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Conformational flexibility tunes the propensity of transthyretin to form fibrils through non-native intermediate states
No full textThe formation of partially unfolded intermediates
through conformational excursions out of the native state is the starting point of many diseases involving protein aggregation. Therapeutic strategies often aim to stabilize the native structure and prevent the formation of intermediates that are also cytotoxic in vivo. However, their transient nature and low population makes it difficult to characterize these intermediates. We have probed the backbone dynamics of transthyretin (TTR) over an extended timescale by using NMR spectroscopy and MD simulations. The location and extent of these motions
indicates that the backbone flexibility of TTR is a cause of
dissociation and destabilization, both of which are responsible for fibril formation. Importantly, approximately 10% of wildtype TTR exists in an intermediate state, which increased to up to 28% for pathogenic TTR mutants, for which the formation of the intermediate state is shown to be energetically more favorable compared to the wild type. This result suggests an important role for the intermediates in TTR amyloidosis
Reduced phosphorylation of Stat3 at Ser-727 mediated by casein kinase 2 — Protein phosphatase 2A enhances Stat3 Tyr-705 induced tumorigenic potential of glioma cells
No full textSignal transducer and activator of transcription 3 (Stat3) is a transcription factor that is involved in cell survival
and proliferation and has been found to be persistently activated inmost human cancers mainly through its phosphorylation at Tyr-705. However, the role and regulation of Stat3 Ser-727 phosphorylation in cancer cells
have not been clearly evaluated. In our findings, correlation studies on the expression of CK2 and Stat3
Ser-727 phosphorylation levels in human glioma patient samples as well as rat orthotopic tumor model show
a degree of negative correlation. Moreover, brain tumor cell lines were treated with various pharmacological
inhibitors to inactivate the CK2 pathway. Here, increased Stat3 Ser-727 phosphorylation upon CK2 inhibition
was observed. Overexpression of CK2 (α, α′ or β subunits) by transient transfection resulted in decreased
Stat3 Ser-727 phosphorylation. Stat3 Tyr-705 residuewas conversely phosphorylated in similar situations. Interestingly, wefound PP2A, a protein phosphatase, to be amediator in the negative regulation of Stat3 Ser-727 phosphorylation by CK2. In vitro assays prove that Ser-727 phosphorylation of Stat3 affects the transcriptional activity of its downstream targets like SOCS3, bcl-xl and Cyclin D1. Stable cell lines constitutively expressing Stat3 S727A mutant showed increased survival, proliferation and invasionwhich are characteristics of a cancer cell. Rat tumor models generated with the Stat3 S727A mutant cell line formed more aggressive tumors when compared to the
Stat3WT expressing stable cell line. Thus, in glioma, reduced Stat3 Ser-727 phosphorylation enhances tumorigenicity which may be regulated in part by CK2–PP2A pathwa
Gold(III) Chloride Catalyzed Synthesis of Chiral Substituted 3- Formyl Furans from Carbohydrates: Application in the Synthesis of 1,5-Dicarbonyl Derivatives and Furo[3,2-c]pyridine
No full textThis report describes a gold(III)-catalyzed efficient
general route to densely substituted chiral 3-formyl furans
under extremely mild conditions from suitably protected 5-
(1-alkynyl)-2,3-dihydropyran-4-one using H2O as a nucleophile.The reaction proceeds through the initial formation of an activated alkyne–gold(III) complex intermediate, followed by either a domino nucleophilic attack/anti-endo-dig cyclization, or the formation of a cyclic oxonium ion with subsequent attack by H2O. To confirm the proposed mechanistic pathway, we employed MeOH as a nucleophile instead of H2O to result in a substituted furo[3,2-c]pyran derivative, as anticipated. The similar furo[3,2-c]pyran skeleton with a hybrid carbohydrate–furan derivative has also been achieved through pyridinium dichromate (PDC) oxidation of a substituted chiral 3-formyl furan. The corresponding protected 5-(1-alkynyl)-2,3-dihydropyran-4-one can be synthesized from the monosaccharides (both hexoses and pentose) following oxidation, iodination, and Sonogashira coupling
sequences. Furthermore, to demonstrate the potentiality of
chiral 3-formyl furan derivatives, a TiBr4-catalyzed reaction of these derivatives has been shown to offer efficient access to 1,5-dicarbonyl compounds, which on treatment with NH4OAc in slightly acidic conditions afforded substituted furo[3,2-c]pyridine
Association of Heme Oxygenase 1 with the Restoration of Liver Function after Damage in Murine Malaria by Plasmodium yoelii
No full textThe liver efficiently restores function after damage induced during malarial infection once the parasites are cleared from the blood. However, the molecular events leading to the restoration of liver function after malaria are still obscure. To study this, we developed a suitable model wherein mice infected with Plasmodium yoelii (45% parasitemia) were treated with the antimalarial
�/�-arteether to clear parasites from the blood and, subsequently, restoration of liver function was monitored. Liver function tests clearly indicated that complete recovery of liver function occurred after 25 days of parasite clearance. Analyses of proinflammatory
gene expression and neutrophil infiltration further indicated that hepatic inflammation, which was induced immediately after parasite clearance from the blood, was gradually reduced. Moreover, the inflammation in the liver after parasite clearance was found to be correlated positively with oxidative stress and hepatocyte apoptosis. We investigated the role of heme oxygenase 1 (HO-1) in the restoration of liver function after malaria because HO-1 normally renders protection against inflammation,
oxidative stress, and apoptosis under various pathological conditions. The expression and activity of HO-1 were found
to be increased significantly after parasite clearance. We even found that chemical silencing of HO-1 by use of zinc protoporphyrin enhanced inflammation, oxidative stress, hepatocyte apoptosis, and liver injury. In contrast, stimulation of HO-1 by cobalt protoporphyrin alleviated liver inflammation and reduced oxidative stress, hepatocyte apoptosis, and associated tissue injury. Therefore, we propose that selective induction of HO-1 in the liver would be beneficial for the restoration of liver function after
parasite clearanc
Biophysical studies on the interaction of isoquinoline alkaloids and analogues with nucleic acids
No full textDeoxyribonucleic acid, generally referred to as DNA by its acronym, is the basis of life, which
contains the information for the development and functioning of all the living organisms.
The DNA research began in 1868, when Swiss physiological chemist Friedrich Miescher first
identified what he called “nuclein” inside the nuclei of human white blood cells. In 1889, R.
Altmann separated nuclein from protein and because of its acidic character he named it nucleic
acid. Phoebus Levene identified the components of DNA and showed that they were linked
in the order phosphate- sugar-base to form units which he referred as nucleotide and
suggested that the DNA molecule consisted of a string of nucleotide units linked together
through the phosphate groups, which is the ‘backbone’ of the molecule. The role of DNA in
heredity was recognized in 1944, when Avery and co-workers published their famous result
that DNA and not proteins were the carriers of genetic information (Avery et al., 1944). The
complementary base-pair rule was found by Chargaff in 1950 (Chargaff et al., 1950). A milestone
in DNA research was the double helix structure which was proposed by Watson and Crick in
1953 based upon the X-ray fiber diffraction data from fibres of DNA obtained by Rosalind
Franklin (Watson and Crick, 1953). From the double helical structure, it was immediately
obvious how information could pass from one generation to the next by synthesis of DNA
complementary strands from parent strands. In 1962 they received the Nobel Prize in Chemistry
for the discovery along with the crystallographer Maurice Wilkins. The first published account
of the directed chemical synthesis of an oligonucleotide occurred in 1955 when Michelson
and Todd reported the preparation of a dithymidinyl nucleotide (Michelson and Todd, 1955).
In the late 1950’s Khorana and his group were able to synthesize oligoribonucleotides that
were used to confirm the Genetic Code. In 1968, Khorana received the Nobel Prize in
Physiology or Medicine for their interpretation of the genetic code and its function in protein
synthesis. Khorana’s method was revolutionary at the time and produced a truly remarkable
feat: the synthesis of an active 72-mer tRNA molecule, which was published in Nature
(Agarwal et al., 1970). However, it was not until the late 1970’s that the development of DNA
research became explosive, when synthetic DNA fragments became commercially available.
Pohl and Jovin first observed the salt-induced cooperative conformational changes of the
synthetic DNA poly(dG-dC).poly(dG-dC) duplex, from a right-handed helix to left handed
helix from the circular dichroism study (Pohl and Jovin, 1972)
Modular Multiantigen T Cell Epitope–Enriched DNA Vaccine Against Human Leishmaniasis
No full textThe leishmaniases are protozoal diseases that severely affect large populations in tropical and subtropical regions.There are only limited treatment options and preventative measures. Vaccines will be important for prevention, control and elimination of leishmaniasis, and could reduce the transmission and burden of disease in endemic populations.We report the development of a DNA vaccine against leishmaniasis that induced T cell–based immunity and is a candidate for clinical trials. The vaccine antigens were selected as conserved in various Leishmania species, different endemic regions, and over time. They were tested with T cells from individuals cured of leishmaniasis, and shown to be immunogenic and to induce CD4+ and CD8+ T cell responses in genetically diverse human populations of different endemic regions. The vaccine proved protective in a rodent model of infection. Thus, the immunogenicity of candidate vaccine antigens in human populations of endemic regions, as well as proof of principle for induction of specific immune responses
and protection against Leishmania infection in mice, provides a viable strategy for T cell vaccine development
Mitochondrial Import Of tRNA: Mechanistic Studies Of Post- Receptor Translocation
No full textMitochondrial Import Of tRNA: Mechanistic Studies Of Post Receptor Translocation
Import of tRNA from the cytosol into the mitochondria of the kinetoplastid protozoon
Leishmania is a multistep process consisting of the binding of tRNA to specific receptors, its
transfer to an intermediate factor and translocation through the membrane. A functional RNA
import complex (RIC) isolated from Leishmania mitochondria or reconstituted by cloned and
expressed protein subunits are being employed in our laboratory to determine molecular basis of
these steps. The RIC from mitochondria of the kinetoplastid protozoon Leishmania tropica
induces translocation of tRNAs across artificial or natural membranes. tRNA import consists of a
number of discrete steps beginning with the binding of the substrate to a receptor subunit RIC1 or
RIC8A, followed by its transfer to a third subunit RIC9. Subsequently the tRNA passes into the
vesicle interior presumably through a membrane embedded translocation channel, the
composition and properties of which are largely unknown. Although the receptor binding and
transfer steps have been characterized in terms of role of specific subunits, little is known about
the final translocation step.
Specifically the objective of my research was to reconstitute functional import pore
complex on lipid bilayers and determine the permeability of such membrane vesicles by RIC
channel under different biochemical and biophysical conditions with tRNA and other different
small molecules, to carry out structural and functional studies of translocation by site directed
mutagenesis of important subunit gene. These experiments provided detailed insights into the
interaction of RIC with mitochondrial and other membranes.
I have shown that subunits RIC6 and RiC9 polymerize on the membrane to form the
hexamer (RIC6)3-(RIC9)3 in presence of RIC4A formed a R3 complex. The resultant complex R3
induced translocation of tRNA when the pH of the medium was lowered to ~6. This process was
independent of ATP and sensitive to the protonophore m-chlorocarbonylcyanide phenylhydrazone
(CCCP), and to the K+ ionophore valinomycin, but resistant to k+/h+ exchanger nigericin,
indicating the requirement of a membrane potrential Δψm generated by transmembrane proton
gradient. Indeed R3 mediated tRNA translocation could be induced at neutral pH by K+ diffusion
potential of 60-90mV (negative inside). However, translocation was independent of tRNA
sequence, and small molecules such as ATP, oligonucleotides, labeled amino acids could be taken
up by R3 liposomes at pH6.0 in contrast to large molecules such as linearized plasmid DNA
which fails the internalization process and shows the size specificity of R3 complex. My results
indicated that the (RIC6)3-(RIC9)3 complex forms a voltage gated pore similar to mitochondrial
protein import channels. To understand the critical residues involves in proton sensing points
mutants of RIC6 subunits were generated in all the 6 cysteines and 6 histidine residues as some of
those residues are involved in proton sensing in a homologous protein rieske fe-s protein of RIC6.
I have found out the critical cysteine and histidines along the protein chain are responsible for
proton sensing. Atomic force microscopy has been used to find out the nature of the reconstituted
channel in vitro. Atomic Force Microscopy of R3 revealed particles with an asymmetric surface
groove of ~20 nm rim diameter and ~1 nm depth
PcrG protects the two long helical oligomerization domains of PcrV, by an interaction mediated by the intramolecular coiled-coil region of PcrG
Get PDFPcrV is a hydrophilic translocator of type three secretion system (TTSS) and a structural component of the functional translocon. C-terminal helix of PcrV is essential for its oligomerization at the needle tip. Conformational changes within PcrV regulate the effector translocation. PcrG is a cytoplasmic regulator of TTSS and forms a high affinity
complex with PcrV. C-terminal residues of PcrG control the effector secretion
Structural and Thermodynamic Studies on the Interaction of Iminium and Alkanolamine Forms of Sanguinarine with Hemoglobin
No full textBinding of the iminium and alkanolamine forms of the benzophenanthridine anticancer alkaloid sanguinarine to
hemoglobin (Hb) was investigated by absorbance, fluorescence, and circular dichroism spectral techniques, and by calorimetry. The binding affinity of the charged iminium was found to be of the order of 106 M−1, higher by one order than that of the neutral alkanolamine, from the analysis of the absorbance data. The fluorescence spectral data revealed that the quenching of Hb fluorescence by both forms of sanguinarine is due to the formation of a complex in the ground state and is of an unusual, static nature. Thermodynamic data revealed that the binding of the iminium form was exothermic in nature while that of the alkanolamine was endothermic; the former case predominantly involved electrostatic and hydrogen bonding interactions but the latter was dominated by mostly hydrophobic interactions. Calculation of the molecular distances (r) between the donor (β- Trp37) and acceptor (iminium and alkanolamine) according to Förster’s theory suggests both forms of the alkaloid to be bound close to β-Trp37 at the α1β2 interface of the protein. The iminium form induced greater secondary structural changes in Hb than
the alkanolamine as revealed by synchronous fluorescence, circular dichroism and three-dimensional fluorescence spectroscopic studies. These results are consistent with a stronger binding of the iminium over the alkanolamine form. Nevertheless, the hydrophobic probe ANS was displaced from hemoglobin more easily by the alkanolamine form than by the iminium. The study showed that Hb binds more strongly to the biologically active iminium form than the alkanolamine, in contrast to the stronger binding of the latter to plasma protein serum albumin. Overall, this study presents insights on the interaction dynamics and energetics of the binding of the two forms of sanguinarine to hemoglobin