Vacuolin, a flotillin/reggie-related protein from Dictyostelium oligomerizes for endosome association

We have analysed the domain structure of vacuolin, a Dictyostelium protein binding to the cytoplasmic surface of late endosomes. Localisation studies using GFP fusions together with a yeast two-hybrid analysis and co-immunoprecipitation experiments reveal that a region close to the C-terminus mediates oligomer formation of the protein through a coiled-coil mechanism which in turn is a prerequisite for the efficient binding to endosomal membranes via a prohibitin (PHB) domain in the middle of the molecule. Overexpression of the coiled-coil domain strongly competes with endogenous vacuolin in the oligomers and reduces the efficiency of membrane targeting. The domain arrangement of vacuolin is most similar to flotillin/reggie, a protein found on late endosomes of mammalian cells.     

Interactions Between Pattern Formation and Domain Growth

In this paper we develop a theoretical framework for investigating pattern formation in biological systems for which the tissue on which the spatial pattern resides is growing at a rate which is itself regulated by the diffusible chemicals that establish the spatial pattern. We present numerical simulations for two cases of interest, namely exponential domain growth and chemically controlled growth. Our analysis reveals that for domains undergoing rapid exponential growth dilution effects associated with domain growth influence both the spatial patterns that emerge and the concentration of chemicals present in the domain. In the latter case, there is complex interplay between the effects of the chemicals on the domain size and the influence of the domain size on the formation of patterns. The nature of these interactions is revealed by a weakly nonlinear analysis of the full system. This yields a pair of nonlinear equations for the amplitude of the spatial pattern and the domain size. The domain is found to grow (or shrink) at a rate that depends quadratically on the pattern amplitude, the particular functional forms used to model the local tissue growth rate and the kinetics of the two diffusible species dictating the resulting behaviour.     

Structural separation of different extracellular activities in aminoacyl-tRNA synthetase-interacting multi-functional protein, p43/AIMP1

AIMP1 (previously known as p43) is first found as a factor associated with a macromolecular tRNA synthetase complex. However, it is also secreted and acts on diverse target cells such as endothelial cells, macrophages, and fibroblasts to control angiogenesis, inflammation, and dermal regeneration, respectively. We previously showed that AIMP1 induces the death of endothelial cell but proliferation of fibroblasts and activates macrophages. In this work, we found that elastase 2-cleaved AIMP1 retained its pro-apoptotic activity to endothelial cells but lost the growth-stimulatory activity to fibroblasts. To determine the functional domains responsible for each activity, we generated several deletion fragments of AIMP1 and compared the activities to the target cells. AIMP1 promoted endothelial cell death and caspase-3 activation through its 101-114 amino acid region, fibroblast proliferation through its 6-46 amino acid region, and endothelial migration through its 114-192 amino acid region as revealed by deletion mapping. Thus, this work revealed that AIMP1 uses different regions for its diverse extracellular activities.     

Membrane topology of human ABC proteins

In this review, we summarize the currently available information on the membrane topology of some key members of the human ABC protein subfamilies, and present the predicted domain arrangements. In the lack of high-resolution structures for eukaryotic ABC transporters this topology is based only on prediction algorithms and biochemical data for the location of various segments of the polypeptide chain, relative to the membrane. We suggest that topology models generated by the available prediction methods should only be used as guidelines to provide a basis of experimental strategies for the elucidation of the membrane topology.     

MELDB: a database for microbial esterases and lipases

MELDB is a comprehensive protein database of microbial esterases and lipases which are hydrolytic enzymes important in the modern industry. Proteins in MELDB are clustered into groups according to their sequence similarities based on a local pairwise alignment algorithm and a graph clustering algorithm (TribeMCL). This differs from traditional approaches that use global pairwise alignment and joining methods. Our procedure was able to reduce the noise caused by dubious alignment in the distantly related or unrelated regions in the sequences. In the database, 883 esterase and lipase sequences derived from microbial sources are deposited and conserved parts of each protein are identified. HMM profiles of each cluster were generated to classify unknown sequences. Contents of the database can be keyword-searched and query sequences can be aligned to sequence profiles and sequences themselves.     

Crystal structure of Thermus caldophilus phosphoglycerate kinase in the open conformation

Phosphoglycerate kinase (PGK) is a key glycolytic enzyme that catalyzes the reversible transfer of a phosphate from 1,3-bisphosphoglycerate to ADP to form 3-phosphoglycerate and ATP in the presence of magnesium. During catalysis, a conformational change occurs that brings the N- and C-domains of PGK closer together. Here we present the 1.8A crystal structure of unliganded PGK from Thermus caldophilus (Tca). Comparison of the structure of TcaPGK (open conformation) with that of Thermotoga maritima (Tma) PGK (closed conformation) revealed that the conformational change reflects a change in the interaction between the domains. We identified Arg148 as a key residue involved in open-to-closed transition. The open conformation of TcaPGK is stabilized by an interdomain salt bridge between Arg148 and Glu375. The binding of 3-PG (or maybe 1,3-BPG) disrupts this salt bridge and, in ternary complex, the formation of new salt bridge between Arg60 and Asp197 stabilizes the closed conformation.     

Drosophila heparan sulfate, a novel design

Heparan sulfate (HS) proteoglycans play critical roles in a wide variety of biological processes such as growth factor signaling, cell adhesion, wound healing, and tumor metastasis. Functionally important interactions between HS and a variety of proteins depend on specific structural features within the HS chains. The fruit fly (Drosophila melanogaster) is frequently applied as a model organism to study HS function in development. Previous structural studies of Drosophila HS have been restricted to disaccharide composition, without regard to the arrangement of saccharide domains typically found in vertebrate HS. Here, we biochemically characterized Drosophila HS by selective depolymerization with nitrous acid. Analysis of the generated saccharide products revealed a novel HS design, involving a peripheral, extended, presumably single, N-sulfated domain linked to an N-acetylated sequence contiguous with the linkage to core protein. The N-sulfated domain may be envisaged as a heparin structure of unusually low O-sulfate content.