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The potency of various synthesized graphene nanocomposites, namely, reduced graphene oxide, silver nanoparticles-reduced graphene oxide and silver nanoparticles-reduced graphene oxide@Lysine, to interact with forensically relevant drugs i.e., nitrazepam and diazepam, was examined under similar physiological conditions using fluorescence spectroscopy. Also, various characterization techniques such as X-ray spectroscopy, Fourier-transform infrared spectroscopy and Raman spectroscopy were employed to confirm the synthesis of graphene and its nanocomposites. The values of binding constant and Stern-Volmer binding constant influenced by temperature were investigated. They were supposed to be related to quenching process between graphene nanocomposites and forensically relevant drugs which were mainly determined by the combination of static and dynamic quenching. Further, the thermodynamic parameters of the nanocomposite were also studied at different temperatures, and [DELTA]G was calculated., Author(s): Garima [sup.1], Varnika Parkash [sup.1], Surinder K. Mehta [sup.2], Shweta Sharma [sup.1] Author Affiliations: (1) grid.261674.0, 0000 0001 2174 5640, Institute of Forensic Sciences and Criminology, Panjab University, , [...]

Calcineurin acts as a calcium-activated phosphatase that dephosphorylates various substrates, including members of the nuclear factor of activated T cells (NFAT) family, to trigger their nuclear translocation and transcriptional activity. However, the detailed mechanism regulating the recruitment of NFATs to calcineurin remains poorly understood. Here, we report that calcineurin A (CNA), encoded by PPP3CB or PPP3CC, is constitutively ubiquitinated on lysine 327, and this polyubiquitin chain is rapidly removed by ubiquitin carboxyl-terminal hydrolase 16 (USP16) in response to intracellular calcium stimulation. The K29-linked ubiquitination of CNA impairs NFAT recruitment and transcription of NFAT-targeted genes. USP16 deficiency prevents calcium-triggered deubiquitination of CNA in a manner consistent with defective maintenance and proliferation of peripheral T cells. T cell-specific USP16 knockout mice exhibit reduced severity of experimental autoimmune encephalitis and inflammatory bowel disease. Our data reveal the physiological function of CNA ubiquitination and its deubiquitinase USP16 in peripheral T cells. Notably, our results highlight a critical mechanism for the regulation of calcineurin activity and a novel immunosuppressive drug target for the treatment of autoimmune diseases., Introduction Calcineurin (protein phosphatase 2B) is the only serine/threonine protein phosphatase regulated by intracellular calcium (1). Calcineurin participates in a variety of signal transduction pathways regulated by cytoplasmic [Ca.sup.2+], and [...]

Deregulation of lysine methylation signalling has emerged as a common aetiological factor in cancer pathogenesis, with inhibitors of several histone lysine methyltransferases (KMTs) being developed as chemotherapeutics (1). The largely cytoplasmic KMT SMYD3 (SET and MYND domain containing protein 3) is overexpressed in numerous human tumours (2-4). However, the molecular mechanism by which SMYD3 regulates cancer pathways and its relationship to tumorigenesis in vivo are largely unknown. Here we show that methylation of MAP3K2 by SMYD3 increases MAP kinase signalling and promotes the formation of Ras-driven carcinomas. Using mouse models for pancreatic ductal adenocarcinoma and lung adenocarcinoma, we found that abrogating SMYD3 catalytic activity inhibits tumour development in response to oncogenic Ras. We used protein array technology to identify the MAP3K2 kinase as a target of SMYD3. In cancer cell lines, SMYD3-mediated methylation of MAP3K2 at lysine 260 potentiates activation of the Ras/Raf/MEK/ERK signalling module and SMYD3 depletion synergizes with a MEK inhibitor to block Rasdriven tumorigenesis. Finally, the PP2A phosphatase complex, a key negative regulator of the MAP kinase pathway, binds to MAP3K2 and this interaction is blocked by methylation. Together, our results elucidate a new role for lysine methylation in integrating cytoplasmic kinase-signalling cascades and establish a pivotal role for SMYD3 in the regulation of oncogenic Ras signalling., The Ras family of oncogenes is activated in a large fraction of human cancers (5). To explore possible connections between KMTs and human cancers driven by activation of Ras, we [...]

Brown adipose tissue (BAT) dissipates chemical energy in the form of heat as a defence against hypothermia and obesity. Current evidence indicates that brown adipocytes arise from [Myf5.sup.+] dermotomal precursors through the action of PR domain containing protein 16 (PRDM16) transcriptional complex (1,2). However, the enzymatic component of the molecular switch that determines lineage specification of brown adipocytes remains unknown. Here we show that euchromatic histone-lysine N-methyltransferase 1 (EHMT1) is an essential BAT-enriched lysine methyltransferase in the PRDM16 transcriptional complex and controls brown adipose cell fate. Loss of EHMT1 in brown adipocytes causes a severe loss of brown fat characteristics and induces muscle differentiation invivo through demethylation of histone 3 lysine 9 (H3K9me2 and 3) of the muscle-selective gene promoters. Conversely, EHMT1 expression positively regulates the BAT-selective thermogenic program by stabilizing the PRDM16 protein. iotably, adipose-specific deletion of EHMT1 leads to a marked reduction of BAT-mediated adaptive thermogenesis, obesity and systemic insulin resistance. These data indicate that EHMT1 is an essential enzymatic switch that controls brown adipose cell fate and energy homeostasis., Obesity develops when energy intake chronically exceeds total energy expenditure. All anti-obesity medications currently approved by the FDA act to repress energy intake, either by suppressing appetite or by inhibiting [...]

Aggregatibacter actinomycetemcomitans is an opportunistic pathogen that resides primarily in the mammalian oral cavity. In this environment, A. actinomycetemcomitans faces numerous host. and microbederived stresses, including intense competition for nutrients and exposure to the host immune system. While it is clear that A. actinomycetemcomitans responds to precise cues that allow it to adapt and proliferate in the presence of these stresses, little is currently known about the regulatory mechanisms that underlie these responses. Many bacteria use noncoding regulatory RNAs (ncRNAs) to rapidly alter gene expression in response to environmental stresses. Although no ncRNAs have been reported in A. actinomycetemcomitans, we propose that they are likely important for colonization and persistence in the oral cavity. Using a bioinformatic and experimental approach, we identified three putative metabolite-sensing riboswitches and nine small regulatory RNAs (sRNAs) in A. actinomycetemcomitans during planktonic and biofilm growth. Molecular characterization of one of the riboswitches revealed that it is a lysine riboswitch and that its target gene, lysT, encodes a novel lysine-specific transporter. Finally, we demonstrated that lysT and the lysT lysine riboswitch are conserved in over 40 bacterial species, including the phylogenetically related pathogen Haemophilus influenzae. doi: 10.1128/JB.00935-10

In comparison to other pseudomonads, Pseudomonas aeruginosa grows poorly in L-lysine as a sole source of nutrient. In this study, the ldcA gene (lysine decarboxylase A; PAl818), previously identified as a member of the ArgR regulon of L-arginine metabolism, was found essential for L-lysine catabolism in this organism. LdcA was purified to homogeneity from a recombinant strain of Escherichia coli, and the results of enzyme characterization revealed that this pyridoxal-5-phosphate-dependent decarboxylase takes L-lysine, but not L-arginine, as a substrate. At an optimal pH of 8.5, cooperative substrate activation by L-lysine was depicted from kinetics studies, with calculated [K]m and [V]max values of 0.73 mM and 2.2 [micro]mole/mg/min, respectively. Contrarily, the ldcA promoter was induced by exogenous L-arginine but not by L-lysine in the wild-type strain PAO1, and the binding of ArgR to this promoter region was demonstrated by electromobility shift assays. This peculiar arginine control on lysine utilization was also noted from uptake experiments in which incorporation of radioactively labeled L-lysine was enhanced in cells grown in the presence of L-arginine but not L-lysine. Rapid growth on L-lysine was detected in a mutant devoid of the main arginine catabolic pathway and with a higher basal level of the intracellular L-arginine pool and hence elevated ArgR-responsive regulons, including ldcA. Growth on L-lysine as a nitrogen source can also be enhanced when the aruH gene encoding an arginine/lysine:pyruvate transaminase was expressed constitutively from plasmids; however, no growth of the ldcA mutant on L-lysine suggests a minor role of this transaminase in L-lysine catabolism. In summary, this study reveals a tight connection of lysine catabolism to the arginine regulatory network, and the lack of lysine-responsive control on lysine uptake and decarboxylation provides an explanation of L-lysine as a poor nutrient for P. aeruginosa. doi: 10.1128/JB.00673-10

The pathway of lysine biosynthesis in the methanococci has not been identified previously. A variant of the diaminopimelic acid (DAP) pathway uses diaminopimelate aminotransferase (DapL) to catalyze the direct conversion of tetrahydrodipicolinate (THDPA) to LL-DAP. Recently, the enzyme DapL (MTH52) was identified in Methanothermobacter thermautotrophicus and shown to belong to the DapLl group. Although the Methanococcus maripaludis genome lacks a gene that can be unambiguously assigned a DapL function based on sequence similarity, the open reading frame MMP1527 product shares 30% amino acid sequence identity with MTH52. A Ammp1527 deletion mutant was constructed and found to be a lysine auxotroph, suggesting that this DapL homolog in methanococci is required for lysine biosynthesis. In cell extracts of the M. maripaludis wild-type strain, the specific activity of DapL using LL-DAP and [alpha]-ketoglutarate as substrates was 24.3 [+ or -] 2.0 nmol [min.sup.-1] mg of [protein.sup.-1]. The gene encoding the DapL homolog in Methanocaidococcus jannaschii (MJ1391) was cloned and expressed in Escherichia coli, and the protein was purified. The maximum activity of MJ1391 was observed at 70[degrees]C and pH 8.0 to 9.0. The apparent [K.sub.m]s of MJ1391 for LL-DAP and [alpha]-ketoglutarate were 82.8 [+ or -] 10 [micro]M and 0.42 [+ or -] 0.02 mM, respectively. MJ1391 was not able to use succinyl-DAP or acetyl- DAP as a substrate. Phyiogenetic analyses suggested that two lateral gene transfers occurred in the DapL genes, one from the archaea to the bacteria in the DapL2 group and one from the bacteria to the archaea in the DapL1 group. These results demonstrated that the DapL pathway is present in marine methanogens belonging to the Methanococcales. doi: 10.1128/JB.00172-10