Structural modulation of insulin by hydrophobic and hydrophilic molecules.

In the bloodstream, insulin interacts with various kinds of molecules, which can alter its structure and modulate its function. In this work, we have synthesized two molecules having extremely hydrophilic and hydrophobic side chains. The effects of hydrophilic and hydrophobic molecules on the binding with insulin have been investigated through a multi-spectroscopic approach. We found that hydrophilic molecules have a slightly higher binding affinity towards insulin. Insulin can bind with the hydrophilic molecules as it binds glucose. The high insulin binding affinity of a hydrophobic molecule indicates its dual nature. The hydrophobic molecule binds at the hydrophobic pocket of the insulin surface, where hydrophilic molecules interact at the polar surface of the insulin. Such binding with the hydrophobic molecule perturbs strongly the secondary structure of the insulin much more in comparison to hydrophilic molecules. Therefore, the stability of insulin decreases in the presence of hydrophobic molecules.

Modulation of amyloid fibrillation of bovine ß-lactoglobulin by selective methionine oxidation.

Deposition of oxidation-modified proteins during normal aging and oxidative stress are directly associated with systemic amyloidoses. Methionine (Met) is believed to be one of the most readily oxidisable amino acid residues of protein. Bovine beta-lactoglobulin (ß-lg), a model globular whey protein, has been presented as a subsequent paradigm for studies on protein aggregation and amyloid formation. Herein, we investigated the effect of t-butyl hydroperoxide (tBHP)-induced oxidation on structure, compactness and fibrillation propensity of ß-lg at physiological pH. Notably, whey protein modification, specifically Met residues, plays an important role in the dairy industry during milk processing and lowering nutritional value and ultimately affecting their technological properties. Several bio-physical studies revealed enhanced structural flexibility and aggregation propensity of oxidised ß-lg in a temperature dependent manner. A molecular docking study is used to predict possible interactions with tBHP and infers selective oxidation of methionine residues at 7, 24 and 107 positions. From our studies, it can be corroborated that specific orientations of Met residues directs the formation of a partially unfolded state susceptible to fibrillation with possible different cytotoxic effects. Our studies have greater implications in deciphering the underlying mechanism of different whey proteins encountering oxidative stress. Our findings are also important to elucidate the understanding of oxidation induced amyloid fibrillation of protein which may constitute a new route to pave the way for a modulatory role of oxidatively stressed proteins in neurological disorders.

Targeting Drugs Against Fibroblast Growth Factor(s)-Induced Cell Signaling.

BACKGROUND: The fibroblast growth factor (FGF) family is comprised of 23 highly regulated monomeric proteins that regulate a plethora of developmental and pathophysiological processes, including tissue repair, wound healing, angiogenesis, and embryonic development. Binding of FGF to fibroblast growth factor receptor (FGFR), a tyrosine kinase receptor, is facilitated by a glycosaminoglycan, heparin. Activated FGFRs phosphorylate the tyrosine kinase residues that mediate induction of downstream signaling pathways, such as RAS-MAPK, PI3K-AKT, PLCγ, and STAT. Dysregulation of the FGF/FGFR signaling occurs frequently in cancer due to gene amplification, FGF activating mutations, chromosomal rearrangements, integration, and oncogenic fusions. Aberrant FGFR signaling also affects organogenesis, embryonic development, tissue homeostasis, and has been associated with cell proliferation, angiogenesis, cancer, and other pathophysiological changes. OBJECTIVE: This comprehensive review will discuss the biology, chemistry, and functions of FGFs, and its current applications toward wound healing, diabetes, repair and regeneration of tissues, and fatty liver diseases. In addition, specific aberrations in FGFR signaling and drugs that target FGFR and aid in mitigating various disorders, such as cancer, are also discussed in detail. CONCLUSION: Inhibitors of FGFR signaling are promising drugs in the treatment of several types of cancers. The clinical benefits of FGF/FGFR targeting therapies are impeded due to the activation of other RTK signaling mechanisms or due to the mutations that abolish the drug inhibitory activity on FGFR. Thus, the development of drugs with a different mechanism of action for FGF/FGFR targeting therapies is the recent focus of several preclinical and clinical studies.

Heparin-Binding Affinity Tag: A Novel Affinity Tag for Simple and Efficient Purification of Recombinant Proteins.

Heparin, a polysulfated polyanionic member of the glycosaminoglycan family, is known to specifically bind to a number of functionally important proteins. Based on the available information on structural specificity of heparin-protein interactions, a novel heparin-binding peptide (HB) affinity tag has been designed to achieve simple and cost-effective purification of target recombinant proteins. The HB-fused recombinant target proteins are purified on a heparin-Sepharose column using a stepwise/continuous sodium chloride gradient. A major advantage of the HB tag is that the HB-fused target proteins can be purified under denaturing conditions in the presence of 8 M urea. In addition, polyclonal antibody directed against the HB tag can be used to specifically detect and quantitate the HB-fused recombinant protein(s). Herein, a step-by-step protocol(s) for the purification of different soluble recombinant target proteins is described. In addition, useful tips to troubleshoot potential problems and also suggestions to successfully adopt the HB-tag-based purification to a wide range of target proteins are provided.

Design of a thrombin resistant human acidic fibroblast growth factor (hFGF1) variant that exhibits enhanced cell proliferation activity.

Acidic fibroblast growth factors (FGF1s) are heparin binding proteins that regulate a wide array of key cellular processes and are also candidates for promising biomedical applications. FGF1-based therapeutic applications are currently limited due to their inherent thermal instability and susceptibility to proteases. Using a wide range of biophysical and biochemical techniques, we demonstrate that reversal of charge on a well-conserved positively charged amino acid, R136, in the heparin binding pocket drastically increases the resistance to proteases, thermal stability, and cell proliferation activity of the human acidic fibroblast growth factor (hFGF1). Two-dimensional NMR data suggest that the single point mutations at position-136 (R136G, R136L, R136Q, R136K, and R136E) did not perturb the backbone folding of hFGF1. Results of the differential scanning calorimetry experiments show that of all the designed R136 mutations only the charge reversal mutation, R136E, significantly increases (ΔTm = 7 °C) the thermal stability of the protein. Limited trypsin and thrombin digestion results reveal that the R136E mutation drastically increases the resistance of hFGF1 to the action of the serine proteases. Isothermal titration calorimetry data show that the R136E mutation markedly decreases the heparin binding affinity of hFGF1. Interestingly, despite lower heparin binding affinity, the cell proliferation activity of the R136E variant is more than double of that exhibited by either the wild type or the other R136 variants. The R136E variant due to its increased thermal stability, resistance to proteases, and enhanced cell proliferation activity are expected to provide valuable clues for the development of hFGF1- based therapeutics for the management of chronic diabetic wounds.

Silver nanoparticle modulates the aggregation of beta-lactoglobulin and induces to form rod-like aggregates.

Silver nanoparticles (SNPs) have been increasingly used in medicines and biomaterials as a drug carriers and diagnostic or therapeutic material due to their smaller size, large surface area and cell penetration ability. Here we report the preparation of SNPs of diameter 10 ±â€¯3 nm by using silver nitrate and sodium borohydride and the interaction of synthesized SNPs with our model protein ß-lactoglobulin (ß-lg) in 10 mM phosphate buffer at pH 7.5 after thermal exposure at 75 °C. Heat exposed ß-lg forms amyloidal fibrillar aggregates whereas this protein aggregates adopt rod-like shape instead of fibrillar structure in presence of SNP under the same conditions. Size of the synthesized SNPs is confirmed by UV-Visible spectroscopy, SEM and TEM. Interactions and subsequent formation of molecular assembly of heat stressed ß-lg with SNP were investigated using Th-T assay and ANS binding assay, DLS, RLS, CD, FT-IR, SEM, TEM. Docking study parallely also support the experimental findings.

Insight into the co-solvent induced conformational changes and aggregation of bovine ß-lactoglobulin.

Many proteins form ordered irreversible aggregates called amyloid fibrils which are responsible for several neurodegenerative diseases. ß-lactoglobulin (ß-lg), an important globular milk protein, self-assembles to form amyloid-like fibrils on heating at low pH. The present study investigated the effects of two commonly used organic solvents acetonitrile (MeCN) and antimicrobial preservative benzyl alcohol (BA) on the conformation and self-assembly of ß-lg at ambient condition. Both MeCN and BA induced a concentration-dependent conformational change showing exposure of hydrophobic patches, loss of tertiary structure and higher α-helical structure at moderate concentrations. In the presence of 50-80% (v/v) MeCN and 1.5-3% (v/v) BA further structural transitions from α-helical to non-native ß-sheet structure were observed with a molten globule-like intermediate at 70% MeCN. These non-native ß-sheet structures have high tendency to form aggregates. The formation of ß-lg self-assembly was confirmed by Thioflavin T studies, Congo red assay, Rayleigh scattering and dynamic light scattering analysis. Transmission electron microscopy studies showed amyloid fibril formation in both MeCN and BA. Our results showed that BA enhances the unfolding and self-assembly of ß-lg at much lower concentration than MeCN. Thus solvent composition forces the protein to achieve the non-native structures which are responsible for protein aggregation.

Amyloid fibril formation by ß-lactoglobulin is inhibited by gold nanoparticles.

The endogenous deposition of protein fibrillar aggregates in the tissues is the cause of several neurodisorders. In the present study the native ß-lactoglobulin (ß-lg) has been driven towards amyloid fibrillar aggregates when it was exposed to heat at 75°C for 1h at pH 7.5. The citrate stabilized gold nanoparticle (AuNPs) of 20nm diameter is shown to inhibit the thermal aggregation of ß-lg. The stability of the ß-lg against heat stress was assessed by studying its aggregation at 75°C, either in presence or in absence of AuNPs. AuNPs stabilizes the monomeric and dimeric forms of the ß-lg inhibiting the nucleation and elongation for the formation of higher aggregates. Incubation of ß-lg with AuNPs at 75°C results in the formation of smaller ragged aggregates. Results show that AuNPs possess the capability of inhibiting the formation of amyloid fibrillar aggregates of ß-lg in a concentration-dependent manner and may thus facilitate the refolding into native like structure. AuNPs thus serve as nano-chaperon to inhibit the protein aggregation. Thus chaperon like activity of AuNP may be exploited in the design of rational therapeutics for the neurodegenerative diseases.