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Institute
- Medizin (1928)
- Biochemie, Chemie und Pharmazie (27)
- Biowissenschaften (27)
- Biochemie und Chemie (23)
- Georg-Speyer-Haus (23)
- Buchmann Institut für Molekulare Lebenswissenschaften (BMLS) (19)
- Zentrum für Arzneimittelforschung, Entwicklung und Sicherheit (ZAFES) (19)
- Frankfurt Institute for Advanced Studies (FIAS) (16)
- Ernst Strüngmann Institut (12)
- MPI für Biophysik (10)
The Na+-F1F0-ATPase operon ofAcetobacterium woodii was recently shown to contain, among eleven atp genes, those genes that encode subunita and b, a gene encoding a 16-kDa proteolipid (subunit c 1), and two genes encoding 8-kDa proteolipids (subunits c 2 andc 3). Because subunits a,b, and c 1 were not found in previous enzyme preparations, we re-determined the subunit composition of the enzyme. The genes were overproduced, and specific antibodies were raised. Western blots revealed that subunits a,b, and c 1 are produced and localized in the cytoplasmic membrane. Membrane protein complexes were solubilized by dodecylmaltoside and separated by blue native-polyacrylamide gel electrophoresis, and the ATPase subunits were resolved by SDS-polyacrylamide gel electrophoresis. N-terminal sequence analyses revealed the presence of subunitsa, c 2, c 3,b, δ, α, γ, β, and ε. Biochemical and immunological analyses revealed that subunitsc 1, c 2, andc 3 are all part of the c-oligomer, the first of a F1F0-ATPase that contains 8- and 16-kDa proteolipids.
Two-person neuroscience (2 PN) is a recently introduced conceptual and methodological framework used to investigate the neural basis of human social interaction from simultaneous neuroimaging of two or more subjects (hyperscanning). In this study, we adopted a 2 PN approach and a multiple-brain connectivity model to investigate the neural basis of a form of cooperation called joint action. We hypothesized different intra-brain and inter-brain connectivity patterns when comparing the interpersonal properties of joint action with non-interpersonal conditions, with a focus on co-representation, a core ability at the basis of cooperation. 32 subjects were enrolled in dual-EEG recordings during a computerized joint action task including three conditions: one in which the dyad jointly acted to pursue a common goal (joint), one in which each subject interacted with the PC (PC), and one in which each subject performed the task individually (Solo).
A combination of multiple-brain connectivity estimation and specific indices derived from graph theory allowed to compare interpersonal with non-interpersonal conditions in four different frequency bands. Our results indicate that all the indices were modulated by the interaction, and returned a significantly stronger integration of multiple-subject networks in the joint vs. PC and Solo conditions. A subsequent classification analysis showed that features based on multiple-brain indices led to a better discrimination between social and non-social conditions with respect to single-subject indices. Taken together, our results suggest that multiple-brain connectivity can provide a deeper insight into the understanding of the neural basis of cooperation in humans.
Cross-frequency coupling (CFC) has been proposed to coordinate neural dynamics across spatial and temporal scales. Despite its potential relevance for understanding healthy and pathological brain function, the standard CFC analysis and physiological interpretation come with fundamental problems. For example, apparent CFC can appear because of spectral correlations due to common non-stationarities that may arise in the total absence of interactions between neural frequency components. To provide a road map towards an improved mechanistic understanding of CFC, we organize the available and potential novel statistical/modeling approaches according to their biophysical interpretability. While we do not provide solutions for all the problems described, we provide a list of practical recommendations to avoid common errors and to enhance the interpretability of CFC analysis.
Spinocerebellar ataxia type 2 (SCA2) is caused by polyglutamine expansion in Ataxin-2 (ATXN2). This factor binds RNA/proteins to modify metabolism after stress, and to control calcium (Ca2+) homeostasis after stimuli. Cerebellar ataxias and corticospinal motor neuron degeneration are determined by gain/loss in ATXN2 function, so we aimed to identify key molecules in this atrophic process, as potential disease progression markers. Our Atxn2-CAG100-Knock-In mouse faithfully models features observed in patients at pre-onset, early and terminal stages. Here, its cerebellar global RNA profiling revealed downregulation of signaling cascades to precede motor deficits. Validation work at mRNA/protein level defined alterations that were independent of constant physiological ATXN2 functions, but specific for RNA/aggregation toxicity, and progressive across the short lifespan. The earliest changes were detected at three months among Ca2+ channels/transporters (Itpr1, Ryr3, Atp2a2, Atp2a3, Trpc3), IP3 metabolism (Plcg1, Inpp5a, Itpka), and Ca2+-Calmodulin dependent kinases (Camk2a, Camk4). CaMKIV–Sam68 control over alternative splicing of Nrxn1, an adhesion component of glutamatergic synapses between granule and Purkinje neurons, was found to be affected. Systematic screening of pre/post-synapse components, with dendrite morphology assessment, suggested early impairment of CamKIIα abundance together with the weakening of parallel fiber connectivity. These data reveal molecular changes due to ATXN2 pathology, primarily impacting excitability and communication.
The subunit composition of the mitochondrial ATP synthase from Saccharomyces cerevisiae was analyzed using blue native gel electrophoresis and high resolution SDS-polyacrylamide gel electrophoresis. We report here the identification of a novel subunit of molecular mass of 6,687 Da, termed subunit j (Su j). An open reading frame of 127 base pairs (ATP18), which encodes for Su j, was identified on chromosome XIII. Su j does not display sequence similarity to ATP synthase subunits from other organisms. Data base searches, however, identified a potential homolog from Schizosaccharomyces pombe with 51% identity to Su j of S. cerevisiae. Su j, a small protein of 59 amino acid residues, has the characteristics of an integral inner membrane protein with a single transmembrane segment. Deletion of the ATP18 gene encoding Su j led to a strain (Deltasu j) completely deficient in oligomycin-sensitive ATPase activity and unable to grow on nonfermentable carbon sources. The presence of Su j is required for the stable expression of subunits 6 and f of the F0 membrane sector. In the absence of Su j, spontaneously arising rho- cells were observed that lacked also ubiquinol-cytochrome c reductase and cytochrome c oxidase activities. We conclude that Su j is a novel and essential subunit of yeast ATP synthase.
Purpose: To evaluate the impact of testing asymptomatic cancer patients, we analyzed all tests for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) before and during radiotherapy at a tertiary cancer center throughout the second wave of the pandemic in Germany. Methods: Results of all real-time polymerase chain reaction (RT-PCR) tests for SARS-CoV 2 performed at our radio-oncology department between 13 October 2020 and 11 March 2021 were included. Clinical data and anamnestic information at the time of testing were documented and examined for (i) the presence of COVID-19-related symptoms and (ii) virus-related anamnesis (high-risk [prior positive test or contact to a positive tested person within the last 14 days] or low-risk [inconspicuous anamnesis within the last 14 days]). Results: A total of 1056 SARS-CoV 2 tests in 543 patients were analyzed. Of those, 1015 tests were performed in asymptomatic patients and 41 tests in patients with COVID-19-associated symptoms. Two of 940 (0.2%) tests in asymptomatic patients with low-risk anamnesis and three of 75 (4.0%) tests in asymptomatic patients with high-risk anamnesis showed a positive result. For symptomatic patients, SARS-CoV 2 was detected in three of 36 (8.3%) low-risk and three of five (60.0%) high-risk tests. Conclusion: To the best of our knowledge, this is the first study evaluating the correlation between individual risk factors and positivity rates of SARS-CoV 2 tests in cancer patients. The data demonstrate that clinical and anamnestic assessment is a simple and effective measure to distinctly increase SARS-CoV 2 test efficiency. This might enable cancer centers to adjust test strategies in asymptomatic patients, especially when test resources are scarce.
Of the 16 non-structural proteins (Nsps) encoded by SARS CoV-2, Nsp3 is the largest and plays important roles in the viral life cycle. Being a large, multidomain, transmembrane protein, Nsp3 has been the most challenging Nsp to characterize. Encoded within Nsp3 is the papain-like protease PLpro domain that cleaves not only the viral protein but also polyubiquitin and the ubiquitin-like modifier ISG15 from host cells. We here compare the interactors of PLpro and Nsp3 and find a largely overlapping interactome. Intriguingly, we find that near full length Nsp3 is a more active protease compared to the minimal catalytic domain of PLpro. Using a MALDI-TOF based assay, we screen 1971 approved clinical compounds and identify five compounds that inhibit PLpro with IC50s in the low micromolar range but showed cross reactivity with other human deubiquitinases and had no significant antiviral activity in cellular SARS-CoV-2 infection assays. We therefore looked for alternative methods to block PLpro activity and engineered competitive nanobodies that bind to PLpro at the substrate binding site with nanomolar affinity thus inhibiting the enzyme. Our work highlights the importance of studying Nsp3 and provides tools and valuable insights to investigate Nsp3 biology during the viral infection cycle.
Many snake venoms are known for their antithrombotic activity. They contain components that specifically target different platelet-activating receptors such as the collagen-binding integrin α2β1 and the von Willebrand factor receptor GPIb. In a search for an α2β1 integrin-blocking component from the venom of the habu snake (Trimeresurus flavoviridis), we employed two independent purification protocols. First, we used the integrin α2A domain, a major collagen-binding domain, as bait for affinity purification of an α2β1 integrin-binding toxin from the crude venom. Second, in parallel, we used classical protein separation protocols and tested for α2β1 integrin-inhibiting capabilities by ELISA. Using both approaches, we identified flavocetin-A as an inhibitor of α2β1 integrin. Hitherto, flavocetin-A has been reported as a GPIb inhibitor. However, flavocetin-A inhibited collagen-induced platelet aggregation even after GPIb was blocked with other inhibitors. Moreover, flavocetin-A antagonized α2β1 integrin-mediated adhesion and migration of HT1080 human fibrosarcoma cells, which lack any GPIb, on collagen. Protein chemical analyses proved that flavocetin-A binds to α2β1 integrin and its α2A domain with high affinity and in a cooperative manner, which most likely is due to its quaternary structure. Kinetic measurements confirmed the formation of a strong complex between integrin and flavocetin-A, which dissociates very slowly. This study proves that flavocetin-A, which has long been known as a GPIb inhibitor, efficiently targets α2β1 integrin and thus blocks collagen-induced platelet activation. Moreover, our findings suggest that the separation of GPIb- and α2β1 integrin-blocking members within the C-type lectin-related protein family is less strict than previously assumed.