Structure - function studies on FIC - mediated AMPylation and deAMPylation by class I Fic proteins

Proteins containing a conserved FIC (filamentation induced by cyclic AMP) domain can be found in all domains of life, where they modify the function of target proteins via post-translational modifications such as AMPylation describing the transfer of AMP onto the threonine, tyrosine, or serine side chain of their respective targets. First studies on AMPylation activity by two bacterial proteins VopS from Vibrio parahaemolyticus and IbpA from Histophilus somni revealed inhibition of RhoGTPases causing disruption of the actin cytoskeleton leading to cell death. Since then, the AMPylation activity of several Fic proteins containing a highly conserved FIC domain has been described. Fic proteins are controlled by the presence of an inhibition motif and depending on its location the enzymes can be separated into three classes. Proteins such as human FICD or NmFic from Neisseria meningitidis have this inhibition motif either on the N-terminus or the C-terminus of the toxin itself and represent class II and class III Fic proteins, respectively. FICD mediates AMPylation of the Hsp70 chaperone BiP in the endoplasmatic reticulum (ER), which leads to BiP’s inactivation when the level of unfolded proteins is low. Recently it was shown that FICD deAMPylates BiP and removes the modification, which recruits the target back into chaperone cycle when the load of unfolded proteins is high. Studies on the class III Fic protein EfFic revealed deAMPylation activity of the protein and that Fic proteins can act as bifunctional enzymes. A conserved glutamate in the inhibition motif of both proteins plays a significant role in regulation of the reactions. Class I Fic proteins such as VbhT from Bartonella schoenbuchensis are inhibited by a small protein antitoxin consisting of the inhibition motif. Another example for class I Fic proteins are the effector proteins of the alpha-proteobacterial genus Bartonella, which have a growing number of species and are studied as model for evolution of bacterial pathogenesis. Bartonella effector proteins (Beps) contain a diverse ensemble of FIC domains, which have evolved in parallel in three Bartonella lineages from a single ancestral toxin-antitoxin module. In research article I we use X-ray crystallography, structural modelling, and phylogenetic analysis to gain more insight into the variety of Beps, which includes nine crystal structures and 99 non-redundant sequences. Minor structural changes of the core FIC domain indicate functional and regulatory variability of Beps. In research article II we show that a recent developed nucleotide quantification assay is a sensitive method to obtain real-time enzymatic progress curves. This assay was chosen to characterize the AMPylation and deAMPylation reaction mediated by the FIC domain of the class I toxin VbhT (VbhT(FIC)) from Bartonella schoenbuchensis, which is regulated by its cognate antitoxin VbhA. Autoradiography assays previously revealed that VbhT(FIC) AMPylates the DNA gyrase subunit B (GyrB) leading to its inactivation and abolishing cell growth, which is inhibited when the antitoxin was present. We show that the VbhT/VbhA toxin-antitoxin complex acts as a bifunctional enzyme causing AMPylation and deAMPylation of a 43 kilodalton (kDa) subunit of GyrB (GyrB43). Mutation of the glutamate in the inhibition motif, which is known to have strong modifying effects, reveals enhanced AMPylation and deAMPylation activity, indicating a different role of the glutamate in the two reactions compared to class II FICD and class III EfFic

Effect of Ni2+ <[Ni hoch 2+]> on Et-1 mediated prostaglandin synthesis in osteoblastic cells

Endothelin-1 ist ein 21 Aminosäure Peptid welches neben seiner Funktion als Vasokonstriktor die Synthese von Prostaglandinen induziert. ET-1 induziert die Synthese von Prostaglandinen über einen G-protein gekoppelten Rezeptor-vermittelte Inositol-1, 4, 5 Triphosphat Signalweg welcher zur Bildung des Prostaglandinvorläufers Arachidonsäure führt. Wie ET-1 induziert auch Bradykinin die Synthese von Prostaglandinen über denselben Signaltransduktionsweg. Interessanterweise wurde gezeigt das Nickel, bekannt als Inhibitor von Kalziumkanälen, in Osteoblasten die bradykinin-vermittelte Prostaglandinsynthese potenziert.Es wurde gezeigt, dass Nickel ebenso die ET-1 induzierte Prostaglandinsynthese potenziert, nämlich in einer dosis-abhängigen Art und Weise. Weiters wurde gezeigt, dass das Enzym COX-1, verantwortlich für die Synthese von Vorläuferprostaglandinen, durch Nickel gehemmt wird. Für weitere Untersuchungen des "Nickel-Effekt" wurden osteoblastische Zellen sowohl mit Cholera Toxin(CTX) als auch mit Pertussis Toxin(PTX) behandelt um verschiedene G-Proteine zu blocken. Interessanterweise wurde im Falle von PTX und CTX der "Nickel-Effekts" um 40-50% reduziert. In Experimenten, in denen verschiedenste Inhibitoren der Signaltransduktionskaskade eingesetzt wurden, wurde gezeigt dass sowohl das Enzym Phospholipase D also auch die Phosphatidinositol-3 ? kinase am potenzierenden Effekt von Nickel beteiligt sein könnten. Zusätzlich wurde versucht mit Hilfe einer immobilisierenden Metalaffinitätschromatographie, SDS ?Page und Westernblot nickel-bindende Proteine zu identifizieren, welchen möglicherweise eine bedeutende Rolle zugeordnet werden kann. Insgesamt deuten die erzielten Ergebnisse darauf, dass sowohl die Phospholipase D also auch die Phosphatidinositol-3 ? kinase in den "Nickel-Effekt" involviert sein könnten. Ebenso kann eine Beteiligung diverser G-Proteine zu diesem Zeitpunkt nicht ausgeschlossen werden.Endothelin-1 is a 21 amino acid peptide that belongs to the superfamily of endothelins. Beside its vasoconstrictive function, ET-1 is known to induce prostaglandin synthesis in various cell types. ET-1 induces prostaglandin synthesis via a GPCR mediated inositol-1, 4, 5 triphosphate pathway which results in the generation of arachidonic acid, a precursor of prostaglandins. It is known that bradykinin induces prostaglandin synthesis via the same signal transduction pathway. It has been shown that nickel, which is known as a potential inhibitor of calcium channels, potentiates the bradykinin - mediated prostaglandin synthesis in an osteoblastic cell line. In this thesis the potentiating effect of nickel on ET-1 mediated prostaglandin synthesis is shown, as well as the further investigation of the so called ?nickel effect?. ET-1 mediated prostaglandin synthesis was potentiated by the addition of nickel in a dose-dependent manner. In contrast, the activity of COX-1 which is responsible for the generation of different prostaglandin precursors was found to be inhibited by nickel concentrations up to 1 mM. Cholera toxin and pertussis toxin are known to inhibit different G-protein subunits. The pre-treatment of cells with cholera toxin and pertussis toxin resulted in a reduction of nickel-potentiated ET-1 mediated prostaglandin synthesis by 40 ? 50 %. Experiments, using different inhibitors of signal transduction cascade parameters showed a possible involvement of phosholipase D as well as an involvement of phosphatidylinositol-3 kinases. In addition, an immobilized metal affinity chromatographic system, as well as western blot analysis was used to identify nickel-binding proteins, which may play a major role concerning the ?nickel effect?. The obtained data indicate an involvement of phospholipase D and phosphatidylinositol-3 kinases concerning the ?nickel effect?, and also a participation of different types of G-proteins can not be excluded at that time.Stefanie Nadja TameggerAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. SpracheGraz, Univ., Masterarb., 2010(VLID)21309

Evolutionary Diversification of Host-Targeted; Bartonella; Effectors Proteins Derived from a Conserved FicTA Toxin-Antitoxin Module

Proteins containing a FIC domain catalyze AMPylation and other post-translational modifications (PTMs). In bacteria, they are typically part of FicTA toxin-antitoxin modules that control conserved biochemical processes such as topoisomerase activity, but they have also repeatedly diversified into host-targeted virulence factors. Among these,; Bartonella; effector proteins (Beps) comprise a particularly diverse ensemble of FIC domains that subvert various host cellular functions. However, no comprehensive comparative analysis has been performed to infer molecular mechanisms underlying the biochemical and functional diversification of FIC domains in the vast Bep family. Here, we used X-ray crystallography, structural modelling, and phylogenetic analyses to unravel the expansion and diversification of Bep repertoires that evolved in parallel in three; Bartonella; lineages from a single ancestral FicTA toxin-antitoxin module. Our analysis is based on 99 non-redundant Bep sequences and nine crystal structures. Inferred from the conservation of the FIC signature motif that comprises the catalytic histidine and residues involved in substrate binding, about half of them represent AMP transferases. A quarter of Beps show a glutamate in a strategic position in the putative substrate binding pocket that would interfere with triphosphate-nucleotide binding but may allow binding of an AMPylated target for deAMPylation or another substrate to catalyze a distinct PTM. The β-hairpin flap that registers the modifiable target segment to the active site exhibits remarkable structural variability. The corresponding sequences form few well-defined groups that may recognize distinct target proteins. The binding of Beps to promiscuous FicA antitoxins is well conserved, indicating a role of the antitoxin to inhibit enzymatic activity or to serve as a chaperone for the FIC domain before translocation of the Bep into host cells. Taken together, our analysis indicates a remarkable functional plasticity of Beps that is mostly brought about by structural changes in the substrate pocket and the target dock. These findings may guide future structure-function analyses of the highly versatile FIC domains

Esterase 22 and beta-glucuronidase hydrolyze retinoids in mouse liver

Excess dietary vitamin A is esterified with fatty acids and stored in the form of retinyl ester (RE) predominantly in the liver. According to the requirements of the body, liver RE stores are hydrolyzed and retinol is delivered to peripheral tissues. The controlled mobilization of retinol ensures a constant supply of the body with the vitamin. Currently, the enzymes catalyzing liver RE hydrolysis are unknown. In this study, we identified mouse esterase 22 (Es22) as potent RE hydrolase highly expressed in the liver, particularly in hepatocytes. The enzyme is located exclusively at the endoplasmic reticulum (ER), implying that it is not involved in the mobilization of RE present in cytosolic lipid droplets. Nevertheless, cell culture experiments revealed that overexpression of Es22 attenuated the formation of cellular RE stores, presumably by counteracting retinol esterification at the ER. Es22 was previously shown to form a complex with β-glucuronidase (Gus). Our studies revealed that Gus colocalizes with Es22 at the ER but does not affect its RE hydrolase activity. Interestingly, however, Gus was capable of hydrolyzing the naturally occurring vitamin A metabolite retinoyl β-glucuronide. In conclusion, our observations implicate that both Es22 and Gus play a role in liver retinoid metabolism