Nano-Formulation of Antioxidants as Effective Inhibitors of γD-Crystallin Aggregation.

The aggregation of crystallin proteins is related to cataracts and age-related macular degeneration. Apart from surgical replacement of the cataract lens, no other alternative treatment is available till date for this ailment. In the current work, we carried out an in-depth investigation of the effect of polyphenol-loaded nano-formulations on the aggregation of γD-crystallin. At first, the protein was allowed to form amorphous aggregates under denaturing conditions. Several polyphenols were then tried to inhibit the aggregation of the protein. Among the polyphenols tested, resveratrol and quercetin were found to be the most effective. Since polyphenols are prone to degradation, they were encapsulated in chitosan nanoparticles in order to provide ambient conditions for them to function effectively. The loading efficiency and polyphenol release kinetics were subsequently tested. Finally, the efficacy of resveratrol/quercetin-loaded chitosan nano-particles as inhibitors of γD-crystallin aggregation was confirmed in a series of experiments demonstrating the potency of the system in the prospective therapeutic intervention of eye ailments concerning self-assembly of γD-crystallin proteins.

An improved strategy of TGFß3 expression in Escherichia coli: Exploiting folding modulators for a switch from misfolded to folded form.

Transforming growth factor beta 3 (TGFß3) exhibits a complex native structure featuring the presence of multiple disulfide bonds forming the active dimer. Consequently, its heterologous expression in microbial system invariably leads to inclusion body (IB) formation. In this study, we observed an interesting phenomenon of switching a significant fraction of misfolded TGFß3 to folded form by modulating the cellular protein folding machinery. We carried out co-expression experiments with chaperones and demonstrated the requirement of a coordinated action of DnaK-DnaJ-GrpE and GroESL, to achieve the native soluble conformation of TGFß3, during over-expression in E. coli. The novelty of this study lies in the fact that orchestration of a group of chaperones to work in concert for efficient folding and assembly of TGFß3-like cytokines has not been widely explored. Additionally, we have also demonstrated that presence of osmolytes (sorbitol or trehalose) in the growth media have an appreciable impact on the solubility of TGFß3. We have further shown a synergism between the effects of molecular chaperone and osmolytes on the solubility of TGFß3. We have confirmed the functionality of soluble TGFß3 by performing binding interactions with its cognate receptor TßRII. Our study delineates the fact that an effective combination of chaperones or optimum concentration of compatible osmolyte, can efficiently abrogate competing aggregation pathways and help attain the native conformation of a cysteine rich cytokine in a facile manner.

Interaction of TGFß3 ligand with its receptors type II (TßRII) and type I (TßRI): A unique mechanism of protein-protein association.

The proper orchestration of transforming growth factor beta (TGFß) mediated signal transduction depends upon a delicate set of interactions between specific ligands and their receptors. Here we present an in-depth profiling of the binding mechanism of TGFß3 ligand with its type II and type I receptors (TßRII and TßRI) using isothermal titration calorimetry (ITC). Studies were carried out in acidic pH as it has great physiological relevance for TGFß3 activity. Our findings reveal an unusual positive enthalpy (∆H) compensated by a large favourable entropy (∆S) during TGFß3-TßRII interaction. In addition to the hydrophobic effect, we propose that a distinct conformational switch from "closed" to "open" form as experienced by TGFß3 on binding to TßRII is contributing significantly to the increase in overall entropy of the system. Binding studies of TGFß3 and TßRII were carried out at different pH values and salt concentrations to gain further insight into the thermodynamics of the interaction. Furthermore, the importance of hydrophobic interactions on the binding affinity of TßRII with TGFß3 was confirmed by two TßRII variants (interfacial). Finally, a distinct shift from entropy to enthalpy dominated interaction was observed upon recruitment of TßRI to the binary complex forming the ternary complex.

Influence of crowding agents on the dynamics of a multidomain protein in its denatured state: a solvation approach.

It is now well appreciated that the crowded intracellular environment significantly modulates an array of physiological processes including protein folding-unfolding, aggregation, and dynamics to name a few. In this work we have studied the dynamics of domain I of the protein human serum albumin (HSA) in its urea-induced denatured states, in the presence of a series of commonly used macromolecular crowding agents. HSA was labeled at Cys-34 (a free cysteine) in domain I with the fluorophore 6-bromoacetyl-2-dimethylaminonaphthalene (BADAN) to act as a solvation probe. In partially denatured states (2-6 M urea), lower crowder concentrations (~ < 125 g/L) induced faster dynamics, while the dynamics became slower beyond 150 g/L of crowders. We propose that this apparent switch in dynamics is an evidence of a crossover from soft (enthalpic) to hard-core (entropic) interactions between the protein and crowder molecules. That soft interactions are also important for the crowders used here was further confirmed by the appreciable shift in the wavelength of the emission maximum of BADAN, in particular for PEG8000 and Ficoll 70 at concentrations where the excluded volume effect is not dominant.

Thinking beyond tradition: Polyphenols as effective refolding modulators.

Despite polyphenols having had proven roles as amyloid alleviators their service has rarely been made use of in protein refolding/renaturation thus far, where aggregation can be a major competing pathway. TGFß3, expressed in inclusion bodies, is a classical example of a protein prone to high rate of aggregation severely limiting its refolding yield owing to its large cysteine content and structural complexity. Here, we have used various polyphenols (EGCG, baicalein, myricetin) either alone or in combination with the pseudo-chaperone beta cyclodextrin, in the refolding buffer. With the help of non-reducing SDS PAGE and size exclusion chromatography, we showed that refolding in the presence of baicalein or EGCG along with ßCD indeed increase the yield of the native protein in a time dependent manner. EGCG expedites the refolding process giving a maximum increase of the refolding yield within 24 h while baicalein takes as long as 48 h for the same. The mechanism of mode of actions of polyphenols during refolding was further delineated by ITC. The effect of polyphenols on the aggregation kinetics and stability of native TGFß3 were also explored. Thus these small molecules provide a promising alternate route in increasing the yield of aggregation prone proteins during refolding.

The central role of cAMP in regulating Plasmodium falciparum merozoite invasion of human erythrocytes.

All pathogenesis and death associated with Plasmodium falciparum malaria is due to parasite-infected erythrocytes. Invasion of erythrocytes by P. falciparum merozoites requires specific interactions between host receptors and parasite ligands that are localized in apical organelles called micronemes. Here, we identify cAMP as a key regulator that triggers the timely secretion of microneme proteins enabling receptor-engagement and invasion. We demonstrate that exposure of merozoites to a low K+ environment, typical of blood plasma, activates a bicarbonate-sensitive cytoplasmic adenylyl cyclase to raise cytosolic cAMP levels and activate protein kinase A, which regulates microneme secretion. We also show that cAMP regulates merozoite cytosolic Ca2+ levels via induction of an Epac pathway and demonstrate that increases in both cAMP and Ca2+ are essential to trigger microneme secretion. Our identification of the different elements in cAMP-dependent signaling pathways that regulate microneme secretion during invasion provides novel targets to inhibit blood stage parasite growth and prevent malaria.

Development and application of in vivo molecular traps reveals that dynein light chain occupancy differentially affects dynein-mediated processes.

The ability to rapidly and specifically regulate protein activity combined with in vivo functional assays and/or imaging can provide unique insight into underlying molecular processes. Here we describe the application of chemically induced dimerization of FKBP to create nearly instantaneous high-affinity bivalent ligands capable of sequestering cellular targets from their endogenous partners. We demonstrate the specificity and efficacy of these inducible, dimeric "traps" for the dynein light chains LC8 (Dynll1) and TcTex1 (Dynlt1). Both light chains can simultaneously bind at adjacent sites of dynein intermediate chain at the base of the dynein motor complex, yet their specific function with respect to the dynein motor or other interacting proteins has been difficult to dissect. Using these traps in cultured mammalian cells, we observed that induction of dimerization of either the LC8 or TcTex1 trap rapidly disrupted early endosomal and lysosomal organization. Dimerization of either trap also disrupted Golgi organization, but at a substantially slower rate. Using either trap, the time course for disruption of each organelle was similar, suggesting a common regulatory mechanism. However, despite the essential role of dynein in cell division, neither trap had a discernable effect on mitotic progression. Taken together, these studies suggest that LC occupancy of the dynein motor complex directly affects some, but not all, dynein-mediated processes. Although the described traps offer a method for rapid inhibition of dynein function, the design principle can be extended to other molecular complexes for in vivo studies.