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Wnt5a–Rac1–NF-kB Homeostatic Circuitry Sustains Innate Immune Functions in Macrophages
No full textMacrophages play a critical role in innate immunity. Differentiation Ags present on macrophages such as CD14 orchestrate the firstline of defense against infection. The basal/homeostatic signaling scheme that keeps macrophages thus groomed for innate immune functions remains unresolved. Wnt5a–Fz5 signaling being a primordial event during cell differentiation, we examined the involvement
of Wnt5a–Fz5 signaling in the maintenance of innate immune functions. In this study, we demonstrate that innate
immune functions of macrophages ensue at least partly through a homeostatic Wnt5a–Fz5–NF-kB (p65) circuit, which is Rac1 dependent. The autocrine/paracrine Wnt5a–Fz5–Rac1–p65 signaling cascade not only maintains basal levels of the immune defense modulating IFNs and CD14; it also supports macrophage survival. Wnt5a–Fz5–Rac1 signaling mediated p65 homeostasis in turn sustains Wnt5a expression in a feed-forward mode. The natural immune response of macrophages to Escherichia coli/LPS and virus is accordingly sustained. The depiction of sustenance of innate immune functions as an outcome of a homeostatic
Wnt5a–p65 axis unfolds previously unidentified details of immune regulation and provides new insight into homeostatic cell signaling
Antibacterial and antiviral evaluation of sulfonoquinovosyldiacylglyceride: a glycolipid isolated from Azadirachta indica leaves
No full textAssessment of antibacterial as well as antiherpes virus activity of sulfonoquinovosyldiacylglyceride (SQDG), a glycolipid, isolated from the leaves of Azadirachta indica has been described. Antimicrobial activity was evaluated
against Gram-positive, Gram-negative bacteria and herpes simplex virus. SQDG showed significant inhibitory activity against Salmonella typhi and two isolates of Shigella dysenteriae with MIC values 32 lg ml �1, while three isolates of Salm. typhi, Escherichia coli and Vibrio cholerae were inhibited at 64 lg ml �1 and have shown zone diameter ranging from 6�2 to 12�3 mm. The growth kinetics study of SQDG on Salm. typhi and Sh. dysenteriae revealed that the growths were completely inhibited at their MIC values within 24 h of exposure. Interestingly, SQDG inhibits herpes simplex virus (HSV) type 1 and 2 with the EC50 of 9�1 and 8�5 lg ml �1, compared with acyclovir (2�2 and
2�8 lg ml �1 against HSV-1 and HSV-2). The selectivity index (SI) was found to be 12�4 against HSV-1 and 13�41 with HSV-2. Furthermore, the expression of proinflammatory cytokines of HSV-infected and SQDG-treated macrophages
using ELISA kit revealed that SQDG significantly downregulated the production of TNF-a, IL-1b, IL-12 and IL-6
Leishmania donovani targets tumor necrosis factor receptor-associated factor (TRAF) 3 for impairing TLR4-mediated host response
No full textIntramacrophage pathogen Leishmania
donovani escapes host immune response by subverting
Toll-like receptor (TLR) signaling, which is critically
regulated by protein ubiquitination. In the present
study, we identified tumor necrosis factor receptorassociated
factor (TRAF) 3, degradative ubiquitination
of which is essential for TLR4 activation, as a target for
Leishmania to deactivate LPS-mediated TLR4 signaling.
We used LPS-treated RAW 264.7 cells and compared
the TLR4-mediated immune response in these cells
with L. donovani and L. donovani � LPS costimulated
macrophages. TRAF3, which was ubiquitinated (2.1-
fold over control) at lys 48 position and subsequently
degraded following LPS treatment, persisted in L.
donovani and L. donovani � LPS costimulated cells due
to defective lys 48 ubiquitination. Lys 63-linked ubiquitinatio
of upstream proteins in the cascade (cIAP1/2 and
TRAF6), mandatory for TRAF3 degradation, was also
reduced postinfection. This may be attributed to reduced
association between ubiquitin-conjugating enzyme
Ubc13 and TRAF6 during infection. Inhibition of
TRAF3 before infection by shRNA in Balb/c mice
showed enhanced IL-12 and TNF-� (10.8- and 8.1-fold
over infected control) and decreased spleen parasite
burden (61.3% suppression, P<0.001), thereby marking
reduction in disease progression. Our findings
identified TRAF3 as a novel molecular regulator exploited
by Leishmania for successful infection.—Gupta,
P., Giri, J., Srivastav, S., Chande, A. G., Mukhopadhyaya,
R., Das, P. K., Ukil, A. Leishmania donovani
targets tumor necrosis factor receptor-associated factor
(TRAF) 3 for impairing TLR4-mediated host response
Mutant Huntingtin Gene Expression and Cell Death: A Mitochondrial Basis for Huntington�s Disease
No full textCellular signalling pathways affecting normal neuronal morphology and mitochondrial quality in two different models of Huntington’s disease (HD), 3-nitropropionic acid (3-NP)-induced HD in rats and 150Q Htt expressing cell line model, were investigated for correcting the errors with the help of pharmacological agents. We used a cell line based model of HD where cells stably express N-terminal end of huntingtin (htt) upon induction, either htt with 16 CAG (taken as control) or 150 CAG (taken as the diseased condition) repeats. This model consistently showed formation of aggregates, loss in ubiquitin proteasomal system (UPS) activity, disruption of normal mitochondrial membrane potential, loss of mitochondrial electron transport chain (ETC) complex activity and cell death. Additionally, we found the imbalance in the mitochondrial dynamics in this model shifted towards the fragmentation of the organelle. For in vivo experiments, we used a chemically induced animal model of HD where the disease pathology was initiated by using 3-NP, an irreversible inhibitor of ETC complex II. This model showed impairment in gait, beam balancing, swimming ability, inability to coordinate fine motor movements and cognition. Histopathology of the brain exhibited lesion formation in the dorsolateral striatum (typical to HD), proliferation of microglia and scarcity of astroglia inside the lesion core. There was a severe loss in normal neuronal morphology with less spine formation in dendrites of the striatal and cortical neurons, mainly because of the altered interaction of profilin2 with β-actin. 3-NP treated HD rats showed increased striatal dopamine and glutamate levels, and serotonin metabolism, without affecting serotonin levels. At the mitochondrial level, a gross inhibition in ETC complex activity, decreased oxygen consumption and intensified mitochondrial fission was discernable.
Melatonin was found to correct cell viability, mitochondrial membrane potential and complex II activity in the cell line model. The mechanism was found to be mediated by the neurohormone’s ability to restore the proteasomal machinery and decreased mitochondrial fission. In the animal model melatonin was not able to protect the striatal lesion formation and the increase in striatal dopamine levels, but showed improvement in the behavioural deficiencies, which is attributed to melatonin’s protective effect on maintaining the dendritic spine density and cerebellar granule cell arborisation. N-acetyl cysteine (NAC) improved cell viability in the cellular model of HD by improving the mitochondrial quality. It was found that NAC treatment reduced the mitochondrial fission by improving the degradation of DRP1 protein. In animal model it was found that NAC administration protected striatum from lesion formation and decreased localization of DRP1 onto the mitochondria. NAC exhibited limited effect on the ubiquitin-proteasome system (UPS) activity and DA
mediated increase in the total DRP1 level. Localization of DRP1 to mitochondria and its oligomerization was found to be crucial for striatum specific neuronal loss.
Quercetin failed to improve cell viability in the cell line model of HD, and was found to be toxic when higher doses (40-100 μM) were administered. A narrow therapeutic window was seen for about 20 μM dose of quercetin, which protected the mHtt-induced cell death on 2nd day of mHtt expression. At this dose an improved UPS activity was obtained on the 4th day of induction, but did not improve mHtt mediated loss in mitochondrial membrane potential. When administered in the animal model of HD, quercetin improved animal behaviour and serotonin metabolism. It was found that quercetin was able to reduce the microglial proliferation inside the striatal lesion core with improved population of astrocytes. It has to be noted that both the models are distinctly different, the cell line model expresses mHtt which triggers the mitochondrial localization of DRP1 and thus imbalances the mitochondrial dynamics. On the other hand, we used a neurotoxin which is specific for ETC complex II inhibition and a potent oxidative stress generator. These effects lead to the increase in DRP1 and decreased Mfn 1/2 level in the animal model. Therefore, both these models together complete all the aspect of disease related mitochondrial abnormalities. Based on these results, it is concluded that mitochondrial fission control and its impact on cell death culminating into the behavioural abnormalities, maintenance of DRP1 homeostasis by UPS activity and finally the oligomerization of the DRP1 protein are the three levels at which therapeutic ventures could be attempted. In the present thesis Htt transgenic cellular model, and a chemical lesioned HD rat model were used for evaluating the therapeutic potential of three different pharmacophores. This kind of a comparative study is for the first time in literature, to the best of the author’s knowledge. In a nutshell, the outcome of the study showed NAC to reproduce all the effects in the cellular model in animal model too, whereas melatonin reproduced most of the effects in cell line model in the animal model, but quercetin failed to show complete congruence in results in cellular and animal models. These unexpected outcome of the study clearly point insufficiency of either of these models, and therefore the best combination could be a chemical lesioned animal model along with a transgenic animal model. This could be true since transgenic animal models are known to suffer from compensatory changes in the gene expression patterns of the counteracting molecules (as for eg. SOD knockout animals have upregulated catalase synthetic machinery!). The limitations of the present study being thus, the author could have adapted to a co-culture regimen (i.e., the neuronal cell line to be cultured with astrocytes to obtain a natural environment) for more meaningful results
Vesicular transport of a ribonucleoprotein to mitochondria
No full textIntracellular trafficking of viruses and proteins commonly occurs via the early endosome in a process involving Rab5. The RNA Import Complex (RIC)-RNA complex is taken up by mammalian cells and targeted to mitochondria. Through RNA interference, it was shown that mito-targeting of the ribonucleoprotein (RNP) was dependent on
caveolin 1 (Cav1), dynamin 2, Filamin A and NSF. Although a minor fraction of the RNP was transported to endosomes in a Rab5- dependent manner, mito-targeting was independent of Rab5 or other endosomal proteins, suggesting that endosomal uptake and mitotargeting occur independently. Sequential immunoprecipitation of the cytosolic vesicles showed the sorting of the RNP away fromCav1 in a process that was independent of the endosomal effector EEA1 but sensitive to nocodazole. However, the RNP was in two types of
vesicle with or without Cav1, with membrane-bound, asymmetrically orientated RIC and entrapped RNA, but no endosomal components, suggesting vesicular sorting rather than escape of free RNP fromendosomes. In vitro, RNP was directly transferred from the Type 2 vesicles to mitochondria. Live-cell imaging captured spherical Cav12
RNP vesicles emerging from the fission of large Cav+ particles. Thus, RNP appears to traffic by a different route than the classical Rab5- dependent pathway of viral transpor
Role of the mTORC1 Complex in Satellite Cell Activation by RNAInduced Mitochondrial Restoration: Dual Control of Cyclin D1 through MicroRNAs
No full textDuring myogenesis, satellite stem cells (SCs) are induced to proliferate and differentiate to myogenic precursors. The role of energy sensors such as the AMP-activated protein kinase (AMPK) and the mammalian Target of Rapamycin (mTOR) in SC activation is unclear. We previously observed that upregulation of ATP through RNA-mediated mitochondrial restoration (MR) accelerates SC activation following skeletal muscle injury. We show here that during regeneration, the AMPK-CRTC2-CREB and Raptor-mTORC-4EBP1 pathways were rapidly activated. The phosho-CRTC2-CREB complex was essential for myogenesis and activated transcription of the critical cell cycle regulator cyclin D1 (Ccnd1). Knockdown (KD) of either mTORC or its subunit
Raptor delayed SC activation without influencing the differentiation program. KD of 4EBP1 had no effect on SC activation but enhanced myofiber size. mTORC1 positively regulated Ccnd1 translation but destabilized Ccnd1 mRNA. These antithetical effects of mTORC1 were mediated by two microRNAs (miRs) targeted to the 3= untranslated region (UTR) of Ccnd1 mRNA: miR-1 was downregulated in mTORC-KD muscle, and depletion of miR-1 resulted in increased levels of mRNA without any effect on Ccnd1 protein. In contrast, miR-26a was upregulated upon mTORC depletion, while anti-miR-26a oligonucleotide specifically stimulated Ccnd1 protein expression. Thus, mTORC may act as a timer of satellite cell proliferation during myogenesis
Comparative proteomics and glycoproteomics of plasma proteins in Indian visceral leishmaniasis
No full textVisceral leishmaniasis (VL) is a deadly parasitic diseases caused by Leishmania donovani; it is a major
health problem in many countries. A lack of proper understanding of the disease biology, poor diagnostic methods and increasing drug resistance are the main reasons for the growing burden of VL infection. Comparative plasma
proteomics are a relatively useful technique that can be used to investigate disease-associated alterations that can
help in understanding host responses against pathogens, and might be useful in disease management and diagnosis.
In this study, a comparative proteomics and glycoproteomics approach using 2DE and 2D-DIGE was employed between early diagnosed VL patients of all age groups and healthy endemic and non-endemic controls in order to aid the recognition of disease-associated alterations in host plasma. Comparative proteomics was performed by the depletion of seven highly abundant plasma proteins. Comparative glycoproteomics was performed by thedepletion of albumin and IgG, followed by purification of plasma glycoproteins using a multi lectin affinity column. From these two approaches, 39 differentially expressed protein spots were identified and sequenced using MALDI-TOF/TOF mass spectrometry. This revealed ten distinct proteins that appeared in multiple spots, suggesting micro-heterogeneity. Among these proteins, alpha-1-antitrypsin, alpha-1-B glycoprotein and amyloid-A1 precursor were up-regulated, whereas
vitamin-D binding protein, apolipoprotein-A-I and transthyretin were down-regulated in VL. Alterations in the levels of these proteins in VL-infected plasma were further confirmed by western blot and ELISA. These proteins may be involved in the survival of parasites, resisting neutrophil elastase, and in their multiplication in macrophages, potentially maintaining endogenous anti-inflammatory and immunosuppressive conditions. Consequently, the results of this study may help in understanding the host response against L.donovani, which could help in the discovery of new drugs and disease management. Finally, these alterations on protein levels might be beneficial in improving early diagnosis considering those as biomarkers in Indian VL
Simultaneous Inhibition of Key Growth Pathways in Melanoma Cells and Tumor Regression by a Designed Bidentate Constrained Helical Peptide
No full textProtein–protein interactions are part of a large number
of signaling networks and potential targets for drug development. However, discovering molecules that can specifically inhibit such interactions is a major challenge.
S100B, a calcium-regulated protein, plays a crucial role
in the proliferation of melanoma cells through protein–
protein interactions. In this article, we report the design
and development of a bidentate conformationally constrained
peptide against dimeric S100B based on a natural tight-binding peptide, TRTK-12. The helical conformation of the peptide was constrained by the substitution of a-amino isobutyric acid—an amino acid having high helical propensity—in positions which do not interact with S100B. A branched bidentate version of the peptide was bound to S100B tightly with a dissociation constant of 8 nM. When conjugated to a cell-penetrating peptide, it caused growth inhibition and rapid apoptosis in melanoma cells. The molecule exerts antiproliferativeaction through simultaneous inhibition of key growth pathways, including reactivation of wild-type p53 and inhibition of Akt and STAT3 phosphorylation. The apoptosis induced by the bidentate constrained helix is caused by direct migration of p53 to mitochondria. Atmoderate intravenous dose, the peptide completely inhibits melanoma growth in a mouse model without any significant observable toxicity. The specificity was shown by lack of ability of a double mutant peptide to cause tumor regression at the same dose level. The methodology described here for direct protein–protein interaction inhibition may be effective for rapid development of inhibitors against relatively weak protein–protein interactions for de novo drug development. VC 2014 Wiley Periodicals,
Inc. Biopolymers (Pept Sci) 102: 344–358, 201
Flavone-resistant Leishmania donovani Overexpresses LdMRP2 Transporter in the Parasite and Activates Host MRP2 on Macrophages to Circumvent the Flavone-mediated Cell Deat
No full textLeishmania donovani adopts several defense mechanisms to become resistant to antileishmanial agents. Laboratory-grown flavone (baicalein)-resistant parasites exhibit efflux of drug by LdABCC2 transporter. Inside the host
macrophage, this parasite up-regulates host MRP2 transporter by an Nrf2-dependent pathway. Resistant Leishmania parasite exploits ABC transporter in the parasite and inside the host
Biophysical studies on the interaction of polyamines with nucleic acids
No full textThe biogenic polyamines spermine (SPM), spermidine (SPD), putrescine (PUT) and cadaverine(CAD) are ubiquitous linear organic molecules present in all eukaryotic and prokaryotic cells. Theyare thought to be essential for the normal functioning of the cells, like cell growth and play essentialroles in a wide variety of cellular regulatory pathways and functions like gene regulation, DNA
packaging, proliferation etc. The binding of biogenic polyamines SPM, SPD, PUT, CAD and theanalogue 1-naphthyl acetyl spermine (NASPM) to different natural DNAs, synthetic DNAs anddouble stranded RNAs has been investigated in this thesis.The comparative binding studies of biogenic polyamines SPM, SPD, PUT and CAD with CT DNA
proved the binding to be strongest for SPM. Conformational studies revealed perturbation of DNA structure on binding of the polyamines; the gross B-form of DNA, however, remained more or less unaltered. Thermal stability of DNA was remarkably enhanced on binding and the binding constants
derived revealed highest affinity for SPM. Interaction of the polyamines were endothermic and entropy driven. The binding affinity of SPM (6.20105 M-1) was much higher than those for the other polyamines. Small but negative heat capacity changes in the four cases suggested the
involvement of significant hydrophobic forces in the complexation. Overall, the affinity varied as SPM > SPD > PUT > CAD. The comparative binding study of these polyamines with three natural DNA of different base
composition i.e. Clostridium perfringens (CP) DNA (27% GC), Escherichia coli (EC) DNA (50% GC) and Micrococcus lysodeikticus (ML) DNA (72% GC), revealed significant changes in the conformation of each of the DNA, but was higher with the DNA with high AT content. Maximum thermal
stabilization was shown by CP DNA followed by EC and ML DNAs. The lower IC50 values (concentration of polyamines required to quench fluorescence of ethidium bromide-DNA complex by 50%) in terms of ethidium displacement and higher binding affinities varied as SPM> SPD>
PUT> CAD. The positive entropy term was higher in the CP DNA compared to the other two DNAs confirming more displacement and release of the water of spine in the minor groove of the AT rich DNA. The values of standard heat capacity change obtained for the systems also supported
a groove binding model. Enthalpy-entropy compensation was observed in each system studied. The polyelectrolytic contribution to the Gibbs energy decreased with the increase in salt concentration, whereas, the non-polyelectrolytic contribution was not affected which showed that electrostatic interaction played a remarkable role in the binding of polyamines to DNA. Thus, polyamines bound
stronger with AT rich CP DNA and least with the GC rich ML DNA and the binding to each DNA was stronger for SPM and varied as SPM> SPD> PUT> CAD. The results proved the AT base
specificity of the polyamines.The binding of the polyamines, SPM, SPD and the polyamine analogue NASPM with synthetic DNA polynucleotides of different base sequences viz. poly(dA).poly(dT), poly(dA-dT).poly(dAdT), poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC) revealed significant conformational perturbations in all the four polynucleotides. Binding of SPM, NASPM, SPD to all the 2
polynucleotides was driven by large positive standard molar entropy changes, clearly revealing entropy driven binding. The strong positive entropy term in these interactions is suggestive of the disruption and release of water molecules from the grooves of the DNA thereby facilitating groove
binding. Higher Go hyd values for the AT polynucleotides indicated that the hydrophobic contribution in the polyamine-AT polynucleotide complexes are much larger as compared to the GC polynucleotides. Thus, even though salt dependent studies revealed electrostatic interaction to be a
prominent contributing force to the Gibbs energy, hydrophobic interaction also apears to play a role in the interaction. The binding affinity of SPM and SPD varied as poly(dA).poly(dT) > poly(dAdT). poly(dA-dT) > poly(dG).poly(dC) > poly(dG-dC).poly(dG-dC). The binding affinity of NASPM varied as poly(dA-dT).poly(dA-dT) > poly(dA).poly(dT) > poly(dG-dC).poly(dG-dC) > poly(dG).poly(dC). Trend in the binding affinity was further confirmed from the binding affinities calculated from the melting stabilization (Tm) values obtained from optical melting and differential
scanning calorimetry studies and was also corroborated from ethidium bromide displacement assay. The results revealed sequence selectivity of polyamines towards the AT sequences over the GC sequences. Furthermore, it proved the ability of NASPM to bind with the AT hetero polynucleotide
with a higher affinity than the other biogenic polyamines. Thus, the results suggest importance of polyamine analogues and its ability to interfere with normal polyamine interactions as a method of cytotoxic activity. RNA targeting is an evolving new approach to anticancer therapeutics and in this context, the capability of polyamines and analogues to target the double stranded RNAs poly(I).poly(C), poly(C).poly(G) and poly(A).poly(U) has been studied to understand the structural and
thermodynamic basis of the binding and the comparative efficacy of the analogue over the the natural polyamines. Circular dichroism spectroscopy revealed structural perturbations for the RNA polynucleotides on binding of the polyamines. Thermal melting results showed enhanced stabilization which varied as SPM > NASPM > SPD. Microcalorimetry results revealed the binding affinity to be
strongest for poly(I).poly(C) and varied as poly(I).poly(C) > poly(C).poly(G) > poly(A).poly(U). The highest affinity was shown by SPM > NASPM > SPD. Ethidium bromide displacement assay reflected the strong ability of SPM to bind to the RNA sequences and corroborated the same trend.
The strong positive entropy term in these interactions is suggestive of the disruption and release of water molecules from the grooves of the RNA thereby facilitating groove binding as suggested for DNAs. The interactions were characterized by total enthalpy–entropy compensation and high standard molar heat capacity values. The standard molar heat capacity change served as an indicator
of dominant hydrophobic effect in the binding process. The binding was found to be influenced by salt concentration suggesting Go pe to be a significant contributor towards the Gibbs energy value.Atomic force microscopy experiments on the interaction of polyamines with double stranded RNA
revealed significant morphological changes leading to compaction and condensation of the linear RNA molecules.
Overall these studies provide an understanding of the underlying mechanism of polyamine interaction with nucleic acids that may enable us to approach a strategy for targeting the polyamine pathway as a means of antiproliferative mechanism