Hexameric oligomerization of mitochondrial peroxiredoxin PrxIIF and formation of an ultrahigh affinity complex with its electron donor thioredoxin Trx-o

Mitochondria from plants, yeast, and animals each contain at least one peroxiredoxin (Prx) that is involved in peroxide detoxification and redox signalling. The supramolecular dynamics of atypical type II Prx targeted to the mitochondrion was addressed in pea. Microcalorimetric (ITC) titrations identified an extremely high-affinity binding between the mitochondrial PsPrxIIF and Trx-o with a KD of 126±14 pM. Binding was driven by a favourable enthalpy change (ΔH= –60.6 kcal mol−1) which was counterbalanced by unfavourable entropy changes (TΔS= –47.1 kcal mol−1). This is consistent with the occurrence of large conformational changes during binding which was abolished upon site-directed mutaganesis of the catalytic C59S and C84S. The redox-dependent interaction was confirmed by gel filtration of mitochondrial extracts and co-immunoprecipitation from extracts. The heterocomplex of PsPrxIIF and Trx-o reduced peroxide substrates more efficiently than free PsPrxIIF suggesting that Trx-o serves as an efficient and specific electron donor to PsPrxIIF in vivo. Other Trx-s tested by ITC analysis failed to interact with PsPrxIIF indicating a specific recognition of PsPrxIIF by Trx-o. PsPrxIIF exists primarily as a dimer or a hexamer depending on the redox state. In addition to the well-characterized oligomerization of classical 2-Cys Prx the results also show that atypical Prx undergo large structural reorganization with implications for protein–protein interaction and function

The Centrosomal Kinase Plk1 Localizes to the Transition Zone of Primary Cilia and Induces Phosphorylation of Nephrocystin-1

Polo-like kinase (Plk1) plays a central role in regulating the cell cycle. Plk1-mediated phosphorylation is essential for centrosome maturation, and for numerous mitotic events. Although Plk1 localizes to multiple subcellular sites, a major site of action is the centrosomes, which supports mitotic functions in control of bipolar spindle formation. In G0 or G1 untransformed cells, the centriolar core of the centrosome differentiates into the basal body of the primary cilium. Primary cilia are antenna-like sensory organelles dynamically regulated during the cell cycle. Whether Plk1 has a role in ciliary biology has never been studied. Nephrocystin-1 (NPHP1) is a ciliary protein; loss of NPHP1 in humans causes nephronophthisis (NPH), an autosomal-recessive cystic kidney disease. We here demonstrate that Plk1 colocalizes with nephrocystin-1 to the transition zone of primary cilia in epithelial cells. Plk1 co-immunoprecipitates with NPHP1, suggesting it is part of the nephrocystin protein complex. We identified a candidate Plk1 phosphorylation motif (D/E-X-S/T-φ-X-D/E) in nephrocystin-1, and demonstrated in vitro that Plk1 phosphorylates the nephrocystin N-terminus, which includes the specific PLK1 phosphorylation motif. Further, induced disassembly of primary cilia rapidly evoked Plk1 kinase activity, while small molecule inhibition of Plk1 activity or RNAi-mediated downregulation of Plk1 limited the first and second phase of ciliary disassembly. These data identify Plk1 as a novel transition zone signaling protein, suggest a function of Plk1 in cilia dynamics, and link Plk1 to the pathogenesis of NPH and potentially other cystic kidney diseases

Phosphorylation of Mouse Immunity-Related GTPase (IRG) Resistance Proteins Is an Evasion Strategy for Virulent Toxoplasma gondii

GTPases of the mouse IRG protein family, mediators of resistance against Toxoplasma gondii in the mouse, are inactivated by a polymorphic kinase of the parasite, resulting in enhanced parasite virulence

Gadoliniumhaltige Kontrastmittel: Ablagerungen im Körper und Gehirn und möglicherweise damit im Zusammenhang stehende langfristige Folgewirkungen – aktueller Stand und Ausblick aus regulatorischer Sicht

Gadoliniumhaltige Kontrastmittel werden zur Kontrastverstärkung bei Magnetresonanztomografien für die Diagnose eines breiten Spektrums von Erkrankungen eingesetzt. Im Allgemeinen sind sie gut verträglich und gelten grundsätzlich als sicher. In seltenen Fällen kann es bei Patienten mit eingeschränkter Nierenfunktion zu einer krankhaften Vermehrung des Bindegewebes der Haut, der Gelenke und inneren Organe kommen, der nephrogenen systemischen Fibrose (NSF). Durch die Einführung von Maßnahmen zur Risikominimierung im Jahr 2010 ist die Anzahl gemeldeter und bestätigter NSF-Fälle stark zurückgegangen. Die kontrastverstärkte MRT galt daher weiterhin als sicheres diagnostisches Verfahren, das auch bei Patienten mit Niereninsuffizienz eingesetzt werden konnte. Im Jahr 2014 wurde erstmals beschrieben, dass es nach Gabe von gadoliniumhaltigen Kontrastmitteln auf MRT-Aufnahmen des Gehirns zu einer Erhöhung der Signalintensität in bestimmten Gehirnbereichen kommen kann. In diesen Bereichen konnte nachgewiesen werden, dass dort Gadolinium abgelagert wurde. Diese Ablagerungen und Signalintensitätserhöhungen betrafen auch Patienten mit gesunder Nierenfunktion. Eine Neubewertung des Sicherheitsprofils und Nutzen-Risiko-Verhältnisses gadoliniumhaltiger Kontrastmittel auf europäischer Ebene führte im Jahr 2017 dazu, dass die Zulassungen einiger Kontrastmittel suspendiert und die Anwendungsgebiete eines Kontrastmittels stark eingeschränkt wurden. Seit Bekanntwerden dieser Problematik häuften sich in den letzten Jahren Meldungen von Patienten, die nach Gabe eines gadoliniumhaltigen Kontrastmittels über klinische Symptome wie Schmerzen, Missempfindungen, Hautveränderungen u.a. berichteten. Von einer Autorengruppe um R.C. Semelka wurde 2016 ein eigenständiges Krankheitsbild postuliert, die sogenannte „gadolinium deposition disease“, die sich mit einem breiten Spektrum unterschiedlicher Symptome manifestiert und direkt auf die Anwendung gadoliniumhaltiger Kontrastmittel zurückzuführen sein soll. Die Existenz eines solchen eigenständigen Krankheitsbildes wird in der wissenschaftlichen Literatur allerdings kontrovers diskutiert. Allein auf Basis der berichteten Fälle ist es momentan äußerst schwierig, eine klinisch eindeutige Beschreibung der möglicherweise vorhandenen Folgewirkungen vorzunehmen, die von den Symptomen der zugrunde liegenden Erkrankung klar abgegrenzt werden können. Beim Menschen wurden bisher auch keine histologischen Veränderungen gefunden, welche die berichtete Symptomatik erklären könnten. Neue Ergebnisse aus tierexperimentellen Studien weisen aber darauf hin, dass es einen Zusammenhang zwischen den berichteten Symptomen und Gewebeveränderungen geben könnte. Im Sinne der Patientensicherheit werden weitere regulatorische Maßnahmen zur Risikominimierung sowie präklinische und klinische Studien angestrebt

Structural Basis of Thermal Stability of the Tungsten Cofactor Synthesis Protein MoaB from Pyrococcus furiosus

Molybdenum and tungsten cofactors share a similar pterin based scaffold, which hosts an ene dithiolate function being essential for the coordination of either molybdenum or tungsten. The biosynthesis of both cofactors involves a multistep pathway, which ends with the activation of the metal binding pterin MPT by adenylylation before the respective metal is incorporated. In the hyperthermophilic organism Pyrococcus furiosus, the hexameric protein MoaB PfuMoaB has been shown to catalyse MPT adenylylation. Here we determined the crystal structure of PfuMoaB at 2.5 resolution and identified key residues of amp; 945;3 helix mediating hexamer formation. Given that PfuMoaB homologues from mesophilic organisms form trimers, we investigated the impact on PfuMoaB hexamerization on thermal stability and activity. Using structure guided mutagenesis, we successfully disrupted the hexamer interface in PfuMoaB. The resulting PfuMoaB H3 variant formed monomers, dimers and trimers as determined by size exclusion chromatography. Circular dichroism spectroscopy as well as chemical cross linking coupled to mass spectrometry confirmed a wild type like fold of the protomers as well as inter subunits contacts. The melting temperature of PfuMoaB H3 was found to be reduced by more than 15 C as determined by differential scanning calorimetry, thus demonstrating hexamerization as key determinant for PfuMoaB thermal stability. Remarkably, while a loss of activity at temperatures higher than 50 C was observed in the PfuMoaB H3 variant, at lower temperatures, we determined a significantly increased catalytic activity. The latter suggests a gain in conformational flexibility caused by the disruption of the hexamerization interfac

Gold Nanoparticles Decorated with Oligo(ethylene glycol) Thiols: Enhanced Hofmeister Effects in Colloid-Protein Mixtures

Oligo(ethylene glycol) (OEG) thiol self-assembled monolayer (SAM) decorated gold nanoparticles (AuNPs) have potential applications in bionanotechnology due to their unique property of preventing the nonspecific absorption of protein on the colloidal surface. For colloid-protein mixtures, a previous study (Zhang et al. J. Phys. Chem. A 2007,111, 12229) has shown that the OEG SAM-coated AuNPs become unstable upon addition of proteins (BSA) above a critical concentration, c*. This has been explained as a depletion effect in the two-component system. Adding salt (NaCl) can reduce the value of c*; that is, reduce the stability of the mixture. In the present work, we study the influence of the nature of the added salt on the stability of this two-component colloid-protein system. It is shown that the addition of various salts does not change the stability of either protein or colloid in solution in the experimental conditions of this work, except that sodium sulfate can destabilize the colloidal solutions. In the binary mixtures, however, the stability of colloid-protein mixtures shows significant dependence on the nature of the salt: chaotropic salts (NaSCN, NaClO 4, NaNO 3, MgCl 2) stabilize the system with increasing salt concentration, while kosmotropic salts (NaCl, Na 2SO 4, NH 4Cl) lead to the aggregation of colloids with increasing salt concentration. These observations indicate that the Hofmeister effect can be enhanced in two-component systems; that is, the modification of the colloidal interface by ions changes significantly the effective depletive interaction via proteins. Real time SAXS measurements confirm in all cases that the aggregates are in an amorphous state. © 2009 American Chemical Society

Long-Term Immunity against Actual Poxviral HLA Ligands as Identified by Differential Stable Isotope Labeling.

Viral peptides are presented by HLA class I on infected cells to activate CD8(+) T cells. Several immunogenic peptides have been identified indirectly by epitope prediction and screening of T cell responses to poxviral vectors, including modified vaccinia virus Ankara (MVA) currently being tested as recombinant or smallpox vaccines. However, for the development of optimal vaccination and immunomonitoring strategies, it is essential to characterize the actual viral HLA ligand repertoire of infected cells. We used an innovative approach to identify naturally processed MVA HLA ligands by differential HPLC-coupled mass spectrometry. We describe 12 viral peptides presented by HLA-A*0201 and 3 by HLA-B*0702. All HLA-A*0201 ligands participated in the memory response of MVA-immune donors, and several were immunogenic in Dryvax vaccinees. Eight epitopes were novel. Viral HLA ligand presentation and viral protein abundance did not correlate. All ligands were expressed early during the viral life cycle, and a pool of three of these mediated stronger protection against a lethal challenge in mice as compared with late epitopes. This highlights the reliability of the comparative mass spectrometry-based technique to identify relevant viral CD8(+) T cell epitopes for optimizing the monitoring of protective immune responses and the development of effective peptide-based vaccines

Phosphoproteomic Analysis Reveals Regulatory Mechanisms at the Kidney Filtration Barrier

aerial view, park as situated on the Mississippi River, 11/13/200

Comparative phosphoproteomic analysis of mammalian glomeruli reveals conserved podocin C-terminal phosphorylation as a determinant of slit diaphragm complex architecture

Glomerular biology is dependent on tightly controlled signal transduction networks that control phosphorylation of signaling proteins such as cytoskeletal regulators or slit diaphragm proteins of kidney podocytes. Cross-species comparison of phosphorylation events is a powerful mean to functionally prioritize and identify physiologically meaningful phosphorylation sites. Here, we present the result of phosphoproteomic analyses of cow and rat glomeruli to allow cross-species comparisons. We discovered several phosphorylation sites with potentially high biological relevance, e.g. tyrosine phosphorylation of the cytoskeletal regulator synaptopodin and the slit diaphragm protein neph-1 (Kirrel). Moreover, cross-species comparisons revealed conserved phosphorylation of the slit diaphragm protein nephrin on an acidic cluster at the intracellular terminus and conserved podocin phosphorylation on the very carboxyl terminus of the protein. We studied a highly conserved podocin phosphorylation site in greater detail and show that phosphorylation regulates affinity of the interaction with nephrin and CD2AP. Taken together, these results suggest that species comparisons of phosphoproteomic data may reveal regulatory principles in glomerular biology. All MS data have been deposited in the ProteomeXchange with identifier PXD001005 ()