Komplexbildung von Protoporphyrinogen IX Oxidase und Ferrochelatase aus dem Cyanobakterium Thermosynechococcus elongatus

Coproporphyrinogen oxidase (CPO), protoporphyrinogen oxidase (PPO) and ferrochelatase (FC) catalyze the three terminal steps of the heme biosynthetic pathway to form protoheme IX. For the protection of the highly toxic intermediate protoporphyrin IX, a protein complex between CPO, PPO and FC was proposed based on the solved crystal structure of tobacco PPO (Koch et al., 2004). It was objective in this thesis to proof the existance and determine the localization of the proposed complex in T. elongatus cells. Co-immunoprecipitation experiments and double-immunogold labelling in combination with electron microscopy were employed. First recombinant T. elongatus HemF (CPO), HemY (PPO) and HemH (FC) were produced and chromatographically purified subsequently, polyclonal rabbit antibodies were raised against the three enzymes. Complex formation of PPO with FC was demonstrated for T. elongatus cell free extracts via co-immunoprecipitation for the first time. These in vitro findings were further confirmed using electron microscopy and double-immunogold labelled PPO and FC. PPO - FC complexes were detected in T. elongatus thylakoid membranes while CPO was not involved in complex formation. Obtained data lay the basis for the in-depth analysis of the cellular arrangement of tetrapyrrole biosynthetic enzymes. The fidelity of protein biosynthesis is dependent on the reliable charging of each tRNA with its cognate amino acid. However, many bacteria lack a glutaminyl-tRNA synthetase. In these organisms tRNAGln is initially mischarged with glutamate by a non-discriminating glutamyl-tRNA synthetase (GluRS). The resulting misacylated Glu-tRNAGln is then converted to Gln-tRNAGln by a tRNA-dependent amidotransferase through the amide addition to the tRNA-bound glutamate. This thesis provided experimental proof that Thermosynechococcus elongatus GluRS is indeed a non-discriminating tRNA synthetase. For this purpose the kinetic parameters for the synthesis of Glu-tRNAGlu and Glu-tRNAGln were determined. Therefore T. elongatus tRNAGlu and tRNAGln were purified. It was shown that T. elongatus GluRS accommodates and charges both tRNAGlu as well as tRNAGln. Obtained results were discussed in the light of solved T. elongatus GluRS crystal structure.Die Biosynthese der Tetrapyrrole ist komplex und umfasst sieben Enzym katalysierte Reaktionen, von denen die biochemische Untersuchung der drei letzten Schritte Hauptgegenstand dieser Dissertation bildeten. Als Modellorganismus wurde das Chlorophyll produzierende Cyanobakterium Thermosynechococcus elongatus genutzt, um die drei terminalen Proteine Coproporphyrinogen IX Oxidase (CPO), Protoporphyrinogen IX Oxidase (PPO) und Ferrochelatase (FC) auf potentielle komplexbildende Enzyme zu untersuchen. In dieser Dissertation wurden alle drei Proteine aus T. elongatus rekombinant in E. coli produziert, gereinigt und auf ihre Funktionalität überprüft. Des Weiteren wurden gegen alle drei gereinigten Proteine Antikörper generiert und gereinigt, die in Immunopräzipitations-, sowie Co-Immunoprecipitationsanalysen zum Nachweis einer Komplexbildung genutzt wurden. Die Ergebnisse ergaben, dass der Komplex aus nur zwei der ehemals postulierten drei Enzyme, nämlich PPO und FC bestand und die CPO nicht an der Komplexbildung beteiligt war. Dazu wurden mikroskopische Feinschnitte von frisch kultivierten T. elongatus-Zellen angefertigt, mit einfach Gold-markierten Antikörpern -gerichtet gegen die einzelnen Proteine- inkubiert und die Lokalisierung der Proteine in der Zelle über elektronenmikroskopische Aufnahmen bestimmt. Komplexe der Proteine wurden mit unterschiedlichen Gold-markierten Antikörpern und Elektronenmikroskopie lokalisiert. T. elongatus besitzt kein Gen für die Glutaminyl-tRNA Synthetase, jedoch nutzt der Organismus auch Glutamin in seinen Proteinen. Für T. elongatus wurde postuliert, dass die Glutamyl-tRNA Synthetase eine ,,Fehlbeladung’’ mit dem Ergebnis einer Glu-tRNAGln durchführt und in einer nachfolgenden Transamidierung zu Gln-tRNAGln korrigiert. In einem anderen Projekt wurden die individuellen tRNAGlu und tRNAGln aus T. elongatus Zellextrakten isoliert und die kinetischen Parameter für die jeweilige Glutamylation der tRNAGlu sowie Fehl-Glutamylation der tRNAGln bestimmt. Die Ergebnisse erlaubten nun eine eindeutige Interpretation der zuvor in der gelösten Kristallstruktur der GluRS aus T. elongatus

Complex formation between protoporphyrinogen IX oxidase and ferrochelatase during haem biosynthesis in Thermosynechococcus elongatus.

International audienceDuring haem and chlorophyll biosynthesis, flavin-dependent protoporphyrinogen IX oxidase catalyses the six-electron oxidation of protoporphyrinogen IX to form protoporphyrin IX. In the following step, iron is inserted into protoporphyrin IX by ferrochelatase. Based on the solved crystal structures of these enzymes, an in silico model for a complex between these two enzymes was proposed to protect the highly photoreactive intermediate protoporphyrin IX. The existence of this complex was verified by two independent techniques. First, co-immunoprecipitation experiments using antibodies directed against recombinantly produced and purified Thermosynechococcus elongatus protoporphyrinogen IX oxidase and ferrochelatase demonstrated their physical interaction. Secondly, protein complex formation was visualized by in vivo immunogold labelling and electron microscopy with T. elongatus cells. Finally, oxygen-dependent coproporphyrinogen III oxidase, which catalyses the formation of protoporphyrinogen IX, was not found to be part of this complex when analysed with the same methodology

Evaluation of Risk Factors for Hepatic Complications after Allogeneic Hematopoietic Stem Cell Transplantation

Background:  Hepatic dysfunction in patients who have undergone allogeneic haematopoietic stem cell transplantation (HSCT) is a major cause of morbidity and mortality. The aim of this study is to evaluate the incidence of post-transplantation hepatic complications in these patients. Methods: A total of 121 patients (age above 15 with no abnormality in their hepatic tests) participated in the study. The influence of a variety of risk factors on the incidence, type, and pattern of hepatic dysfunction as well as the length of hospital stay related to these complications were studied. Results:  As a whole, 76 patients (62%)—44 males and 32 females—were diagnosed with hepatic dysfunction after transplantation. As many as 31(25%) of the patients showed increased measures in their hepatic enzyme, while 45(37%) of them ended up with both abnormal enzyme measure and clinical symptoms including diarrhoea, skin rash, jaundice, and anorexia. The hepatic dysfunction rates owing to drug toxicity and GVHD (21.5% and 16.5%, respectively) proved to be the highest in our study. Analysing risk factors, the immunosuppressive regimen could affect the type of hepatic dysfunction—i.e., less patients with GVHD were found in the group who received ATG in their regimen (p-value =0.034). Conclusion: According to these findings, the immunosuppressive regimen can play a role in preventing the incidence of GVHD. Less occurrence of hepatic complications, especially GVHD, may lead to less clinical symptoms and time of hospital stay

Crystal structure of a non-discriminating glutamyl-tRNA synthetase.

Error-free protein biosynthesis is dependent on the reliable charging of each tRNA with its cognate amino acid. Many bacteria, however, lack a glutaminyl-tRNA synthetase. In these organisms, tRNA(Gln) is initially mischarged with glutamate by a non-discriminating glutamyl-tRNA synthetase (ND-GluRS). This enzyme thus charges both tRNA(Glu) and tRNA(Gln) with glutamate. Discriminating GluRS (D-GluRS), found in some bacteria and all eukaryotes, exclusively generates Glu-tRNA(Glu). Here we present the first crystal structure of a non-discriminating GluRS from Thermosynechococcus elongatus (ND-GluRS(Tel)) in complex with glutamate at a resolution of 2.45 A. Structurally, the enzyme shares the overall architecture of the discriminating GluRS from Thermus thermophilus (D-GluRS(Tth)). We confirm experimentally that GluRS(Tel) is non-discriminating and present kinetic parameters for synthesis of Glu-tRNA(Glu) and of Glu-tRNA(Gln). Anticodons of tRNA(Glu) (34C/UUC36) and tRNA(Gln) (34C/UUG36) differ only in base 36. The pyrimidine base of C36 is specifically recognized in D-GluRS(Tth) by the residue Arg358. In ND-GluRS(Tel) this arginine residue is replaced by glycine (Gly366) presumably allowing both cytosine and the bulkier purine base G36 of tRNA(Gln) to be tolerated. Most other ND-GluRS share this structural feature, leading to relaxed substrate specificity

Alzheimer disease macrophages shuttle amyloid-beta from neurons to vessels, contributing to amyloid angiopathy

Neuronal accumulation of oligomeric amyloid-β (Αβ) is considered the proximal cause of neuronal demise in Alzheimer disease (AD) patients. Blood-borne macrophages might reduce Aβ stress to neurons by immigration into the brain and phagocytosis of Αβ. We tested migration and export across a blood-brain barrier model, and phagocytosis and clearance of Αβ by AD and normal subjects’ macrophages. Both AD and normal macrophages were inhibited in Αβ export across the blood-brain barrier due to adherence of Aβ-engorged macrophages to the endothelial layer. In comparison to normal subjects’ macrophages, AD macrophages ingested and cleared less Αβ, and underwent apoptosis upon exposure to soluble, protofibrillar, or fibrillar Αβ. Confocal microscopy of stained AD brain sections revealed oligomeric Aβ in neurons and apoptotic macrophages, which surrounded and infiltrated congophilic microvessels, and fibrillar Aβ in plaques and microvessel walls. After incubation with AD brain sections, normal subjects’ monocytes intruded into neurons and uploaded oligomeric Aβ. In conclusion, in patients with AD, macrophages appear to shuttle Aβ from neurons to vessels where their apoptosis may release fibrillar Aβ, contributing to cerebral amyloid angiopathy

Functional definition of the tobacco protoporphyrinogen IX oxidase substrate-binding site

PPO (protoporphyrinogen IX oxidase) catalyses the flavin-dependent six-electron oxidation of protogen (protoporphyrinogen IX) to form proto (protoporphyrin IX), a crucial step in haem and chlorophyll biosynthesis. The apparent K(m) value for wild-type tobacco PPO2 (mitochondrial PPO) was 1.17 μM, with a V(max) of 4.27 μM·min(−1)·mg(−1) and a catalytic activity k(cat) of 6.0 s(−1). Amino acid residues that appear important for substrate binding in a crystal structure-based model of the substrate docked in the active site were interrogated by site-directed mutagenesis. PPO2 variant F392H did not reveal detectable enzyme activity indicating an important role of Phe(392) in substrate ring A stacking. Mutations of Leu(356), Leu(372) and Arg(98) increased k(cat) values up to 100-fold, indicating that the native residues are not essential for establishing an orientation of the substrate conductive to catalysis. Increased K(m) values of these PPO2 variants from 2- to 100-fold suggest that these residues are involved in, but not essential to, substrate binding via rings B and C. Moreover, one prominent structural constellation of human PPO causing the disease variegate porphyria (N67W/S374D) was successfully transferred into the tobacco PPO2 background. Therefore tobacco PPO2 represents a useful model system for the understanding of the structure–function relationship underlying detrimental human enzyme defects