The leucine-rich repeat as a protein recognition motif

Leucine-rich repeats (LRRs) are 20-29-residue sequence motifs present in a number of proteins with diverse functions. The primary function of these motifs appears to be to provide a versatile structural framework for the formation of protein-protein interactions. The past two years have seen an explosion of new structural information on proteins with LRRs. The new structures represent different LRR subfamilies and proteins with diverse functions, including GTPase-activating protein rna 1 p from the ribonuclease-inhibitor-like subfamily; spliceosomal protein U2A', Rab geranylgeranyltransferase, internalin B, dynein light chain 1 and nuclear export protein TAP from the SDS22-like subfamily; Skp2 from the cysteine-containing subfamily; and YopM from the bacterial subfamily. The new structural information has increased our understanding of the structural determinants of LRR proteins and our ability to model such proteins with unknown structures, and has shed new light on how these proteins participate in protein-protein interactions.

Chemistry, properties, and phylogenetic implications of the methylated carrageenans from red algae of the genus Areschougia (Areschougiaceae, Gigartinales, Rhodophyta)

The three Australian-endemic species comprising the genus Aresehougia have been examined to determine the structure of their nonfibrillar wall components. The polysaccharide extracted from the most widely distributed species, A. congesta (Turner) J. Agardh, was shown by compositional analyses, Fourier transform infrared (FTIR) spectroscopy, linkage analysis, and C-13-NMR spectroscopy to be a carrageenan composed predominantly of the repeating disaccharides 6'-O-methylcarrabiose 2,4'-disulfate, carrabiose 2,4-disulfate (the repeating unit of L-carrageenan), 4',6'-O-(1-carboxyethylidene)carrabiose 2-sulfate, and 6'-O-methylcarrabiose 2-sulfate. The carrageenan also contained small amounts of 4-linked Galp residues, some bearing methyl ether substitution at O-3 and some possibly bearing sulfate ester and/or glycosyl substitutions at O-3. The A. congesta carrageenan had unique rheological properties, its gels having some similarities to those of commercial iota -carrageenan but with the viscosity of commercial lambda -carrageenan. Polysaccharides from A. ligulata Harvey ex J. Agardh and A. stuartii Harvey were shown by constituent sugar and FTIR analyses to be sulfated galactans rich in mono-O-methylgalactose. The carrageenan structures of Areschougia spp. were consistent with those of the genera Rhabdonia, Erythroclonium, and Austroclonium, the other genera constituting the family Areschougiaceae.

Cross-talk between caveolae and glycosylphosphatidylinositol-rich domains

Most mammalian cells have in their plasma membrane at least two types of lipid microdomains, non-invaginated lipid rafts and caveolae. Glycosylphosphatidylinositol (GPI)-anchored proteins constitute a class of proteins that are enriched in rafts but not caveolae at steady state. We have analyzed the effects of abolishing GPI biosynthesis on rafts, caveolae, and cholesterol levels. GPI-deficient cells were obtained by screening for resistance to the pore-forming toxin aerolysin, which uses this class of proteins as receptors. Despite the absence of GPI-anchored proteins, mutant cells still contained lipid rafts, indicating that GPI-anchored proteins are not crucial structural elements of these domains. Interestingly, the caveolae-specific membrane proteins, caveolin-1 and 2, were up-regulated in GPI-deficient cells, in contrast to flotillin-I and GM1, which were expressed at normal levels. Additionally, the number of surface caveolae was increased. This effect was specific since recovery of GPI biosynthesis by gene recomplementation restored caveolin expression and the number of surface caveolae to wild type levels. The inverse correlation between the expression of GPI-anchored proteins and caveolin-1 was confirmed by the observation that overexpression of caveolin-1 in wild type cells led to a decrease in the expression of GPI-anchored proteins. In cells lacking caveolae, the absence of GPI-anchored proteins caused an increase in cholesterol levels, suggesting a possible role of GPI-anchored proteins in cholesterol homeostasis, which in some cells, such as Chinese hamster ovary cells, can be compensated by caveolin up-regulation.

Assessment of the ability to model proteins with leucine-rich repeats in light of the latest structural information

The three-dimensional structures of leucine-rich repeat (LRR) -containing proteins from five different families were previously predicted based on the crystal structure of the ribonuclease inhibitor. using an approach that combined homology-based modeling, structure-based sequence alignment of LRRs, and several rational assumptions. The structural models have been produced based on very limited sequence similarity, which, in general. cannot yield trustworthy predictions. Recently, the protein structures from three of these five families have been determined. In this report we estimate the quality of the modeling approach by comparing the models with the experimentally determined structures. The comparison suggests that the general architecture, curvature, interior/exterior orientations of side chains. and backbone conformation of the LRR structures can be predicted correctly. On the other hand. the analysis revealed that, in some cases. it is difficult to predict correctly the twist of the overall super-helical structure. Taking into consideration the conclusions from these comparisons, we identified a new family of bacterial LRR proteins and present its structural model. The reliability of the LRR protein modeling suggests that it would be informative to apply similar modeling approaches to other classes of solenoid proteins.

Characteristic zirconium-rich coring structures in Mg-Zr alloys

The most characteristic feature of the microstructure of a magnesium alloy that contains more than a few tenths per cent soluble zirconium is the zirconium-rich cores that exist in most grains. The morphology, distribution and composition of cores observed in a Mg-0.56%Zr alloy and the small particles present in them were investigated. (C) 2002 Acta Materialia Inc. Published by Elsevier Science Ltd. All rights reserved.

Phytophthora sojae avirulence genes Avr4 and Avr6 are located in a 24kb, recombination-rich region of genomic DNA

A cross between two different races (race 7 x race 25) of the soybean root and stem rot pathogen Phytophthora sojae was analyzed to characterize the genomic region flanking two cosegregating avirulence genes, Anur4 and Anur6. Both genes cosegregated in the ratio of 82:17 (avirulent:virulent) in an F-2 population, suggestive of a single locus controlling both phenotypes. A chromosome walk was commenced from RAPD marker OPE7.1C, 2.0 cM distant from the Anur4/6 locus. Three overlapping cosmids were isolated which included genetic markers that flank the Anur4/6 locus. The chromosome walk spanned a physical distance of 67 kb which represented a genetic map distance of 22.3cM, an average recombination frequency of 3.0kb/cM and 11.7-fold greater than the predicted average recombination frequency of 35.3 kb/cM for the entire P. sojae genome. Six genes (cDNA clones) expressed from the Anur4/6 genomic region encompassed by the cosmid contig were identified. Single nucleotide polymorphisms and restriction fragment length polymorphisms showed these six genes were closely linked to the Anur4/6 locus. Physical mapping of the cDNA clones within the cosmid contig made it possible to deduce the precise linkage order of the cDNAs. None of the six cDNA clones appear to be candidates for Anur4/6. We conclude that two of these cDNA clones flank a physical region of approximately 24 kb and 4.3 cM that appears to include the Anur4/6 locus. (C) 2003 Elsevier Inc. All rights reserved.

Autosomal dominant hypocalcemia: A novel activating mutation (E604K) in the cysteine-rich domain of the calcium-sensing receptor

We report a novel activating mutation (E604K) of the calcium-sensing receptor in a family with autosomal dominant hypocalcemia. Whereas all affected individuals exhibited marked hypocalcemia, some cases with untreated hypocalcemia exhibited seizures in infancy, whereas others were largely asymptomatic from birth into adulthood. The missense mutation E604K (G2182A, GenBank accession no. U20759), which affects an amino acid residue in the C terminus of the cysteine-rich domain of the extracellular head, co-segregated with hypocalcemia in all seven individuals for whom DNA was available. Two unaffected, normocalcemic members of the family did not exhibit the mutation. The molecular impact of the mutation on two key components of the signaling response was assessed in HEK-293 cells transiently transfected with cDNA corresponding to either the wild-type calcium-sensing receptor or the E604K mutation derived by site-directed mutagenesis. There was a significant leftward shift in the concentration response curves for the effects of extracellular Ca2+ on both intracellular Ca2+ mobilization (determined by aequorin luminescence) and MAPK activity (determined by luciferase expression). The C terminus of the cysteine-rich domain of the extracellular head may normally act to suppress receptor activity in the presence of low extracellular Ca2+ concentrations.