Biochemistry http://hdl.handle.net/2262/57 2013-05-12T23:45:08Z 2013-05-12T23:45:08Z RAKOVICH, TATSIANA KELLY, VINCENT PATRICK BERNSTEIN, IIANA Boland, Colin http://hdl.handle.net/2262/64120 2012-07-03T15:08:22Z 2011-01-01T00:00:00Z

Title: Queuosine deficiency in eukaryotes compromises tyrosine production through increased tetrahydrobiopterin oxidation. Author: RAKOVICH, TATSIANA; KELLY, VINCENT PATRICK; BERNSTEIN, IIANA; Boland, Colin Abstract: Queuosine is a modified pyrrolopyrimidine nucleoside found in the anticodon loop of transfer RNA acceptors for the amino acids tyrosine, asparagine, aspartic acid, and histidine. Since it is exclusively synthesised by bacteria, higher eukaryotes must salvage queuosine or its nucleobase queuine from food and the gut microflora. Previously, animals made deficient in queuine died within 18 days of withdrawing tyrosine-a non-essential amino acid-from the diet [Marks T, Farkas WR (1997) Biochem Biophys Res Commun 230:233-7]. Here we show that human HepG2 cells deficient in queuine and mice made deficient in queuosine modified transfer RNA, by disruption of the tRNA guanine transglycosylae (TGT) enzyme, are compromised in their ability to produce tyrosine from phenylalanine. This has similarities to the disease phenylketonuria, which arises from mutation in the enzyme phenylalanine hydroxylase or from a decrease in the supply of its cofactor tetrahydrobiopterin (BH4). Immunoblot and kinetic analysis of liver from TGT deficient animals indicate normal expression and activity of phenylalanine hydroxylase. By contrast, BH4 levels are significantly decreased in the plasma and both plasma and urine show a clear elevation in dihydrobiopterin, an oxidation product of BH4, despite normal activity of the salvage enzyme dihydrofolate reductase. Our data suggest that queuosine modification limits BH4 oxidation in vivo and thereby potentially impacts on numerous physiological processes in eukaryotes. Description: IN_PRESS

2011-01-01T00:00:00Z MILLS, KINGSTON http://hdl.handle.net/2262/63990 2012-06-26T09:00:45Z 2011-11-18T00:00:00Z

Title: TLR-dependent T cell activation in autoimmunity. Author: MILLS, KINGSTON Abstract: Autoimmune disease can develop as a result of a breakdown in immunological tolerance, leading to the activation of self-reactive T cells. There is an established link between infection and human autoimmune diseases. Furthermore, experimental autoimmune diseases can be induced by immunization with autoantigens that are administered together with complete Freund’s adjuvant containing killed Mycobacteria tuberculosis, which in some cases can be replaced with individual pathogen-associated molecular patterns (PAMPs). Exogenous PAMPs and endogenous danger signals from necrotic cells bind to pathogen recognition receptors, including Toll-like receptors, and activate signaling pathways in innate immune cells and on T cells, leading to inflammatory cytokine production and T cell activation, and this is now considered to be a major factor in the development of autoimmunity. Description: PUBLISHED

2011-11-18T00:00:00Z HARRIS, JAMES CREAGH, EMMA LAVELLE, EDWARD http://hdl.handle.net/2262/62632 2012-03-12T17:11:29Z 2011-01-01T00:00:00Z

Title: Autophagy controls IL-1ß secretion by targeting pro-IL-1ß for degradation Author: HARRIS, JAMES; CREAGH, EMMA; LAVELLE, EDWARD Abstract: Autophagy is a key regulator of cellular homeostasis that can be activated by pathogen-associated molecules and recently has been shown to influence IL-1β secretion by macrophages. However, the mechanisms behind this are unclear. Here, we describe a novel role for autophagy in regulating the production of IL-1β in antigen-presenting cells. After treatment of macrophages with Toll-like receptor ligands, pro-IL-1β was specifically sequestered into autophagosomes, whereas further activation of autophagy with rapamycin induced the degradation of pro-IL-1β and blocked secretion of the mature cytokine. Inhibition of autophagy promoted the processing and secretion of IL-1β by antigen-presenting cells in an NLRP3- and TRIF-dependent manner. This effect was reduced by inhibition of reactive oxygen species but was independent of NOX2. Induction of autophagy in mice in vivo with rapamycin reduced serum levels of IL-1β in response to challenge with LPS. These data demonstrate that autophagy controls the production of IL-1β through at least two separate mechanisms: by targeting pro-IL-1β for lysosomal degradation and by regulating activation of the NLRP3 inflammasome. Description: PUBLISHED

2011-01-01T00:00:00Z KHAN, AMIR RAFIQ http://hdl.handle.net/2262/62419 2012-02-29T14:54:30Z 2012-01-01T00:00:00Z

Title: Crystal Structure of NAD-dependent Peptoniphilus Asaccharolyticus Glutamate Dehydrogenase Reveals Determinants of Cofactor Specificity Author: KHAN, AMIR RAFIQ Abstract: Glutamate dehydrogenases (EC 1.4.1.2–4) catalyse the oxidative deamination of l-glutamate to α-ketoglutarate using NAD(P) as a cofactor. The bacterial enzymes are hexamers and each polypeptide consists of an N-terminal substrate-binding (Domain I) followed by a C-terminal cofactor-binding segment (Domain II). The reaction takes place at the junction of the two domains, which move as rigid bodies and are presumed to narrow the cleft during catalysis. Distinct signature sequences in the nucleotide-binding domain have been linked to NAD vs. NADP specificity, but they are not unambiguous predictors of cofactor preferences. Here, we have determined the crystal structure of NAD–specific Peptoniphilus asaccharolyticus glutamate dehydrogenase in the apo state. The poor quality of native crystals was resolved by derivatization with selenomethionine, and the structure was solved by single-wavelength anomalous diffraction methods. The structure reveals an open catalytic cleft in the absence of substrate and cofactor. Modeling of NAD+ in domain II suggests that a hydrophobic pocket and polar residues contribute to nucleotide specificity. Mutagenesis and isothermal titration calorimetry studies of a critical glutamate at the P7 position of the core fingerprint confirms its role in NAD binding. Finally, the cofactor binding site is compared with bacterial and mammalian enzymes to understand how the amino acid sequences and three-dimensional structures may distinguish between NAD vs. NADP recognition. Description: PUBLISHED

2012-01-01T00:00:00Z COLL, REBECCA O'NEILL, LUKE ANTHONY JOHN http://hdl.handle.net/2262/61966 2012-01-31T16:33:05Z 2011-01-01T00:00:00Z

Title: The Cytokine Release Inhibitory Drug CRID3 Targets ASC Oligomerisation in the NLRP3 and AIM2 Inflammasomes Author: COLL, REBECCA; O'NEILL, LUKE ANTHONY JOHN Abstract: BACKGROUND: The Inflammasomes are multi-protein complexes that regulate caspase-1 activation and the production of the pro-inflammatory cytokine IL-1β. Previous studies identified a class of diarylsulfonylurea containing compounds called Cytokine Release Inhibitory Drugs (CRIDs) that inhibited the post-translational processing of IL-1β. Further work identified Glutathione S-Transferase Omega 1 (GSTO1) as a possible target of these CRIDs. This study aimed to investigate the mechanism of the inhibitory activity of the CRID CP-456,773 (termed CRID3) in light of recent advances in the area of inflammasome activation, and to clarify the potential role of GSTO1 in the regulation of IL-1β production. METHODOLOGY AND RESULTS: In murine bone marrow derived macrophages, CRID3 inhibited IL-1β secretion and caspase 1 processing in response to stimulation of NLRP3 and AIM2 but not NLRC4. CRID3 also prevented AIM2 dependent pyroptosis in contrast to the NLRP3 inhibitors glyburide and parthenolide, which do not inhibit AIM2 activation. Confocal microscopy and Western blotting assays indicated that CRID3 inhibited the formation of ASC complexes or 'specks' in response to NLRP3 and AIM2 stimulation. Co-immunoprecipitation assays show that GSTO1 interacted with ASC. SIGNIFICANCE: These results identify CRID3 as a novel inhibitor of the NLRP3 and AIM2 inflammasomes and provide an insight into the mechanism of action of this small molecule. In addition GSTO1 may be a component of the inflammasome that is required for ASC complex formation. Description: PUBLISHED

2011-01-01T00:00:00Z HOKAMP, KARSTEN O'FARRELLY, CLIONA http://hdl.handle.net/2262/61963 2012-01-31T15:47:24Z 2011-01-01T00:00:00Z

Title: Genome-wide transcriptional profiling of peripheral blood leukocytes from cattle infected with Mycobacterium bovis reveals suppression of host immune genes Author: HOKAMP, KARSTEN; O'FARRELLY, CLIONA Abstract: Background: Mycobacterium bovis is the causative agent of bovine tuberculosis (BTB), a pathological infection with significant economic impact. Recent studies have highlighted the role of functional genomics to better understand the molecular mechanisms governing the host immune response to M. bovis infection. Furthermore, these studies may enable the identification of novel transcriptional markers of BTB that can augment current diagnostic tests and surveillance programmes. In the present study, we have analysed the transcriptome of peripheral blood leukocytes (PBL) from eight M. bovis-infected and eight control non-infected age-matched and sex-matched Holstein-Friesian cattle using the Affymetrix(R) GeneChip(R) Bovine Genome Array with 24,072 gene probe sets representing more than 23,000 gene transcripts. Results: Control and infected animals had similar mean white blood cell counts. However, the mean number of lymphocytes was significantly increased in the infected group relative to the control group (P = 0.001), while the mean number of monocytes was significantly decreased in the BTB group (P = 0.002). Hierarchical clustering analysis using gene expression data from all 5,388 detectable mRNA transcripts unambiguously partitioned the animals according to their disease status. In total, 2,960 gene transcripts were differentially expressed (DE) between the infected and control animal groups (adjusted P-value threshold [less than or equal to] 0.05); with the number of gene transcripts showing decreased relative expression (1,563) exceeding those displaying increased relative expression (1,397). Systems analysis using the Ingenuity(R) Systems Pathway Analysis (IPA) Knowledge Base revealed an over-representation of DE genes involved in the immune response functional category. More specifically, 64.5% of genes in the affects immune response subcategory displayed decreased relative expression levels in the infected animals compared to the control group. Conclusions: This study demonstrates that genome-wide transcriptional profiling of PBL can distinguish active M. bovis-infected animals from control non-infected animals. Furthermore, the results obtained support previous investigations demonstrating that mycobacterial infection is associated with host transcriptional suppression. These data support the use of transcriptomic technologies to enable the identification of robust, reliable transcriptional markers of active M. bovis infection. Description: PUBLISHED

2011-01-01T00:00:00Z CUNNINGHAM, COLM http://hdl.handle.net/2262/61670 2012-01-13T16:01:21Z 2012-01-01T00:00:00Z

Title: Systemic inflammation induces acute working memory deficits in the primed brain; relevance for delirium Author: CUNNINGHAM, COLM Abstract: Delirium is an acute, severe neuropsychiatric syndrome, characterized by cognitive deficits, that is highly prevalent in aging and dementia and is frequently precipitated by peripheral infections. Delirium is poorly understood and the lack of biologically relevant animal models has limited basic research. Here we hypothesized that synaptic loss and accompanying microglial priming during chronic neurodegeneration in the ME7 mouse model of prion disease predisposes these animals to acute dysfunction in the region of prior pathology upon systemic inflammatory activation. Lipopolysaccharide (LPS; 100 μg/kg) induced acute and transient working memory deficits in ME7 animals on a novel T-maze task, but did not do so in normal animals. LPS-treated ME7 animals showed heightened and prolonged transcription of inflammatory mediators in the central nervous system (CNS), compared with LPS-treated normal animals, despite having equivalent levels of circulating cytokines. The demonstration that prior synaptic loss and microglial priming are predisposing factors for acute cognitive impairments induced by systemic inflammation suggests an important animal model with which to study aspects of delirium during dementia. Description: PUBLISHED

2012-01-01T00:00:00Z DUNNE, AISLING http://hdl.handle.net/2262/61576 2012-01-09T13:22:38Z 2011-01-01T00:00:00Z

Title: Inflammasome activation: from inflammatory disease to infection Author: DUNNE, AISLING Abstract: The recognition of pathogen-derived molecules by the innate immune system is mediated by a number of receptors, including members of the TLR (Toll-like receptor), RLH [RIG (retinoic acid-inducible gene)-like helicase] and the NLR (NOD-like receptor) families. NLRs in particular are also involved in the recognition of host-derived ‘danger’-associated molecules which are produced under conditions of cellular stress or injury. Activation of these receptors leads to the assembly of high-molecular-mass complexes called inflammasomes which in turn leads to the generation of active caspase 1 and to the production of mature IL-1β (interleukin 1β). The discovery that NLRP3 (NLR-related protein 3) can recognize host-derived particulate matter such as uric acid and cholesterol crystals has led to this inflammasome being implicated in a number of inflammatory diseases, including gout, atherosclerosis and Type 2 diabetes. In addition, aberrant NLRP3 activation has also been observed in a number of heritable disorders now referred to as cryopyrinopathies. On the other hand, a number of studies have reported that recognition of both viral and bacterial products by NLRs is required for effective pathogen clearance. The present review discusses both aspects of NLR activation and will highlight the role of additional inflammasome complexes in sensing infection. Description: PUBLISHED

2011-01-01T00:00:00Z DAVEY, GAVIN http://hdl.handle.net/2262/61047 2011-12-01T15:27:42Z 2011-01-01T00:00:00Z

Title: Synergizing metabolic flux analysis and nucleotide sugar metabolism to understand the control of glycosylation of recombinant protein in CHO cells. Author: DAVEY, GAVIN Abstract: BACKGROUND: The glycosylation of recombinant proteins can be altered by a range of parameters including cellular metabolism, metabolic flux and the efficiency of the glycosylation process. We present an experimental set-up that allows determination of these key processes associated with the control of N-linked glycosylation of recombinant proteins. RESULTS: Chinese hamster ovary cells (CHO) were cultivated in shake flasks at 0 mM glutamine and displayed a reduced growth rate, glucose metabolism and a slower decrease in pH, when compared to other glutamine-supplemented cultures. The N-linked glycosylation of recombinant human chorionic gonadotrophin (HCG) was also altered under these conditions; the sialylation, fucosylation and antennarity decreased, while the proportion of neutral structures increased. A continuous culture set-up was subsequently used to understand the control of HCG glycosylation in the presence of varied glutamine concentrations; when glycolytic flux was reduced in the absence of glutamine, the glycosylation changes that were observed in shake flask culture were similarly detected. The intracellular content of UDP-GlcNAc was also reduced, which correlated with a decrease in sialylation and antennarity of the N-linked glycans attached to HCG. CONCLUSIONS: The use of metabolic flux analysis illustrated a case of steady state multiplicity, where use of the same operating conditions at each steady state resulted in altered flux through glycolysis and the TCA cycle. This study clearly demonstrated that the control of glycoprotein microheterogeneity may be examined by use of a continuous culture system, metabolic flux analysis and assay of intracellular nucleotides. This system advances our knowledge of the relationship between metabolic flux and the glycosylation of biotherapeutics in CHO cells and will be of benefit to the bioprocessing industry. Description: PUBLISHED

2011-01-01T00:00:00Z LI, DIANFAN CAFFREY, MARTIN http://hdl.handle.net/2262/61017 2011-11-30T12:38:25Z 2011-01-01T00:00:00Z

Title: Lipid cubic phase as a membrane mimetic for integral membrane protein enzymes Author: LI, DIANFAN; CAFFREY, MARTIN Abstract: The lipidic cubic mesophase has been used to crystallize important membrane proteins for high-resolution structure determination. To date, however, no integral membrane enzymes have yielded to this method, the in meso. For a crystal structure to be meaningful the target protein must be functional. Using the in meso method with a membrane enzyme requires that the protein is active in the mesophase that grows crystals. Because the cubic phase is sticky and viscous and is bicontinuous topologically, quantitatively assessing enzyme activity in meso is a challenge. Here, we describe a procedure for characterizing the catalytic properties of the integral membrane enzyme, diacylglycerol kinase, reconstituted into the bilayer of the lipidic cubic phase. The kinase activity of this elusive crystallographic target was monitored spectrophotometrically using a coupled assay in a high-throughput, 96-well plate format. In meso, the enzyme exhibits classic Michaelis–Menten kinetics and works with a range of lipid substrates. The fact that the enzyme and its lipid substrate and product remain confined to the porous mesophase while its water-soluble substrate and product are free to partition into the aqueous bathing solution suggests a general and convenient approach for characterizing membrane enzymes that function with lipids in a membrane-like environment. The distinctive rheology of the cubic phase means that a procedural step to physically separate substrate from product is not needed. Because of its open, bicontinuous nature, the cubic phase offers the added benefit that the protein is accessible for assay from both sides of the membrane. Description: PUBLISHED

2011-01-01T00:00:00Z