Implications of molecular diversity of chitin and its derivatives

Chitin is a long unbranched polysaccharide, made up of β-1,4-linked N-acetylglucosamine which forms crystalline fiber-like structure. It is present in the fungal cell walls, insect and crustacean cuticles, nematode eggshells, and protozoa cyst. We provide a critical appraisal on the chemical modifications of chitin and its derivatives in the context of their improved efficacy in medical applications without any side effect. Recent advancement in nanobiotechnology has helped to synthesize several chitin derivatives having significant biological applications. Here, we discuss the molecular diversity of chitin and its applications in enzyme immobilization, wound healing, packaging material, controlled drug release, biomedical imaging, gene therapy, agriculture, biosensor, and cosmetics. Also, we highlighted chitin and its derivatives as an antioxidant, antimicrobial agent, anticoagulant material, food additive, and hypocholesterolemic agent. We envisage that chitin and chitosan-based nanomaterials with their potential applications would augment nanobiotechnology and biomedical industries.

     

Adipogenic function of tetranectin mediated by enhancing mitotic clonal expansion via ERK signaling

Tetranectin (TN), an adipogenic serum protein, enhances adipocyte differentiation, however, its functional mechanism has yet to be elucidated. In the present study, we investigated the adipogenic function of TN by using medium containing TN-depleted fetal bovine serum (TN-del-FBS) and recombinant mouse TN (mTN). The adipocyte differentiation of 3T3-L1 cells was significantly enhanced by mTN supplementation essentially at differentiation induction, which indicated a potential role of the protein in the early differentiation phase. The adipogenic effect of mTN was more significant with insulin in the differentiation induction cocktail, implicating their close functional relationship. mTN enhanced not only the proliferation of growing cells, but also mitotic clonal expansion (MCE) that is a prerequisite for adipocyte differentiation in the early phase. Consistently, mTN increased the phosphorylation of ERK in the early phase of adipocyte differentiation. Results of this study demonstrate that the adipogenic function of mTN is mediated by enhancing MCE via ERK signaling.     

Application of cellulases from an alkalothermophilic Thermomonospora sp. in biopolishing of denims

Use of cellulase for denim washing is a standard eco-friendly technique to achieve desirable appearance and softness for cotton fabrics and denims. But enzymatic washing of denim till date involved acid cellulase (Trichoderma reesei) and neutral cellulase (Humicola isolens) the use of which has a drawback of backstaining of the indigo dye on to the fabric. Though it has been suggested that pH is a major factor in controlling backstaining there are no reports on use of cellulase under alkaline conditions for denim washing. In this study for the first time an alkali stable endoglucanase from alkalothermophilic Thermomonospora sp. (T-EG) has been used for denim biofinishing under alkaline conditions. T-EG is effective in removing hairiness with negligible weight loss and imparting softness to the fabric. Higher abrasive activity with lower backstaining was a preferred property for denim biofinishing exhibited by T-EG. The activities were comparable to acid and neutral cellulases that are being regularly used. The enzyme was also effective under non-buffering conditions which is an added advantage for use in textile industry. A probable mechanism of enzymatic finishing of cotton fabric has been represented based on the unique properties of T-EG.     

Structural insights into Rab21 GTPase activation mechanism by molecular dynamics simulations

Rab proteins belong to the family of monomeric GTPases which are involved in the cellular membrane trafficking. Rab21 protein exists in interchangeable GTP- and GDP-bound states. Rabs switch between two active and inactive conformations like other GTPases. The inactive form of Rab is bound to GDP while its active form is bounded with the GTP. Interexchange between active and inactive form is mediated by the GDP/GTP exchange factor (GEF) which catalyses the conversion from GDP-bound to GTP-bound form, thereby activating the Rab. While the GTP hydrolysis of Rabs is regulated by a GTPase-activating protein (GAP) which causes Rab inactivation. Here, we report the structural flexibility of the Rab21-GTP and Rab21-GDP complexes by docking and molecular dynamics (MD) simulations. Structural analysis of exchange mechanisms of the co-factors complexed with Rab21 reveals that Cys29, Thr33, His48, Gln78 and Lys133 are essentially important in the activation of proteins. Furthermore, a significant change in the orientation of the interacting co-factors, with slight variation in the free energy of binding was observed. Complexation of GEF with Rab21-GTP and Rab21-GDP reveal a flipping of the switchable residues. Finally, 50 ns MD simulations confirm that the GTP-bound Rab21 complex is thermodynamically more favoured than the corresponding GDP-bound complex. This study provides a detailed understanding of the structural elements involved in the conformational changes of Rab21.     

Protein unfolding studies of thiol-proteinase inhibitor from goat (<Emphasis Type="Italic">Capra hircus</Emphasis>) muscle in the presence of urea and GdnHCl as denaturants

In recent years, many advances have been made in the understanding of functional and structural characteristics of protein evolution from denaturant-based studies that subject the protein to a change in the microenvironment. This paper reports the chemical denaturation of purified goat muscle cystatin (GMC) a thiol-proteinase inhibitor, using urea and guanidine hydrochloride (GdnHCl). The subtle conformational changes of GMC were monitored by intrinsic fluorescence, extrinsic fluorescence, and CD spectroscopic techniques. Further, the activity of GMC as a function of increasing concentration of denaturants was also studied. It was found that increasing the concentration of GdnHCl significantly enhances the inactivation and unfolding of the inhibitor (GMC). In urea-induced denaturation, the intrinsic and extrinsic fluorescence intensity reveals significant structural changes in the inhibitor. Further, it was found that at low concentrations of urea, up to 0.5–1.0 M, there was quenching of fluorescence intensity compared with the native form and a red shift of 5 nm was observed up to 5–8 M. The results presented in this paper suggest that GdnHCl-induced denaturation of GMC follows a simple two-state rule in which native → denatured state transition occurs in a single step. However denaturation with urea proceeds through an intermediate or non-native state.     

Insight into Temperature Dependence of GTPase Activity in Human Guanylate Binding Protein-1

Interferon-γ induced human guanylate binding protein-1(hGBP1) belongs to a family of dynamin related large GTPases. Unlike all other GTPases, hGBP1 hydrolyzes GTP to a mixture of GDP and GMP with GMP being the major product at 37°C but GDP became significant when the hydrolysis reaction was carried out at 15°C. The hydrolysis reaction in hGBP1 is believed to involve with a number of catalytic steps. To investigate the effect of temperature in the product formation and on the different catalytic complexes of hGBP1, we carried out temperature dependent GTPase assays, mutational analysis, chemical and thermal denaturation studies. The Arrhenius plot for both GDP and GMP interestingly showed nonlinear behaviour, suggesting that the product formation from the GTP-bound enzyme complex is associated with at least more than one step. The negative activation energy for GDP formation and GTPase assay with external GDP together indicate that GDP formation occurs through the reversible dissociation of GDP-bound enzyme dimer to monomer, which further reversibly dissociates to give the product. Denaturation studies of different catalytic complexes show that unlike other complexes the free energy of GDP-bound hGBP1 decreases significantly at lower temperature. GDP formation is found to be dependent on the free energy of the GDP-bound enzyme complex. The decrease in the free energy of this complex at low temperature compared to at high is the reason for higher GDP formation at low temperature. Thermal denaturation studies also suggest that the difference in the free energy of the GTP-bound enzyme dimer compared to its monomer plays a crucial role in the product formation; higher stability favours GMP but lower favours GDP. Thus, this study provides the first thermodynamic insight into the effect of temperature in the product formation of hGBP1.     

Glycine betaine may have opposite effects on protein stability at high and low pH values

The compatible osmolyte glycine betaine (GB) is the most efficient osmoprotectant and best excluder from the protein surface. It can reverse protein aggregation and correct mutant protein defects and counter the harmful effects of urea and salts in vivo and in vitro. In this study we have investigated the pH dependence of the stabilizing effect of GB on three different proteins, namely, α-lactalbumin (α-LA), lysozyme and ribonuclease-A (RNase-A). We show here that (a) GB stabilizes RNase-A at all pH values, and (b) GB has opposite effects on two proteins at high pH and low pH values, namely, α-LA and lysozyme. This conclusion was reached by determining T_m (midpoint of denaturation), ΔH_m (denaturational enthalpy change at T_m), ΔC_p (constant-pressure heat capacity change) and ΔG_D^o (denaturational Gibbs energy change at 25 °C) of proteins in the presence of different GB concentrations. Another conclusion of this study is that ΔH_m and ΔC_p are not significantly changed in the presence of GB. This study suggests that other methylated glycine osmolytes may also behave in the same manner.     

Salt Potentiates Methylamine Counteraction System to Offset the Deleterious Effects of Urea on Protein Stability and Function

Cellular methylamines are osmolytes (low molecular weight organic compounds) believed to offset the urea’s harmful effects on the stability and function of proteins in mammalian kidney and marine invertebrates. Although urea and methylamines are found at 2:1 molar ratio in tissues, their opposing effects on protein structure and function have been questioned on several grounds including failure to counteraction or partial counteraction. Here we investigated the possible involvement of cellular salt, NaCl, in urea-methylamine counteraction on protein stability and function. We found that NaCl mediates methylamine counteracting system from no or partial counteraction to complete counteraction of urea’s effect on protein stability and function. These conclusions were drawn from the systematic thermodynamic stability and functional activity measurements of lysozyme and RNase-A. Our results revealed that salts might be involved in protein interaction with charged osmolytes and hence in the urea-methylamine counteraction.