Biology subjects

Maiti, N. C.

Publications and source records attributed to Maiti, N. C..

4 recordsLinked to original sources

bioRxiv2024

Dengue Protease-Mediated Activation and Polarization of Macrophages

Controlled activation of macrophages to achieve desired phenotypes in vivo and in vitro could lead to possible treatments for several inflammatory and proliferative diseases. This investigation uniquely established that dengue protease enhances the polarization and activation of human and murine macrophages following extracellular exposure. It was observed that macrophages have a robust response to extracellularly administered purified dengue NS2B/NS3 protease; it stimulated the macrophages in classical M1 directions and enhanced both the mitogen-activated protein kinases (MAPK) and c-Jun N-terminal kinases (JNK). The cells exhibited an increase in the intracellular levels of proinflammatory cytokines such as IL-2, IL-6, IL-12, and IFN-{gamma} and an elevation of phosphorylated versions of Akt, P38 MARK, SAPK, and ERK. In tandem, this investigation also established that the heightened responses of stimulated cells were associated with an increase in the cellular reactive oxygen species (ROS) level and the nuclear translocation of the NF-{kappa}B (P65) protein. The cell viability assay showed that NS2B/NS3 protease exerts no major toxic effect on macrophages after 24 hours of treatment, even at a dosage (20 g/ml) which was four times higher than the effective dose (5 g/ml). Remarkably, we also observed that the native form of the viral protease, which drives its enzyme activities, had no bearing on the antigenic qualities of the enzyme. Thus, our study highlighted the efficacy of dengue viral NS2B/NS3 protease as a non-toxic ex vivo macrophage activating/polarizing agent and may serve a vital role in macrophage-based cell therapy in the near future. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/624958v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@d29cd1org.highwire.dtl.DTLVardef@1357e96org.highwire.dtl.DTLVardef@109cc52org.highwire.dtl.DTLVardef@45536b_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2020

Structure Specific Neuro-toxicity of α-Synuclein Oligomer

Parkinsons disease (PD) is linked to -synuclein (aS) aggregation and deposition of amyloid in the substantia nigra region of the brain tissues. Recent reports suggested that oligomeric assembly structure could be neurotoxic to neuronal cells. In the current investigation we produced two distinct classes of aS oligomers and link the protein conformation state and stability to neuronal cell toxicity. Natural oligomers (NO) enriched with alpha-helical folds are produced in storage of aS at below -20{degrees}C for 7 days. Induced oligomer (IO), often observed in the aggregation pathway of aS were made incubating the protein solution at 37{degrees}C. Natural oligomers remained stable and did not transform into {beta}-sheet rich amyloid fiber and exhibited higher toxicity (80% cell death) compared to induced oligomers. Natural oligomers were ovular shape and the size ranged between 4-5.5 nm. It maintained significant number ([~] 60%) of residues in -helical conformational space. However, initiation of hydrophobic zipping with beta sheet conformation was evidenced in induced oligomer (IO) and a lesser number residues (45%) remained with preference to -helical secondary structure. Hydrophobic collapse leads the transformation of IO into thermodynamically most stable {beta}-sheet rich amyloid fibril. Molten globule like secondary structure stabilized by H-bonding in natural oligomers caused enhanced stability and cellular toxicity compared to induced oligomer. Thus off-pathway/natural oligomers could be plausible reason of neuronal cell death and possible cause of Parkinsons disease.

biophysics↗

bioRxiv2020

Mechanistic Studies of the Stabilization of Insulin Helical Structure by Coomassie Brilliant Blue

Human insulin (HI) is an essential protein hormone and its biological activity mostly depends on folded and active conformation in the monomeric state. The present investigation established that Coomassie Brilliant Blue G-250 (CBBG), a small multicyclic hydroxyl compound can reversibly bind to the hormonal protein dimer and maintained most of -helical folds crucial for biological function of the enzyme. The solution-state 1D NMR and isothermal calorimetric analysis showed a sub-micromolar binding affinity of the molecule to HI. 2D NOESY NMR established that the HI dimer undergoes residue level local conformational change upon binding to CBBG. The chemical shift perturbation and the NOE parameters of active protons of amino acid residues throughout the polypeptides further suggested that CBBG upon binding the protein stabilize -helixes of both the A and B subunits of the hormonal protein. The changes in Gibbs free energy ({triangleup}G) of the binding was of ~-11.1 kcal/mol and suggested a thermodynamically favourable process. The changes in enthalpy ({triangleup}H) and entropy term (T{triangleup}S) were -57.2 kcal/mol and -46.1 kcal/mol, respectively. The negative changes in entropy and the NOE transfer effectiveness of several residues in the presence of CBBG molecules indicated that the binding was an enthalpy driven favourable equilibrium process. The NMR-based atomic resolution data and molecular docking studies confirmed that the CBBG binds to HI at the dimeric stage and prevents the availability of the crucial residue segments that partake directly in further oligomerization and subsequent fibrillation. Extended computational analysis based on chemical shift perturbation of protons of active residues further established receptor-ligand based pharmacophore model comprised of 5 hydrophobic and a hydrogen bond acceptor features that can anchor the residues at the A and B chains of HI and inhibit the partial unfolding and hydrophobic collapse to nucleate the fibrillation. Taken together, the results demonstrated that CBBG and their close analogues might be useful to develop a formulation that will maintain the active and functional form of the hormonal protein for a significantly longer time. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/267799v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@b2c363org.highwire.dtl.DTLVardef@39b7b6org.highwire.dtl.DTLVardef@14c4d71org.highwire.dtl.DTLVardef@704f00_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2019

Deciphering the structural intricacy in virulence effectors for proton-motive force mediated unfolding and type-III protein secretion

Many gram-negative pathogenic bacteria use type III secretion system (T3SS) to inject virulence effectors directly into the cytosol of targeted host cells. Given that the protein unfolding requisite for secretion via nano-size pore of T3SS injectisome is an energetically unfavorable process, "How do pathogenic bacteria unfold and secrete hundreds of toxic proteins in seconds" remain largely unknown. In this study, first, from an in-depth analysis of folding and stability of T3SS effector ExoY, we show that the proton-concentration gradient ([~]pH 5.8-6.0) generated by proton-motive force (PMF) can significantly amortize tertiary structural folding and stability of effectors without significant entropic cost. Strikingly, it was found that the lower energetic cost associated with the global unfolding of ExoY is mainly due to its weakly folded geometry and abundance of geometrical frustrations stemming from buried water molecules and native-like folded intermediates in the folded cores. From in-silico structural analysis of 371 T3SS effectors, it can be curtained that T3SS effectors belong to typical class (disorder globules) of IDPs and have evolved similar conserved intrinsic structural archetypes to mediate early-stage unfolding. The slower folding kinetics in effector proteins requisite for efficient T3SS-mediated secretion mostly stems from reduced hydrophobic density and enhanced polar-polar repulsive interactions in their sequence landscapes. Lastly, the positively evolved histidine-mediated stabilizing interactions and gate-keeper residues in effector proteins shed light on collaborative role of evolved structural chemistry in T3SS effectors and PMF in the spatial-temporal regulation of effector folding and stability essential for maintaining balance in secretion and function trade-off.

biophysics↗