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Studies of Protein Folding and Misfolding Using Fluorescence Correlation Spectroscopy (FCS) and other Biophysical Methods
No full textParkinson‟s disease (PD) is a multifactorial movement disorder (resting of tremor, rigidity,
Bradykinesia), in which both genetic as well as environmental factors play important roles.
Several evidences have been accumulated which implies that the aggregation of α-synuclein is a
critical factor in PD manifestation. Rare familial cases of PD have been associated with
mutations in α-synuclein even though the function of α-synuclein is still completely unknown.
The primary structure of purified α-synuclein, exists as a “natively unfolded” protein although a
recent report raises some controversies over the native structure of protein. A number of different
conformational states of α-synuclein including partially-folded (key intermediate in aggregation
and fibrillation), oligomeric species, as well as fibrillar and amorphous aggregates have been
observed. A number of factors that either accelerate or inhibit the rate of fibrillation have been
observed in vitro. Accelerators include environmental factors (certain pesticides and metals),
molecular crowding and various natural and synthetic charged polymers. α‑synuclein (α‑syn)
aggregation can take place either in the cytoplasm or when in association with the cellular
membrane. In the cytosol, unfolded monomers interact to form oligomers of varying
morphologies that eventually gets converted in to fibrils. The accumulation of these amyloid
fibrils leads to the formation of intracellular inclusions called Lewy bodies. Membrane bound
monomeric α‑syn adopts a predominantly α‑helical conformation and it undergoes a
conformational change via oligomerization to eventually form membrane bound β‑sheet‑rich
structures that self‑associate to form trans‑membrane amyloid pores and fibrils. During α‑syn
fibrillogenesis and aggregation, the intermediate species (oligomers and amyloid fibrils) are
highly toxic, affecting mitochondrial function, endoplasmic reticulum–Golgi trafficking, protein
degradation and/or synaptic transmission, all of which are thought to induce the process of
neurodegeneration.
We have aimed to establish the aggregation mechanism of alpha synuclein in membrane as well
as in cytosol. With the help of mainly single molecule fluorescence spectroscopy, a number of
heterogeneous intermediates are indentified in the presence of membrane or membrane mimics.
In this case, the secondary and tertiary structural changes, dynamic fluctuations (extended and
compact), and accumulation of aggregation prone species are observed in order to understand the
structural features and contribution of such conformations in the formation of amyloid
aggregates. Interestingly, the aggregation mechanism and the structural features of amyloid
fibrils in WT and familial mutants of alpha synuclein are different. This indicates that there is a
correlation between conformational changes in intermediates and the rate of aggregation.
But in the case of cytosol, the stability and aggregation mechanism of a protein cannot be
measured with the help of conventional methods as the viscosity and available space (i.e.;
excluded volume) severely alters and hence affects the single molecule detection. Here we have
developed a strategy that employs „two-diffusion fit‟ and „effective medium model‟ to evaluate
the conformation of proteins in the presence of viscous medium like polyacrylamide gel. With
the help of this model, we were able to measure the conformation of proteins with accuracy even
under viscous conditions. We could also explain the stability of protein as a function of excluded
volume effect. The structural deviation of a-syn from that of globular proteins have been shown
to deviate more under viscous conditions than under solution conditions, which could be
explained more clearly by using this method. The proper monitoring of heterogeneous
conformations that are generated during the aggregation process of alpha synuclein in membrane
and cytosol could be enabled which may pave way for newer therapeutics applications and may
help in revealing the mystery of protein stability in cellular environments
IRAK-M regulates the inhibition of TLR-mediated macrophage immune response during late in vitro Leishmania donovani infection
No full textIntramacrophage protozoan parasite Leishmania donovani, causative agent of visceral leishmaniasis, escapes Toll-like receptor (TLR) dependent early host immune response
by inducing the deubiquitinating enzyme A20, which is sustained up to 6 h postinfection only. Therefore, Leishmania must apply other means to deactivate late host responses. Here, we elucidated the role of IL-1 receptor-associated kinaseM(IRAK-M), a negative regulator
of TLR signaling, in downregulating macrophage proinflammatory response during late hours of in vitro infection. Our data reveal a sharp decline in IRAK1 and IRAK4 phosphorylation at 24 h postinfection along with markedly reduced association of IRAK1–TNF receptor associated factor 6, which is mandatory for TLR activation. In contrast, IRAK-M was induced after A20 levels decreased and reached a maximum at 24 h postinfection. IRAK-M induction coincided with increased stimulation of TGF-β, a hallmark cytokine of visceral infection. TGF-β-dependent signaling-mediated induction of SMAD family of proteins, 2, 3, and 4 plays important roles in transcriptional upregulation of IRAK-M. In infected macrophages, siRNA-mediated silencing of IRAK-M displayed enhanced IRAK1
and IRAK4 phosphorylation with a concomitant increase in downstream NF-κB activity and reduced parasite survival. Taken together, the results suggest that IRAK-M may be
targeted by L. donovani to inhibit TLR-mediated proinflammatory response late during in vitro infectio
Bioremediation by alkaline protease (AkP) from edible mushroom Termitomyces clypeatus: optimization approach based on statistical design and characterization for diverse applications
No full textBACKGROUND: Bioremediation using enzymes has become an attractive approach for removing hazardous chemicals from the environment. The present study investigated theproduction, optimization andapplications of an extracellular alkalineprotease(AkP) fromthe edible mushroom Termitomyces clypeatus focusing on bioremediation.
RESULTS: For optimized enzyme production, the variables significantly influencing the protease activitywere screened through the 11 factorial Plackett–Burman design. The optimum values of the selected variables and their mutual interactions were determined through response surfacemethodology using the Box–Behnken experimental design. Overall a 10-fold increase in enzyme activity (1830 UmL-1)was obtained which closely matched the yield predicted by the statisticalmodel (1778.63 UmL-1)
with R2 =0.9819. AkP could efficiently dehair the goat skin, detached bird feather vanes from its shaft and reduce BOD, COD and pH of tannery waste effluent. The AkP also demonstrated bactericidal property against Staphylococcus aureus as detected by MIC and flow cytometry analyses.
CONCLUSION: As an ecofriendly alternative, the enzyme showed significant promise for bioremediation and industrial applications through time-saving bioprocesses. This is first report of alkaline protease from T. clypeatus or from a fungal source with wide-ranging application potential
DNA Topoisomerases and their Interacting Proteins as Targets for development of Therapeutics in Cancer
No full textThe present dissertation is divided into three Chapters. In the first Chapter of the work we found that majority of colon cancer cell lines have defects in the G2
decatenation checkpoint function to different extents and such decatenation checkpoint defective cell lines are comparatively more sensitive to ICRF193 (a
catalytic inhibitor of topo I
Sequence and expression variations in 23 genes involved in mitochondrial and non-mitochondrial apoptotic pathways and risk of oral leukoplakia and cancer
No full textOral cancer is usually preceded by pre-cancerous lesion and related to tobacco abuse. Tobacco carcinogens
damage DNA and cells harboring such damaged DNA normally undergo apoptotic death, but cancer cells are
exceptionally resistant to apoptosis. Here we studied association between sequence and expression variations
in apoptotic pathway genes and risk of oral cancer and precancer. Ninety nine tag SNPs in 23 genes, involved
in mitochondrial and non-mitochondrial apoptotic pathways,were genotyped in 525 cancer and 253 leukoplakia
patients and 538 healthy controls using Illumina Golden Gate assay. Six SNPs (rs1473418 at BCL2; rs1950252 at
BCL2L2; rs8190315 at BID; rs511044 at CASP1; rs2227310 at CASP7 and rs13010627 at CASP10) significantlymodified
risk of oral cancer but SNPs only at BCL2, CASP1and CASP10 modulated risk of leukoplakia. Combination of
SNPs showed a steep increase in risk of cancerwith increase in “effective” number of risk alleles. In silico analysis
of published data set and our unpublished RNAseq data suggest that change in expression of BID and CASP7 may
have affected risk of cancer. In conclusion, three SNPs, rs1473418 in BCL2, rs1950252 in BCL2L2 and rs511044 in
CASP1, are being implicated for the first time in oral cancer. Since SNPs at BCL2, CASP1 and CASP10modulated risk
of both leukoplakia and cancer, so, they should be studied inmore details for possible biomarkers in transition of
leukoplakia to cancer. This study also implies importance of mitochondrial apoptotic pathway gene (such as
BCL2) in progression of leukoplakia to oral cancer
Doubly chloro bridged dimeric copper(II) complex: magneto-structural correlation and anticancer activity
No full textWe have synthesized and structurally characterized a new doubly chloro bridged dimeric copper(II) complex, [Cu2(μ-Cl)2(HL)2Cl2] (1) based on a Schiff base ligand, 5-[(pyridin-2-ylmethylene)-amino]-pentan-1-ol). Single crystal X-ray diffraction shows the presence of dinuclear copper(II) centres in a square pyramidal geometry linked by obtuse double chloro bridge. The magnetic study illustrated that
weak antiferromagnetic interactions (J = −0.47 cm−1) prevail in complex 1 which is well supported by magneto-structural correlation. This compound adds to the library of doubly chloro bridged copper(II) complexes in the regime of spin state cross over. DFT calculations have been conducted within a brokensymmetry(BS) framework to investigate the exchange interaction further which depicts that the approximate spin projection technique yields the best corroboration of the experimental J value. Spin density
plots show the presence of an ∼0.52e charge residing on the copper atom along with a substantial charge on bridging and peripheral chlorine atoms. The potential of complex 1 to act as an anticancer agent is thoroughly examined on a series of liver cancer cell lines and screening shows the HepG2 cell line exhibits maximum cytotoxicity by phosphatidyl serine exposure in the outer cell membrane associated with ROS generation and mitochondrial depolarization with increasing time in the in vitro
model system
Metabolic reconfiguration of the central glucose metabolism: a crucial strategy of Leishmania donovani for its survival during oxidative stress
No full textUnderstanding the mechanism that allows the intracellular protozoan parasite Leishmania donovani (Ld) to respond to reactive oxygen species (ROS) is of increasing therapeutic importance because of the continuing resistance toward antileishmanial drugs and for determining the illusive survival strategy of these parasites. A shift in primary carbon metabolism is the fastest response to oxidative stress. A 14CO2 evolution study, expression of glucose transporters together with consumption assays, indicated a shift in metabolic flux of the parasites from glycolysis toward pentose phosphate pathway(PPP) when exposed to different oxidants in vitro/ex vivo. Changes in gene expression, protein levels, and enzyme activities all pointed to a metabolic reconfiguration of the central glucose metabolism in response to oxidants. Generation of glucose-6-phosphate dehydrogenase (G6PDH) (∼5-fold) and transaldolase (TAL) (∼4.2-fold) overexpressing Ld cells reaffirmed that lethal doses of ROS were counterbalanced by effective manipulation of NADPH:NADP+ ratio and stringent maintenance of reduced thiol content. The extent of protein carbonylation and accumulation of lipid peroxidized products were also found to be less in overexpressed cell lines. Interestingly, the LD50 of sodiumantimony gluconate (SAG),amphotericin-B (AmB), and miltefosine were significantly high toward overexpressing parasites. Consequently, this study illustrates that Ld strategizes a metabolic reconfiguration for replenishment of NADPH pool to
encounter oxidative challenges.—Ghosh, A. K., Sardar,
A. H., Mandal, A., Saini, S., Abhishek, K., Kumar, A.,
Purkait, B., Singh, R., Das, S., Mukhopadhyay, R., Roy,
S., Das, P. Metabolic reconfiguration of the central
glucose metabolism: a crucial strategy of Leishmania
donovani for its survival during oxidative stress
Attenuation of the Early Events of α‑Synuclein Aggregation: A Fluorescence Correlation Spectroscopy and Laser Scanning Microscopy Study in the Presence of Surface-Coated Fe3O4 Nanoparticles
No full textThe aggregation of α-synuclein (A-syn) has been implicated in the pathogenesis of Parkinson’s disease (PD). Although the early events of aggregation and not the matured amyloid fibrils are believed to be responsible for the toxicity, it has been difficult to probe the formation of early oligomers
experimentally. We studied the effect of Fe3O4 nanoparticle (NP) in the early stage of aggregation of A-syn using fluorescence correlation spectroscopy (FCS) and laser scanning microscopy. The binding between the monomeric protein and NPs was also studied using FCS at single-molecule resolution. Our data showed that the addition of bare Fe3O4 NPs accelerated the rate of early aggregation, and it did not bind the monomeric A-syn. In contrast, L-lysine (Lys)-coated Fe3O4 NPs showed strong binding with the monomeric A-syn, inhibiting the early events of aggregation. Lys-coated Fe3O4 NPs showed significantly less cell toxicity compared with bare Fe3O4 NPs and can be explored as a possible strategy to develop therapeutic application against PD. To the best of our knowledge, this report is the first example of using a small molecule to attenuate the early (and arguably the most relevant in terms of PD pathogenesis) events of A-syn aggregation
Isolation, structural elucidation and cytotoxicity evaluation of a new pentahydroxy-pimarane diterpenoid along with other chemical constituents from Aerva lanata
No full textAerva lanata possesses various useful medicinal and pharmaceutical activities.Phytochemical investigation of the plant has now led to the isolation of a new
2a,3a,15,16,19-pentahydroxy pimar-8(14)-ene diterpenoid (1) together with 12 other known compounds identified as b-sitosterol (2), b-sitosterol-3-O-b-D-glucoside (3),
canthin-6-one (4), 10-hydroxycanthin-6-one (aervine, 5), 10-methoxycanthin-6-one (methylaervine, 6), b-carboline-1-propionic acid (7), 1-O-b-D-glucopyranosyl- (2S,3R,8E)-2-[(20R)-2-hydroxylpalmitoylamino]-8-octadecene-1,3-diol (8), 1-O-(b-Dglucopyranosyl)-(2S,3S,4R,8Z)-2-[(20R)-20-hydroxytetracosanoylamino]-8(Z)-octadene-1,3,4-triol (9), (2S,3S,4R,10E)-2-[(20R)-20-hydroxytetracosanoylamino]-10-octadecene-1,3,4-triol (10), 60-O-(400-hydroxy-trans-cinnamoyl)-kaempferol-3-O-b-Dglucopyranoside
(tribuloside, 11), 3-cinnamoyltribuloside (12) and sulfonoquinovosyldiacylglyceride (13). Among these, six compounds (8–13) are reported for the first
time from this plant. Cytotoxicity evaluation of the compounds against five cancer cell lines (CHO, HepG2, HeLa, A-431 and MCF-7) shows promising IC50 values for
compounds 4, 6 and 12