Contribution of Specific Residues of the β-Solenoid Fold to HET-s Prion Function,Amyloid Structure and Stability

The [Het-s] prion of the fungus Podospora anserina represents a good model system for studying the structure-function relationship in amyloid proteins because a high resolution solid-state NMR structure of the amyloid prion form of the HET-s prion forming domain (PFD) is available. The HET-s PFD adopts a specific β-solenoid fold with two rungs of β-strands delimiting a triangular hydrophobic core. A C-terminal loop folds back onto the rigid core region and forms a more dynamic semi-hydrophobic pocket extending the hydrophobic core. Herein, an alanine scanning mutagenesis of the HET-s PFD was conducted. Different structural elements identified in the prion fold such as the triangular hydrophobic core, the salt bridges, the asparagines ladders and the C-terminal loop were altered and the effect of these mutations on prion function, fibril structure and stability was assayed. Prion activity and structure were found to be very robust; only a few key mutations were able to corrupt structure and function. While some mutations strongly destabilize the fold, many substitutions in fact increase stability of the fold. This increase in structural stability did not influence prion formation propensity in vivo. However, if an Ala replacement did alter the structure of the core or did influence the shape of the denaturation curve, the corresponding variant showed a decreased prion efficacy. It is also the finding that in addition to the structural elements of the rigid core region, the aromatic residues in the C-terminal semi-hydrophobic pocket are critical for prion propagation. Mutations in the latter region either positively or negatively affected prion formation. We thus identify a region that modulates prion formation although it is not part of the rigid cross-β core, an observation that might be relevant to other amyloid models.     

The Iberian Peninsula as a potential source for the plant species pool in Germany under projected climate change

The application of niche-based modelling techniques to plant species has not been explored for the majority of taxa in Europe, primarily due to the lack of adequate distributional data. However, it is of crucial importance for conservation adaptation decisions to assess and quantify the likely pool of species capable of colonising a particular region under altered future climate conditions. We here present a novel method that combines the species pool concept and information about shifts in analogous multidimensional climate space. This allows us to identify regions in Europe with a current climate which is similar to that projected for future time periods in Germany. We compared the extent and spatial location of climatically analogous European regions for three projected greenhouse gas emission scenarios in Germany for the time period 2071–2080 (+2.4°C, +3.3°C, +4.5°C average increase in mean annual temperature) to those of the recent past in Europe (1961–90). Across all three scenarios, European land areas which are characterised by climatic conditions analogue to those found in Germany decreased from 14% in 1961–1990 to ca. 10% in 2071–2080. All scenarios show disappearing current climate types in Germany, which can mainly be explained with a general northwards shift of climatically analogous regions. We estimated the size of the potential species pool of these analogous regions using floristic inventory data for the Iberian Peninsula as 2,354 plant species. The identified species pool in Germany indicates a change towards warmth and drought adapted southern species. About one-third of the species from the Iberian analogous regions are currently already present in Germany. Depending on the scenario used, 1,372 (+2.4°C average change of mean annual temperature), 1,399 (+3.3°C) and 1,444 (+4.5°C) species currently not found in Germany, occur in Iberian regions which are climatically analogous to German 2071–80 climate types. We believe that our study presents a useful approach to illustrate and quantify the potential size and spatial distribution of a pool of species potentially colonising new areas under changing climatic conditions.     

The self‐sufficient P450 RhF expressed in a whole cell system selectively catalyses the 5‐hydroxylation of diclofenac

P450 monooxygenases are able to catalyze the highly regio‐ and stereoselective oxidations of many organic molecules. However, the scale‐up of such bio‐oxidations remains challenging due to the often‐low activity, level of expression and stability of P450 biocatalysts. Despite these challenges they are increasingly desirable as recombinant biocatalysts, particularly for the production of drug metabolites. Diclofenac is a widely used anti‐inflammatory drug that is persistent in the environment along with the 4'‐ and 5‐hydroxy metabolites. Here we have used the self‐sufficient P450 RhF (CYP116B2) from Rhodococcus sp. in a whole cell system to reproducibly catalyze the highly regioselective oxidation of diclofenac to 5‐hydroxydiclofenac. The product is a human metabolite and as such is an important standard for environmental and toxicological analysis. Furthermore, access to significant quantities of 5‐hydroxydiclofenac has allowed us to demonstrate further oxidative degradation to the toxic quinoneimine product. Our studies demonstrate the potential for gram‐scale production of human drug metabolites through recombinant whole cell biocatalysis.     

ANTIFEEDING DEFENSE IN BALTIC MACROALGAE: INDUCTION BY DIRECT GRAZING VERSUS WATERBORNE CUES1

The prevalence of antigrazing defense induction and the cues triggering induction in marine macroalgae are generally not well understood. We examined the capacity of defense and the mechanisms of regulation in five common perennial macroalgal species from the Baltic Sea, Furcellaria lumbricalis (Huds.) J. V. Lamour., Delesseria sanguinea (Huds.) J. V. Lamour., Phyllophora pseudoceranoides (S. G. Gmel.) Newroth et A. R. A. Taylor, Fucus serratus L., and Fucus evanescens C. Agardh. Specifically, we investigated whether direct feeding and/or waterborne cues from feeding on neighboring conspecifics decreased the palatability of the tested algae. Direct feeding by the local isopod Idotea baltica triggered the induction of chemical defense in Fur. lumbricalis, D. sanguinea, P. pseudoceranoides, F. serratus, and F. evanescens. Conversely, we did not find any evidence for waterborne cues associated with feeding to trigger defense induction in neighboring conspecifics.     

Driving membrane curvature in clathrin-dependent and clathrin-independent endocytosis

Cellular activity depends to a large extent on membrane bilayer dynamics. Many processes, such as organelle biogenesis and vesicular transport, rely on alterations in membrane structure and shape. It is now widely accepted that intracellular membrane curvature generation and remodelling is mediated and regulated by protein action, and the mechanisms behind the processes are currently being revealed. Here, we will briefly discuss the key principles of membrane deformation and focus on different endocytic events that use various kinds of proteins to shape the plasma membrane into transport carriers. The entry routes are adopted to make sure that a vast variety of molecules on the cell surface can be regulated by endocytosis. The principles for membrane sculpting of endocytic carriers can be viewed either from a perspective of rigid coat budding or of flexible opportunistic budding. We will discuss these principles and their implications, focusing on clathrin-dependent and -independent carrier formation and the proteins involved in the respective pathways.     

Superresolution imaging of biological nanostructures by spectral precision distance microscopy

For the improved understanding of biological systems on the nanoscale, it is necessary to enhance the resolution of light microscopy in the visible wavelength range beyond the limits of conventional epifluorescence microscopy (optical resolution of about 200 nm laterally, 600 nm axially). Recently, various far-field methods have been developed allowing a substantial increase of resolution ("superresolution microscopy", or "lightoptical nanoscopy"). This opens an avenue to 'nano-image' intact and even living cells, as well as other biostructures like viruses, down to the molecular detail. Thus, it is possible to combine light optical spatial nanoscale information with ultrastructure analyses and the molecular interaction information provided by molecular cell biology. In this review, we describe the principles of spectrally assigned localization microscopy (SALM) of biological nanostructures, focusing on a special SALM approach, spectral precision distance/position determination microscopy (SPDM) with physically modified fluorochromes (SPDM(Phymod) . Generally, this SPDM method is based on high-precision localization of fluorescent molecules, which can be discriminated using reversibly bleached states of the fluorophores for their optical isolation. A variety of application examples is presented, ranging from superresolution microscopy of membrane and cytoplasmic protein distribution to dual-color SPDM of nuclear proteins. At present, we can achieve an optical resolution of cellular structures down to the 20-nm range, with best values around 5 nm (～1/100 of the exciting wavelength).     

Presenilin-dependent intramembrane proteolysis of CD44 leads to the liberation of its intracellular domain and the secretion of an Abeta-like peptide

Alzheimer's disease (AD)-associated gamma-secretase is a presenilin (PS)- dependent proteolytic activity involved in the intramembraneous cleavage of the beta-amyloid precursor protein, Notch, LDL receptor-related protein, E-cadherin, and ErbB-4. This cut produces the corresponding intracellular domains (ICD), which are required for nuclear signaling of Notch and probably ErbB-4, the beta-amyloid precursor protein, E-cadherin, and the LDL receptor-related protein as well. We have now investigated CD44, a cell surface adhesion molecule, which also undergoes an intramembraneous cleavage to liberate its ICD. We demonstrate that this cleavage requires a PS-dependent gamma-secretase activity. A loss-of-function PS1 mutation, a PS1/PS2 knockout, as well as two independent and highly specific gamma-secretase inhibitors, abolish this cleavage. Surprisingly, small peptides similar to the amyloid beta-peptide (Abeta) are generated by an additional cut in the middle of the transmembrane region of CD44. Like Abeta, these CD44 beta-peptides are generated in a PS-dependent manner. These findings therefore suggest a dual intramembraneous cleavage mechanism mediated by PS proteins. The dual cleavage mechanism is required for nuclear signaling as well as removal of remaining transmembrane domains, a general function of PS in membrane protein metabolism.     

"It's been good, not drinking": Alaska Native narratives of lifetime sobriety

Alcohol abuse is closely connected with so much hurt and pain in northern communities that it had to be addressed in this session. Much of what is done in the way of prevention and treatment of alcohol abuse originates from outside indigenous cultures. However, many Native people have either remained sober or become sober without ever going into a formal treatment program. Ironically, until very recently, little research effort has gone into understanding the backgrounds and attitudes of this population. “The People Awakening Project,” a collaborative effort between a group of Alaska Natives interested in sobriety and the University of Alaska Fairbanks, has changed that. Although the project is not finished, this presentation provides a clear sense of how the research is being conducted, what kinds of data are emerging from it, and what some of the preliminary results look like. Chase Hensel gave the original presentation in Quebec City. Svenne Haakenson and Gerry Mohatt, who are heavily involved in the project, join him in authoring this written version.     

The N‐Terminal Sequences of Four Major Hydrogenosomal Proteins Are Not Essential for Import into Hydrogenosomes of Trichomonas vaginalis

The human pathogen Trichomonas vaginalis harbors hydrogenosomes, organelles of mitochondrial origin that generate ATP through hydrogen‐producing fermentations. They contain neither genome nor translation machinery, but approximately 500 proteins that are imported from the cytosol. In contrast to well‐studied organelles like Saccharomyces mitochondria, very little is known about how proteins are transported across the two membranes enclosing the hydrogenosomal matrix. Recent studies indicate that—in addition to N‐terminal transit peptides—internal targeting signals might be more common in hydrogenosomes than in mitochondria. To further characterize the extent to which N‐terminal and internal motifs mediate hydrogenosomal protein targeting, we transfected Trichomonas with 24 hemagglutinin (HA) tag fusion constructs, encompassing 13 different hydrogenosomal and cytosolic proteins of the parasite. Hydrogenosomal targeting of these proteins was analyzed by subcellular fractionation and independently by immunofluorescent localization. The investigated proteins include some of the most abundant hydrogenosomal proteins, such as pyruvate ferredoxin oxidoreductase (PFO), which possesses an amino‐terminal targeting signal that is processed on import into hydrogenosomes, but is shown here not to be required for import into hydrogenosomes. Our results demonstrate that the deletion of N‐terminal signals of hydrogenosomal precursors generally has little, if any, influence upon import into hydrogenosomes. Although the necessary and sufficient signals for hydrogenosomal import recognition appear complex, targeting to the organelle is still highly specific, as demonstrated by the finding that six HA‐tagged glycolytic enzymes, highly expressed under the same promoter as other constructs studied here, localized exclusively to the cytosol and did not associate with hydrogenosomes.