Mutations in SECISBP2 result in abnormal thyroid hormone metabolism

Incorporation of selenocysteine (Sec), through recoding of the UGA stop codon, creates a unique class of proteins. Mice lacking tRNA(Sec) die in utero, but the in vivo role of other components involved in selenoprotein synthesis is unknown, and Sec incorporation defects have not been described in humans. Deiodinases (DIOs) are selenoproteins involved in thyroid hormone metabolism. We identified three of seven siblings with clinical evidence of abnormal thyroid hormone metabolism. Their fibroblasts showed decreased DIO2 enzymatic activity not linked to the DIO2 locus. Systematic linkage analysis of genes involved in DIO2 synthesis and degradation led to the identification of an inherited Sec incorporation defect, caused by a homozygous missense mutation in SECISBP2 (also called SBP2). An unrelated child with a similar phenotype was compound heterozygous with respect to mutations in SECISBP2. Because SBP2 is epistatic to selenoprotein synthesis, these defects had a generalized effect on selenoproteins. Incomplete loss of SBP2 function probably causes the mild phenotype.     

Structural characterization of the molecular platform for type III secretion system assembly

Type III secretion systems (TTSSs) are multi-protein macromolecular 'machines' that have a central function in the virulence of many Gram-negative pathogens by directly mediating the secretion and translocation of bacterial proteins (termed effectors) into the cytoplasm of eukaryotic cells. Most of the 20 unique structural components constituting this secretion apparatus are highly conserved among animal and plant pathogens and are also evolutionarily related to proteins in the flagellar-specific export system. Recent electron microscopy experiments have revealed the gross 'needle-shaped' morphology of the TTSS, yet a detailed understanding of the structural characteristics and organization of these protein components within the bacterial membranes is lacking. Here we report the 1.8-A crystal structure of EscJ from enteropathogenic Escherichia coli (EPEC), a member of the YscJ/PrgK family whose oligomerization represents one of the earliest events in TTSS assembly. Crystal packing analysis and molecular modelling indicate that EscJ could form a large 24-subunit 'ring' superstructure with extensive grooves, ridges and electrostatic features. Electron microscopy, labelling and mass spectrometry studies on the orthologous Salmonella typhimurium PrgK within the context of the assembled TTSS support the stoichiometry, membrane association and surface accessibility of the modelled ring. We propose that the YscJ/PrgK protein family functions as an essential molecular platform for TTSS assembly.     

Cell-specific mitotic defect and dyserythropoiesis associated with erythroid band 3 deficiency

Most eukaryotic cell types use a common program to regulate the process of cell division. During mitosis, successful partitioning of the genetic material depends on spatially coordinated chromosome movement and cell cleavage. Here we characterize a zebrafish mutant, retsina (ret), that exhibits an erythroid-specific defect in cell division with marked dyserythropoiesis similar to human congenital dyserythropoietic anemia. Erythroblasts from ret fish show binuclearity and undergo apoptosis due to a failure in the completion of chromosome segregation and cytokinesis. Through positional cloning, we show that the ret mutation is in a gene (slc4a1) encoding the anion exchanger 1 (also called band 3 and AE1), an erythroid-specific cytoskeletal protein. We further show an association between deficiency in Slc4a1 and mitotic defects in the mouse. Rescue experiments in ret zebrafish embryos expressing transgenic slc4a1 with a variety of mutations show that the requirement for band 3 in normal erythroid mitosis is mediated through its protein 4.1R-binding domains. Our report establishes an evolutionarily conserved role for band 3 in erythroid-specific cell division and illustrates the concept of cell-specific adaptation for mitosis.     

The study of macromolecular complexes by quantitative proteomics

We describe a generic strategy for determining the specific composition, changes in the composition, and changes in the abundance of protein complexes. It is based on the use of isotope-coded affinity tag (ICAT) reagents and mass spectrometry to compare the relative abundances of tryptic peptides derived from suitable pairs of purified or partially purified protein complexes. In a first application, the genuine protein components of a large RNA polymerase II (Pol II) preinitiation complex (PIC) were distinguished from a background of co-purifying proteins by comparing the relative abundances of peptides derived from a control sample and the specific complex that was purified from nuclear extracts by a single-step promoter DNA affinity procedure. In a second application, peptides derived from immunopurified STE12 protein complexes isolated from yeast cells in different states were used to detect quantitative changes in the abundance of the complexes, and to detect dynamic changes in the composition of the samples. The use of quantitative mass spectrometry to guide identification of specific complex components in partially purified samples, and to detect quantitative changes in the abundance and composition of protein complexes, provides the researcher with powerful new tools for the comprehensive analysis of macromolecular complexes.     

Geochemical evidence for efficient aquifer isolation over geological timeframes

Aquitards-layers of rock having low permeability-have been suggested as potential long-term reservoirs for toxic materials such as nuclear or chemical waste. But information about the isolation properties of aquitard layers is essential to evaluate whether they can indeed be used safely as reservoirs. Here we investigate the long-term mobility of groundwaters between two aquifers surrounding an aquitard layer in the eastern recharge area of the Paris basin, France, using helium isotopes as a geochemical tracer. The deeper Trias sandstone aquifer, which lies above the crystalline basement, accumulates radiogenic (4)He and primordial (3)He from large regions of the crust and mantle at rates comparable to the degassing of the whole crust and of mid-ocean ridges. We show that the overlying carbonate Dogger aquifer, which is separated from the Trias aquifer by an aquitard layer consisting of a approximately 600 m succession of shales and clays, is stagnant and has been extremely well isolated from the Trias over the past several million years. This finding, together with previous studies at the centre of the Paris basin, shows that diffusive mass transfer across aquitards is negligible and that cross-formational flow in basins takes place preferentially in faulted areas.     

Kremen proteins are Dickkopf receptors that regulate Wnt/beta-catenin signalling

The Wnt family of secreted glycoproteins mediate cell cell interactions during cell growth and differentiation in both embryos and adults. Canonical Wnt signalling by way of the beta-catenin pathway is transduced by two receptor families. Frizzled proteins and lipoprotein-receptor-related proteins 5 and 6 (LRP5/6) bind Wnts and transmit their signal by stabilizing intracellular beta-catenin. Wnt/beta-catenin signalling is inhibited by the secreted protein Dickkopf1 (Dkk1), a member of a multigene family, which induces head formation in amphibian embryos. Dkk1 has been shown to inhibit Wnt signalling by binding to and antagonizing LRP5/6. Here we show that the transmembrane proteins Kremen1 and Kremen2 are high-affinity Dkk1 receptors that functionally cooperate with Dkk1 to block Wnt/beta-catenin signalling. Kremen2 forms a ternary complex with Dkk1 and LRP6, and induces rapid endocytosis and removal of the Wnt receptor LRP6 from the plasma membrane. The results indicate that Kremen1 and Kremen2 are components of a membrane complex modulating canonical Wnt signalling through LRP6 in vertebrates.     

Identification of the gene (BBS1) most commonly involved in Bardet-Biedl syndrome,a complex human obesity syndrome

Bardet-Biedl syndrome (BBS, OMIM 209900) is a genetic disorder with the primary features of obesity, pigmentary retinopathy, polydactyly, renal malformations, mental retardation and hypogenitalism. Individuals with BBS are also at increased risk for diabetes mellitus, hypertension and congenital heart disease. What was once thought to be a homogeneous autosomal recessive disorder is now known to map to at least six loci: 11q13 (BBS1), 16q21 (BBS2), 3p13 p12 (BBS3), 15q22.3 q23 (BBS4), 2q31 (BBS5) and 20p12 (BBS6). There has been considerable interest in identifying the genes that underlie BBS, because some components of the phenotype are common. Cases of BBS mapping ro BBS6 are caused by mutations in MKKS; mutations in this gene also cause McKusick-Kaufman syndrome (hydrometrocolpos, post-axial polydactyly and congenital heart defects). In addition, we recently used positional cloning to identify the genes underlying BBS2 (ref. 16) and BBS4 (ref. 17). The BBS6 protein has similarity to a Thermoplasma acidophilum chaperonin, whereas BBS2 and BBS4 have no significant similarity to chaperonins. It has recently been suggested that three mutated alleles (two at one locus, and a third at a second locus) may be required for manifestation of BBS (triallelic inheritance). Here we report the identification of the gene BBS1 and show that a missense mutation of this gene is a frequent cause of BBS. In addition, we provide data showing that this common mutation is not involved in triallelic inheritance.