The Gelsolin Pathogenic D187N Mutant Exhibits Altered Conformational Stability and Forms Amyloidogenic Oligomers.

Gelsolin is an actin-severing protein that attains an open functional conformation in the presence of Ca2+ or low pH. Mutations (D187N/Y) in the second domain of gelsolin trigger the proteolytic pathway producing amyloidogenic fragments that form the pathological hallmark of gelsolin amyloidosis and lattice corneal dystrophy type 2 (LCD2). Here, we show that the D187N mutant gelsolin in a Ca2+ depleted, low pH-activated, open conformation could assemble into amyloidogenic oligomers without necessarily undergoing the specific proteolytic step. Although both wild-type (WT) and mutant proteins exhibit closely overlapping globular shapes at physiological conditions, the latter exhibits subjugated actin depolymerization, loss of thermodynamic stability, and folding cooperativity. Mutant gelsolin displayed aberrant conformational unwinding and formed structural conformers with high associative properties at low pH conditions. A SAXS intensity profile and Guinier analysis of these conformers showed the formation of unusual, higher order aggregates. Extended incubation at low pH resulted in the formation of thioflavin T and Congo red positive, ß-sheet rich aggregates with a fibrillar, amyloid-like morphology visible under electron and atomic force microscopy. Mass spectrometric analysis of disaggregated end-stage fibrils displayed peptide fragments encompassing the entire protein sequence, indicating the involvement of full length mutant gelsolin in fibril formation. Atomistic and REMD simulations indicated a larger increase in solvent accessibility and loss of fold architecture in mutant gelsolin at low pH as compared to WT gelsolin. Our findings support the existence of a secondary oligomerization-dependent aggregation pathway associated with gelsolin amyloidosis and can pave the way for better therapeutic strategies.

Evaluation of novobiocin and telmisartan for anti-CHIKV activity.

Chikungunya has re-emerged as an epidemic with global distribution and high morbidity, necessitating the need for effective therapeutics. We utilized already approved drugs with a good safety profile used in other diseases for their new property of anti-chikungunya activity. It provides a base for a fast and efficient approach to bring a novel therapy from bench to bedside by the process of drug-repositioning. We utilized an in-silico drug screening with FDA approved molecule library to identify inhibitors of the chikungunya nsP2 protease, a multifunctional and essential non-structural protein required for virus replication. Telmisartan, an anti-hypertension drug, and the antibiotic novobiocin emerged among top hits on the screen. Further, SPR experiments revealed strong in-vitro binding of telmisartan and novobiocin to nsP2 protein. Additionally, small angle x-ray scattering suggested binding of molecules to nsP2 and post-binding compaction and retention of monomeric state in the protein-inhibitor complex. Protease activity measurement revealed that both compounds inhibited nsP2 protease activity with IC50 values in the low micromolar range. More importantly, plaque formation assays could show the effectiveness of these drugs in suppressing virus propagation in host cells. We propose novobiocin and telmisartan as potential inhibitors of chikungunya replication. Further research is required to establish the molecules as antivirals of clinical relevance against chikungunya.

L-Asparaginase of Leishmania donovani: Metabolic target and its role in Amphotericin B resistance.

Emergence of Amphotericin B (AmB) resistant Leishmania donovani has posed major therapeutic challenge against the parasite. Consequently, combination therapy aimed at multiple molecular targets, based on proteome wise network analysis has been recommended. In this regard we had earlier identified and proposed L-asparaginase of Leishmania donovani (LdAI) as a crucial metabolic target. Here we report that both LdAI overexpressing axenic amastigote and promastigote forms of L. donovani survives better when challenged with AmB as compared to wild type strain. Conversely, qRT-PCR analysis showed an upregulation of LdAI in both forms upon AmB treatment. Our data demonstrates the importance of LdAI in imparting immediate protective response to the parasite upon AmB treatment. In the absence of structural and functional information, we modeled LdAI and validated its solution structure through small angle X-ray scattering (SAXS) analysis. We identified its specific inhibitors through ligand and structure-based approach and characterized their effects on enzymatic properties (Km, Vmax, Kcat) of LdAI. We show that in presence of two of the inhibitors L1 and L2, the survival of L. donovani is compromised whereas overexpression of LdAI in these cells restores viability. Taken together, our results conclusively prove that LdAI is a crucial metabolic enzyme conferring early counter measure against AmB treatment by Leishmania.