To Degrade or not to Degrade : Protein Handling by the Proteasome

The proteasome, a large multi-subunit protease that degrades proteins, must be able to distinguish between substrates deemed for destruction and accessory proteins that should be spared. The shuttle factor Rad23 operates in close proximity to the proteasome in order to deliver proteins for their degradation. While its cargo is efficiently degraded, the shuttle factor itself remains unharmed. Rad23 is protected by its C-terminal ubiquitin-associated (UBA)-2 domain that has been identified to be a cisacting stabilization signal. A major aim of the work presented in this dissertation has been to decipher the mode of action responsible for this protective effect. Another protein that is targeted to the proteasome but resists degradation was studied in Paper I. UBB+1 is an aberrant product of the ubiquitin precursor gene UBB and comprises ubiquitin and a 19 amino acid-long C-terminal extension. Even though UBB+1 structurally resembles a ubiquitin fusion degradation (UFD) substrate, a class of proteins that is targeted for degradation by a non-cleavable N-terminal ubiquitin moiety, it is poorly degraded. We found that designed UFD substrates with equally short C-terminal extensions were stable. Extending their C-termini to 25 amino acids converted them into short-lived proteins, suggesting that the proteasome requires an unstructured polypeptide of a minimum length from which unfolding of the substrate may be initiated. In Paper II we further investigated the impact of unstructured polypeptide sequences on proteasomal degradation. We found that C-terminal initiation sites for proteasomal unfolding rendered the degradation of UFD substrates independent of the ubiquitin-binding chaperone Cdc48 and polyubiquitylation. This observation suggests that substrates bypassing the unfoldase activity of Cdc48 require an unstructured initiation site for their efficient degradation and implies that the chaperone complex acting upstream of the proteasome can be the primary determinant for the polyubiquitin-dependency of proteasomal degradation. Consistently, we found that the ubiquitin-like (UbL) domains of Rad23 and Dsk2, which interact with the proteasome in a polyubiquitin-independent manner, only sufficed to target proteins carrying initiation sites for degradation. The impact of C-terminal unstructured polypeptide sequences on proteasomal degradation prompted us to investigate the role of structural integrity in UBA-mediated protection in Paper III. To this end, we used the C-terminal UBA domain of the human protein p62 and took advantage of naturally occurring mutant domains that are linked to Paget s disease of bone. We found that not only the wild-type UBA domain could delay degradation of reporter substrates in yeast but also mutant domains that were thermally stable but impaired in their ubiquitin binding. This suggests that the UBA-mediated protective effect depends rather on the structural integrity than ubiquitin binding capability. Our finding that proteins targeted to the proteasome through a UbL domain require an unstructured initiation site for efficient degradation turned out to be of significance for understanding the molecular mechanism behind Rad23 protection and encouraged us to study in Paper IV whether the protective UBA domains interfered with unfolding. Strikingly, introduction of C-terminal unstructured polypeptides turned the shuttle factor Rad23 into an efficiently degraded proteasome substrate. Positioning the UBA2 domain C-terminally to an adjacent unstructured polypeptide inhibited degradation, whereas non-protective UBA domains were able to function as initiation sites themselves. In summary, we provide evidence that the protective effect of UBA domains is mediated by preventing initiation of degradation by the proteasome. These molecular insights help explain how proteasomes decide which proteins to degrade and which proteins to spare and thus how shuttle factors can deliver substrates to the proteasome without themselves becoming subject to degradation

Sind die Erhöhungen der Lebensmittelpreise gerechtfertigt?

Die seit einiger Zeit einsetzenden Preissteigerungen für Getreide, Milch und Milcherzeugnisse verunsichern die Verbraucher, waren Nahrungsmittelpreise doch über Jahrzehnte stabil. Welche Entwicklungen haben zu den Preisanhebungen geführt? Für Stefan Tangermann, OECD, sind die Märkte für Agrarprodukte und damit auch diejenigen für Lebensmittel in der EU "in ein neues Zeitalter eingetreten". Die Agrarpolitik sei reformiert worden, und damit seien die Agrarpreise in der EU zurückgegangen. Gleichzeitig seien die Weltmarktpreise für Agrarprodukte deutlich angestiegen. Beide Preisbewegungen hätten sich jetzt getroffen, und zum ersten Mal führten die Entwicklungen an den weltweiten Agrarmärkten auch in der EU zu Preisbewegungen. Auch Ursula Heinen, Bundesministerium für Ernährung, Landwirtschaft und Verbraucherschutz, weist darauf hin, dass die Funktion des Preises als Indikator für Knappheit auf den Agrarmärkten wieder stärker zum Tragen kommt und Produktionsentscheidungen in der Landwirtschaft vom Marktpreis und nicht von staatlich festgelegten Stützpreisen bestimmt werden. In der Gesellschaft sei ein Umdenken notwendig: "Wer Gutes erwartet - und die hier produzierten Lebensmittel erfüllen diese Erwartungen - muss auch bereit sein, hierfür einen angemessenen Preis zu zahlen." Christian Thorun, Verbraucherzentrale Bundesverband, schätzt die Erhöhung der Lebensmittelpreise "von der Tendenz her berechtigt, in der Höhe nicht immer nachvollziehbar" ein. Bernhard Brümmer, Universität Göttingen, weist darauf hin, dass zwar die Entwicklung, die insbesondere seit dem zweiten Quartal dieses Jahres zu beobachten sei, zu mehr als einer Verdopplung der (nominalen) Preise seit 2005 geführt habe. Es sei aber auch klar, dass damit bei weitem noch nicht die Preisspitzen beispielsweise der siebziger Jahre erreicht worden seien. Es handele sich bei den heute geltenden Preisen keineswegs um historische Höchststände.Nahrungsmitelpreis, Agrarpolitik, EU-Staaten

Direct evidence for attention-dependent influences of the frontal eye-fields on feature-responsive visual cortex

Voluntary selective attention can prioritize different features in a visual scene. The frontal eye-fields (FEF) are one potential source of such feature-specific top-down signals, but causal evidence for influences on visual cortex (as was shown for "spatial" attention) has remained elusive. Here, we show that transcranial magnetic stimulation (TMS) applied to right FEF increased the blood oxygen level-dependent (BOLD) signals in visual areas processing "target feature" but not in "distracter feature"-processing regions. TMS-induced BOLD signals increase in motion-responsive visual cortex (MT+) when motion was attended in a display with moving dots superimposed on face stimuli, but in face-responsive fusiform area (FFA) when faces were attended to. These TMS effects on BOLD signal in both regions were negatively related to performance (on the motion task), supporting the behavioral relevance of this pathway. Our findings provide new causal evidence for the human FEF in the control of nonspatial "feature"-based attention, mediated by dynamic influences on feature-specific visual cortex that vary with the currently attended propert

Conceptualizing Micromobility

While micromobility has seen a significant rise of interest across policy, industry and academia, a detailed conceptualisation of it has so far been missing from the scientific literature. This paper develops a multi-dimensional conceptualisation of micromobility, in conjunction with a new socio-technical definition. To do so, it reviews related concepts; it analyses how the term micromobility has been used; and it critically engages with existing definitions most frequently cited in this literature. Building on these insights, we develop a multi-dimensional conceptualization of micromobility. Our definition of micromobility covers a wide range of mobility options that can typically be manoeuvred by one human without motor assistance, at least for short distances, and that are ‘micro’ in terms of energy demand, environmental impact, and use of road space, relative to automobility. According to our conceptualisation, micromobility modes comprise fully human powered, partially motor assisted and fully powered options. They typically do not exceed 25 kilometres per hour (or 45 for faster ones) and weigh (often significantly) less than 350 kilogram, while often providing some (public) health benefits. Trip lengths are typically less than 15 kilometres and daily distance travelled less than 80 kilometres. This new definition has relevance for future transport and mobility scholarship, as well as policy and evaluation. Advantages of a new and widely accepted definition and conceptualisation of micromobility could include more robust design standards, legislation, as well as evaluation metrics and methods, all leading to greater understanding of, and attention paid to, this form of mobility. This paper highlights the important role that micromobilities could play in moving beyond automobility, to create more sustainable and just mobility futures

Parietal stimulation decouples spatial and feature-based attention

Everyday visual scenes contain a vast quantity of information, only a fraction of which can guide our behavior. Properties such as the location, color and orientation of stimuli help us extract relevant information from complex scenes (Treisman and Gelade, 1980; Livingstone and Hubel, 1987). But how does the brain coordinate the selection of such different stimulus characteristics? Neuroimaging studies have revealed significant regions of overlapping activity in frontoparietal cortex during attention to locations and features, suggesting a global component to visual selection (Vandenberghe et al., 2001; Corbetta and Shulman, 2002; Giesbrecht et al., 2003; Slagter et al., 2007). At the same time, the neural consequences of spatial and feature-based attention differ markedly in early visual areas (Treue and Martinez-Trujillo, 2007), implying that selection may rely on more specific top-down processes. Here we probed the balance between specialized and generalized control by interrupting preparatory attention in the human parietal cortex with transcranial magnetic stimulation (TMS). We found that stimulation of the supramarginal gyrus (SMG) impaired spatial attention only, whereas TMS of the anterior intraparietal sulcus (aIPS) disrupted spatial and feature-based attention. The selection of different stimulus characteristics is thus mediated by distinct top-down mechanisms, which can be decoupled by cortical interference

Nutritional Control of DNA Replication Initiation through the Proteolysis and Regulated Translation of DnaA

Bacteria can arrest their own growth and proliferation upon nutrient depletion and under various stressful conditions to ensure their survival. However, the molecular mechanisms responsible for suppressing growth and arresting the cell cycle under such conditions remain incompletely understood. Here, we identify post-transcriptional mechanisms that help enforce a cell-cycle arrest in Caulobacter crescentus following nutrient limitation and during entry into stationary phase by limiting the accumulation of DnaA, the conserved replication initiator protein. DnaA is rapidly degraded by the Lon protease following nutrient limitation. However, the rate of DnaA degradation is not significantly altered by changes in nutrient availability. Instead, we demonstrate that decreased nutrient availability downregulates dnaA translation by a mechanism involving the 5' untranslated leader region of the dnaA transcript; Lon-dependent proteolysis of DnaA then outpaces synthesis, leading to the elimination of DnaA and the arrest of DNA replication. Our results demonstrate how regulated translation and constitutive degradation provide cells a means of precisely and rapidly modulating the concentration of key regulatory proteins in response to environmental inputs.National Institutes of Health (U.S.) (Grant 5R01GM082899

Methylxanthines enhance the effects of cocoa flavanols on cardiovascular function: randomized, double-masked controlled studies

Background: Cocoa flavanol intake, especially that of (−)-epicatechin, has been linked to beneficial effects on human cardiovascular function. However, cocoa also contains the methylxanthines theobromine and caffeine, which may also affect vascular function. Objective: We sought to determine whether an interaction between cocoa flavanols and methylxanthines exists that influences cocoa flavanol–dependent vascular effects. Design: Test drinks that contained various amounts of cocoa flavanols (0–820 mg) and methylxanthines (0–220 mg), either together or individually, were consumed by healthy volunteers (n = 47) in 4 different clinical studies—3 with a randomized, double-masked crossover design and 1 with 4 parallel crossover studies. Vascular status was assessed by measuring flow-mediated vasodilation (FMD), brachial pulse wave velocity (bPWV), circulating angiogenic cells (CACs), and blood pressure before and 2 h after the ingestion of test drinks. Results: Although cocoa flavanol intake increased FMD 2 h after intake, the consumption of cocoa flavanols with methylxanthines resulted in a greater enhancement of FMD. Methylxanthine intake alone did not result in statistically significant changes in FMD. Cocoa flavanol ingestion alone decreased bPWV and diastolic blood pressure and increased CACs. Each of these changes was more pronounced when cocoa flavanols and methylxanthines were ingested together. It is important to note that the area under the curve of the plasma concentration of (−)-epicatechin metabolites over time was higher after the co-ingestion of cocoa flavanols and methylxanthines than after the intake of cocoa flavanols alone. Similar results were obtained when pure (−)-epicatechin and the methylxanthines theobromine and caffeine were consumed together. Conclusion: A substantial interaction between cocoa flavanols and methylxanthines exists at the level of absorption, in which the methylxanthines mediate an increased plasma concentration of (−)-epicatechin metabolites that coincides with enhanced vascular effects commonly ascribed to cocoa flavanol intake

Motor outcome, executive functioning, and health‐related quality of life of children, adolescents, and young adults after ventricular assist device and heart transplantation

Objective The aim of the current study is to measure long‐term executive function, motor outcome, and QoL in children, adolescents, and young adults after VAD and Htx. Methods Patients were examined during routine follow‐up. Investigation tools were used as follows: Examination for MND of motor outcomes, Epitrack® for attention and executive functioning, and Kidscreen‐52 and EQ‐5D‐5L questionnaires for QoL. Additional data were retrospectively obtained by an analysis of patient medical records. Results Out of 145 heart transplant recipients at the department of pediatric cardiology of the University Hospital Munich, 39 were implanted with a VAD between 1992 and 2016. Seventeen (43.6%) patients died before or after Htx; 22 (56.4%) patients were included in our study. Mean age at transplant was 9.52 years (range: 0.58‐24.39 years, median 9), and the mean follow‐up time after Htx was 6.18 years (range: 0.05‐14.60 years, median 5.82). MND examination could be performed in 13 patients (normal MND: n = 11, simple MND: n = 1, complex MND: n = 1). Executive functioning was tested in 15 patients. Two (13.3%) patients had good results, six (40%) average results, three (20%) borderline results, and four (26.7%) impaired results. QoL (Kidscreen n = 7, EQ‐5D‐5L n = 8) was similar to a healthy German population. Conclusion Motor outcome, executive functioning and QoL in survivors of VAD bridging therapy and Htx can be good, though underlying diseases and therapies are associated with a high risk of cerebral ischemic or hemorrhagic complications

Poly(2-dimethylamino ethylmethacrylate)-Based Polymers To Camouflage Red Blood Cell Antigens

peer reviewedPoly(2-dimethylamino-ethylmethacrylate) (PDMAEMA) is a cationic polymer when dissolved in a 7.4 pH fluid. Owing to its ionic nature, this polycation interacts with the negatively charged cell membrane surface of red blood cells (RBCs). The electrostatic self-assembly of PDMAEMA on RBCs membrane can be employed for inducing the formation of a polymeric shield camouflaging blood group antigens on RBCs as a valuable strategy for developing “universal RBCs” for blood transfusion. The purpose of this research was to evaluate the camouflaging ability of PDMAEMA homopolymers and PDMAEMA-copoly(nethylene glycol) copolymers differing in molecular weight and architecture. Surprisingly, the PDMAEMAs caused a partially masking, no masking, and sensitization of the same RBCs population. The MW and architecture of the polymers as well as temperature of PDMAEMA-RBCs treatment influenced the results observed. Herein, the very particular reactivity of PDMAEMAs and RBCs is analyzed and discussed