Crosslinking-MS analysis reveals RNA polymerase I domain architecture and basis of rRNA cleavage

RNA polymerase (Pol) I contains a 10-subunit catalytic core that is related to the core of Pol II and includes subunit A12.2. In addition, Pol I contains the heterodimeric subcomplexes A14/43 and A49/34.5, which are related to the Pol II subcomplex Rpb4/7 and the Pol II initiation factor TFIIF, respectively. Here we used lysine-lysine crosslinking, mass spectrometry (MS) and modeling based on five crystal structures, to extend the previous homology model of the Pol I core, to confirm the location of A14/43 and to position A12.2 and A49/34.5 on the core. In the resulting model of Pol I, the C-terminal ribbon (C-ribbon) domain of A12.2 reaches the active site via the polymerase pore, like the C-ribbon of the Pol II cleavage factor TFIIS, explaining why the intrinsic RNA cleavage activity of Pol I is strong, in contrast to the weak cleavage activity of Pol II. The A49/34.5 dimerization module resides on the polymerase lobe, like TFIIF, whereas the A49 tWH domain resides above the cleft, resembling parts of TFIIE. This indicates that Pol I and also Pol III are distantly related to a Pol II-TFIIS-TFIIF-TFIIE comple

Serum-Konzentration des „Brain-derived neurotrophic factors“ bei Depressionen im Zusammenhang mit körperlicher Aktivität

Das Neurotrophin „Brain-derived neurotrophic factor“ (BDNF) scheint eine bedeutende Rolle bei der Pathophysiologie und Therapie von Depressionen zu spielen. Es konnte bereits anhand einiger Studien gezeigt werden, dass BDNF bei Depressiven im peripheren und zentralen Nervensystem in verminderten Konzentrationen messbar ist und invers mit dem Schweregrad der Depression korreliert. In dieser Arbeit wurden diese Zusammenhänge erneut überprüft und der erweiterten Betrachtung unterzogen, ob die BDNF-Konzentration mit habitueller körperlicher Aktivität zusammenhängt. In der Literatur wurde ein positiver Einfluss von Sport auf die BDNF-Konzentration bei Gesunden sowie Depressiven bereits vielfach beschrieben und bei Letzteren wegen des ebenfalls positiven Einflusses auf die klinische Symptomatik als Therapieoption eingesetzt. Die Auswertung im Rahmen dieser Arbeit bezog sich darauf, ob die habituelle Aktivität (d.h. ohne forciertes Sportprogramm im Studienrahmen) ebenfalls Auswirkungen auf die BDNF-Konzentration und / oder depressive Symptomatik zeigt. Des Weiteren wurden bereits identifizierte Störfaktoren erneut evaluiert sowie explorative Berechnungen bezüglich darüber hinausgehender Einflussfaktoren der BDNF-Konzentration durchgeführt. Methodik: Es wurden 80 Depressive in die Patientengruppe sowie 41 Gesunde in die Kontrollgruppe integriert, deren BDNF-Konzentration im Serum gemessen wurde. Der klinische Schweregrad der Depression wurde anhand der Hamilton Depression Rating Scale sowie des Beck Depression Inventory II evaluiert. Der eigens für diese Studie entwickelte Fragebogen zur körperlichen Aktivität wurde zur retrospektiven Erfassung der alltäglichen, sportlichen und beruflichen Bewegung der Studienteilnehmer verwendet. Ergebnisse: Es konnte eine signifikant verminderte BDNF-Konzentration bei Depressiven im Vergleich zu gesunden Kontrollprobanden gezeigt werden. Des Weiteren zeigte sich ein inverser Zusammenhang des Schweregrades der Major Depression mit der BDNF-Konzentration mit großer Nähe zum definierten Signifikanzniveau. Ein Zusammenhang der BDNF-Konzentration mit habitueller körperlicher Aktivität konnte hier nicht belegt werden, allerdings zeigte sich tendenziell ein inverser Zusammenhang zwischen Letzterer und dem Beck Depression Inventory II. Es konnten auch demografische und anamnestische Einflussfaktoren der BDNF-Konzentration identifiziert werden. Dabei erweitert diese Arbeit die bisherigen Kenntnisse über Störfaktoren, welche im Falle des Alters sowie Nikotinkonsums belegt werden konnten, um den Hinweis auf eine Abhängigkeit der BDNF-Konzentration von Arbeitslosigkeit und dem Beziehungsstatus. Schlussfolgerung: Diese Arbeit stützt bisherige Forschungsergebnisse bzgl. der verminderten BDNF-Konzentration bei Major-Depressionen und ihrer inversen Korrelation mit der klinischen Symptomatik. Bei der Interpretation des negativen Ergebnisses bzgl. des Zusammenhangs von habitueller Aktivität mit BDNF müssen die Limitationen durch die Verwendung eines retrospektiven und darüber hinaus nicht validierten Fragebogens beachtet werden.The neurotrophine „Brain-derived neurotrophic factor“ (BDNF) seems to play an important role in the pathophysiology and therapy of depressions. It was shown in existing studies, that BDNF in depressive subjects is measured in lower concentrations, both in the central and peripheral nervous system, and that it correlates inversely with the severity of the depression. In this thesis, these relationships were validated, and it was tested if habitual physical activity is also correlated with the BDNF-concentration. A positive influence of sports on the BDNF-concentration in healthy and depressive subjects is already repeatedly described in the literature and in the latter case, because of the positive influence on the clinical symptomatology, deployed as a therapy-option. This thesis examined if the habitual activity (i.e. without an enforced sports program within this study) shows an additional influence on the BDNF-concentration and / or the depressive symptomatology. Already known confounding factors were evaluated again and explorative calculations regarding further influencing factors on the BDNF-concentration were conducted. Methodology: 80 depressive subjects were assigned to the patient group, as well as 41 healthy subjects to the control group, whose BDNF-concentration was measured in the serum. The clinical severity of the depression was evaluated with the Hamilton Depression Rating Scale as well as the Beck Depression Inventory II. The specifically for this study devised questionnaire for habitual physical activity was used for the retrospective acquisition of everyday-, sportive- and occupational exercise. Results: A significantly reduced BDNF-concentration in depressive subjects compared to healthy ones was shown. Additionally the inverse relationship between the severity of the major depression and the BDNF-concentration showed great proximity to the defined level of significance. A connection between the BDNF-concentration and the habitual physical activity could not be validated in this study, but tendencies of an inverse relationship between the latter and the Beck Depression Inventory II were visible. Demographical and anamnestic influence factors on the BDNF-concentration were identified as well. Thereby, this thesis expands the preliminary findings about confounding factors, which have already been validated in the cases of age and nicotine-consumption, by indications of a correlation between BDNF-concentration and unemployment and the relationship status. Conclusion: This thesis supports the prior research findings concerning the reduced BDNF-concentration in major depressions and its inverse correlation with the clinical symptomatology. A relation between BDNF-concentration and habitual physical activity was not confirmed. However, interpreting these results, one must keep in mind the limitations caused by the utilization of a retrospective and not validated questionnaire

RNA polymerase III subunit architecture and implications for open promoter complex formation

Transcription initiation by eukaryotic RNA polymerase (Pol) III relies on the TFIIE-related subcomplex C82/34/31. Here we combine crosslinking and hydroxyl radical probing to position the C82/34/31 subcomplex around the Pol III active center cleft. The extended winged helix (WH) domains 1 and 4 of C82 localize to the polymerase domains clamp head and clamp core, respectively, and the two WH domains of C34 span the polymerase cleft from the coiled-coil region of the clamp to the protrusion. The WH domains of C82 and C34 apparently cooperate with other mobile regions flanking the cleft during promoter DNA binding, opening, and loading. Together with published data, our results complete the subunit architecture of Pol III and indicate that all TFIIE-related components of eukaryotic and archaeal transcription systems adopt an evolutionarily conserved location in the upper part of the cleft that supports their functions in open promoter complex formation and stabilization

An AP-MS- and BioID-compatible MAC-tag enables comprehensive mapping of protein interactions and subcellular localizations

Protein-protein interactions govern almost all cellular functions. These complex networks of stable and transient associations can be mapped by affinity purification mass spectrometry (AP-MS) and complementary proximity-based labeling methods such as BioID. To exploit the advantages of both strategies, we here design and optimize an integrated approach combining AP-MS and BioID in a single construct, which we term MAC-tag. We systematically apply the MAC-tag approach to 18 subcellular and 3 sub-organelle localization markers, generating a molecular context database, which can be used to define a protein's molecular location. In addition, we show that combining the AP-MS and BioID results makes it possible to obtain interaction distances within a protein complex. Taken together, our integrated strategy enables the comprehensive mapping of the physical and functional interactions of proteins, defining their molecular context and improving our understanding of the cellular interactome.Peer reviewe

Role of Serum Brain Derived Neurotrophic Factor and Central N-Acetylaspartate for Clinical Response under Antidepressive Pharmacotherapy

Background: The predictive therapeutic value of brain derived neurotrophic factor (BDNF) and its changes associated with the use of specific antidepressants are still unclear. In this study, we examined BDNF as a peripheral and NAA as a central biomarker over the time course of antidepressant treatment to specify both of their roles in the response to the medication and clinical outcome. Methods: We examined serum BDNF (ELISA kit) in a sample of 76 (47 female and 29 male) depressed patients in a naturalistic setting. BDNF was assessed before medication and subsequently after two, four and six weeks of antidepressant treatment. Additionally, in fifteen patients, N-acetylaspartate (NAA) was measured in the anterior cingulate cortex (ACC) with magnetic resonance spectroscopy (MRS). Over a time course of six weeks BDNF and NAA were also examined in a group of 41 healthy controls. Results: We found significant lower serum BDNF concentrations in depressed patients compared to the sample of healthy volunteers before and after medication. BDNF and clinical symptoms decreased significantly in the patients over the time course of antidepressant treatment. Serum BDNF levels at baseline predicted the symptom outcome after eight weeks. Specifically, responders and remitters had lower serum BDNF at baseline than the nonresponders and nonremitters. NAA was slightly decreased but not significantly lower in depressed patients when compared with healthy controls. During treatment period, NAA showed a tendency to increase. Limitations: A relative high drop-out rate and possibly, a suboptimal observation period for BDNF. Conclusion: Our data confirm serum BDNF as a biomarker of depression with a possible role in response prediction. However, our findings argue against serum BDNF increase being a prerequisite to depressive symptom reduction

Crosslinking-MS analysis reveals RNA polymerase I domain architecture and basis of rRNA cleavage

RNA polymerase (Pol) I contains a 10-subunit catalytic core that is related to the core of Pol II and includes subunit A12.2. In addition, Pol I contains the heterodimeric subcomplexes A14/43 and A49/34.5, which are related to the Pol II subcomplex Rpb4/7 and the Pol II initiation factor TFIIF, respectively. Here we used lysine-lysine crosslinking, mass spectrometry (MS) and modeling based on five crystal structures, to extend the previous homology model of the Pol I core, to confirm the location of A14/43 and to position A12.2 and A49/34.5 on the core. In the resulting model of Pol I, the C-terminal ribbon (C-ribbon) domain of A12.2 reaches the active site via the polymerase pore, like the C-ribbon of the Pol II cleavage factor TFIIS, explaining why the intrinsic RNA cleavage activity of Pol I is strong, in contrast to the weak cleavage activity of Pol II. The A49/34.5 dimerization module resides on the polymerase lobe, like TFIIF, whereas the A49 tWH domain resides above the cleft, resembling parts of TFIIE. This indicates that Pol I and also Pol III are distantly related to a Pol II–TFIIS–TFIIF–TFIIE complex

xiNET: Cross-link network maps with residue resolution:Cross-link network maps with residue resolution

xiNET is a visualization tool for exploring cross-linking/mass spectrometry results. The interactive maps of the cross-link network that it generates are a type of node-link diagram. In these maps xiNET displays: (1) residue resolution positional information including linkage sites and linked peptides; (2) all types of cross-linking reaction product; (3) ambiguous results; and, (4) additional sequence information such as domains. xiNET runs in a browser and exports vector graphics which can be edited in common drawing packages to create publication quality figures. Availability: xiNET is open source, released under the Apache version 2 license. Results can be viewed by uploading data to http://crosslinkviewer.org/ or by downloading the software from http://github.com/colin-combe/crosslink-viewer and running it locally