Oscillations in MAPK cascade triggered by two distinct designs of coupled positive and negative feedback loops

ABSTRACT: BACKGROUND: Feedback loops, both positive and negative are embedded in the Mitogen Activated Protein Kinase (MAPK) cascade. In the three layer MAPK cascade, both feedback loops originate from the terminal layer and their sites of action are either of the two upstream layers. Recent studies have shown that the cascade uses coupled positive and negative feedback loops in generating oscillations. Two plausible designs of coupled positive and negative feedback loops can be elucidated from the literature; in one design the positive feedback precedes the negative feedback in the direction of signal flow and vice-versa in another. But it remains unexplored how the two designs contribute towards triggering oscillations in MAPK cascade. Thus it is also not known how amplitude, frequency, robustness or nature (analogous/digital) of the oscillations would be shaped by these two designs. RESULTS: We built two models of MAPK cascade that exhibited oscillations as function of two underlying designs of coupled positive and negative feedback loops. Frequency, amplitude and nature (digital/analogous) of oscillations were found to be differentially determined by each design. It was observed that the positive feedback emerging from an oscillating MAPK cascade and functional in an external signal processing module can trigger oscillations in the target module, provided that the target module satisfy certain parametric requirements. The augmentation of the two models was done to incorporate the nuclear-cytoplasmic shuttling of cascade components followed by induction of a nuclear phosphatase. It revealed that the fate of oscillations in the MAPK cascade is governed by the feedback designs. Oscillations were unaffected due to nuclear compartmentalization owing to one design but were completely abolished in the other case. CONCLUSION: The MAPK cascade can utilize two distinct designs of coupled positive and negative feedback loops to trigger oscillations. The amplitude, frequency and robustness of the oscillations in presence or absence of nuclear compartmentalization were differentially determined by two designs of coupled positive and negative feedback loops. A positive feedback from an oscillating MAPK cascade was shown to induce oscillations in an external signal processing module, uncovering a novel regulatory aspect of MAPK signal processing.     

Methimazole complexes of platinum(II): Synthesis, characterization and redox behavior

A variety of platinum(II) complexes of methimazole (2-mercapto-1-methylimidazole; HImS = neutral form and ImS = thiolate form), coordinated in both thione and thiolate forms, have been isolated by reacting methimazole with [PtCl(terpy)]Cl (terpy = 2,2′:6′,2″ terpyridine), [PtCl₂(bipy)] (bipy = bipyridine), [PtCl₂(o-phen)] (o-phen = o-phenanthroline), [PtCl₂(CH₃CN)₂] and [PtCl₂(COD)] (COD = 1,5-cyclooctadiene). These complexes were characterized by electronic absorption, IR and NMR (¹H, ¹³C, ¹⁹⁵Pt) spectroscopies. Molecular structure of [Pt(bipy)(HImS)₂]Cl₂·3H₂O (3a·3H₂O) has been established by single crystal X-ray crystallography. Platinum thiolate complex, [Pt(ImS)₂(HImS)₂] (5), could be obtained by treatment of [Pt(HImS)₄]Cl₂ with sodium methoxide in methanol. The solution of 5 in organic solvents yielded bi- and tri-nuclear platinum complexes. The effect of diimine ligands on oxidation of methimazole moiety in the complexes has been studied by electrochemical oxidation and pulse radiolytic oxidation employing specific one-electron oxidant, radical.     

Characterization of Leishmania donovani aquaporins shows presence of subcellular aquaporins similar to tonoplast intrinsic proteins of plants

Leishmania donovani, a protozoan parasite, resides in the macrophages of the mammalian host. The aquaporin family of proteins form important components of the parasite-host interface. The parasite-host interface could be a potential target for chemotherapy. Analysis of L. major and L. infantum genomes showed the presence of five aquaporins (AQPs) annotated as AQP9 (230aa), AQP putative (294aa), AQP-like protein (279aa), AQP1 (314aa) and AQP-like protein (596aa). We report here the structural modeling, localization and functional characterization of the AQPs from L. donovani. LdAQP1, LdAQP9, LdAQP2860 and LdAQP2870 have the canonical NPA-NPA motifs, whereas LdAQP putative has a non-canonical NPM-NPA motif. In the carboxyl terminal to the second NPA box of all AQPs except AQP1, a valine/alanine residue was found instead of the arginine. In that respect these four AQPs are similar to tonoplast intrinsic proteins in plants, which are localized to intracellular organelles. Confocal microscopy of L. donovani expressing GFP-tagged AQPs showed an intracellular localization of LdAQP9 and LdAQP2870. Real-time PCR assays showed expression of all aquaporins except LdAQP2860, whose level was undetectable. Three-dimensional homology modeling of the AQPs showed that LdAQP1 structure bears greater topological similarity to the aquaglyceroporin than to aquaporin of E. coli. The pore of LdAQP1 was very different from the rest in shape and size. The cavity of LdAQP2860 was highly irregular and undefined in geometry. For functional characterization, four AQP proteins were heterologously expressed in yeast. In the fps1Δ yeast cells, which lacked the key aquaglyceroporin, LdAQP1 alone displayed an osmosensitive phenotype indicating glycerol transport activity. However, expression of LdAQP1 and LdAQP putative in a yeast gpd1Δ strain, deleted for glycerol production, conferred osmosensitive phenotype indicating water transport activity or aquaporin function. Our analysis for the first time shows the presence of subcellular aquaporins and provides structural and functional characterization of aquaporins in Leishmania donovani.     

Impact of extracellular matrix derived from osteoarthritis subchondral bone osteoblasts on osteocytes: role of integrinβ1 and focal adhesion kinase signaling cues

Introduction

Our recent study indicated that subchondral bone pathogenesis in osteoarthritis (OA) is associated with osteocyte morphology and phenotypic abnormalities. However, the mechanism underlying this abnormality needs to be identified. In this study we investigated the effect of extracellular matrix (ECM) produced from normal and OA bone on osteocytic cells function.

Methods

De-cellularized matrices, resembling the bone provisional ECM secreted from primary human subchondral bone osteoblasts (SBOs) of normal and OA patients were used as a model to study the effect on osteocytic cells. Osteocytic cells (MLOY4 osteocyte cell line) cultured on normal and OA derived ECMs were analyzed by confocal microscopy, scanning electron microscopy (SEM), cell attachment assays, zymography, apoptosis assays, qRT-PCR and western blotting. The role of integrinβ1 and focal adhesion kinase (FAK) signaling pathways during these interactions were monitored using appropriate blocking antibodies.

Results

The ECM produced by OA SBOs contained less mineral content, showed altered organization of matrix proteins and matrix structure compared with the matrices produced by normal SBOs. Culture of osteocytic cells on these defective OA ECM resulted in a decrease of integrinβ1 expression and the de-activation of FAK cell signaling pathway, which subsequently affected the initial osteocytic cell’s attachment and functions including morphological abnormalities of cytoskeletal structures, focal adhesions, increased apoptosis, altered osteocyte specific gene expression and increased Matrix metalloproteinases (MMP-2) and -9 expression.

Conclusion

This study provides new insights in understanding how altered OA bone matrix can lead to the abnormal osteocyte phenotypic changes, which is typical in OA pathogenesis.     

Pyrroloquinoline-quinone: a reactive oxygen species scavenger in bacteria

Transgenic Escherichia coli expressing pyrroloquinoline-quinone (PQQ) synthase gene from Deinococcus radiodurans showed superior survival during Rose Bengal induced oxidative stress. Such cells showed significantly low levels of protein carbonylation as compared to non-transgenic control. In vitro, PQQ reacted with reactive oxygen species with rate constants comparable to other well known antioxidants, producing non-reactive molecular products. PQQ also protected plasmid DNA and proteins from the oxidative damage caused by gamma-irradiation in solution. The data suggest that radioprotective/oxidative stress protective ability of PQQ in bacteria may be consequent to scavenging of reactive oxygen species per se and induction of other free radical scavenging mechanism.     

Virodhamine,an endocannabinoid,induces megakaryocyte differentiation by regulating MAPK activity and function of mitochondria

Endocannabinoids are well‐known regulators of neurotransmission by activating the cannabinoid (CB) receptors. Endocannabinoids are being used extensively for the treatment of various neurological disorders such as Alzheimer's and Parkinson's diseases. Although endocannabinoids are well studied in cell survival, proliferation, and differentiation in various neurological disorders and several cancers, the functional role in the regulation of blood cell development is less examined. In the present study, virodhamine, which is an agonist of CB receptor‐2, was used to examine its effect on megakaryocytic development from a megakaryoblastic cell. We observed that virodhamine increases cell adherence, cell size, and cytoplasmic protrusions. Interestingly, we have also observed large nucleus and increased expression of megakaryocytic marker (CD61), which are the typical hallmarks of megakaryocytic differentiation. Furthermore, the increased expression of CB2 receptor was noticed in virodhamine‐induced megakaryocytic cells. The effect of virodhamine on megakaryocytic differentiation could be mediated through CB2 receptor. Therefore, we have studied virodhamine induced molecular regulation of megakaryocytic differentiation; mitogen‐activated protein kinase (MAPK) activity, mitochondrial function, and reactive oxygen species (ROS) production were majorly affected. The altered mitochondrial functions and ROS production is the crucial event associated with megakaryocytic differentiation and maturation. In the present study, we report that virodhamine induces megakaryocytic differentiation by triggering MAPK signaling and ROS production either through MAPK effects on ROS‐generating enzymes or by the target vanilloid receptor 1‐mediated regulation of mitochondrial function.     

The mechanism of substrate release by the aspartate transporter GltPh: insights from simulations

Glutamate transporters regulate excitatory amino acid neurotransmission across neuronal and glial cell membranes by coupling the translocation of their substrate (aspartate or glutamate) into the intracellular (IC) medium to the energetically favorable transport of sodium ions or other cations. The first crystallographically resolved structure of this family, the archaeal aspartate transporter, Glt(Ph), has served as a structural paradigm for elucidating the mechanism of substrate translocation by these transporters. Two helical hairpins, HP2 and HP1, at the core domains of the three subunits that form this membrane protein have been proposed to act as the respective extracellular and IC gates for substrate intake and release. Molecular dynamics simulations using the outward-facing structure have confirmed that the HP2 loop acts as an EC gate. The mechanism of substrate release at atomic scale, however, remained unknown due to the lack of structural data until the recent determination of the inward-facing structure of Glt(Ph). In the present study, we use this recently resolved structure to simulate the release of substrate to the cytoplasm and the roles of HP1 and HP2 in this process. The highly flexible HP2 loop is observed to serve as an activator (or initiator) prompting the release of a gatekeeper Na(+) to the cytoplasm and promoting the influx of water molecules from the cytoplasm, which effectively disrupt substrate-protein interactions and drive the dislodging of the substrate from its binding site. The completion of substrate release and exit, however, entails the opening of the highly stable HP1 loop as well. Overall, the unique conformational flexibility of the HP2 loop, the dissociation of a Na(+), the hydration of binding pocket, and final yielding of the HP1 loop 3-Ser motif emerge as the successive events controlling the release of the bound substrate to the cell interior by glutamate transporters.