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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 first line 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
PARP1–TDP1 coupling for the repair of topoisomerase I–induced DNA damage
No full textPoly(ADP-ribose) polymerases (PARP) attach poly(ADP-ribose) (PAR) chains to various proteins including themselves and chromatin. Topoisomerase I(Top1) regulates DNA supercoiling and is the targetof camptothecin and indenoisoquinoline anticancer drugs, as it forms Top1 cleavage complexes
(Top1cc) that are trapped by the drugs. Endogenous
and carcinogenic DNA lesions can also trap Top1cc.
Tyrosyl-DNA phosphodiesterase 1 (TDP1), a key
repair enzyme for trapped Top1cc, hydrolyzes the
phosphodiester bond between the DNA 30-end and
the Top1 tyrosyl moiety. Alternative repair pathways
for Top1cc involve endonuclease cleavage. However,
it is unknown what determines the choice between
TDP1 and the endonuclease repair pathways. Here
we show that PARP1 plays a critical role in this
process. By generating TDP1 and PARP1 doubleknockout
lymphoma chicken DT40 cells, we demonstrate
that TDP1 and PARP1 are epistatic for the
repair of Top1cc. The N-terminal domain of TDP1
directly binds the C-terminal domain of PARP1, and
TDP1 is PARylated by PARP1. PARylation stabilizes
TDP1 together with SUMOylation of TDP1. TDP1
PARylation enhances its recruitment to DNA
damage sites without interfering with TDP1 catalytic
activity. TDP1–PARP1 complexes, in turn recruit X-ray
repair cross-complementing protein 1 (XRCC1). This work identifies PARP1 as a key component driving
the repair of trapped Top1cc by TDP1
Female Reproductive Aging Is Master-Planned at the Level of Ovary
No full textThe ovary receives a finite pool of follicles during fetal life. Atresia remains the major form of follicular expenditure at all stages since development of ovary. The follicular reserve, however, declines at an exponential rate leading to accelerated rate of decay during the
years preceding menopause. We examined if diminished follicle reserve that characterizes ovarian aging impacts the attrition rate. Premature ovarian aging was induced in rats by intraembryonicinjection of galactosyltransferase-antibody on embryonic day 10. On post-natal day
35 of the female litters, either a wedge of fat (sham control) or a wild type ovary collected from 25-day old control rats, was transplanted under the ovarian bursa in both sides.Follicular growth and atresia, and ovarian microenvironment were evaluated in the follicledeficient
host ovary and transplanted ovary by real time RT-PCR analysis of growth differentiation factor-9, bone morphogenetic protein 15, and kit ligand, biochemical evaluation of ovarian lipid peroxidation, superoxide dismutase (SOD) and catalase activity, and western
blot analysis of ovarian pro- and anti-apoptotic factors including p53, bax, bcl2, and caspase demonstrated that the rate of follicular atresia, which was highly preponderant in
the follicle-deficient ovary of the sham-operated group, was significantly prevented in thepresence of the transplanted ovary. As against the follicle-deficient ovary of the shamoperated group, the follicle-deficient host ovary as well as the transplanted ovary in the
ovary-transplanted group exhibited stimulated follicle growth with increased expression of anti-apoptotic factors and down regulation of pro-apoptotic factors. Both the host andtransplanted ovaries also had significantly lower rate of lipid peroxidation with increased SOD and catalase activity. We conclude that the declining follicular reserve is perhaps the immediate thrust that increases the rate of follicle depletion during the final phase of ovarian
life when the follicle reserve wanes below certain threshold size
Understanding The Dynamics Of Annexin A6 In Cardiomyocytes
No full textDeregulations in signaling pathways that control cell-size are prominent in maladaptive cardiomyocyte hypertrophy, a major risk factor in heart failure.Present work explores the intracellular dynamics of annexin A6 (Anxa6), a
protein differentially expressed in cardiomyocyte hypertrophy. Induction of prohypertrophic signaling by acute and chronic exposure of H9c2(2-1) cardiomyocytes to chemical agonists Phenylephrine (PE), Angiotensin II (Ang II) or Isoproterenol (Iso) resulted in profound increment in cell-size and hypertrophy associated phenotypes, as revealed by microscopic and biochemical examinations. Chronic treatments also compromised mitochondrial dynamics
and activated mitochondrial pathways of apoptosis. Anxa6 was upregulated in a phasic fashion in course of acute treatments and in a more linear fashion in
course of chronic treatments. Biophysical and microscopic experiments revealed an oscillatory cytosolic dynamics of the protein in hypertrophied cells that resulted in a differential localization pattern. Anxa6 was progressively distributed from perinuclear compartment to the plasma membrane under acute treatment. Chronic treatments concentrated the protein more to the perinuclear space. In
either scenario, the appearance was granular punctate. Ectopic expression of Anxa6 at controlled levels significantly protected the cardiomyocytes from
hypertrophy-associated increase in cell size and conserved mitochondrial dynamics. However, such expression also rendered the cells vulnerable to apoptosis. Anxa6 knockdown augmented hypertrophic responses and severely
abrogated mitochondrial dynamics in cardiomyocytes. At molecular levels,Anxa6 was found to interact with the anti-hypertrophic candidate ANP, proapoptotic nuclear enzyme Poly-(ADP-ribose) polymerase 1 (Parp1) and the
survival kinase Akt. Capability to interact with such diversely functional candidates enabled Anxa6 to participate in multiple scaffolding events
simultaneously, thereby exhibiting the oscillatory pattern of dynamics. Mutagenesis studies confirmed that Ca2+ was essential for Anxa6 interactions, which occasionally required cholesterol. The Anxa6-ANP interaction was crucial
for ANP mediated counter hypertrophic responses whereas Anxa6-Parp1 and Anxa6-Akt interactions have implications in apoptosis and mitochondrial dynamics. Structurally, interactions of Anxa6 were mediated mostly by its Nterminus
whereas C-terminus was more catalytic, presumably serving to bind Ca2+ required for the interactions. Together, present study depicts a novel mechanistic spectrum of Anxa6 dynamics that helps to regulate equilibrium state
of the homeostatic machineries that dictate life and death decisions in cardiomyocytes
Regulation of Toxin Delivery System and other Genes of Helicobacter pylori on Adherence to the Human Gastric Cell Line
No full textHelicobacter pylori is a gram negative, spiral shaped bacteria persistently colonizes the gastric mucosa of approximately one half of the world’s population leading to chronic gastric inflammation, peptic ulcer disease, gastric cancer or MALT lymphoma in some individuals. The major toxins of H. pylori are CagA and VacA. CagA is encoded by cagA gene of ~40kb Cag pathogenicity island (Cag-PAI). Motility and chemotaxis is the major factor for H. pylori to colonize the human stomach as strains deficient in many flagellar biosynthesis genes are not competent to colonize. Adherence of H. pylori to the gastric epithelial cell line AGS strongly induces expression of the major virulence gene cagA. In this study, we demonstrate that cagA upregulation in AGS-adhered H. pylori is dependent on the flagellar hook-length control protein FliK. The fliK gene was strongly induced in AGS-adhered H. pylori and practically no upregulation of cagA was observed in a fliK mutant following adherence to AGS cells. In a ΔfliK mutant, the anti- σ28 factor FlgM sequesters σ28 within the cell and the concentration of free, functional σ28 is lower in the ΔfliK mutant than in the wild type strain. To examine whether σ28 had a role in cagA upregulation in AGS-adhered H. pylori, a fliA mutant deficient in 28 was constructed. Using the fliA mutant it was shown that cagA upregulation in AGSadhered H. pylori was mediated by 28-RNAP that initiates transcription from a putative promoter downstream of the designated 80-RNAP promoter. Direct binding of 28 to the cagA promoter was demonstrated by chromatin immunoprecipitation and the transcription start site was identified by 5RACE. The 28-dependent cagA promoter was active specifically in AGS-adhered H. pylori. These results indicate that H. pylori has evolved to integrate expression of the major virulence gene cagA with the flagellar regulatory circuit, essential for colonization of the human host
Binding of the Iminium and Alkanolamine Forms of Sanguinarine to Lysozyme: Spectroscopic Analysis, Thermodynamics, and Molecular Modeling Studies
No full textSanguinarine (SGR) exists in charged iminium (SGRI) and neutral alkanolamine (SGRA) forms. The binding of
these two forms to the protein lysozyme (Lyz) was investigated by fluorescence, UV−vis absorbance and circular dichroism spectroscopy, and in silico molecular docking approaches. Binding thermodynamics were studied by microcalorimetry. Both forms of sanguinarine quenched the intrinsic fluorescence of Lyz, but the quenching efficiencies varied on the basis of binding that was derived after correction for an inner-filter effect. The equilibrium binding constants at 25 ± 1.0 °C for the iminium and alkanolamine forms were 1.17 × 105 and 3.32 × 105 M−1, respectively, with approximately one binding site for both forms of the protein. Conformational changes of the protein in the presence of SGR were confirmed by absorbance, circular dichroism, threedimensional
fluorescence, and synchronous fluorescence spectroscopy. Microcalorimetry data revealed that SGRI binding is
endothermic and predominantly involves electrostatic and hydrophobic interactions, whereas SGRA binding is exothermic and dominated by hydrogen-bonding interactions. The molecular distances (r) of 3.27 and 3.04 nm between the donor (Lyz) and the SGRI and SGRA acceptors, respectively, were calculated according to Förster’s theory. These data suggested that both forms were bound near the Trp-62/63 residues of Lyz. Stronger binding of SGRA than SGRI was apparent from the results of both structural and thermodynamic experiments. Molecular docking studies revealed that the putative binding site for the SGR
analogues resides at the catalytic site. The docking results are in accordance with the spectroscopic and thermodynamic data, further validating the stronger binding of SGRA over SGRI to Lyz. The binding site is situated near a deep crevice on the protein surface and is close to several crucial amino acid residues, including Asp-52, Glu-35, Trp-62, and Trp-63. This study advances our knowledge of the structural nature and thermodynamic aspects of binding between the putative anticancer alkaloid sanguinarine and lysozyme
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.
© 2014 Society of Chemical Industr
Synthesis, Characterization, and Biological Evaluation of 99mTc(CO)3-Labeled Peptides for Potential Use as Tumor Targeted Radiopharmaceuticals
No full textDuring the past decade, several peptides containing Arg-Gly-Asp sequence have been conjugated with different chelating agents for labeling with various radionuclides for the diagnosis of tumor development. In this study, we report the synthesis of two tetrapeptides (Asp-Gly-Arg-His and Asp-Gly-Arg-Cys) and one hexapeptide [Asp-Gly-Arg-D-Tyr-Lys-His] by changing the amino acid sequence of the Arg-Gly-Asp motif. Peptide synthesis was initiated from aspartic acid. Aspartic acid placed at C-terminal end of the peptide chain can be conjugated with different drug molecules facilitating
their transport to the site of action. The peptides were
synthesized in excellent yield and labeled using freshly
prepared [99mTc(CO)3(H2O)3]+ intermediate. A complexation
yield of over 97% was achieved under mild conditions even at low ligand concentrations of 10�2 M.
Radiolabeled peptides were characterized by HPLC and were found to be substantially stable in saline, in His solution as well as in rat serum and tissue (kidney, liver) homogenates. Internalization studies using Ehrlich ascites carcinoma cell line showed rapid and significant internalization (30–35% at 30 min of incubation attaining
maximum value of about 40–60% after 2–4 h incubation). A good percentage of quick internalization was also observed in avb3-receptor-positive B16F10 mouse melanoma cell line (14–16% after 30 min of incubation and 25–30% after 2–4 h incubation). Imaging and biodistribution studies were performed in Swiss albino mice bearing Ehrlich ascites tumor in right thigh.
Radiolabeled peptides exhibited fast blood clearance and rapid elimination through the urinary systems. 99mTc(CO)3-tetra-Pep2 exhibited remarkable localization at tumor site (1.15%, 1.17%, and 1.37% ID/g at 2, 4, and 6 h p.i., respectively) which could be due to slow clearance of the radiolabeled peptide from blood in comparison with the other two radiolabeled peptides. However, 99mTc(CO)3-hexa-Pep exhibited the highest tumor to muscle and tumor to blood
ratios among the three. The preliminary results with these amino acid–based peptides are encouraging enough to carry out further experiments for targeting tumor
Sequence Complexity of Amyloidogenic Regions in Intrinsically Disordered Human Proteins
No full textAn amyloidogenic region (AR) in a protein sequence plays a significant role in protein aggregation and amyloid formation. We have investigated the sequence complexity of AR that is present in intrinsically disordered human proteins. More than 80% human proteins in the disordered protein databases (DisProt+IDEAL) contained one or more ARs. With decrease of protein disorder, AR content in the protein sequence was decreased. A probability density distribution analysis and discrete analysis of AR sequences showed that ,8% residue in a protein sequence was in AR and the region was in average 8 residues long. The residues in the AR were high in sequence complexity and it seldom overlapped with low complexity regions (LCR), which was largely abundant in disorder proteins. The sequences in the AR showed mixed conformational adaptability towards a-helix, b-sheet/strand and coil conformations
