Juvenile hormones (JHs) are key regulators of both metamorphosis and adult reproductive processes. The role of two key enzymes in the biosynthetic pathway of JH were examined: Farnesoic Acid O-Methyltransferase (FAMeT) and Juvenile Hormone Acid Methyltransferase (JHAMT). In crustaceans, FAMeT has been found to methylate farnesoic acid (FA), producing methyl farnesoate (MF) prior to epoxidation at the penultimate stage of JH biosynthesis. JHAMT was discovered more recently in the silkworm Bombyx mori and converts epoxidated FA (JHacids) to active JH through methylation using S-adenosyl-L-methionine (SAM). The aim of the proposed research is to examine the influence of a) decreasing the amount of FAMeT produced using an enhancer trapping P-element and b) increasing the levels of JHAMT and FAMeT in specific tissues using GAL4 overexpression techniques. Immunohistochemical analysis was used to confirm the presence of FAMeT in the CA of D. melanogaster ring glands. Analysis of MF, JHIII and JHB3 release in wild type and mutant stocks in the presence and absence of Drome AST (PISCF-type) suggest that Drosophila FAMeT has little if any effect on the sesquiterpenoid biosynthesis. Drome-AST appears to have a select effect on JHB3 biosynthesis and not MF or JHIII. Analysis of JHB3 release from larval and adult flies ubiquitously overexpressing JHAMT showed a significant increase when compared to wildtype (p<0.01 and p<0.0001 respectively). No significant difference was seen in JHB3 release in flies ubiquitously overexpressing FAMeT. A significant increase in hatching success was seen in flies overexpressing FAMeT in the larval ring gland and oocytes (p<0.05) whereas no significant decrease was seen in JHAMT-overexpressing flies during development. A significant extension of lifespan was also seen when FAMeT was overexpressed in the border and follicle cells of the oocyte (p<0.0001). The direct role of JHAMT in JHB3 synthesis has been demonstrated. The involvement of FAMeT and JHAMT in development and longevity may require other interacting proteins to elicit an effect, which is a limiting factor in overexpression experiments of the two enzymes. Additionally, this is the first example of AST action within D. melanogaster. / Thesis (Master, Biology) -- Queen's University, 2007-09-27 20:08:23.69

MiRNR molekulės yra 21-24 nukleotidų ilgio dvigrandės RNR molekulės. Jos pačios nieko nekoduoja. Viena svarbiausių jų funkcijų yra genų raiškos valdymas potranskripciniame lygmenyje. Jas koduoja daugelis organizmų. miRNR brendimas skiriasi priklausomai nuo ląstelės tipo (gyvūninė ar augalinė). Augalų ląstelėse yra papildomas etapas, kuriu metu dvigrandė miRNR yra metilinama HEN1 baltymo. HEN1 metiltransferazė yra neseniai augaluose identifikuotas baltymas. HEN1 baltymas yra didelis (106,6 kDa), daugiadomeninis baltymas, kurio savybės mažai ištirtos. Jis vienintelis sugeba modifikuoti mažąsias RNR molekules pernešdama metilo grupę nuo kofaktoriaus S-adenozil-L-metionino ant 3&#8242; galinio nt 2&#8242; -OH grupės. HEN1 baltymas yra labai specifiškas savo substratui, kurį atpažįsta ne pagal nt seką, o pagal jo antrinę struktūrą ir ilgį. (Chen, 2005) Pagal homologiją su kitais baltymais HEN1 buvo nustatyti konservatyvūs motyvai. (Č. Venclovas) N-galinėje dalyje nustatyti du RNR prijungiantys (dvRNR-PD ir La- tipo) motyvai. Naudojant sutrumpintus baltymus ir išanalizavus juos elektroforetinio judrumo poslinkio PAA gelyje metodu buvo nustatyta, kad dvRNR-PD atlieka RNR prijungimo funkciją. dvRNR-PD buvo sumodeliuota erdvinė struktūra. Išanalizavus ją buvo nustatytos keturios aminorūgščių liekanos (12K, 22K, 69K ir 70K), kurios gali būti svarbios formuojantis baltymo ir RNR kompleksui. Ištyrus mutantinius baltymus, turinčius šių lizinų pakaitas buvo nustatyta, kad 12-as lizinas... [toliau žr. visą tekstą] / MiRNAs are 21-24 nt length double-stranded RNA molecules which do not encode anything. They play mayor role in post-transcriptional gene regulation level. These molecules are encoded by various organisms. miRNA maturation depends on the type of the cell (animal or plant). There is an additional step in plants when double stranded miRNA is methylated by HEN1 protein. HEN1 methyltransferase is a novel protein identified in plants. HEN1 protein has high molecular weight (106,6 kDa) and it is multistructural. Its features are not yet investigated. This is the only protein which modifies small RNA molecules by transferring methyl group from cofactor S-adenosil-L-methyonin to the 2&#8242; -OH group of the last 3&#8242; nt. HEN1 proteins is very specific to its substrate and recognizes it not by the nt sequence but by the molecule length and structure. After analysis of homologous proteins HEN1 conservative motives were identified. (Č. Venclovas) Two RNA binding motives (dsRNA-BM and La-type) were identified in the N-terminal region. By using shortened proteins and analyzing those by electrophoretic gel mobility in PAA gel shift it was find out that dsRNA-BM is responsible for binding miRNA. Theoretical structure model was made for dsRNA-BM. After analyzing it four amino acid residues (12K, 22K, 69K, and 70K) which can be important for the formation of protein and RNA complex were identified. After examination of mutant proteins with changed lysines it was determined that 12th... [to full text]

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Nicotinamide N-methyltransferase (NNMT) is a new regulator of energy homeostasis. Its expression is increased in models of obesity and diabetes. An enhanced NNMT level is also caused by an adipose tissue-specific knockout of glucose transporter type 4 (GLUT4) in mice, whereas the overexpression of this glucose transporter reduced the NNMT expression. Furthermore, the knockdown of the enzyme prevents mice from diet-induced obesity (DIO) and the recently developed small molecule inhibitors for NNMT reverses the DIO. These previous findings demonstrated the exclusive role of NNMT in adipose tissue and further make it to a promising target in obesity treatment. However, the regulation mechanism of this methyltransferase is not yet clarified. The first part of the thesis focus on the investigation whether pro-inflammatory signals are responsible for the enhanced NNMT expression in obese adipose tissue because a hallmark of this tissue is a low-level chronic inflammation. Indeed, the NNMT mRNA in our study was elevated in obese patients compared with the control group, whereas the GLUT4 mRNA expression does not differ between lean and obese humans. To analyze whether pro inflammatory signals, like interleukin (IL 6) and tumor necrosis factor α (TNF-α), regulate NNMT expression 3T3-L1 adipocytes were treated with these cytokines. However, IL 6, TNF α, and leptin, which is an alternative activator of the JAK/STAT pathway, did not affect the NNMT protein or mRNA level in differentiated 3T3-L1 adipocytes. The mRNA and protein levels were measured by quantitative polymerase chain reaction (qPCR) and western blotting. In the second part of this study, 3T3-L1 adipocytes were cultivated with varying glucose concentrations to show whether NNMT expression depends on glucose availability. Further studies with activators and inhibitors of AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin (mTOR) signaling pathways were used to elucidate the regulation mechanism of the enzyme. The glucose deprivation of differentiated 3T3-L1 adipocytes led to a 2-fold increase in NNMT expression. This effect was confirmed by the inhibition of the glucose transports with phloretin as well as the inhibition of glycolysis with 2-deoxyglucose (2-DG). AMPK serves as an intracellular energy sensor and the pharmacological activation of it enhanced the NNMT expression. This increase was also caused by the inhibition of mTOR. Conversely, the activation of mTOR using MHY1485 prevented the effect of glucose deprivation on NNMT. Furthermore, the NNMT up-regulation was also blocked by the different autophagy inhibitors. Taken together, NNMT plays a critical role in autophagy in adipocytes, because an inhibition of this process prevented the augmented NNMT expression during glucose starvation. Moreover, the effect on NNMT protein and mRNA level depends on AMPK and mTOR. However, pro-inflammatory signals did not affect the expression. Further in vivo studies have to clarify whether AMPK activation and mTOR inhibition as well as autophagy are responsible for the increased NNMT levels in obese adipose tissue. In future this methyltransferase emerges as an awesome therapeutic target for obesity. / NNMT ist ein neuer Regler der Energiehomöostase. Seine Expression ist in Adipositas- und Diabetesmodellorgansimen erhöht. Ein verstärktes NNMT Level wird auch durch einen fettgewebs-spezifischen GLUT4 Knockout in Mäusen hervorgerufen, wobei die Überexpression des Glukosetransporters die NNMT Expression reduziert. Des Weiteren schützt der Knockdown von NNMT die Mäuse vor Diät-induzierter Adipositas und die kürzlich entwickelten kleinen Molekülinhibitoren gegen NNMT kehren eine durch die Ernährung bedingte Adipositas wieder um. Neuere Erkenntnisse zeigen die exklusive Rolle von NNMT im Fettgewebe auf und machen das Enzym so zu einem vielversprechenden Target für die Adipositastherapie. Jedoch ist der Regulationsmechanismus dieser Methyltransferase noch nicht geklärt. Der erste Teil der Arbeit befasst sich mit der Untersuchung, ob pro-inflammatorische Signale verantwortlich sind für die erhöhten NNMT Expression im adipösen Fettgewebe, da sich dieses Gewebe durch eine chronische Inflammation auszeichnet. Tatsächlich war die mRNA in unserer Studie verstärkt exprimiert in adipösen Patienten im Vergleich zur Kontrollgruppe, wobei die GLUT4 mRNA Expression zwischen Schlanken und Adipösen nicht verändert war. Um zu untersuchen, ob pro-inflammatorische Signale, wie IL 6 und TNF α, die NNMT Expression regulieren, wurden 3T3-L1 Adipozyten mit diesen Zytokinen behandelt. Jedoch beeinflussten IL 6, TNF α und Leptin, welches ein weiterer Aktivator des JAK/STAT Signalweges ist, NNMT Protein oder mRNA Level in differenzierten 3T3 L1 Adipozyten nicht. Die mRNA und Protein Level wurden mittels qPCR und Western Blot analysiert. Im zweiten Teil dieser Studie wurden 3T3 L1 Adipozyten mit unterschiedlichen Glukosekonzentrationen kultiviert, um zu zeigen, ob die NNMT Expression von der Glukoseverfügbarkeit abhängig ist. Für die Untersuchung des genauen Regulationsmechanismus von NNMT, wurden weitere Studien mit Aktivatoren und Inhibitoren der AMPK und mTOR Signalwege durchgeführt. Der Glukosemangel führte zu einem 2-fachen Anstieg der NNMT Expression in differenzierten 3T3-L1 Adipozyten. Dieser Effekt wurde bestätigt durch die Inhibierung der Glukosetransporter mit Phloretin sowie durch die Inhibierung der Glykolyse mit 2-DG. AMPK ist ein intrazellulärer Energiesensor und dessen pharmakologische Aktvierung erhöhte die NNMT Expression. Dieser Anstieg wurde auch verursacht durch die Inhibierung von mTOR. Hingegen verhinderte die Aktivierung von mTOR mithilfe von MHY1485 den Effekt auf NNMT während des Glukoseentzugs. Des Weiteren wurde die Auswirkungen auf NNMT durch Autophagieinhibitoren unterbunden. Zusammenfassend spielt NNMT eine kritische Rolle für die Autophagie in Adipozyten, da eine Inhibierung des Prozesses die erhöhte NNMT Expression während eines Glukoseentzugs verhinderte. Darüber hinaus ist der Effekt auf die NNMT Protein und mRNA Level abhängig von AMPK and mTOR. Jedoch beeinflussten pro-inflammatorische Signale die Expression nicht. Weitere in vivo Studien müssen klären, ob eine AMPK Aktivierung und eine mTOR Inhibierung sowie die Autophagie in Adipozyten verantwortlich sind für die verstärkte NNMT Expression im adipösen Fettgewebe. Zukünftig wird sich NNMT als ein beeindruckendes Target für die Adipositastherapie herausstellen.

No description available.

Ribosomes form the core of the protein biosynthesis machinery and are essential to life. Ribosome biogenesis is a complex cellular process involving transcription of rRNA, pre-rRNA processing, rRNA modification and simultaneous assembly of ribosomal proteins. RNA nucleotide modification is observed in all domains of life. While there is enormous conservation of ribosome structure, very few post-transcriptional rRNA modifications have been conserved throughout evolution. A notable example of such rare conservation is the dimethylation of two adjacent adenosines in the 3’-terminal helix, a highly conserved region of the small subunit rRNA. Enzymes that carry out these dimethylations are equally conserved and are collectively known as the KsgA/Dim1 family of methyltransferases. The first member of the family, KsgA, was identified in E. coli as the determinant for resistance to the aminoglycoside antibiotic Kasugamycin. Orthologs have since been described in organisms of wide spread evolutionary origins as well as in eukaryotic cellular organelles, thus underscoring the unprecedented conservation of this family of enzymes and the resultant rRNA modification. The higher evolutionary orthologs of KsgA have adopted secondary roles in ribosome biogenesis in addition to their dimethyltransferase role. The eukaryotic ortholog, Dim1, is essential for proper processing of the primary rRNA transcript. Recently, KsgA has been speculated to function as a late stage ribosome biogenesis factor and a ΔksgA genotype in E. coli has been linked to cold sensitivity and altered ribosomal profiles. This report focuses on the biochemical characterization of KsgA and its interaction with the 30S subunit. We have established the salt conditions required for optimal KsgA methyltransferase activity while confirming that KsgA recognizes a translationally inactive conformation of 30S subunit in vitro. Our study of the functional conservation of KsgA/Dim1 enzymes in the bacterial system revealed that KsgA and the evolutionarily higher orthologs could recognize a common ribosomal substrate. This indicates that the recognition elements of both, the protein and the small subunit, have remained largely unchanged during the course of evolution. Finally, based on our site directed mutagenesis and biochemical studies, we report that KsgA binds to structural components of 16S rRNA other than the helix containing the target nucleosides.

Thesis advisor: Steven D. Bruner / The enediyne antitumor antibiotics are produced by complex biosynthetic machinery in acetomycetes. This dissertation will focus on the study of three enzymes involved in key steps in the biosynthesis of two enediynes, neocarzinostatin and C-1027. Neocarzinostatin is biosynthesized by a number of enzymes that synthesize and decorate the enediyne core and the peripheral moieties. NcsB1 is one enzyme involved in functionalizing the naphthoic acid portion of neocarzinostatin, a key group involved in binding to target DNA duplexes. The enzyme has been shown to be a promiscuous (<italic>S</italic>)-adenosylmethionine-dependent <italic>O</italic>-methyltransferase responsible for methylating a variety of hydroxynaphthoic acids. Multiple crystal structures of NcsB1 cocomplexed to substrate and/or cofactor have been solved. These structures revealed a displacement of the C-terminal domain when not bound to substrate, a movement that likely opens up the active site for naphthoate binding. Additionally, the ternary complex structure of 1,4-dihydroxynaphthoic acid, (<italic>S</italic>)-adenosylhomocysteine, and NcsB1 was solved and showed a rotation of this alternate substrate in the binding pocket, allowing for methylation. These results led us to probe NcsB1 activity using active site mutants, demonstrating altered substrate specificity and revealing key residues in substrate binding. The final step of neocarzinostatin biosynthesis involves multiple enzymes that convergently assemble the multiple biosynthetic intermediates to form the chromophore. NcsB2, originally proposed to catalyze the attachment of the naphthoic acid moiety to the enediyne core, has been characterized <italic>in vitro</italic>. Studies into its substrate specificity as an adenylation domain led to a revised biosynthetic pathway of 2-hydroxy-7-methoxy-5-methyl naphthoic acid. Instead of catalyzing the attachment of an enzyme bound naphthoic acid to the enediyne core, NcsB2 was found to act as a CoA-ligase, activating a variety of naphthoic acids and forming a naphthoyl-CoA intermediate. The results of these studies present an outstanding opportunity to produce novel analogs of neocarzinostatin by manipulating its biosynthesis. C-1027 is an architecturally similar enediyne that is also biosynthesized in a convergent route. C-1027 is a member of a class of enediynes that contains a functionalized &beta;-tyrosine derived from L-tyrosine. The first catalytic step towards this beta-tyrosine moiety is achieved by <italic>Sg</italic>TAM, a tyrosine aminomutase that catalyzes a 2,3-amino shift on L-tyrosine to form (<italic>S</italic>)-&beta;-tyrosine. The first X-ray crystal structure of <italic>Sg</italic>TAM was recently solved by our group, revealing structural homology to ammonia lyases. Through site-directed mutagenesis, X-ray crystallography, and biochemical analysis, residues that influence the mechanism by which <italic>Sg</italic>TAM catalyzes this difficult transformation were explored. From these studies, the enzymatic base and other pertinent residues involved in catalysis have been identified. In addition, residues that close the tunnel leading to the active site, thought to play a key role in mutase activity, were probed. Further study of rational mutants of <italic>Sg</italic>TAM will allow us to engineer its activity to alter its substrate specificity and the type of product it produces. / Thesis (PhD) — Boston College, 2009. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Ime4 is the catalytic subunit of a conserved methyltransferase (MTase) complex found in yeast, S. cerevisiae. This complex is responsible for creating the RNA modification N6- methyladenosine (m6A), the most common post-transcriptional modification in higher eukaryotes. There is evidence to suggest that m6A is an important mediator of gene expression control within the cell and has been associated with a diverse array of phenotypic effects, notably as a conserved determinant of cell fate. The MTase complex is known to be a nuclear protein, the compartment where it is believed to carry out most of its methylation activity. Recently, the nuclear localization signals (NLS) of the subunits of the human MTase complex were experimentally identified, whereas the NLSs of the yeast MTase complex remain unknown. Here, we have experimentally identified the amino acid sequence 517RKYQEFMKSKTGTSHTGTKKIDKK540, located within the C-terminal region, as a putative bipartite NLS for Ime4.

Post-translational histone modifications (PTMs) such as methylation of lysine residues influence chromatin structure and function. PTMs are involved in different cellular processes such as DNA replication, transcription and cell differentiation. Deregulations of PTM patterns are responsible for a variety of human diseases including acute leukemia. DOT1 enzymes are highly conserved histone methyltransferases that are responsible for methylation of lysine 79 on histone H3 (H3K79). Most eukaryotes contain one single DOT1 enzyme, whereas African trypanosomes have two homologues, DOT1A and DOT1B, which methylate H3K76 (H3K76 is homologous to H3K79 in other organisms). DOT1A is essential and mediates mono- and di-methylations, whereas DOT1B additionally catalyzes tri-methylation of H3K76. However, a mechanistic understanding how these different enzymatic activities are achieved is lacking. This thesis exploits the fact that trypanosomes possess two DOT1 enzymes with different catalytic properties to understand the molecular basis for the differential product-specificity of DOT1 enzymes. A trypanosomal nucleosome reconstitution system was established to analyze methyltransferase activity under defined in vitro conditions. Homology modeling allowed the identification of critical residues within and outside the catalytic center that modulate product-specificity. Exchange of these residues transferred the product-specificity from one enzyme to the other and revealed regulatory domains adjacent to the catalytic center. This work provides the first evidence that few specific residues in DOT1 enzymes are crucial to catalyze methyl-state-specific reactions. These results have also consequences for the functional understanding of homologous enzymes in other eukaryotes. / Posttranslationale Histonmodifizierungen (PTMs), wie beispielsweise die Methylierung von Lysinseitenketten, beeinflussen maßgeblich die Struktur und Funktion von Chromatin. PTMs spielen eine wichtige Rolle in verschiedensten zellulären Prozessen, darunter DNA Replikation, Transkription oder Zelldifferenzierung. Darüber hinaus liegt ein verändertes PTM-Muster einer Vielzahl humaner Erkrankungen zugrunde, wie z.B. der akuten myeloischen Leukämie. DOT1-Enzyme sind hochkonservierte Histonmethyltransferasen, die für die Methylierung von Lysin 79 in Histon H3 (H3K79) verantwortlich sind. Im Gegensatz zu den meisten Eukaryoten, die lediglich ein einziges DOT1-Enzym besitzen, finden sich zwei homologe Proteine in afrikanischen Trypanosomen (DOT1A und DOT1B), die Lysin 76 in Histon H3 (H3K76) methylieren (H3K76 ist homolog zu H3K79 in anderen Organismen). DOT1A ist essentiell und katalysiert Mono- und Di-Methylierungen, wohin gegen DOT1B darüber hinaus eine Trimethylierung an H3K76 setzen kann. Derzeit fehlt jegliches mechanistische Verständnis darüber, wie beide Enzyme diese unterschiedliche Produktspezifität erreichen. Die vorliegende Dissertation macht sich den Umstand zunutze, dass Trypanosomen zwei DOT1-Methyltransferasen mit unterschiedlichen katalytischen Eigenschaften besitzen, um Einblicke in die molekulare Grundlage der unterschiedlichen Produktspezifität zu erlangen. Zunächst wurde ein Rekonstitutionssystem für Nukleosomen aus Trypanosomen etabliert, das es ermöglichte die Methyltransferase-Aktivitäten unter definierten in vitro Bedingungen zu analysieren. Homologiemodelle erlaubten die Identifikation von wichtigen Aminosäurepositionen innerhalb und außerhalb des katalytischen Zentrums der Enzyme, die einen Einfluss auf die Produktspezifität haben. Ein Austausch der Aminosäuren an diesen Positionen führte zu einer Umwandlung der Produktspezifität und offenbarte gleichzeitig DOT1A- und DOT1B-spezifische regulatorische Domänen, die an das katalytische Zentrum angrenzen. Diese Arbeit liefert erste Hinweise, dass wenige maßgebliche Aminosäuren in DOT1-Enzymen für den H3K76-Methylierungsgrad während der Katalyse entscheidend sind. Darüber hinaus haben die hier dargestellten Ergebnisse ebenfalls Konsequenzen für das funktionale Verständnis der homologen Enzyme in anderen Eukaryoten.

Thesis supervisor: Steven Clegg. Includes bibliographic references (p. 123-131).