Mechanism of intramembrane proteolysis investigated with purified rhomboid proteases

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.     

Model membrane studies for characterization of different antibiotic activities of lipopeptides from Pseudomonas

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.     

Bone Morphogenetic Protein 7 (BMP-7) Influences Tendon-Bone Integration In Vitro

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.     

Cutting proteins within lipid bilayers: rhomboid structure and mechanism

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.     

Comparing methods for measuring the rate of spread of invading populations

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.