Traumatic brain injury stimulates hippocampal catechol-O-methyl transferase expression in microglia.

Outcome following traumatic brain injury (TBI) is in large part determined by the combined action of multiple processes. In order to better understand the response of the central nervous system to injury, we utilized an antibody array to simultaneously screen 507 proteins for altered expression in the injured hippocampus, a structure critical for memory formation. Array analysis indicated 41 candidate proteins have altered expression levels 24h after TBI. Of particular interest was catechol-O-methyl transferase (COMT), an enzyme involved in metabolizing catecholamines released following neuronal activity. Altered catecholamine signaling has been observed after brain injury, and may contribute to the cognitive dysfunctions and behavioral deficits often experienced after TBI. Our data shows that COMT expression in the injured ipsilateral hippocampus was elevated for at least 14 d after controlled cortical impact injury. We found strong co-localization of COMT immunoreactivity with the microglia marker Iba1 near the injury site. Since dopamine transporter expression has been reported to be down-regulated after brain injury, COMT-mediated catecholamine metabolism may play a more prominent role in terminating catecholamine signaling in injured areas.

Aldosterone-induced Sgk1 relieves Dot1a-Af9-mediated transcriptional repression of epithelial Na+ channel alpha.

Aldosterone plays a major role in the regulation of salt balance and the pathophysiology of cardiovascular and renal diseases. Many aldosterone-regulated genes--including that encoding the epithelial Na+ channel (ENaC), a key arbiter of Na+ transport in the kidney and other epithelia--have been identified, but the mechanisms by which the hormone modifies chromatin structure and thus transcription remain unknown. We previously described the basal repression of ENaCalpha by a complex containing the histone H3 Lys79 methyltransferase disruptor of telomeric silencing alternative splice variant a (Dot1a) and the putative transcription factor ALL1-fused gene from chromosome 9 (Af9) as well as the release of this repression by aldosterone treatment. Here we provide evidence from renal collecting duct cells and serum- and glucocorticoid-induced kinase-1 (Sgk1) WT and knockout mice that Sgk1 phosphorylated Af9, thereby impairing the Dot1a-Af9 interaction and leading to targeted histone H3 Lys79 hypomethylation at the ENaCalpha promoter and derepression of ENaCalpha transcription. Thus, Af9 is a physiologic target of Sgk1, and Sgk1 negatively regulates the Dot1a-Af9 repressor complex that controls transcription of ENaCalpha and likely other aldosterone-induced genes.

CROSSTALK BETWEEN R1175 METHYLATION AND Y1173 PHOSPHORYLATION NEGATIVELY MODULATES EGFR-MEDIATED ERK ACTIVATION

Post-translational protein modifications are critical regulators of protein functions as they expand the signaling potentials of the modified proteins, leading to diverse physiological consequences. Currently, increasing evidence suggests that protein methylation is as important as other post-translational modifications in the regulation of various biological processes. This drives us to ask whether methylation is involved in the EGFR (epidermal growth factor receptor) signaling, a biological process extensively regulated by multiple post-translational modifications including phosphorylation, glycosylation and ubiquitination. We found that EGFR R1175 is methylated by a protein arginine methyltransferase named PRMT5. During EGFR activation, PRMT5-mediated R1175 methylation specifically enhances EGF-induced EGFR autophosphorylation at Y1173 residue. This novel modification crosstalk increases SHP1 recruitment to EGFR and suppresses EGFR-mediated ERK activation, resulting in inhibition of cell proliferation, migration, and invasion of EGFR-expressing cells. Based on these findings, we provide the first link between arginine methylation and tyrosine phosphorylation and identify R1175 methylation as an inhibitory modification specifically against EGFR-mediated ERK activation.

An mRNA-Derived Noncoding RNA Targets and Regulates the Ribosome.

The structural and functional repertoire of small non-protein-coding RNAs (ncRNAs) is central for establishing gene regulation networks in cells and organisms. Here, we show that an mRNA-derived 18-nucleotide-long ncRNA is capable of downregulating translation in Saccharomyces cerevisiae by targeting the ribosome. This 18-mer ncRNA binds to polysomes upon salt stress and is crucial for efficient growth under hyperosmotic conditions. Although the 18-mer RNA originates from the TRM10 locus, which encodes a tRNA methyltransferase, genetic analyses revealed the 18-mer RNA nucleotide sequence, rather than the mRNA-encoded enzyme, as the translation regulator. Our data reveal the ribosome as a target for a small regulatory ncRNA and demonstrate the existence of a yet unkown mechanism of translation regulation. Ribosome-targeted small ncRNAs are found in all domains of life and represent a prevalent but so far largely unexplored class of regulatory molecules.

An mRNA-derived ncRNA targets and regulates the ribosome

Small non-protein-coding RNA (ncRNA) molecules have been recognized recently as major contributors to regulatory networks in controlling gene expression in a highly efficient manner. While the list of validated ncRNAs that regulate crucial cellular processes grows steadily, not a single ncRNA has been identified that directly interacts and regulates the ribosome during protein biosynthesis (with the notable exceptions of 7SL RNA and tmRNA). All of the recently discovered regulatory ncRNAs that act on translation (e.g. microRNAs, siRNAs or antisense RNAs) target the mRNA rather than the ribosome. This is unexpected, given the central position the ribosome plays during gene expression. To investigate whether such a class of regulatory ncRNAs does exist we performed genomic screens for small ribosome-associated RNAs in various model organisms of all three domains [1,2]. Here we focus on the functional characterisation of an 18 nucleotide long ncRNA candidate derived from an open reading frame (ORF) of an annotated S. cerevisiae gene, which encodes a tRNA methyltransferase. Yeast cells lacking this tRNA methyltransferase showed clear growth defects in high salt containing media. Genetic analysis showed that the absence of the mRNA-derived ncRNA rather than the absence of the tRNA methyltransferase activity is responsible for the observed phenotype. Since we performed a screen for small ribosome-associated RNAs we examined the regulatory potential of the synthetic 18mer during translation in vitro and in vivo. Metabolic labeling experiments in the presence of the synthetic 18mer RNA revealed an inhibitory potential on the global protein biosynthesis rate. In vitro translation and northern blot analysis further strengthen the hypothesis, that this RNA is a ribosome-associated regulatory ncRNA. Our studies in pro- and eukaryotic model organisms reveal the ribosome as a novel target for small regulatory ncRNAs in all domains of life. Ribosome-bound ncRNAs are capable of fine tuning translation and might represent a so far largely unexplored class of regulatory ncRNAs.

Emergence of Klebsiella pneumoniae co-producing NDM-1, OXA-48, CTX-M-15, CMY-16, QnrA and ArmA in Switzerland

Extensively drug-resistant (XDR) Klebsiella pneumoniae isolates usually carry a single carbapenemase (e.g. KPC, NDM, OXA-48-like). Here we describe an XDR K. pneumoniae of sequence type 101 that was detected in the screening rectal swab of a patient transferred from the intensive care unit of a hospital located in Belgrade (Serbia) to Bern University Hospital (Switzerland). The isolate was resistant to all antibiotics with the exception of colistin [minimum inhibitory concentration] (MIC≤0.125μg/mL), tigecycline (MIC=0.5μg/mL) and fosfomycin (MIC=2μg/mL). The isolate co-possessed class B (NDM-1) and class D (OXA-48) carbapenemases, class A extended-spectrum β-lactamase (CTX-M-15), class C cephalosporinase (CMY-16), ArmA 16S rRNA methyltransferase, substitutions in GyrA and ParC, loss of OmpK35 porin, as well as other genes conferring resistance to quinolones (qnrA), tetracyclines [tet(A)], sulfonamides (sul1, sul2), trimethoprim (dfrA12, dfrA14), rifampicin (arr-1), chloramphenicol (cmlA1, floR) and streptomycin (aadA1). The patient was placed under contact isolation precautions preventing the spread of this nearly untreatable pathogen.

An mRNA-derived ncRNA targets and regulates the ribosome

Small non-protein-coding RNA (ncRNA) molecules are key players in controlling gene expression at multiple steps in all domains of life. While the list of validated ncRNAs that regulate crucial cellular processes grows steadily (such as micro RNAs and small-interfering RNAs), not a single ncRNA has been identified that directly interacts and regulates the ribosome during protein biosynthesis (with the notable exceptions of 7SL RNA and tmRNA). This is unexpected, given the central position the ribosome plays during gene expression. To investigate whether such a class of regulatory ncRNAs does exist we performed genomic screens for small ribosome-associated RNAs in various model organisms of all three domains [1,2]. Here we show that an mRNA-derived 18 nucleotide long ncRNA is capable of down-regulating translation in Saccharomyces cerevisiae by directly targeting the ribosome [3]. This 18-mer ncRNA binds to polysomes upon salt stress and is crucial for efficient growth under hyperosmotic conditions. Although the 18-mer RNA originates from the TRM10 locus, which encodes a tRNA methyltransferase, genetic analyses revealed the 18-mer RNA nucleotide sequence, rather than the mRNA-encoded enzyme, as the translation regulator under these stress conditions. Our data reveal the ribosome as a target for small regulatory ncRNAs and unveil the existence of a novel mechanism of translation regulation. Analogous genomic screens in organisms spanning all three domains of life demonstrate the existence of thousands of ncRNA candidates putatively regulating the ribosome. We therefore anticipate that ribosome-bound ncRNAs are capable of fine tuning translation and might represent a so far largely unexplored class of regulatory ncRNAs.

An mRNA-derived ncRNA targets and regulates the ribosome

Small non-protein-coding RNAs (ncRNAs) are key players in controlling gene expression. The advantage of ncRNA regulators is their almost immediate availability since they act on the RNA level. The list of validated ncRNAs regulating translation, such as micro RNAs, is growing steadily, however, they almost exclusively target the mRNA rather than the ribosome. This is unexpected given the central position the ribosome plays. Here we show that an mRNA-derived 18 nucleotide long ncRNA is capable of down-regulating translation in Saccharomyces cerevisiae by targeting the ribosome. This 18-mer ncRNA binds to polysomes upon salt stress and is crucial for efficient growth. Although the 18-mer RNA originates from the TRM10 locus, which encodes a tRNA methyltransferase, genetic analyses revealed the 18-mer RNA nucleotide sequence as the translation regulator. Our data reveal the ribosome as a target for a small regulatory ncRNA and demonstrate the existence of a yet unknown mechanism of translation regulation.

Prognostic and predictive roles of MGMT protein expression and promoter methylation in sporadic pancreatic neuroendocrine neoplasms.

BACKGROUND/AIMS O(6)-methylguanine-methyltransferase (MGMT) is an important enzyme of DNA repair. MGMT promoter methylation is detectable in a subset of pancreatic neuroendocrine neoplasms (pNEN). A subset of pNEN responds to the alkylating agent temozolomide (TMZ). We wanted to correlate MGMT promoter methylation with MGMT protein loss in pNEN, correlate the findings with clinico-pathological data and determine the role of MGMT to predict response to TMZ chemotherapy. METHODS We analysed a well-characterized collective of 141 resected pNEN with median follow-up of 83 months for MGMT protein expression and promoter methylation using methylation-specific PCR (MSP). A second collective of 10 metastasized, pretreated and progressive patients receiving TMZ was used to examine the predictive role of MGMT by determining protein expression and promoter methylation using primer extension-based quantitative PCR. RESULTS In both collectives there was no correlation between MGMT protein expression and promoter methylation. Loss of MGMT protein was associated with an adverse outcome, this prognostic value, however, was not independent from grade and stage in multivariate analysis. Promoter hypermethylation was significantly associated with response to TMZ. CONCLUSION Loss of MGMT protein expression is associated with adverse outcome in a surgical series of pNET. MGMT promoter methylation could be a predictive marker for TMZ chemotherapy in pNEN, but further, favourably prospective studies will be needed to confirm this result and before this observation can influence clinical routine.

Cilengitide treatment of newly diagnosed glioblastoma patients does not alter patterns of progression

The integrin antagonist cilengitide has been explored as an adjunct with anti-angiogenic properties to standard of care temozolomide chemoradiotherapy (TMZ/RT → TMZ) in newly diagnosed glioblastoma. Preclinical data as well as anecdotal clinical observations indicate that anti-angiogenic treatment may result in altered patterns of tumor progression. Using a standardized approach, we analyzed patterns of progression on MRI in 21 patients enrolled onto a phase 2 trial of cilengitide added to TMZ/RT → TMZ in newly diagnosed glioblastoma. Thirty patients from the experimental treatment arm of the EORTC/NCIC pivotal TMZ trial served as a reference. MRIcro software was used to map location and extent of initial preoperative and recurrent tumors on MRI of both groups into the same stereotaxic space which were then analyzed using an automated tool of image analysis. Clinical and outcome data of the cilengitide-treated patients were similar to those of the EORTC/NCIC trial except for a higher proportion of patients with a methylated O(6)-methylguanyl-DNA-methyltransferase gene promoter. Analysis of recurrence pattern revealed neither a difference in the size of the recurrent tumor nor in the distance of the recurrences from the preoperative tumor location between groups. Overall frequencies of distant recurrences were 20 % in the reference group and 19 % (4/21 patients) in the cilengitide group. Compared with TMZ/RT → TMZ alone, the addition of cilengitide does not alter patterns of progression. This analysis does not support concerns that integrin antagonism by cilengitide may induce a more aggressive phenotype at progression, but also provides no evidence for an anti-invasive activity of cilengitide in patients with newly diagnosed glioblastoma.

Methylation of ribosomal RNA by NSUN5 is a conserved mechanism modulating organismal lifespan

Several pathways modulating longevity and stress resistance converge on translation by targeting ribosomal proteins or initiation factors, but whether this involves modifications of ribosomal RNA is unclear. Here, we show that reduced levels of the conserved RNA methyltransferase NSUN5 increase the lifespan and stress resistance in yeast, worms and flies. Rcm1, the yeast homologue of NSUN5, methylates C2278 within a conserved region of 25S rRNA. Loss of Rcm1 alters the structural conformation of the ribosome in close proximity to C2278, as well as translational fidelity, and favours recruitment of a distinct subset of oxidative stress-responsive mRNAs into polysomes. Thus, rather than merely being a static molecular machine executing translation, the ribosome exhibits functional diversity by modification of just a single rRNA nucleotide, resulting in an alteration of organismal physiological behaviour, and linking rRNA-mediated translational regulation to modulation of lifespan, and differential stress response.

miR-125b controls apoptosis and temozolomide resistance by targeting TNFAIP3 and NKIRAS2 in glioblastomas.

Diffusely infiltrating gliomas are among the most prognostically discouraging neoplasia in human. Temozolomide (TMZ) in combination with radiotherapy is currently used for the treatment of glioblastoma (GBM) patients, but less than half of the patients respond to therapy and chemoresistance develops rapidly. Epigenetic silencing of the O(6)-methylguanine-DNA methyltransferase (MGMT) has been associated with longer survival in GBM patients treated with TMZ, but nuclear factor κB (NF-κB)-mediated survival signaling and TP53 mutations contribute significantly to TMZ resistance. Enhanced NF-κB is in part owing to downregulation of negative regulators of NF-κB activity, including Tumor necrosis factor alpha-induced protein 3 (TNFAIP3) and NF-κB inhibitor interacting RAS-like 2 (NKIRAS2). Here we provide a novel mechanism independent of TP53 and MGMT by which oncogenic miR-125b confers TMZ resistance by targeting TNFAIP3 and NKIRAS2. GBM cells overexpressing miR-125b showed increased NF-κB activity and upregulation of anti-apoptotic and cell cycle genes. This was significantly associated with resistance of GBM cells to TNFα- and TNF-related inducing ligand-induced apoptosis as well as resistance to TMZ. Conversely, overexpression of anti-miR-125b resulted in cell cycle arrest, increased apoptosis and increased sensitivity to TMZ, indicating that endogenous miR-125b is sufficient to control these processes. GBM cells overexpressing TNFAIP3 and NKIRAS2 were refractory to miR-125b-induced apoptosis resistance as well as TMZ resistance, indicating that both genes are relevant targets of miR-125b. In GBM tissues, high miR-125b expression was significantly correlated with nuclear NF-κB confirming that miR-125b is implicated in NF-κB signaling. Most remarkably, miR-125b overexpression was clearly associated with shorter overall survival of patients treated with TMZ, suggesting that this microRNA is an important predictor of response to therapy.

The special Sm core structure of the U7 snRNP: far-reaching significance of a small nuclear ribonucleoprotein

The polypeptide composition of the U7 small nuclear ribonucleoprotein (snRNP) involved in histone messenger RNA (mRNA) 3' end formation has recently been elucidated. In contrast to spliceosomal snRNPs, which contain a ring-shaped assembly of seven so-called Sm proteins, in the U7 snRNP the Sm proteins D1 and D2 are replaced by U7-specific Sm-like proteins, Lsm10 and Lsm11. This polypeptide composition and the unusual structure of Lsm11, which plays a role in histone RNA processing, represent new themes in the biology of Sm/Lsm proteins. Moreover this structure has important consequences for snRNP assembly that is mediated by two complexes containing the PRMT5 methyltransferase and the SMN (survival of motor neurons) protein, respectively. Finally, the ability to alter this polypeptide composition by a small mutation in U7 snRNA forms the basis for using modified U7 snRNA derivatives to alter specific pre-mRNA splicing events, thereby opening up a new way for antisense gene therapy.

Characterization of the Drosophila protein arginine methyltransferases DART1 and DART4

The role of arginine methylation in Drosophila melanogaster is unknown. We identified a family of nine PRMTs (protein arginine methyltransferases) by sequence homology with mammalian arginine methyltransferases, which we have named DART1 to DART9 ( Drosophila arginine methyltransferases 1-9). In keeping with the mammalian PRMT nomenclature, DART1, DART4, DART5 and DART7 are the putative homologues of PRMT1, PRMT4, PRMT5 and PRMT7. Other DART family members have a closer resemblance to PRMT1, but do not have identifiable homologues. All nine genes are expressed in Drosophila at various developmental stages. DART1 and DART4 have arginine methyltransferase activity towards substrates, including histones and RNA-binding proteins. Amino acid analysis of the methylated arginine residues confirmed that both DART1 and DART4 catalyse the formation of asymmetrical dimethylated arginine residues and they are type I arginine methyltransferases. The presence of PRMTs in D. melanogaster suggest that flies are a suitable genetic system to study arginine methylation.

Role of cytochrome P450 in DNA damage produced by treatment of colon cells with 1,2-dimethylhydrazine

The study of colon cancer has taken advantage of the development of a model in animals in which tumors in the colon are easily induced by chemical treatment. When 1,2-dimethylhydrazine (DMH) is injected into rats tumor growth is observed in colon in preference to other tissues. This observation led us to investigate the Cytochrome P450 system in colon and its participation in the particular “colon sensitivity” to DMH. It has been established that the Cytochrome P450 system participates in the metabolism of DMH and the methyl carbonium product of Cytochrome P450 activation of DMH is responsible for DNA damage which is considered an initial step to carcinogenesis. The Cytochrome P450 system is a reasonable place to search for an explanation of this organotropic effect of DMH and we feel that the knowledge obtained from this study can take us closer to understanding the development of colonic malignancy. In our study we used a human colon cell line (LS174T) treated with DMH. The Cytochrome P450 system in the cells was manipulated with inducers of different isoforms of Cytochrome P450. The effect of DMH on colon cells was measured by determination of O-6-methylguanine which is a DNA adduct derived from the metabolism of this chemical and is associated with development of tumors. Our results support the hypothesis that Cytochrome P450 plays an important role in the damage to cellular DNA by DMH. This damage is increased after induction of Cytochromes P450 1A1 and 2E1. The effect of inhibition of the methyltransferase and glutathione systems on protection against DMH damage in colon demonstrated the importance of the protective role of the former and the lack of effective protection of the latter system. ^