphatase and the lipid bilayer of resealed ghost cell is exclusively via glycosylphosphatidylinositol (GPI) anchor of the enzyme. An important point shown is that an enzyme bound to resealed ghost cell does not lose the ability to hydrolyze ATP, pyrophosphate and p-nitrophenyl phosphate (PNPP), but the presence of a ghost membrane, as a support of the enzyme, affects its kinetic properties. Moreover, calcium ions stimulate and phosphate ions inhibit the PNPPase activity of alkaline phosphatase present in resealed ghost cells.     

Efficient construction of a diabody using a refolding system: anti-carcinoembryonic antigen recombinant antibody fragment

Recombinant fragments of the variable region of antibodies are useful in many experimental and clinical applications. However, it can be difficult to obtain these materials in soluble form after their expression in bacteria. Here, we report an efficient procedure for preparing several variable-domain fragments (Fv), single-chain Fv (scFv), and a diabody (the smallest functional bispecific antibody) of anti-carcinoembryonic antigen (CEA) antibody by overexpression in Escherichia coli in inclusion bodies, using a refolding system to obtain renatured proteins. Two types of refolded Fv were prepared: (i) Heavy and light chains of the immunoglobulin variable regions (VH and VL, respectively) were coexpressed with a dicistronic expression vector (designated Fv(co)); (ii) VH and VL were expressed separately, mixed stoichiometrically, and refolded (designated Fv(mix)). All samples refolded with high efficiency; Fv(co), Fv(mix), scFv, and the bispecific diabody bound to several CEA-positive cell lines, exactly as did soluble Fv fragments secreted by E. coli (Fv(sol)) and the parent IgG. The refolded fragments inhibited binding of the parent IgG to CEA-positive cell lines, indicating that their epitope is identical to that of IgG. The bispecific diabody, which combined variable-region fragments of anti-CEA antibody with variable-region fragments of anti-CD3 antibody, was also prepared using the refolding system. This refolded diabody could bind to lymphokine-activated killer cells. In addition, its cytotoxicity toward human bile duct carcinoma TFK-1 and other several other CEA-positive cell lines was concentration-dependent. Taken together, our results suggest that a refolding procedure can be used to prepare various functional antibody fragments (Fv, scFv, and diabody).     

Enantioselective sequential transformations by use of metal complexes: Tandem-Knoevenagel-hetero-Diels–Alder reactions with new chiral Lewis acids

Enantioselective reaction of the aldehydes 1a-g and the 1,3-dicarbonyl compound 2 in the presence of the chiral Lewis acid 5 , derived from diacetone glucose, leads in a sequential transformation consisting of a Knoevenagel condensation and an intramolecular Diels Alder reaction to the cycloadducts 4a-g with an ee value up to 88%. The selectivity is strongly dependent upon the temperature and solvent giving best results at room temperature in isodurene; in agreement with the principle of isoinversion, the ee values decrease at lower and higher temperatures. © 1993 Wiley-Liss, Inc.     

Periodinates: a new class of protein tyrosine phosphatase inhibitors

A series of periodinates has been synthesized and tested as protein tyrosine phosphatase substrates. Their potency is comparable to or higher than that of vanadates but much lower than that of peroxovanadates.     

Alkaline fixation-resistant acid phosphatases in human tissues: histochemical evidence for a new type of acid phosphatase in endothelial, endometrial and neuronal sites

The effect of pH during formalin fixation on acid phosphatases in human tissues was studied. Lysosomal-type acid phosphatase was sensitive to alkaline fixation, being completely inactive after fixation at pH 9.0. Prostatic and tartrate-resistant osteoclastic/macrophagic types were alkaline fixation-resistant, as was an acid phosphatase localized in endothelium, endometrial stromal cells and intestinal nerves. The latter activity was further separable into fluoride- and tartrate-sensitive beta-glycerophosphatase and fluoride-sensitive, tartrate-resistant alpha-naphthyl phosphatase. The activities appeared to represent either different, tightly associated enzymes or separate activity centres of a single enzyme. Alkaline fixation-resistant alpha-naphthyl phosphatase at endothelial, endometrial and neuronal sites was also well demonstrated in unfixed or neutral formalin-fixed sections as tartrate-resistant activity similar to classical tartrate-resistant acid phosphatase, but these phosphatases appear to be antigenically different. Alkaline fixation-resistant acid phosphatase showed a restricted tissue distribution both in endothelium (mainly in vessels of abdominal organs) and at neuronal sites (only in intestinal nerves). Alkaline fixation-resistant acid phosphatase appears to represent a previously unknown or uncharacterized enzyme activity whose chemical properties could not be classified as any previously known type of acid or other phosphatases.     

Synchronizing a multicellular system by external input: an artificial control strategy

MOTIVATION: Although there are significant advances on elucidating the collective behaviors on biological organisms in recent years, the essential mechanisms by which the collective rhythms arise remain to be fully understood, and further how to synchronize multicellular networks by artificial control strategy has not yet been well explored. RESULTS: A control strategy is developed to synchronize gene regulatory networks in a multicellular system when spontaneous synchronization cannot be achieved. We first construct an impulsive control system to model the process of periodically injecting coupling substances with constant or random impulsive control amounts into the common extracellular medium, and further study its effects on the dynamics of individual cells. We derive the threshold of synchronization induced by the periodic substance input. Therefore, we can synchronize the multicellular network to a specific collective behavior by changing the frequency and amplitude of the periodic stimuli. Moreover, a two-stage scheme is proposed to facilitate the synchronization in this paper. We show that the presence of the external input may also initiate different dynamics. The multicellular network of coupled repressilators is used to show the effectiveness of the proposed method. The results not only provide a perspective to understand the interactions between external stimuli and intrinsic physiological rhythms, but also may lead to development of realistic artificial control strategy and medical therapy. AVAILABILITY: CONTACT: aihara@sat.t.u-tokyo.ac.jp.     

Rubredoxin refolding on nanostructured hydrophobic surfaces: Evidence for a new type of biomimetic chaperones

Rubredoxins (Rds) are small proteins containing a tetrahedral Fe(SCys)₄ site. Folded forms of metal free Rds (apoRds) show greatly impaired ability to incorporate iron compared with chaotropically unfolded apoRds. In this study, formation of the Rd holoprotein (holoRd) on addition of iron to a structured, but iron‐uptake incompetent apoRd was investigated in the presence of polystyrene nanoparticles (NP). In our rationale, hydrophobic contacts between apoRd and the NP surface would expose protein regions (including ligand cysteines) buried in the structured apoRd, allowing iron incorporation and folding to the native holoRd. Burial of the hydrophobic regions in the folded holoRd would allow its detachment from the NP surface. We found that both rate and yield of holoRd formation increased significantly in the presence of NP and were influenced by the NP concentration and size. Rates and yields had an optimum at “catalytic” NP concentrations (0.2 g/L NP) when using relatively small NP (46 nm diameter). At these optimal conditions, only a fraction of the apoRd was bound to the NP, consistent with the occurrence of turnover events on the NP surface. Lower rates and yields at higher NP concentrations or when using larger NP (200 nm) suggest that steric effects and molecular crowding on the NP surface favor specific “iron‐uptake‐competent” conformations of apoRd on the NP surface. This bio‐mimetic chaperone system may be applicable to other proteins requiring an unfolding step before cofactor‐triggered refolding, particularly when over‐expressed under limited cofactor accessibility. Proteins 2014; 82:3154–3162. © 2014 Wiley Periodicals, Inc.     

Alkaline phosphatase and dipeptidylpeptidase IV staining of tissue components of skeletal muscle: a comparative study.

A combined alkaline phosphatase (AP) and dipeptidlypeptidase IV (DPP IV) staining reaction has demonstrated enzymatic heterogeneity of the arterial and venous segments of capillaries in rat skeletal muscle. This study compared the staining reactions of skeletal muscles in many commonly used laboratory animals, including the axolotl, chick, quail, Monodelphys, rat, mouse, hamster, guinea pig, rabbit, dog, monkey, and human. DPP IV activity was found in the venous ends of the capillaries and in the endothelium of some larger veins in many of the species but was never demonstrated in the arterial side of the circulation. AP was found in the arterial ends of capillaries in all species except the axolotl, and it was also found in the endothelium of larger arteries of most species. AP activity was absent in venous endothelium of all species except for birds and Monodelphys. DPP IV activity was found in the perineurium of intramuscular nerves of most species, and AP activity was commonly seen in tendons and intramuscular connective tissue. The interspecies variability found in this study shows that care must be taken in comparing experimental data involving this technique from one species to another, but within a species the technique allows a fine level of discrimination between functionally distinct compounds of skeletal muscle tissue.