Obtaining a high activity subtilisin preparation by controlled thermal stress in n-octane.

The use of enzymes in organic solvents has considerably widened their repertoire of applications. Such low water containing media also offer the possibility of carrying out enzymatic reactions at higher temperatures and enhancing reaction yields. The utility of such preparations is limited by the damage caused to the protein structure during freeze-drying. This work investigates the result of exposing the proteolytic enzyme subtilisin to high temperature in low water containing n-octane on its activity in aqueous and non-aqueous media. Exposing subtilisin at 90 °C for 5 h led to 18-fold improvement in its transesterification activity even at the normal assay temperature (37 °C) when compared with the untreated enzyme. The use of n-octane as the reaction medium was important as it helped to retain the three-dimensional architecture of the enzyme and should be considered while designing strategies for obtaining high activity preparations of other enzymes. Structural analysis using differential scanning fluorimetry showed that the enzyme lost its structure after heating in aqueous medium but retained it when heated in organic solvent. The simplicity and general applicability of the strategy should make it useful for obtaining highly active preparations of other enzymes as well.

Glycerol 3-phosphate dehydrogenase regulates heat shock response in Saccharomyces cerevisiae.

The aim of this work was to identify elements of adaptive regulatory mechanism for basal level of yeast histone deacetylase Sir2. Heat shock response (HSR) was altered in the absence of the NAD-dependent glycerol 3-phosphate dehydrogenase (Gpd1). Increase in HSR was lower in ΔGpd1 cells than wild-type cells. An inverse correlation existed between Gpd1 and Sir2; Sir2-deleted cells showed higher expression of Gpd1 while deletion of Gpd1 led to higher expression of Sir2. In the absence of Gpd1, basal activity of Sir2 promoter was higher and was increased further upon heat shock, suggesting higher Sir2 levels. No interaction between Gpd1 and Sir2 was detected without or with heat shock using immunoprecipitation. The results show that Gpd1 regulates HSR in yeast cells and likely blocks its uncontrolled activation. As uncontrolled stress adversely affects the cellular adaptive response, Gpd1 may be a component of the cell's catalogue to ensure a balanced response to unmitigated thermal stress.

Effect of chemical chaperones in improving the solubility of recombinant proteins in Escherichia coli.

The recovery of active proteins from inclusion bodies usually involves chaotrope-induced denaturation, followed by refolding of the unfolded protein. The efficiency of renaturation is low, leading to reduced yield of the final product. In this work, we report that recombinant proteins can be overexpressed in the soluble form in the host expression system by incorporating compatible solutes during protein expression. Green fluorescent protein (GFP), which was otherwise expressed as inclusion bodies, could be made to partition off into the soluble fraction when sorbitol and arginine, but not ethylene glycol, were present in the growth medium. Arginine and sorbitol increased the production of soluble protein, while ethylene glycol did not. Production of ATP increased in the presence of sorbitol and arginine, but not ethylene glycol. A control experiment with fructose addition indicated that protein solubilization was not due to a simple ATP increase. We have successfully reproduced these results with the N-terminal domain of HypF (HypF-N), a bacterial protein which forms inclusion bodies in Escherichia coli. Instead of forming inclusion bodies, HypF-N could be expressed as a soluble protein in the presence of sorbitol, arginine, and trehalose in the expression medium.

Saccharomyces cerevisiae Fpr1 functions as a chaperone to inhibit protein aggregation.

Peptidyl prolyl isomerases (PPIases) accelerate the rate limiting step of protein folding by catalyzing cis/trans isomerization of peptidyl prolyl bonds. The larger PPIases have been shown to be multi-domain proteins, with functions other than isomerization of the proline-containing peptide bond. Recently, a few smaller PPIases have also been described for their ability to stabilize folding intermediates. The yeast Fpr1 (FK506-sensitive proline rotamase) is a homologue of the mammalian prolyl isomerase FKBP12 (FK506-binding protein of 12 kDa). Its ability to stabilize stressed cellular proteins has not been reported yet. We had earlier reported upregulation of Fpr1 in yeast cells exposed to proteotoxic stress conditions. In this work, we show that yeast Fpr1 exhibits characteristics typical of a general chaperone of the proteostasis network. Aggregation of mutant huntingtin fragment was higher in Fpr1-deleted as compared to parental yeast cells. Overexpression of Fpr1 led to reduced protein aggregation by decreasing the amount of oligomers and diverting the aggregation pathway towards the formation of detergent-soluble species. This correlated well with higher survival of these cells. Purified and enzymatically active yeast Fpr1 was able to inhibit aggregation of mutant huntingtin fragment and luciferase in vitro in a concentration-dependent manner; suggesting a direct action for aggregation inhibitory action of Fpr1. Overexpression of yeast Fpr1 was able to protect E. coli cells against thermal shock. This work establishes the role of Fpr1 in the protein folding network and will be used for the identification of novel pharmacological leads in disease conditions.

Nucleic acid therapeutics: a focus on the development of aptamers.

INTRODUCTION: Aptamers provide exciting opportunities for the development of specific and targeted therapeutic approaches. AREAS COVERED: In this review, the authors discuss different therapeutic options available with nucleic acids, including aptamers, focussing on similarities and differences between them. The authors concentrate on case studies with specific aptamers, which exemplify their distinct advantages. The reasons for failure, wherever available, are deliberated upon. Attempts to accelerate the in vitro selection process have been discussed. Challenges with aptamers in terms of their specificity and targeted delivery and strategies to overcome these are described. Examples of precise regulation of systemic half-life of aptamers using antidotes are discussed. EXPERT OPINION: Despite their nontoxic nature, a variety of reasons limit the therapeutic potential of aptamers in the clinic. The analysis of adverse effects observed with the pegnivacogin/anivamersen pair has highlighted the need to screen for preexisting PEG antibodies in any clinical trial involving pegylated molecules. Surprisingly, and promisingly, the ability of nucleic acid therapeutics to breach the blood brain barrier seems achievable. The recognition of specific motifs, e.g. G-quadruplex in thrombin-binding aptamers, or a 'nucleation' zone while designing aptamer-antidote pairs, is likely to accelerate the discovery of therapeutically efficacious molecules.

Converting Enzymes into Tools of Industrial Importance.

BACKGROUND: Enzymes have applications in numerous biotechnological products and processes that are commonly used in the production of food and beverages, cleaning supplies, clothing, paper products, transportation fuels, pharmaceuticals, and monitoring devices. Enzymes, however, are optimized to function under physiological conditions. Any change in reaction conditions results in their activity as well as stability being compromised. Hence, most of the natural biomolecules are not suitable for industrial applications. Modifications are required to develop efficient and successful reagents as per demand. Protein engineering can be applied to cope up with these situations. METHODS: This review describes some of the novel uses/unusual properties of enzymes as biological catalysts. It explains the different ways in which enzymes can be and have been used under non-native conditions. Different strategies have been discussed regarding stabilization of enzyme as well optimum conditions of its uses in different industries. The following patents databases were consulted: European Patent Office (EPO), the United States Patent and Trademark Office (USPTO), Patent scope Search International and National Patent Collections (WIPO) and Google Patents. RESULTS: The review illustrates the width of the umbrella of applications covered by biocatalysts. Employing the tools of solvent and protein engineering, viz. non-aqueous media, additives, immobilization, mutagenesis, to name a few; biotechnology has been able to make enzyme catalyzed processes an essential components of the industrialist's armoury. CONCLUSION: The article lists a number of successful examples, both of patented technology as well as biocatalysts which are currently being used in the industry, to highlight the accomplishments of technologies which have been adopted till now for making enzyme technology industrially viable.

A Sensitive, Rapid, and Specific Technique for the Detection of Collagenase Using Zymography.

In-gel zymography is a commonly employed tool to identify active enzymes in a quantitative and qualitative manner. In this work, apart from the incorporation of substrate which is traditionally employed in zymography, the identification of collagenase by incubation of the enzyme resolved on a polyacrylamide gel with substrate solution is described. The two methods are quite fast and result in specific detection of bacterial collagenase.

Design, synthesis and biological evaluation of 5-benzylidene-2-iminothiazolidin-4-ones as selective GSK-3ß inhibitors.

In this work, iminothiazolidin-4-one derivatives were explored as selective GSK-3ß inhibitors. Molecular docking analysis was carried to design a series of compounds, which were synthesized using substituted thiourea, 2-bromoacetophenones and benzaldehydes. Out of the twenty five compounds synthesized during this work, the in vitro evaluation against GSK-3 led to the identification of nine compounds with activity in lower nano-molar range (2-85 nM). Further, in vitro evaluation against CDK-2 showed five compounds to be selective towards GSK-3.

Effect of osmolytes on the fibrillation of HypF-N.

We have studied the effect of a series of stabilizing and destabilizing osmolytes on the fibrillation pattern of a model amyloidogenic protein, HypF-N. Under mildly denaturing conditions, HypF-N forms cross ß-sheet structures, characteristic of amyloid fibrils. In the presence of all stabilizing osmolytes except proline, fibrillation of HypF-N is inhibited. Notably, fibrillation kinetics is retarded at subdenaturing concentrations of chaotropes. In case of proline, fibrillation of HypF-N is accelerated. Thus, the changes during exposure of a protein to denaturing conditions in the presence of osmolyes cannot be extrapolated from their role as anti-fibrillation agents.

Correlation between Al(3+) -induced thermal stability and inhibition of fibrillation of N-terminal domain of the hydrogenase maturation factor.

Fibrillation can be induced in proteins by altering solvent conditions. Stabilization of the protofibrillar structure arrests formation of longer fibers. Thermal stability and fibrillation of N-terminal domain of the hydrogenase maturation factor (HypF-N) were studied in the presence of a series of metal ions. Only Al(3+) was able to reverse the thermal denaturation of HypF-N upon heating. On being exposed to denaturing conditions, the native protein formed fibrillar structure under moderately denaturing conditions, whereas in the presence of Al(3+) , the protein was found to retain its native conformation. Under strongly denaturing conditions, only Al(3+) was able to stabilize the protein in the fibrillar state. Spectrofluorimetric analysis revealed that Al(3+) alone was able to stabilize the partially unfolded intermediate state of HypF-N. Based on the similarity in observations, we propose a link between reversal of thermal instability of HypF-N and its ability to form an intermediate structure in the presence of Al(3+) . Al(3+) stabilizes the partially unfolded state in the N↔I↔U equilibrium so that upon heating, the three-dimensional structure of the protein is not lost completely. Kinetic analysis confirmed that Al(3+) interacts with an early structure on the aggregation landscape and delays fibrillation. Under mildly denaturing state, HypF-N is able to recover its native conformation in the presence of Al(3+) and under strongly denaturing conditions, the protein does not acquire a completely disordered structure. Instead, it forms an ordered ß-sheet-rich structure.

Length of polyglutamine tract affects secondary and tertiary structures of huntingtin protein.

The role of polyglutamine (polyQ) tract on protein stability and disease pathology remains ambiguous. We monitored the unfolding/refolding patterns of huntingtin proteins with varying polyQ lengths. In the presence of urea, minor differences in unfolding and refolding efficiencies were observed. However, in the presence of guanidinium hydrochloride, the protein with a longer polyQ stretch was able to regain its secondary but not tertiary structure on step-wise removal of denaturant. Thus, in case of Huntington's disease, the higher aggregation propensity of the mutant protein is likely to be due to the lower stability of the protein due to elongated polyQ tract.

Effect of disaccharides on the stabilization of bovine trypsin against detergent and autolysis.

Osmolytes have been reported to stabilize biomolecules and even whole organisms against exposure to adverse environmental conditions. In this work, we report for the first time the use of some of these osmolytes, viz., the disaccharides trehalose and sucrose, in the stabilization of bovine trypsin against exposure to the anionic detergent sodium dodecyl sulfate and autolysis. Exposure of trypsin to SDS at a molar ratio of 1:45 led to decrease in trypsin activity by 61%. In the presence of 1 M sucrose and 1 M trehalose, the residual trypsin activity was found to increase to that of original enzyme activity. These two disaccharides were also found to slow down the rate of autolysis, resulting in residual activities of 80 and 88%, respectively, after incubation for 24 h. Active site titration showed retention of the fraction of active sites in the presence of trehalose. Fluorescence and CD spectroscopies were used to decipher the probable mechanism of this protective role of the disaccharides. Although complete resumption of secondary structure was not seen in the presence of the two disaccharides, the spectra of trypsin in the presence of stabilizers resembled the spectrum of native trypsin and were significantly different from the spectrum of detergent-denatured enzyme. Correlating the data obtained from spectroscopy with those obtained from activity assay, we propose that the retention of secondary structure of the enzyme is largely responsible for the retention of the functionally active form of trypsin.