Raman Spectroscopic Insights of Phase-Separated Insulin Aggregates.

Phase-separated protein accumulation through the formation of several aggregate species is linked to the pathology of several human disorders and diseases. Our current investigation envisaged detailed Raman signature and structural intricacy of bovine insulin in its various forms of aggregates produced in situ at an elevated temperature (60 °C). The amide I band in the Raman spectrum of the protein in its native-like conformation appeared at 1655 cm-1 and indicated the presence of a high content of α-helical structure as prepared freshly in acidic pH. The disorder content (turn and coils) also was predominately present in both the monomeric and oligomeric states and was confirmed by the presence shoulder amide I maker band at ∼1680 cm-1. However, the band shifted to ∼1671 cm-1 upon the transformation of the protein solution into fibrillar aggregates as produced for a longer time of incubation. The protein, however, maintained most of its helical conformation in the oligomeric phase; the low-frequency backbone α-helical conformation signal at ∼935 cm-1 was similar to that of freshly prepared aqueous protein solution enriched in helical conformation. The peak intensity was significantly weak in the fibrillar aggregates, and it appeared as a good Raman signature to follow the phase separation and the aggregation behavior of insulin and similar other proteins. Tyrosine phenoxy moieties in the protein may maintained its H-bond donor-acceptor integrity throughout the course of fibril formation; however, it entered in more hydrophobic environment in its journey of fibril formation. In addition, it was noticed that oligomeric bovine insulin maintained the orientation/conformation of the disulfide bonds. However, in the fibrillar state, the disulfide linkages became more strained and preferred to maintain a single conformation state.

Structure specific neuro-toxicity of α-synuclein oligomer.

Parkinson's disease (PD) is linked to α-synuclein (aS) aggregation and deposition of amyloid in the substantia nigra region of the brain tissues. In the current investigation we produced two distinct classes of aS oligomer of differed protein conformation, stability and compared their toxic nature to cultured neuronal cells. Lyophilized oligomer (LO) was produced in storage of aS at-20 °C for 7 days and it was enriched with loosely hold molten globule like structure with residues having preferences for α-helical conformational space. The size of the oligomer was 4-5.5 nm under AFM. This kind of oligomer exhibited potential toxicity towards neuronal cell lines and did not transform into compact ß-sheet rich amyloid fiber even after incubation at 37 °C for several days. Formation of another type of oligomer was often observed in the lag phase of aS fibrillation that often occurred at an elevated temperature (37 °C). This kind of heat induced oligomer (IO) was more hydrophobic and relatively less toxic to neuronal cells compared to lyophilized oligomer (LO). Importantly, initiation of hydrophobic zipping of aS caused the transformation of IO into thermodynamically stable ß-sheet rich amyloid fibril. On the other hand, the presence of molten globule like conformation in LO, rendered greater toxicity to cultured neuronal cells.

Single point mutations at the S129 residue of α-synuclein and their effect on structure, aggregation, and neurotoxicity.

Parkinson's disease is an age-related neurological disorder, and the pathology of the disease is linked to different types of aggregates of α-synuclein or alpha-synuclein (aS), which is an intrinsically disordered protein. The C-terminal domain (residues 96-140) of the protein is highly fluctuating and possesses random/disordered coil conformation. Thus, the region plays a significant role in the protein's solubility and stability by an interaction with other parts of the protein. In the current investigation, we examined the structure and aggregation behavior of two artificial single point mutations at a C-terminal residue at position 129 that represent a serine residue in the wild-type human aS (wt aS). Circular Dichroism (CD) and Raman spectroscopy were performed to analyse the secondary structure of the mutated proteins and compare it to the wt aS. Thioflavin T assay and atomic force microscopy imaging helped in understanding the aggregation kinetics and type of aggregates formed. Finally, the cytotoxicity assay gave an idea about the toxicity of the aggregates formed at different stages of incubation due to mutations. Compared to wt aS, the mutants S129A and S129W imparted structural stability and showed enhanced propensity toward the α-helical secondary structure. CD analysis showed proclivity of the mutant proteins toward α-helical conformation. The enhancement of α-helical propensity lengthened the lag phase of fibril formation. The growth rate of ß-sheet-rich fibrillation was also reduced. Cytotoxicity tests on SH-SY5Y neuronal cell lines established that the S129A and S129W mutants and their aggregates were potentially less toxic than wt aS. The average survivability rate was ∼40% for cells treated with oligomers (presumably formed after 24 h of incubation of the freshly prepared monomeric protein solution) produced from wt aS and ∼80% for cells treated with oligomers obtained from mutant proteins. The relative structural stability with α-helical propensity of the mutants could be a plausible reason for their slow rate of oligomerization and fibrillation, and this was also the possible reason for reduced toxicity to neuronal cells.

Coomassie brilliant blue G-250 acts as a potential chemical chaperone to stabilize therapeutic insulin.

Our studies show Coomassie Brilliant Blue G-250 as a promising chemical chaperone that stabilises the α-helical native human insulin conformers, disrupting their aggregation. Furthermore, it also increases the insulin secretion. This multipolar effect coupled with its non-toxic nature could be useful for developing highly bioactive, targeted and biostable therapeutic insulin.

Dabrafenib, idelalisib and nintedanib act as significant allosteric modulator for dengue NS3 protease.

Dengue virus (DENV) encodes a unique protease (NS3/NS2B) essential for its maturation and infectivity and, it has become a key target for anti-viral drug design to treat dengue and other flavivirus related infections. Present investigation established that some of the drug molecules currently used mainly in cancer treatment are susceptible to bind non-active site (allosteric site/ cavity) of the NS3 protease enzyme of dengue virus. Computational screening and molecular docking analysis found that dabrafenib, idelalisib and nintedanib can bind at the allosteric site of the enzyme. The binding of the molecules to the allosteric site found to be stabilized via pi-cation and hydrophobic interactions, hydrogen-bond formation and π-stacking interaction with the molecules. Several interacting residues of the enzyme were common in all the five serotypes. However, the interaction/stabilizing forces were not uniformly distributed; the π-stacking was dominated with DENV3 proteases, whereas, a charged/ionic interaction was the major force behind interaction with DENV2 type proteases. In the allosteric cavity of protease from DENV1, the residues Lys73, Lys74, Thr118, Glu120, Val123, Asn152 and Ala164 were involved in active interaction with the three molecules (dabrafenib, idelalisib and nintedanib). Molecular dynamics (MD) analysis further revealed that the molecules on binding to NS3 protease caused significant changes in structural fluctuation and gained enhanced stability. Most importantly, the binding of the molecules effectively perturbed the protein conformation. These changes in the protein conformation and dynamics could generate allosteric modulation and thus may attenuate/alter the NS3 protease functionality and mobility at the active site. Experimental studies may strengthen the notion whether the binding reduce/enhance the catalytic activity of the enzyme, however, it is beyond the scope of this study.

Impact of porous nanomaterials on inhibiting protein aggregation behaviour.

Aggregation of intrinsically disordered as well as the ordered proteins under certain premises or physiological conditions leads to pathological disorder. Here we have presented a detailed investigation on the effect of a porous metallic (Au) and a non-metallic (Si) nanomaterial on the formation of ordered (fiber-like/amyloid) and disordered (amorphous) aggregates of proteins. Porous nanogold (PNG) was found to reduce the amyloid aggregation of insulin but does not have much impact on the lag phase in the aggregation kinetics, whereas porous nano-silica (PNS) was found both to decrease the amount of aggregation as well as prolong the lag phase of amyloid fiber formation from insulin. On the other hand, both the porous nanoparticles are found to decrease the extent of amorphous aggregation (with slight improvement for PNS) of pathogenic huntingtin (Htt) protein in Huntington's disease cell model. This is a noted direct observation in controlling and understanding protein aggregation diseases which may help us to formulate nanotherapeutic drugs for future clinical applications.

Order, Disorder, and Reorder State of Lysozyme: Aggregation Mechanism by Raman Spectroscopy.

Lysozyme, like many other well-folded globular proteins, under stressful conditions produces nanoscale oligomer assembly and amyloid-like fibrillar aggregates. With engaging Raman microscopy, we made a critical structural analysis of oligomer and other assembly structures of lysozyme obtained from hen egg white and provided a quantitative estimation of a protein secondary structure in different states of its fibrillation. A strong amide I Raman band at 1660 cm-1 and a N-Cα-C stretching band at ∼930 cm-1 clearly indicated the presence of a substantial amount of α-helical folds of the protein in its oligomeric assembly state. In addition, analysis of the amide III region and Raman difference spectra suggested an ample presence of a PPII-like secondary structure in these oligomers without causing major loss of α-helical folds, which is found in the case of monomeric samples. Circular dichroism study also revealed the presence of typical α-helical folds in the oligomeric state. Nonetheless, most of the Raman bands associated with aromatic residues and disulfide (-S-S-) linkages broadened in the oligomeric state and indicated a collapse in the tertiary structure. In the fibrillar state of assembly, the amide I band became much sharper and enriched with the ß-sheet secondary structure. Also, the disulfide bond vibration in matured fibrils became much weaker compared to monomer and oligomers and thus confirmed certain loss/cleavage of this bond during fibrillation. The Raman band of tryptophan and tyrosine residues indicated that some of these residues experienced a greater hydrophobic microenvironment in the fibrillar state than the protein in the oligomeric state of the assembly structure.

Astrakurkurone, a sesquiterpenoid from wild edible mushroom, targets liver cancer cells by modulating Bcl-2 family proteins.

Induction of apoptosis is the target of choice for modern chemotherapeutic treatment of cancer, where lack of potent "target-specific" drugs has led to extensive research on anticancer compounds from natural sources. In our study, we have used astrakurkurone, a triterpene isolated from wild edible mushroom, Astraeus hygrometricus. We have discussed the structure and stability of astrakurkurone employing single-crystal X-ray crystallography and studied its potential apoptogenicity in hepatocellular carcinoma (HCC) cells. Our experiments reveal that it is cytotoxic against the HCC cell lines (Hep 3B and Hep G2) at significantly low doses. Further investigations indicated that astrakurkurone acts by inducing apoptosis in the cells, disrupting mitochondrial membrane potential and inducing the expression of Bcl-2 family proteins, for example, Bax, and the downstream effector caspases 3 and 9. A molecular docking study also predicted direct interactions of the drug with antiapoptotic proteins Bcl-2 and Bcl-xL. Thus, astrakurkurone could become a valuable addition to the conventional repertoire of future anticancer drugs. © 2019 IUBMB Life, 1-11, 2019.

Structural Insight of Amyloidogenic Intermediates of Human Insulin.

Engaging Raman spectroscopy as a primary tool, we investigated the early events of insulin fibrilization and determined the structural content present in oligomer and protofibrils that are formed as intermediates in the fibril formation pathway. Insulin oligomer, as obtained upon incubation of zinc-free insulin at 60 °C, was mostly spherical in shape, with a diameter of 3-5 nm. Longer incubation produced "necklace"-like beaded protofibrillar assembly species. These intermediates eventually transformed into 5-8 nm thick fibers with smooth surface texture. A broad amide I band in the Raman spectrum of insulin monomer appeared at 1659 cm-1, with a shoulder band at 1676 cm-1. This signature suggested the presence of major helical and extended secondary structure of the protein backbone. In the oligomeric state, the protein maintained its helical imprint (∼50%) and no substantial increment of the compact cross-ß-sheet structure was observed. A nonamide helix signature band at 940 cm-1 was present in the oligomeric state, and it was weakened in the fibrillar structure. The 1-anilino-8-naphthalene-sulfonate binding study strongly suggested that a collapse in the tertiary structure, not the major secondary structural realignment, was the dominant factor in the formation of oligomers. In the fibrillar state, the contents of helical and disordered secondary structures decreased significantly and the ß-sheet amount increased to ∼62%. The narrow amide I Raman band at 1674 cm-1 in the fibrillar state connoted the formation of vibrationally restricted highly organized ß-sheet structure with quaternary realignment into steric-zipped species.

Envisaging the Structural Elevation in the Early Event of Oligomerization of Disordered Amyloid ß Peptide.

In Alzheimer's disease (AD), amyloid ß (Aß) protein plays a detrimental role in neuronal injury and death. Recent in vitro and in vivo studies suggest that soluble oligomers of the Aß peptide are neurotoxic. Structural properties of the oligomeric assembly, however, are largely unknown. Our present investigation established that the 40-residue-long Aß peptide (Aß40) became more helical, ordered, and compact in the oligomeric state, and both the helical and ß-sheet components were found to increase significantly in the early event of oligomerization. The band-selective two-dimensional NMR analysis suggested that majority of the residues from sequence 12 to 22 gained a higher-ordered secondary structure in the oligomeric condition. The presence of a significant amount of helical conformation was confirmed by Raman bands at 1650 and 1336 cm-1. Other residues remained mostly in the extended polyproline II (PPII) and less compact ß-conformation space. In the event of maturation of the oligomers into an amyloid fiber, both the helical content and the PPII-like structural components declined and ∼72% residues attained a compact ß-sheet structure. Interestingly, however, some residues remained in the collagen triple helix/extended 2.51-helix conformation as evidenced by the amide III Raman signature band at 1272 cm-1. Molecular dynamics analysis using an optimized potential for liquid simulation force field with the peptide monomer indicated that some of the residues may have preferences for helical conformation and this possibly contributed in the event of oligomer formation, which eventually became a ß-sheet-rich amyloid fiber.

Formulation and antitumorigenic activities of nanoencapsulated nifetepimine: A promising approach in treating triple negative breast carcinoma.

Triple negative breast cancer (TNBC) is one of the most common invasive malignancies among women, associated with poor prognosis. Standard chemotherapy targets all dividing cells, resulting in dose-limiting toxicities. In this study, we demonstrated a strategy of encapsulating a hydrophobic synthetic compound, nifetepimine, having anticancer properties, in poly (lactic-co-glycolic acid) nanoparticles to increase selectivity of drug to cancerous cells with minimum toxicity towards normal cells. Nanoencapsulated nifetepimine (30-100nm) having loading and encapsulation efficiency of 7.45% and 75% respectively, was successfully internalized inside TNBC cells upon sustained release resulting in apoptosis. An in vivo bio-distribution study indicated that nanonifetepimine selectively accumulated into breast tumor sites of mice, primarily due to prolonged blood circulation time and binding of nifetepimine to epidermal growth factor receptor that remains overexpressed in most of the TNBC tumors. Moreover, we observed significant reduction in breast tumor volume with improved survival implying high tumor targetability of nanonifetepimine.

Hydrogen bonding plays a significant role in the binding of coomassie brilliant blue-R to hemoglobin: FT-IR, fluorescence and molecular dynamics studies.

An analog of coomassie brilliant blue-R (CBB-R) was recently found to act as an antagonist to ATP-sensitive purinergic receptors (P2X7R) and has potential to be used in medicine. With the aim of understanding its transportation and distribution through blood, in this investigation, we measured the binding parameters of CBB-R with bovine hemoglobin (BHG). The molecule specifically bound to a single binding site of the protein with a stoichiometric ratio of 1 : 1 and the observed binding constant Ka was 3.5, 2.5, 2.0 and 1.5 × 10(5) M(-1) at 20 °C, 27 °C, 37 °C and 45 °C, respectively. The measured respective ΔG(0) values of the binding at four temperatures were -30.45, -22.44, -18.04 and -11.95 kJ mol(-1). The ΔH(0) (change in enthalpy) and ΔS(0) (change in entropy) values were -23.6 kJ mol(-1) and -70.66 J mol(-1) respectively in the binding process. The negative value of ΔH(0) and ΔS(0) indicated that the binding of the molecule was thermodynamically favorable. The best energy structure in the molecular docking analysis revealed that CBB-R preferred to be intercalated in the cavity among the α2, ß1 and ß2 subunits and the binding location was 7.4 Å away from Trp37 in the ß2 subunit. The binding of the molecule with the protein was stabilized by hydrogen bonds involving the side chain of two amino acid residues. The residues were Lys104 and Glu101 in the ß2 subunit. The binding was further stabilized via hydrogen bond formation between the amide group of the peptide backbone (residue Tyr145 of the ß1 subunit) and CBB-R. A shift of the amide I (-C=O stretching) band frequency of ∼8 cm(-1) to low energy was ascribed to the hydrogen bond interaction involving the polypeptide carbonyl of the protein and the CBB-R molecule. In addition, two π-cation interactions between Lys99 of the α2 subunit and Lys104 of the ß2 subunit and CBB-R contributed favorably in the binding processes. No substantial change in the soret and Q absorption bands of BHG could be observed in the presence of CBB-R. It indicated that the oxygen binding domain or the heme proximity was not blocked or substantially perturbed due to the binding of CBB-R. The circular dichroism and the molecular dynamics analysis further established that the binding interaction caused no significant alteration in the protein long range secondary structure.