Role of the loop L-4,L-5 in allosteric regulation in mtHsp70s: in vivo significance of domain communication and its implications in protein translocation

Mitochondrial Hsp70 (mtHsp70) is essential for a vast repertoire of functions, including protein import, and requires effective interdomain communication for efficient partner-protein interactions. However, the in vivo functional significance of allosteric regulation in eukaryotes is poorly defined. Using integrated biochemical and yeast genetic approaches, we provide compelling evidence that a conserved substrate-binding domain (SBD) loop, L-4,L-5, plays a critical role in allosteric communication governing mtHsp70 chaperone functions across species. In yeast, a temperature-sensitive L-4,L-5 mutation (E467A) disrupts bidirectional domain communication, leading to compromised protein import and mitochondrial function. Loop L-4,L-5 functions synergistically with the linker in modulating the allosteric interface and conformational transitions between SBD and the nucleotide-binding domain (NBD), thus regulating interdomain communication. Second-site intragenic suppressors of E467A isolated within the SBD suppress domain communication defects by conformationally altering the allosteric interface, thereby restoring import and growth phenotypes. Strikingly, the suppressor mutations highlight that restoration of communication from NBD to SBD alone is the minimum essential requirement for effective in vivo function when primed at higher basal ATPase activity, mimicking the J-protein-bound state. Together these findings provide the first mechanistic insights into critical regions within the SBD of mtHsp70s regulating interdomain communication, thus highlighting its importance in protein translocation and mitochondrial biogenesis.

Single network adaptive critic aided dynamic inversion for optimal regulation and command tracking with online adaptation for enhanced robustness

To combine the advantages of both stability and optimality-based designs, a single network adaptive critic (SNAC) aided nonlinear dynamic inversion approach is presented in this paper. Here, the gains of a dynamic inversion controller are selected in such a way that the resulting controller behaves very close to a pre-synthesized SNAC controller in the output regulation sense. Because SNAC is based on optimal control theory, it makes the dynamic inversion controller operate nearly optimal. More important, it retains the two major benefits of dynamic inversion, namely (i) a closed-form expression of the controller and (ii) easy scalability to command tracking applications without knowing the reference commands a priori. An extended architecture is also presented in this paper that adapts online to system modeling and inversion errors, as well as reduced control effectiveness, thereby leading to enhanced robustness. The strengths of this hybrid method of applying SNAC to optimize an nonlinear dynamic inversion controller is demonstrated by considering a benchmark problem in robotics, that is, a two-link robotic manipulator system. Copyright (C) 2013 John Wiley & Sons, Ltd.

Interferon-gamma induced cell death: Regulation and contributions of nitric oxide, cJun N-terminal kinase, reactive oxygen species and peroxynitrite

Interferon-gamma (Ifn gamma), a known immunomodulatory cytokine, regulates cell proliferation and survival. In this study, the mechanisms leading to the selective susceptibility of some tumor cells to Ifn gamma were deciphered. Seven different mouse tumor cell lines tested demonstrated upregulation of MHC class I to variable extents with Ifn gamma; however, only the cell lines, H6 hepatoma and L929 fibrosarcoma, that produce higher amounts of nitric oxide (NO) and reactive oxygen species (ROS) are sensitive to Ifn gamma-induced cell death. NO inhibitors greatly reduce Ifn gamma-induced ROS; however, ROS inhibitors did not affect the levels of Ifn gamma-induced NO, demonstrating that NO regulates ROS. Consequently, NO inhibitors are more effective, compared to ROS inhibitors, in reducing Ifn gamma-induced cell death. Further analysis revealed that Ifn gamma induces peroxynitrite and 3-nitrotyrosine amounts and a peroxynitrite scavenger, FeTPPS, reduces cell death. Ifn gamma treatment induces the phosphorylation of c-jun N-terminal kinase (Jnk) in H6 and L929 but not CT26, a colon carcinoma cell line, which is resistant to Ifn gamma-mediated death. Jnk activation downstream to NO leads to induction of ROS, peroxynitrite and cell death in response to Ifn gamma. Importantly, three cell lines tested, i.e. CT26, EL4 and Neuro2a, that are resistant to cell death with Ifn gamma alone become sensitive to the combination of Ifn gamma and NO donor or ROS inducer in a peroxynitrite-dependent manner. Overall, this study delineates the key roles of NO as the initiator and Jnk, ROS, and peroxynitrite as the effectors during Ifn gamma-mediated cell death. The implications of these findings in the Ifn gamma-mediated treatment of malignancies are discussed. (C) 2014 Elsevier B.V. All rights reserved.

Interferon-gamma induced cell death: Regulation and contributions of nitric oxide, cJun N-terminal kinase, reactive oxygen species and peroxynitrite

Interferon-gamma (Ifn gamma), a known immunomodulatory cytokine, regulates cell proliferation and survival. In this study, the mechanisms leading to the selective susceptibility of some tumor cells to Ifn gamma were deciphered. Seven different mouse tumor cell lines tested demonstrated upregulation of MHC class I to variable extents with Ifn gamma; however, only the cell lines, H6 hepatoma and L929 fibrosarcoma, that produce higher amounts of nitric oxide (NO) and reactive oxygen species (ROS) are sensitive to Ifn gamma-induced cell death. NO inhibitors greatly reduce Ifn gamma-induced ROS; however, ROS inhibitors did not affect the levels of Ifn gamma-induced NO, demonstrating that NO regulates ROS. Consequently, NO inhibitors are more effective, compared to ROS inhibitors, in reducing Ifn gamma-induced cell death. Further analysis revealed that Ifn gamma induces peroxynitrite and 3-nitrotyrosine amounts and a peroxynitrite scavenger, FeTPPS, reduces cell death. Ifn gamma treatment induces the phosphorylation of c-jun N-terminal kinase (Jnk) in H6 and L929 but not CT26, a colon carcinoma cell line, which is resistant to Ifn gamma-mediated death. Jnk activation downstream to NO leads to induction of ROS, peroxynitrite and cell death in response to Ifn gamma. Importantly, three cell lines tested, i.e. CT26, EL4 and Neuro2a, that are resistant to cell death with Ifn gamma alone become sensitive to the combination of Ifn gamma and NO donor or ROS inducer in a peroxynitrite-dependent manner. Overall, this study delineates the key roles of NO as the initiator and Jnk, ROS, and peroxynitrite as the effectors during Ifn gamma-mediated cell death. The implications of these findings in the Ifn gamma-mediated treatment of malignancies are discussed. (C) 2014 Elsevier B.V. All rights reserved.

Regulation of Off-Network Pricing in a Nonneutral Network

Representatives of several Internet service providers (ISPs) have expressed their wish to see a substantial change in the pricing policies of the Internet. In particular, they would like to see content providers (CPs) pay for use of the network, given the large amount of resources they use. This would be in clear violation of the ``network neutrality'' principle that had characterized the development of the wireline Internet. Our first goal in this article is to propose and study possible ways of implementing such payments and of regulating their amount. We introduce a model that includes the users' behavior, the utilities of the ISP and of the CPs, and, the monetary flow that involves the content users, the ISP and CP, and, in pUrticular, the CP's revenues from advertisements. We consider various game models and study the resulting equilibria; they are all combinations of a noncooperative game (in which the ISPs and CPs determine how much they will charge the users) with a ``cooperative'' one on how the CP and the ISP share the payments. We include in our model a possible asymmetric weighting parameter (that varies between zero to one). We also study equilibria that arise when one of the CPs colludes with the TSP. We also study two dynamic game models as well as the convergence of prices to the equilibrium values.

Direct regulation of topoisomerase activity by a nucleoid-associated protein

The topological homeostasis of bacterial chromosomes is maintained by the balance between compaction and the topological organization of genomes. Two classes of proteins play major roles in chromosome organization: the nucleoid-associated proteins (NAPs) and topoisomerases. The NAPs bind DNA to compact the chromosome, whereas topoisomerases catalytically remove or introduce supercoils into the genome. We demonstrate that HU, a major NAP of Mycobacterium tuberculosis specifically stimulates the DNA relaxation ability of mycobacterial topoisomerase I (TopoI) at lower concentrations but interferes at higher concentrations. A direct physical interaction between M. tuberculosis HU (MtHU) and TopoI is necessary for enhancing enzyme activity both in vitro and in vivo. The interaction is between the amino terminal domain of MtHU and the carboxyl terminal domain of TopoI. Binding of MtHU did not affect the two catalytic trans-esterification steps but enhanced the DNA strand passage, requisite for the completion of DNA relaxation, a new mechanism for the regulation of topoisomerase activity. An interaction-deficient mutant of MtHU was compromised in enhancing the strand passage activity. The species-specific physical and functional cooperation between MtHU and TopoI may be the key to achieve the DNA relaxation levels needed to maintain the optimal superhelical density of mycobacterial genomes.

Genome wide chromatin occupancy of mrhl RNA and its role in gene regulation in mouse spermatogonial cells

Mrhl RNA is a nuclear lncRNA encoded in the mouse genome and negatively regulates Wnt signaling in spermatogonial cells through p68/Ddx5 RNA helicase. Mrhl RNA is present in the chromatin fraction of mouse spermatogonial Gc1-Spg cells and genome wide chromatin occupancy of mrhl RNA by ChOP (Chromatin oligo affinity precipitation) technique identified 1370 statistically significant genomic loci. Among these, genes at 37 genomic loci also showed altered expression pattern upon mrhl RNA down regulation which are referred to as GRPAM (Genes Regulated by Physical Association of Mrhl RNA). p68 interacted with mrhl RNA in chromatin at these GRPAM loci. p68 silencing drastically reduced mrhl RNA occupancy at 27 GRPAM loci and also perturbed the expression of GRPAM suggesting a role for p68 mediated mrhl RNA occupancy in regulating GRPAM expression. Wnt3a ligand treatment of Gc1-Spg cells down regulated mrhl RNA expression and also perturbed expression of these 27 GRPAM genes that included genes regulating Wnt signaling pathway and spermatogenesis, one of them being Sox8, a developmentally important transcription factor. We also identified interacting proteins of mrhl RNA associated chromatin fraction which included Pc4, a chromatin organizer protein and hnRNP A/B and hnRNP A2/B1 which have been shown to be associated with lincRNA-Cox2 function in gene regulation. Our findings in the Gc1-Spg cell line also correlate with the results from analysis of mouse testicular tissue which further highlights the in vivo physiological significance of mrhl RNA in the context of gene regulation during mammalian spermatogenesis.

Do gamma oscillations play a role in cerebral cortex?

Gamma rhythm (which has a center frequency between 30 and 80 Hz) is modulated by cognitive mechanisms such as attention and memory, and has been hypothesized to play a role in mediating these processes by supporting communication channels between cortical areas or encoding information in its phase. We highlight several issues related to gamma rhythms, such as low and inconsistent power, its dependence on low-level stimulus features, problems due to conduction delays, and contamination due to spike-related activity that makes accurate estimation of gamma phase difficult. Gamma rhythm could be a potentially useful signature of excitation-inhibition interactions in the brain, but whether it also provides a mechanism for information processing or coding remains an open question.

Architectural plan of transcriptional regulation in Mycobacterium tuberculosis

Transcriptional regulation enables adaptation in bacteria. Typically, only a few transcriptional events are well understood, leaving many others unidentified. The recent genome-wide identification of transcription factor binding sites in Mycobacterium tuberculosis has changed this by deciphering a molecular road-map of transcriptional control, indicating active events and their immediate downstream effects.

Lateralisation abnormalities in obsessive-compulsive disorder: a line bisection study

Objective Asymmetry in brain structure and function is implicated in the pathogenesis of psychiatric disorders. Although right hemisphere abnormality has been documented in obsessive-compulsive disorder (OCD), cerebral asymmetry is rarely examined. Therefore, in this study, we examined anomalous cerebral asymmetry in OCD patients using the line bisection task. Methods A total of 30 patients with OCD and 30 matched healthy controls were examined using a reliable and valid two-hand line bisection (LBS) task. The comparative profiles of LBS scores were analysed using analysis of covariance. Results Patients with OCD bisected significantly less number of lines to the left and had significant rightward deviation than controls, indicating right hemisphere dysfunction. The correlations observed in this study suggest that those with impaired laterality had more severe illness at baseline. Conclusions The findings of this study indicate abnormal cerebral lateralisation and right hemisphere dysfunction in OCD patients.

Simultaneously targeting inflammatory response and parasite sequestration in brain to treat Experimental Cerebral Malaria

Malaria afflicts around 200 million people annually, with a mortality number close to 600,000. The mortality rate in Human Cerebral Malaria (HCM) is unacceptably high (15-20%), despite the availability of artemisinin-based therapy. An effective adjunct therapy is urgently needed. Experimental Cerebral Malaria (ECM) in mice manifests many of the neurological features of HCM. Migration of T cells and parasite-infected RBCs (pRBCs) into the brain are both necessary to precipitate the disease. We have been able to simultaneously target both these parameters of ECM. Curcumin alone was able to reverse all the parameters investigated in this study that govern inflammatory responses, CD8(+) T cell and pRBC sequestration into the brain and blood brain barrier (BBB) breakdown. But the animals eventually died of anemia due to parasite build-up in blood. However, arteether-curcumin (AC) combination therapy even after the onset of symptoms provided complete cure. AC treatment is a promising therapeutic option for HCM.

Systematic Analysis of Mycobacterial Acylation Reveals First Example of Acylation-mediated Regulation of Enzyme Activity of a Bacterial Phosphatase

Protein lysine acetylation is known to regulate multiple aspects of bacterial metabolism. However, its presence in mycobacterial signal transduction and virulence-associated proteins has not been studied. In this study, analysis of mycobacterial proteins from different cellular fractions indicated dynamic and widespread occurrence of lysine acetylation. Mycobacterium tuberculosis proteins regulating diverse physiological processes were then selected and expressed in the surrogate host Mycobacterium smegmatis. The purified proteins were analyzed for the presence of lysine acetylation, leading to the identification of 24 acetylated proteins. In addition, novel lysine succinylation and propionylation events were found to co-occur with acetylation on several proteins. Protein-tyrosine phosphatase B (PtpB), a secretory phosphatase that regulates phosphorylation of host proteins and plays a critical role in Mycobacterium infection, is modified by acetylation and succinylation at Lys-224. This residue is situated in a lid region that covers the enzyme's active site. Consequently, acetylation and succinylation negatively regulate the activity of PtpB.

Regulation of Acetate Metabolism and Acetyl Co-a Synthetase 1 (ACS1) Expression by Methanol Expression Regulator 1 (Mxr1p) in the Methylotrophic Yeast Pichia pastoris

Methanol expression regulator 1 (Mxr1p) is a zinc finger protein that regulates the expression of genes encoding enzymes of the methanol utilization pathway in the methylotrophic yeast Pichia pastoris by binding to Mxr1p response elements (MXREs) present in their promoters. Here we demonstrate that Mxr1p is a key regulator of acetate metabolism as well. Mxr1p is cytosolic in cells cultured in minimal medium containing a yeast nitrogen base, ammonium sulfate, and acetate (YNBA) but localizes to the nucleus of cells cultured in YNBA supplemented with glutamate or casamino acids as well as nutrient-rich medium containing yeast extract, peptone, and acetate (YPA). Deletion of Mxr1 retards the growth of P. pastoris cultured in YNBA supplemented with casamino acids as well as YPA. Mxr1p is a key regulator of ACS1 encoding acetyl-CoA synthetase in cells cultured in YPA. A truncated Mxr1p comprising 400 N-terminal amino acids activates ACS1 expression and enhances growth, indicating a crucial role for the N-terminal activation domain during acetate metabolism. The serine 215 residue, which is known to regulate the expression of Mxr1p-activated genes in a carbon source-dependent manner, has no role in the Mxr1p-mediated activation of ACS1 expression. The ACS1 promoter contains an Mxr1p response unit (MxRU) comprising two MXREs separated by a 30-bp spacer. Mutations that abrogate MxRU function in vivo abolish Mxr1p binding to MxRU in vitro. Mxr1p-dependent activation of ACS1 expression is most efficient in cells cultured in YPA. The fact that MXREs are conserved in genes outside of the methanol utilization pathway suggests that Mxr1p may be a key regulator of multiple metabolic pathways in P. pastoris.

Regulation of Growth, Cell Shape, Cell Division, and Gene Expression by Second Messengers (p)ppGpp and Cyclic Di-GMP in Mycobacterium smegmatis

The alarmone (p)ppGpp regulates transcription, translation, replication, virulence, lipid synthesis, antibiotic sensitivity, biofilm formation, and other functions in bacteria. Signaling nucleotide cyclic di-GMP (c-di-GMP) regulates biofilm formation, motility, virulence, the cell cycle, and other functions. In Mycobacterium smegmatis, both (p) ppGpp and c-di-GMP are synthesized and degraded by bifunctional proteins Rel(Msm) and DcpA, encoded by rel(Msm) and dcpA genes, respectively. We have previously shown that the Delta rel(Msm) and Delta dcpA knockout strains are antibiotic resistant and defective in biofilm formation, show altered cell surface properties, and have reduced levels of glycopeptidolipids and polar lipids in their cell wall (K. R. Gupta, S. Kasetty, and D. Chatterji, Appl Environ Microbiol 81:2571-2578, 2015, http://dx.doi.org/10.1128/AEM.03999-14). In this work, we have explored the phenotypes that are affected by both (p) ppGpp and c-di-GMP in mycobacteria. We have shown that both (p) ppGpp and c-di-GMP are needed to maintain the proper growth rate under stress conditions such as carbon deprivation and cold shock. Scanning electron microscopy showed that low levels of these second messengers result in elongated cells, while high levels reduce the cell length and embed the cells in a biofilm-like matrix. Fluorescence microscopy revealed that the elongated Delta rel(Msm) and Delta dcpA cells are multinucleate, while transmission electron microscopy showed that the elongated cells are multiseptate. Gene expression analysis also showed that genes belonging to functional categories such as virulence, detoxification, lipid metabolism, and cell-wall-related processes were differentially expressed. Our results suggests that both (p) ppGpp and c-di-GMP affect some common phenotypes in M. smegmatis, thus raising a possibility of cross talk between these two second messengers in mycobacteria. IMPORTANCE Our work has expanded the horizon of (p) ppGpp and c-di-GMP signaling in Gram-positive bacteria. We have come across a novel observation that M. smegmatis needs (p) ppGpp and c-di-GMP for cold tolerance. We had previously shown that the Delta rel(Msm) and Delta dcpA strains are defective in biofilm formation. In this work, the overproduction of (p) ppGpp and c-di-GMP encased M. smegmatis in a biofilm-like matrix, which shows that both (p) ppGpp and c-di-GMP are needed for biofilm formation. The regulation of cell length and cell division by (p) ppGpp was known in mycobacteria, but our work shows that c-di-GMP also affects the cell size and cell division in mycobacteria. This is perhaps the first report of c-di-GMP regulating cell division in mycobacteria.