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Integrative "Omics"-Approach Discovers Dynamic and Regulatory Features of Bacterial Stress Responses
(2013)
Bacteria constantly face stress conditions and therefore mount specific responses to ensure adaptation and survival. Stress responses were believed to be predominantly regulated at the transcriptional level. In the phototrophic bacterium Rhodobacter sphaeroides the response to singlet oxygen is initiated by alternative sigma factors. Further adaptive mechanisms include post-transcriptional and post-translational events, which have to be considered to gain a deeper understanding of how sophisticated regulation networks operate. To address this issue, we integrated three layers of regulation: (1) total mRNA levels at different time-points revealed dynamics of the transcriptome, (2) mRNAs in polysome fractions reported on translational regulation (translatome), and (3) SILAC-based mass spectrometry was used to quantify protein abundances (proteome). The singlet oxygen stress response exhibited highly dynamic features regarding short-term effects and late adaptation, which could in part be assigned to the sigma factors RpoE and RpoH2 generating distinct expression kinetics of corresponding regulons. The occurrence of polar expression patterns of genes within stress-inducible operons pointed to an alternative of dynamic fine-tuning upon stress. In addition to transcriptional activation, we observed significant induction of genes at the post-transcriptional level (translatome), which identified new putative regulators and assigned genes of quorum sensing to the singlet oxygen stress response. Intriguingly, the SILAC approach explored the stress-dependent decline of photosynthetic proteins, but also identified 19 new open reading frames, which were partly validated by RNA-seq. We propose that comparative approaches as presented here will help to create multi-layered expression maps on the system level ("expressome"). Finally, intense mass spectrometry combined with RNA-seq might be the future tool of choice to re-annotate genomes in various organisms and will help to understand how they adapt to alternating conditions.
We identified eukaryotic translation elongation factor 1A (eEF1A) Raf-mediated phosphorylation sites and defined their role in the regulation of eEF1A half-life and of apoptosis of human cancer cells. Mass spectrometry identified in vitro S21 and T88 as phosphorylation sites mediated by B-Raf but not C-Raf on eEF1A1 whereas S21 was phosphorylated on eEF1A2 by both B- and C-Raf. Interestingly, S21 belongs to the first eEF1A GTP/GDP-binding consensus sequence. Phosphorylation of S21 was strongly enhanced when both eEF1A isoforms were preincubated prior the assay with C-Raf, suggesting that the eEF1A isoforms can heterodimerize thus increasing the accessibility of S21 to the phosphate. Overexpression of eEF1A1 in COS 7 cells confirmed the phosphorylation of T88 also in vivo. Compared with wt, in COS 7 cells overexpressed phosphodeficient (A) and phospho-mimicking (D) mutants of eEF1A1 (S21A/D and T88A/D) and of eEF1A2 (S21A/D), resulted less stable and more rapidly proteasome degraded. Transfection of S21 A/D eEF1A mutants in H1355 cells increased apoptosis in comparison with the wt isoforms. It indicates that the blockage of S21 interferes with or even supports C-Raf induced apoptosis rather than cell survival. Raf-mediated regulation of this site could be a crucial mechanism involved in the functional switching of eEF1A between its role in protein biosynthesis and its participation in other cellular processes.
We identified eukaryotic translation elongation factor 1A (eEF1A) Raf-mediated phosphorylation sites and defined their role in the regulation of eEF1A half-life and of apoptosis of human cancer cells. Mass spectrometry identified in vitro S21 and T88 as phosphorylation sites mediated by B-Raf but not C-Raf on eEF1A1 whereas S21 was phosphorylated on eEF1A2 by both B-and C-Raf. Interestingly, S21 belongs to the first eEF1A GTP/GDP-binding consensus sequence. Phosphorylation of S21 was strongly enhanced when both eEF1A isoforms were preincubated prior the assay with C-Raf, suggesting that the eEF1A isoforms can heterodimerize thus increasing the accessibility of S21 to the phosphate. Overexpression of eEF1A1 in COS 7 cells confirmed the phosphorylation of T88 also in vivo. Compared with wt, in COS 7 cells overexpressed phosphodeficient (A) and phospho-mimicking (D) mutants of eEF1A1 (S21A/D and T88A/D) and of eEF1A2 (S21A/D), resulted less stable and more rapidly proteasome degraded. Transfection of S21 A/D eEF1A mutants in H1355 cells increased apoptosis in comparison with the wt isoforms. It indicates that the blockage of S21 interferes with or even supports C-Raf induced apoptosis rather than cell survival. Raf-mediated regulation of this site could be a crucial mechanism involved in the functional switching of eEF1A between its role in protein biosynthesis and its participation in other cellular processes.
Context:
Adrenal tumors have a prevalence of around 2% in the general population. Adrenocortical carcinoma (ACC) is rare but accounts for 2–11% of incidentally discovered adrenal masses. Differentiating ACC from adrenocortical adenoma (ACA) represents a diagnostic challenge in patients with adrenal incidentalomas, with tumor size, imaging, and even histology all providing unsatisfactory predictive values.
Objective:
Here we developed a novel steroid metabolomic approach, mass spectrometry-based steroid profiling followed by machine learning analysis, and examined its diagnostic value for the detection of adrenal malignancy.
Design:
Quantification of 32 distinct adrenal derived steroids was carried out by gas chromatography/mass spectrometry in 24-h urine samples from 102 ACA patients (age range 19–84 yr) and 45 ACC patients (20–80 yr). Underlying diagnosis was ascertained by histology and metastasis in ACC and by clinical follow-up [median duration 52 (range 26–201) months] without evidence of metastasis in ACA. Steroid excretion data were subjected to generalized matrix learning vector quantization (GMLVQ) to identify the most discriminative steroids.
Results:
Steroid profiling revealed a pattern of predominantly immature, early-stage steroidogenesis in ACC. GMLVQ analysis identified a subset of nine steroids that performed best in differentiating ACA from ACC. Receiver-operating characteristics analysis of GMLVQ results demonstrated sensitivity = specificity = 90% (area under the curve = 0.97) employing all 32 steroids and sensitivity = specificity = 88% (area under the curve = 0.96) when using only the nine most differentiating markers.
Conclusions:
Urine steroid metabolomics is a novel, highly sensitive, and specific biomarker tool for discriminating benign from malignant adrenal tumors, with obvious promise for the diagnostic work-up of patients with adrenal incidentalomas.
Das ANP/GC-A-System spielt durch die Produktion des sekundären Botenstoffs cGMP eine wichtige Rolle bei der Regulation des Blutdruckes und des Blutvolumens. Bei Patienten mit Herzhypertrophie oder Herzinsuffizienz sind die ANP-Plasmakonzentrationen erhöht, aber die GC-A-vermittelten Effekte stark reduziert, was auf einen Defekt des Signalsystems hinweist. Studien an metabolisch markierten GC-A-überexprimierenden HEK 293-Zellen zeigten, dass der GC-A-Rezeptor im basalen Zustand stark phosphoryliert und die homologe bzw. heterologe Desensitisierung wahrscheinlich mit einer Dephosphorylierung verbunden ist. Die Desensitisierung stellt einen Mechanismus dar, der in vivo zu einem Funktionsverlust des Rezeptors beitragen könnte. Im Rahmen dieser Arbeit konnten mittels Massenspektrometrie sieben Phosphorylierungsstellen in der Kinasehomologen Domäne aus FLAG-GC-A exprimierenden HEK 293-Zellen detektiert werden: Ser487, Ser497, Thr500, Ser502, Ser506, Ser510 und Thr513. Die massenspektrometrische relative Quantifizierung basierend auf der Multiple-Reaction-Monitoring (MRM)-Methode zeigte bei ANP-induzierter, homologer Desensitisierung eine Dephosphorylierung der Phosphorylierungsstellen Ser497, Thr500, Ser502, Ser506, Ser510 und Thr513, was mit bereits publizierten Daten übereinstimmt, und einen starken Anstieg der Phosphorylierung an Ser487. Nach Inkubation mit Angiotensin II, welches eine heterologe Desensitisierung hervorruft, wurde eine Reduzierung aller Phosphorylierungen verzeichnet, die zudem stärker ausgeprägt war als bei der ANP-abhängigen Desensitisierung. Die Funktion der neu identifizierten Phosphorylierung an Ser487 wurde mittels Mutagenese analysiert. Die Substitution des Serins durch Alanin, welche den unphosphorylierten Zustand nachstellt, resultierte in einer Rezeptoraktivität und desensitisierung vergleichbar zum GC-A Wildtyp-Rezeptor. Wurde hingegen Serin gegen Glutamat getauscht, um den phosphorylierten Zustand zu imitieren, konnte der Rezeptor weder aktiviert noch desensitisiert werden. Diese Ergebnisse bestätigen vorherige Studien, dass die GC-A-Rezeptorantwort auf ANP durch die Phosphorylierungen reguliert wird. Allerdings scheint bei der homologen Desensitisierung die Phosphorylierung an der Position Ser487 eine Rolle zu spielen, da sie die Aktivität des Rezeptors inhibiert. Die Identifizierung und Charakterisierung dieser Phosphorylierungsstelle trägt zum Verständnis des Mechanismus der homologen Desensitierung bei. Zusätzlich konnten einige der beschriebenen Phosphorylierungen in Zellsystemen detektiert werden, die die GC-A endogen exprimieren. Dadurch sind unter physiologischen Bedingungen Analysen der Mechanismen möglich, die bei der Aktivierung und Deaktivierung der GC-A involviert sind und somit wichtige pathophysiologische Konsequenzen haben können.
In this thesis we have used Drosophila melanogaster as a model organism to investigate proteins and their putative interacting partners that are directly or indirectly involved in the release of neurotransmitters at the synapse. We have used molecular techniques to investigate conserved synaptic proteins, synapsin and synapse associated protein of 47 kD (SAP47), and a putative interaction partner of SAP47, tubulin binding chaperone E-like (TBCEL). SAP47 and synapsins are highly conserved synaptic vesicle associated proteins in Drosophila melanogaster. To further investigate the role and function of Sap47 and Syn genes, we had earlier generated the null mutants by P-element mutagenesis (Funk et al., 2004; Godenschwege et al., 2004). Western blots and ELISA of brain homogenates from Sap47156 null mutants showed the presence of up-regulated phospho-synapsin in comparison to wild-type (CS) and the presence of up-regulated phospho-synapsin was partially abolished when a pan-neuronal rescue of SAP47 was performed by the Gal4- UAS technique. Thus, the results suggest a qualitative and quantitative modulation of synapsin by SAP47. At the transcript level, we did not observe any difference in content of Syn transcript in Sap47156 and wild-type CS flies. The question of a direct molecular interaction between SAP47 and synapsin was investigated by co-immunoprecipitation (Co-IP) experiments and we did not find any stable interactions under the several IP conditions we tested. The possibility of Sap47 as a modifier of Syn at the genetic level was investigated by generating and testing homozygous double null mutants of Sap47 and Syn. The Syn97, Sap47156 double mutants are viable but have a reduced life span and decreased locomotion when compared to CS. In 2D-PAGE analysis of synapsins we identified trains of spots corresponding to synapsins, suggesting that synapsin has several isoforms and each one of them is posttranslationally modified. In an analysis by Blue native-SDS-PAGE (BN-SDS-2D- PAGE) and Western blot we observed synapsin and SAP47 signals to be present at 700-900 kDa and 200-250 kDa, respectively, suggesting that they are part of large but different complexes. We also report the possibility of Drosophila synapsin forming homo- and heteromultimers, which has also been reported for synapsins of vertebrates. In parallel to the above experiments, phosphorylation of synapsins in Drosophila was studied by IP techniques followed by 1D-SDS gel electrophoresis and mass spectrometry (in collaboration with S. Heo and G. Lubec). We identified and verified 5 unique phosphorylation sites in Drosophila synapsin from our MS analysis. Apart from phosphorylation modifications we identified several other PTMs which have not been verified. The significance of these phosphorylations and other identified PTMs needs to be investigated further and their implications for synapsin function and Drosophila behavior has to be elucidated by further experiments. In a collaborative project with S. Kneitz and N. Nuwal, we investigated the effects of Sap156 and Syn97 mutations by performing a whole Drosophila transcriptome microarray analysis of the individual null mutants and the double mutants (V2 and V3). We obtained several candidates which were significantly altered in the mutants. These genes need to be investigated further to elucidate their interactions with Sap47 and Syn. In another project, we investigated the role and function of Drosophila tubulin- binding chaperone E-like (Tbcel, CG12214). The TBCEL protein has high homology to vertebrate TBCE-like (or E-like) which has high sequence similarity to tubulin-binding chaperone E (TBCE) (hence the name TBCE-Like). We generated an anti-TBCEL polyclonal antiserum (in collaboration with G. Krohne). According to flybase, the Tbcel gene has only one exon and codes for two different transcripts by alternative transcription start sites. The longer transcript RB is present only in males whereas the shorter transcript RA is present only in females. In order to study the gene function we performed P- element jump-out mutagenesis to generate deletion mutants. We used the NP4786 (NP) stock which has a P(GawB) insertion in the 5’ UTR of the Tbcel gene. NP4786 flies are homozygous lethal due to a second-site lethality as the flies are viable over a deficiency (Df) chromosome (a deletion of genomic region spanning the Tbcel gene and other upstream and downstream genes). We performed the P-element mutagenesis twice. In the first trial we obtained only revertants and the second experiment is still in progress. In the second attempt, jump-out was performed over the deficiency chromosome to prevent homologous chromosome mediated double stranded DNA repair. During the second mutagenesis an insertion stock G18151 became available. These flies had a P-element insertion in the open reading frame (ORF) of the Tbcel gene but was homozygous viable. Western blots of fresh tissue homogenates of NP/Df and G18151 flies probed with anti-TBCEL antiserum showed no TBCEL signal, suggesting that these flies are Tbcel null mutants. We used these flies for further immunohistochemical analyses and found that TBCEL is specifically expressed in the cytoplasm of cyst cells of the testes and is associated with the tubulin of spermatid tails in wild-type CS, whereas in NP/Df and G18151 flies the TBCEL staining in the cyst cells was absent and there was a disruption of actin investment cones. We also found enrichment of TBCEL staining around the actin investment cone. These results are also supported by the observation that the enhancer trap expression of the NP4786 line is localised to the cyst cells, similar to TBCEL expression. Also, male fertility of NP/Df and G18151 flies was tested and they were found to be sterile with few escapers. Thus, these results suggest that TBCEL is involved in Drosophila spermatogenesis with a possible role in the spermatid elongation and individualisation process.
Conjugation of reactive intermediates of drugs with proteins or DNA may result in toxic effects such as hepatotoxicity, agranulocytosis, allergies, tumors, etc. From 1975 to 1999, 2.9% of drugs were withdrawn from the market due to such severe adverse drug reactions. Thus, formation of chemically reactive intermediates is a widely discussed problem in drug development processes. Early detection of potentially toxic compounds is required for drug discovery and drug development. Conjugation of such electrophilic compounds with glutathione (GSH) is one of the most important detoxifying reactions in vivo. Processing of these GSH-conjugates ultimately leads to the formation of renally cleared mercapturic acids, which may also be oxidized to sulfoxides. Thus, mercapturic acids may be generated and detected in vitro and non-invasively in vivo in urine to assess the reactivity of a compound in early stages of drug development processes. Therefore, the aim of this work was to develop and evaluate a HPLC-MS/MS screening method for simple and rapid detection and characterization of known and unknown mercapturic acids and application of the method to several different matrices. Based on the common constant neutral loss (CNL) of 129 Da of all mercapturic acids tested (in negative ion mode), a CNL survey scan was performed using a linear ion trap instrument and was combined with two enhanced product ion (EPI) scans with different collision energies to characterize the detected signals. The CNL resulted from the cleavage between the sulfur and the carbon atom in the N-acetyl-L-cysteine moiety. After optimization of the experimental parameters, the detection limits of the reference substances in rat urine ranged from 0.3 to 15.5 pmol on column (i.e. 20 ng/ml to 800 ng/ml). For in vitro evaluation of the method, the model compounds acetaminophen, diclofenac, bifonazole, clozapine, troglitazone, carbamazepine, and bisphenol A were screened for formation of reactive intermediates and, hence, detection of the corresponding mercapturic acids. To determine possible species- and tissue-specific toxicities, the model compounds were incubated with stimulated neutrophils and with liver microsomes from rats and humans. Species-specific differences were observed in incubations of acetaminophen and diclofenac with rat and human hepatic microsomes. Tissue-specific differences in biotransformation of the model compounds in incubations with human neutrophils and human liver microsomes were observed for diclofenac, carbamazepine, clozapine, and bifonazole. The developed HPLC-MS/MS method was also evaluated in vivo by analysis of rat and human urine. Drug-related mercapturic acids were detected in urine of rats orally treated with acetaminophen (20 mg/kg and 640 mg/kg b.w.) or diclofenac (10 mg/kg and 20 mg/kg b.w.). Human urine samples were analyzed before and after oral administration of a clinically used dose of 500 mg and 50 mg of acetaminophen. Besides detection of the mercapturic acid of N-acetylbenzoquinoneimine (AAP-MA), a second mercapturic acid with m/z 327 occurred dose-dependently in rat and human urine samples after administration of acetaminophen. Further investigations on identification of this metabolite using authentic compounds and comparing their MS/MS mass spectra demonstrated oxidation of AAP-MA to stereoisomeric sulfoxides in vivo. For diclofenac, a novel mercapturic acid with m/z 441 was detected in rat urine samples that was identical to a metabolite obtained in incubations with human neutrophils before. The in vivo formation of this diclofenac metabolite is described here for the first time. In addition, three endogenously formed mercapturic acids were detected and identified. In conclusion, the results of the in vitro and in vivo evaluation demonstrate the advantages of the rapid and generic HPLC-MS/MS screening method for the detection of mercapturic acids, that can be obtained with a minimum of sample preparation and a high throughput in diverse matrices.
Im Rahmen der vorliegenden Arbeit wurden analytische Methoden zur Bestimmung des Verunreinigungsprofils von Erythromycin entwickelt, die der bestehenden Ph.Eur.-Methode überlegen sind. Die neue HPLC-Methode ist in der Lage, alle verwandten Verbindungen mit angemessener Präzision nachzuweisen und zu quantifizieren. Mit Hilfe der Massenspektrometrie konnten alle Hauptkomponenten und verwandten Verbindungen der Base, ihrer Ester und Salze eindeutig identifiziert und quantifiziert werden. Zudem konnten zwei neue verwandte Verbindungen von Erythromycin gefunden und als N,N-Didemethylerythromycin A und Anhydroerythromycin F identifiziert werden.
Im Mittelpunkt dieser Arbeit stehen Untersuchungen zur Bildung des Pigments Neuromelanin, das die Ursache für die dunkle Farbgebung der humanen Substantia nigra pars compacta ist. Eine Beteiligung von Neuromelanin an den pathobiochemischen Ereignissen bei Parkinson-Krankheit erklärt das klinische Interesse an Neuromelanin. Die Untersuchungsmöglichkeiten von Neuromelanin sind limitiert: einerseits ist eine chemische Strukturaufklärung aufgrund der Unlöslichkeit dieses amorphen Polymers kaum zu bewerkstelligen, andererseits wird mangels geeigneter biologischer Testsysteme ein Einblick in die Biogenese von Neuromelanin verwehrt. Zurzeit wird die Bildung von Neuromelanin anhand der beiden konkurrierenden Hypothesen als Autoxidation von Dopamin oder durch Beteiligung eines Enzyms („Tyrosinase-Konzept“) erklärt. In dieser Arbeit wurden beide hypothetischen Ansätze bearbeitet, wobei einer enzymatischen Biogenese von Neuromelanin die Präferenz gegeben wird. Zur globalen Untersuchung von Neuromelanin-Granula wurde nun erstmals eine Isolierung der Pigment-haltigen Organelle vorgestellt, die die Basis für eine umfassende Proteomanalyse mittels 1-D-SDS-PAGE und ESI-Tandem-Massenspektrometrie bildete. Mit diesem methodischen Ansatz wurden ingesamt 73 Proteinen identifiziert. Diese waren vor allem lysosomalen Proteinen zuordenbar, z.B. charakteristischen Membranproteinen (LAMP-1), sämtlichen Proteasen, Proteinen des Metabolismus von (Glyco-)Lipiden und Glycoproteinen, aber auch Proteinen des Cytosols und des vesikulären Verkehrs. Entscheidend war die Anwesenheit von Proteinen des Endoplasmatischen Reticulums (ER); Calnexin gilt als ein melanogenes Chaperon, das nicht in Lysosomen vorkommt, dagegen aber in Lysosomen-verwandten Organellen. Im Vergleich mit bereits existierenden Proteinprofilen von Lysosomen und Lysosomen-verwandten Organellen zeigten die in Neuromelanin-Granula identifizierten lysosomalen Proteine und Proteine des ER, dass diese Organellen der humanen Substantia nigra keine konventionellen Lysosomen sind, sondern mit hoher Wahrscheinlichkeit der Gruppe der Lysosomen-verwandten Organellen zuzuordnen sind.