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1.
Intramembrane proteases have the unusual property of cleaving peptide bonds within the lipid bilayer, an environment not obviously suited to a water-requiring hydrolysis reaction. These enzymes include site-2 protease, gamma-secretase/presenilin, signal peptide peptidase and the rhomboids, and they have a wide range of cellular functions. All have multiple transmembrane domains and, because of their high hydrophobicity, have been difficult to purify. We have now developed an in vitro assay to monitor rhomboid activity in the detergent solubilised state. This has allowed us to isolate for the first time a highly pure rhomboid with catalytic activity. Our results suggest that detergent-solubilised rhomboid activity mimics its activity in biological membranes in many aspects. Analysis of purified mutant proteins suggests that rhomboids use a serine protease catalytic dyad instead of the previously proposed triad. This analysis also suggests that other conserved residues participate in subsidiary functions like ligand binding and water supply. We identify a motif shared between rhomboids and the recently discovered derlins, which participate in translocation of misfolded membrane proteins. 相似文献
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Reder-Christ K Schmidt Y Dörr M Sahl HG Josten M Raaijmakers JM Gross H Bendas G 《Biochimica et biophysica acta》2012,1818(3):566-573
Lipopeptides (LPs) are a structurally diverse class of amphipathic natural products that were in the past mainly known for their surfactant properties. However, the recent discovery of their antimicrobial and cytotoxic bioactivities have fueled and renewed the interest in this compound class. Propelled by the antimicrobial potential of this compound class, in this study a range of six underinvestigated LPs from Pseudomonads were examined with respect to their antibiotic activities towards bacteria. The assays revealed that only the glycosylated lipodipeptide SB-253514, produced by Pseudomonas strain SH-C52, showed significant antibacterial activity. Since the bioactivity of LPs is commonly attributed to membrane interactions, we analyzed the molecular interactions between the LPs and bacteria-like lipid model membranes in more detail via complementary biophysical approaches. Application of the quartz crystal microbalance (QCM) showed that all LPs possess a high binding affinity towards the model membranes. Despite their similar membrane affinity, monolayer studies displayed different tendencies of LPs to incorporate into the membrane. The degree of membrane incorporation could be correlated with specific structural features of the investigated LPs, such as distance between the peptidic macrocycle and the fatty acid, but did not fully reflect their respective antibacterial activity. Cyclic voltammetry (CV) experiments further demonstrated that SB-253514 showed no membrane permeabilization effects at inhibitory concentrations. Collectively, these results suggests that the antibacterial activity of SB-253514 cannot be explained by an unspecific detergent-like mechanism generally proposed for amphiphilic molecules but instead appears to occur via a defined structural target. 相似文献
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Kjartan
stbye Marius Hagen Hassve Ana‐Maria Peris Tamayo Mari Hagenlund Thomas Vogler Kim Prbel 《Evolutionary Applications》2020,13(6):1240-1261
The origin of species is a central topic in biology. Ecological speciation might be a driver in adaptive radiation, providing a framework for understanding mechanisms, level, and rate of diversification. The Arctic charr Salvelinus alpinus L. is a polymorphic species with huge morphological and life‐history diversity in Holarctic water systems. We studied adaptive radiation of Arctic charr in the 460‐m‐deep Lake Tinnsjøen to (a) document eco‐morphology and life‐history traits of morphs, (b) estimate reproductive isolation of morphs, and (c) illuminate Holarctic phylogeography and lineages colonizing Lake Tinnsjøen. We compared Lake Tinnsjøen with four Norwegian outgroup populations. Four field‐assigned morphs were identified in Lake Tinnsjøen: the planktivore morph in all habitats except deep profundal, the dwarf morph in shallow‐moderate profundal, the piscivore morph mainly in shallow‐moderate profundal, and a new undescribed abyssal morph in the deep profundal. Morphs displayed extensive life‐history variation in age and size. A moderate‐to‐high concordance was observed among morphs and four genetic clusters from microsatellites. mtDNA suggested two minor endemic clades in Lake Tinnsjøen originating from one widespread colonizing clade in the Holarctic. All morphs were genetically differentiated at microsatellites (FST: 0.12–0.20), associated with different mtDNA clade frequencies. Analyses of outgroup lakes implied colonization from a river below Lake Tinnsjøen. Our findings suggest postglacial adaptive radiation of one colonizing mtDNA lineage with niche specialization along a depth–temperature–productivity–pressure gradient. Concordance between reproductive isolation and habitats of morphs implies ecological speciation as a mechanism. Particularly novel is the extensive morph diversification with depth into the often unexplored deepwater profundal habitat, suggesting we may have systematically underestimated biodiversity in lakes. In a biological conservation framework, it is imperative to protect endemic below‐species‐level biodiversity, particularly so since within‐species variation comprises an extremely important component of the generally low total biodiversity observed in the northern freshwater systems. 相似文献
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Markus Oberpaul Stephan Brinkmann Michael Marner Sanja Mihajlovic Benedikt Leis Maria A. Patras Christoph Hartwig Andreas Vilcinskas Peter E. Hammann Till F. Schäberle Marius Spohn Jens Glaeser 《Microbial biotechnology》2022,15(2):415-430
High-throughput platforms facilitating screening campaigns of environmental samples are needed to discover new products of natural origin counteracting the spreading of antimicrobial resistances constantly threatening human and agricultural health. We applied a combination of droplet microfluidics and fluorescence-activated cell sorting (FACS)-based technologies to access and assess a microbial environmental sample. The cultivation performance of our microfluidics workflow was evaluated in respect to the utilized cultivation media by Illumina amplicon sequencing of a pool of millions of droplets, respectively. This enabled the rational selection of a growth medium supporting the isolation of microbial diversity from soil (five phyla affiliated to 57 genera) including a member of the acidobacterial subgroup 1 (genus Edaphobacter). In a second phase, the entire diversity covered by 1071 cultures was used for an arrayed bioprospecting campaign, resulting in > 6000 extracts tested against human pathogens and agricultural pests. After redundancy curation by using a combinatorial chemical and genomic fingerprinting approach, we assigned the causative agents present in the extracts. Utilizing UHPLC-QTOF-MS/MS-guided fractionation and microplate-based screening assays in combination with molecular networking the production of bioactive ionophorous macrotetrolides, phospholipids, the cyclic lipopetides massetolides E, F, H and serratamolide A and many derivatives thereof was shown. 相似文献
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Tim Schwarting Philipp Lechler Johannes Struewer Marius Ambrock Thomas Manfred Frangen Steffen Ruchholtz Ewgeni Ziring Michael Frink 《PloS one》2015,10(2)
IntroductionSuccessful graft ingrowth following reconstruction of the anterior cruciate ligament is governed by complex biological processes at the tendon-bone interface. The aim of this study was to investigate in an in vitro study the effects of bone morphogenetic protein 7 (BMP-7) on tendon-bone integration.ResultsIn both models, positive effects of BMP-7 on ALP enzyme activity were observed (p<0.001). Additionally, similar results were noted for LDH activity and lactate concentration. BMP-7 stimulation led to a significant increase in OCN expression. Whereas the effects of BMP-7 on tendon monoculture peaked during an early phase of the experiment (p<0.001), the cocultures showed a maximal increase during the later stages (p<0.001). The histological analysis showed a stimulating effect of BMP-7 on extracellular matrix formation. Organized ossification zones and calcium carbonate-like structures were only observed in the BMP-stimulated cell cultures.DiscussionThis study showed the positive effects of BMP-7 on the biological process of tendon-bone integration in vitro. Histological signs of improved mineralization were paralleled by increased rates of osteoblast-specific protein levels in primary bovine osteoblasts and fibroblasts.ConclusionOur findings indicated a role for BMP-7 as an adjuvant therapeutic agent in the treatment of ligamentous injuries, and they emphasized the importance of the transdifferentiation process of tendinous fibroblasts at the tendon-bone interface. 相似文献
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Rhomboids were only discovered to be novel proteases in 2001, but progress on understanding this newest family of intramembrane proteases has been rapid. They are now the best characterized of these rather mysterious enzymes that cleave transmembrane domains within the lipid bilayer. In particular, the biochemical analysis of solubilized rhomboids and, most recently, a flurry of high-resolution crystal structures, have led to real insight into their enzymology. Long-standing questions about how it is possible for a water-requiring proteolytic reaction to occur in the lipid bilayer are now answered for the rhomboids. Intramembrane proteases, which control many medically important biological processes, have made the transition from rather heretical outsiders to novel enzymes that are becoming well understood. 相似文献
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Measuring rates of spread during biological invasions is important for predicting where and when invading organisms will spread in the future as well as for quantifying the influence of environmental conditions on invasion speed. While several methods have been proposed in the literature to measure spread rates, a comprehensive comparison of their accuracy when applied to empirical data would be problematic because true rates of spread are never known. This study compares the performances of several spread rate measurement methods using a set of simulated invasions with known theoretical spread rates over a hypothetical region where a set of sampling points are distributed. We vary the density and distribution (aggregative, random, and regular) of the sampling points as well as the shape of the invaded area and then compare how different spread rate measurement methods accommodate these varying conditions. We find that the method of regressing distance to the point of origin of the invasion as a function of time of first detection provides the most reliable method over adverse conditions (low sampling density, aggregated distribution of sampling points, irregular invaded area). The boundary displacement method appears to be a useful complementary method when sampling density is sufficiently high, as it provides an instantaneous measure of spread rate, and does not require long time series of data. 相似文献