SRP-mediated protein targeting: structure and function revisited

The signal recognition particle (SRP) and its membrane-bound receptor (SR) deliver membrane proteins and secretory proteins to the translocation channel in the plasma membrane (or the endoplasmic reticulum). The general outline of the SRP pathway is conserved in all three kingdoms of life. During the past decade, structure determination together with functional studies has brought our understanding of the SRP-mediated protein transport to an almost molecular level. An impressive amount of new information especially on the prokaryotic SRP is integrated into the current picture of the SRP pathway.     

Transgenic mice reveal novel activities of growth hormone in wound repair, angiogenesis, and myofibroblast differentiation

An increasing number of patients are being treated with growth hormone (GH) for the enhancement of body growth but also as an anti-aging strategy. However, the side effects of GH have been poorly defined. In this study we determined the effect of GH on wound repair and its mechanisms of action at the wound site. For this purpose, we performed wound healing studies in transgenic mice overexpressing GH. Full thickness incisional and excisional wounds of transgenic animals developed extensive, highly vascularized granulation tissue. However, wound bursting strength was not increased. Wound closure was strongly delayed as a result of enhanced granulation tissue formation and impaired wound contraction. The latter effect is most likely due to a significantly reduced number of myofibroblasts at the wound site. By using in vitro studies with stressed collagen lattices, we identified GH as an inhibitor of transforming growth factor beta-induced myofibroblast differentiation, resulting in a reduction in fibroblast contractile activity. These results revealed novel roles of GH in angiogenesis and myofibroblast differentiation, which are most likely not mediated via insulin-like growth factors at the wound site. Furthermore, our data suggested that systemic GH treatment is detrimental for wound healing in healthy individuals.     

Mouse lysozyme-M knockout mice reveal how the self-determinant hierarchy shapes the T cell repertoire against this circulating self antigen in wild-type mice

We have studied T cell tolerance to defined determinants within ML-M using wild-type (WT; ML-M(+/+)) and LysMcre (ML-M(-/-)) C3H (H-2(k)) mice to determine the relative contribution of ML-M-derived epitopes vs those from other self Ags in selection of the ML-M-specific T cell repertoire. ML-M was totally nonimmunogenic in WT mice, but was rendered immunogenic in LysMcre mice. Most of the response to ML-M in LysMcre mice was directed to the immunodominant determinant region 105-119. This determinant is spontaneously displayed (without adding exogenous ML-M) by macrophages of WT, but not LysMcre, mice and is stimulatory for peptide 105-119 (p105-119)-primed T cells. Moreover, neonatal tolerization of LysMcre mice with p105-119 or ML-M abrogated the T cell response to subsequent challenge with ML-M or p105-119. Furthermore, p95-109 and p110-125 of ML-M were immunogenic in LysMcre mice, but not in WT mice, thereby representing subdominant, tolerance-inducing epitopes of ML-M. As expected, the T cell repertoire to cryptic ML determinants in WT mice was also intact in LysMcre mice. Furthermore, the pattern of response to the related homologue of ML-M, hen eggwhite lysozyme, was similar in these two groups of mice. Thus, several codominant T cell determinants within ML-M contribute significantly to tolerance induction, and the anti-cryptic T cell repertoire to ML-M was positively selected on non-ML-M self ligands. These results reveal that the induction of self tolerance to a multideterminant protein follows the quantitative hierarchy of self-determinant expression and are of relevance in understanding the pathogenesis of autoimmunity.     

Structural analysis of the human Golgi-associated plant pathogenesis related protein GAPR-1 implicates dimerization as a regulatory mechanism

The plant pathogenesis related proteins group 1 (PR-1) and a variety of related mammalian proteins constitute a PR-1 protein family that share sequence and structural similarities. GAPR-1 is a unique family member as thus far it is the only PR-1 family member that is not co-translationally targeted to the lumen of the endoplasmic reticulum before trafficking to either vacuoles or secretion. Here we report that GAPR-1 may form dimers in vitro and in vivo, as determined by yeast two-hybrid screening, biochemical and biophysical assays. The 1.55A crystal structure demonstrates that GAPR-1 is structurally homologous to the other PR-1 family members previously solved (p14a and Ves V 5). Through an examination of inter-molecular interactions between GAPR-1 molecules in the crystal lattice, we propose a number of the highly conserved amino acid residues of the PR-1 family to be involved in the regulation of dimer formation of GAPR-1 with potential implications for other PR-1 family members. We show that mutagenesis of these conserved amino acid residues leads to a greatly increased dimer population. A recent report suggests that PR-1 family members may exhibit serine protease activity and further examination of the dimer interface of GAPR-1 indicates that a catalytic triad similar to that of serine proteases may be formed across the dimer interface by residues from both molecules within the dimer.     

Reconstituted TOM core complex and Tim9/Tim10 complex of mitochondria are sufficient for translocation of the ADP/ATP carrier across membranes

Precursor proteins of the solute carrier family and of channel forming Tim components are imported into mitochondria in two main steps. First, they are translocated through the TOM complex in the outer membrane, a process assisted by the Tim9/Tim10 complex. They are passed on to the TIM22 complex, which facilitates their insertion into the inner membrane. In the present study, we have analyzed the function of the Tim9/Tim10 complex in the translocation of substrates across the outer membrane of mitochondria. The purified TOM core complex was reconstituted into lipid vesicles in which purified Tim9/Tim10 complex was entrapped. The precursor of the ADP/ATP carrier (AAC) was found to be translocated across the membrane of such lipid vesicles. Thus, these components are sufficient for translocation of AAC precursor across the outer membrane. Peptide libraries covering various substrate proteins were used to identify segments that are bound by Tim9/Tim10 complex upon translocation through the TOM complex. The patterns of binding sites on the substrate proteins suggest a mechanism by which portions of membrane-spanning segments together with flanking hydrophilic segments are recognized and bound by the Tim9/Tim10 complex as they emerge from the TOM complex into the intermembrane space.     

Inhibin/activin subunits alpha, beta-A and beta-B are differentially expressed in normal human endometrium throughout the menstrual cycle

Inhibins are dimeric glycoproteins composed of an alpha () subunit and one of two possible beta (-) subunits (A or B). The aims of this study were to assess the frequency and tissue distribution patterns of the inhibin subunits in normal human endometrium. Samples from human endometrium from proliferative phase (PP; n=32), early secretory phase (ES; n=10) and late secretory phase (LS; n=12) were obtained. Immunohistochemistry, immunofluorescence and a statistical analysis were performed. All three inhibin subunits were expressed by normal endometrium by immunohistochemistry and immunofluorescence. Inhibin- was primarily detected in glandular epithelial cells, while inhibin- subunits were additionally localised in stromal tissue. Inhibin- staining reaction increased significantly between PP and ES (P<0.05), PP and LS (P<0.01), and ES and LS (P<0.02). Inhibin-A and -B were significant higher in LS than PP (P<0.05) and LS than ES (P<0.05). All three inhibin subunits were expressed by human endometrium varying across the menstrual cycle. This suggests substantial functions in human implantation of inhibin- subunit, while stromal expression of the subunits could be important in the paracrine signalling for adequate endometrial maturation. The distinct expression in human endometrial tissue suggests a synthesis of inhibins into the lumen and a predominant secretion of activins into the stroma.I. Mylonas and U. Jeschke contributed equally to this work     

Redistribution of adhering junctions in human endometrial epithelial cells during the implantation window of the menstrual cycle

The human uterine epithelium is characterised by remarkable plasticity with cyclic changes in differentiation that are controlled by ovarian steroid hormones to optimise conditions for embryo implantation. To understand whether and how cell-cell adhesion is affected, the localisation of junction proteins was studied throughout the menstrual cycle. Expression patterns were examined by immunofluorescence in 36 human endometrial specimens of different cycle stages. Antibodies against the desmosomal proteins desmoplakin 1/2 (Dp 1/2) and desmoglein 2 (Dsg 2), the adherens junction proteins E-cadherin and β-catenin and also the common junctional linker protein plakoglobin showed a strong subapical staining during the proliferative phase until the early luteal phase (day 20). In the mid- to late luteal phase, however, these junctional proteins redistributed over the entire lateral plasma membranes. In contrast, tight junction proteins (ZO-1, claudin 4) remained at their characteristic subapical position throughout the menstrual cycle. mRNA levels of Dp 1/2, E-cadherin and ZO-1 obtained by real time RT-PCR were not significantly changed during the menstrual cycle. The observed redistribution of desmosomes and adherens junctions coincides with the onset of the so called implantation window of human endometrium. We propose that this change is controlled by ovarian steroids and prepares the endometrium for successful trophoblast invasion.     

Fungal Growth and Biomass Development is Boosted by Plants in Snow-Covered Soil

Soil microbial communities follow distinct seasonal cycles which result in drastic changes in processes involving soil nutrient availability. The biomass of fungi has been reported to be highest during winter, but is fungal growth really occurring in frozen soil? And what is the effect of plant cover on biomass formation and on the composition of fungal communities? To answer these questions, we monitored microbial biomass N, ergosterol, and the amount of fungal hyphae during summer and winter in vegetated and unvegetated soils of an alpine primary successional habitat. The winter fungal communities were identified by rDNA ITS clone libraries. Winter soil temperatures ranged between -0.6°C and -0.1°C in snow-covered soil. We found distinct seasonal patterns for all biomass parameters, with highest biomass concentrations during winter in snow-covered soil. The presence of plant cover had a significant positive effect on the amount of biomass in the soil, but the type of plant cover (plant species) was not a significant factor. A mean hyphal ingrowth of 5.6 m g(-1) soil was detected in snow-covered soil during winter, thus clearly proving fungal growth during winter in snow-covered soil. Winter fungal communities had a typical species composition: saprobial fungi were dominating, among them many basidiomycete yeasts. Plant cover had no influence on the composition of winter fungal communities.     

The Relationship Between Foraging,Learning Abilities and Neophobia in Two Species of Darwin’s Finches

The ability to unlearn a previously established association is an important component of behavioural flexibility and may vary according to species ecology. Previously, two closely related sympatric Darwin’s finches were found to differ in their learning abilities. Small tree finches (Camarhynchus parvulus) outperformed woodpecker finches (Cactospiza pallida) in reversal learning but performed worse in an operant task. We attributed this difference to the habit of woodpecker finches to engage in long bouts of energetic pecking during extractive foraging. Persistently repeating one action without reward could favour performance in operant tasks but also limit behavioural flexibility. Here, we tested whether perseverance is the reason for woodpecker finches’ depressed reversal learning performance. Two new reversal conditions allowed the disentanglement of two sources of error in reversal learning: perseverant choice of the previously rewarded stimulus and failure to respond to the previously non‐rewarded stimulus. For the within‐species comparison, we predicted that woodpecker finches should find it more difficult to learn to avoid the previously rewarded stimulus than learning to choose the previously non‐rewarded stimulus. For the species comparison, we predicted the woodpecker finches should make more errors of perseverance than small tree finches. As performance could also be influenced by reaction to novelty, we compared neophobic responses between species and related them to reversal learning proficiency. We found no significant difference in reversal learning in the predicted direction, but found a negative correlation between neophobia and reversal learning at the inter‐ and the intraspecific level, which points towards a general relationship between reaction to novelty and flexibility.