L23 protein functions as a chaperone docking site on the ribosome

During translation, the first encounter of nascent polypeptides is with the ribosome-associated chaperones that assist the folding process--a principle that seems to be conserved in evolution. In Escherichia coli, the ribosome-bound Trigger Factor chaperones the folding of cytosolic proteins by interacting with nascent polypeptides. Here we identify a ribosome-binding motif in the amino-terminal domain of Trigger Factor. We also show the formation of crosslinked products between Trigger Factor and two adjacent ribosomal proteins, L23 and L29, which are located at the exit of the peptide tunnel in the ribosome. L23 is essential for the growth of E. coli and the association of Trigger Factor with the ribosome, whereas L29 is dispensable in both processes. Mutation of an exposed glutamate in L23 prevents Trigger Factor from interacting with ribosomes and nascent chains, and causes protein aggregation and conditional lethality in cells that lack the protein repair function of the DnaK chaperone. Purified L23 also interacts specifically with Trigger Factor in vitro. We conclude that essential L23 provides a chaperone docking site on ribosomes that directly links protein biosynthesis with chaperone-assisted protein folding.     

Genome-wide meta-analyses of nonsyndromic cleft lip with or without cleft palate identify six new risk loci

We have conducted the first meta-analyses for nonsyndromic cleft lip with or without cleft palate (NSCL/P) using data from the two largest genome-wide association studies published to date. We confirmed associations with all previously identified loci and identified six additional susceptibility regions (1p36, 2p21, 3p11.1, 8q21.3, 13q31.1 and 15q22). Analysis of phenotypic variability identified the first specific genetic risk factor for NSCLP (nonsyndromic cleft lip plus palate) (rs8001641; P(NSCLP) = 6.51 × 10(-11); homozygote relative risk = 2.41, 95% confidence interval (CI) 1.84-3.16).     

Genome-wide in situ exon capture for selective resequencing

Increasingly powerful sequencing technologies are ushering in an era of personal genome sequences and raising the possibility of using such information to guide medical decisions. Genome resequencing also promises to accelerate the identification of disease-associated mutations. Roughly 98% of the human genome is composed of repeats and intergenic or non-protein-coding sequences. Thus, it is crucial to focus resequencing on high-value genomic regions. Protein-coding exons represent one such type of high-value target. We have developed a method of using flexible, high-density microarrays to capture any desired fraction of the human genome, in this case corresponding to more than 200,000 protein-coding exons. Depending on the precise protocol, up to 55-85% of the captured fragments are associated with targeted regions and up to 98% of intended exons can be recovered. This methodology provides an adaptable route toward rapid and efficient resequencing of any sizeable, non-repeat portion of the human genome.     

Knockout of the Bcmo1 gene results in an inflammatory response in female lung, which is suppressed by dietary beta-carotene

Beta-carotene 15,15′-monooxygenase 1 knockout (Bcmo1 ^?/?) mice accumulate beta-carotene (BC) similarly to humans, whereas wild-type (Bcmo1 ^+/+) mice efficiently cleave BC. Bcmo1 ^?/? mice are therefore suitable to investigate BC-induced alterations in gene expression in lung, assessed by microarray analysis. Bcmo1 ^?/? mice receiving control diet had increased expression of inflammatory genes as compared to BC-supplemented Bcmo1 ^?/? mice and Bcmo1 ^+/+ mice that received either control or BC-supplemented diets. Differential gene expression in Bcmo1 ^?/? mice was confirmed by real-time quantitative PCR. Histochemical analysis indeed showed an increase in inflammatory cells in lungs of control Bcmo1 ^?/? mice. Supported by metabolite and gene-expression data, we hypothesize that the increased inflammatory response is due to an altered BC metabolism, resulting in an increased vitamin A requirement in Bcmo1 ^?/? mice. This suggests that effects of BC may depend on inter-individual variations in BC-metabolizing enzymes, such as the frequently occurring human polymorphisms in BCMO1.     

Embryonic lethality caused by mutations in basement membrane collagen of C. elegans

Basement membranes are specialized forms of extracellular matrix with important functions in development. A major structural component of basement membranes is type IV collagen, a heterotrimer of two alpha 1(IV) and one alpha 2(IV) chains, which forms a complex, polygonal network associated with other basement membrane components. Here we report that the alpha 1(IV) collagen chain of Caenorhabditis elegans is encoded by the genetic locus emb-9. Mutations in emb-9 cause temperature-sensitive lethality during late embryogenesis. We have identified single nucleotide alterations that substitute glutamic acid for glycine in the triple-helical Gly-X-Y repeat region of the alpha 1(IV) collagen in three emb-9 mutant strains. These results are direct evidence that defects in basement membranes can disrupt embryonic development and form a basis for the genetic analysis of basement membrane function.     

Neuropeptide modulation of single calcium and potassium channels detected with a new patch clamp configuration

Calcium channel activity is crucial for secretion and synaptic transmission, but it has been difficult to study Ca2+ channel modulation because survival and regulation of some of these channels require cytoplasmic constituents that are lost with the formation of cell-free patches. Here we report a new patch clamp configuration in which activity and regulation of channels are maintained after removal from cells. A pipette containing the pore-forming agent nystatin is sealed onto a cell and withdrawn to form an enclosed vesicle. The resulting perforated vesicle, formed from pituitary tumour cells, contains Ca2+ and K+ channels. Ca2(+)-activated K+ channels in the vesicle are activated by cyclic AMP analogues, and by a neuropeptide (thyrotropin-releasing hormone) that stimulates phosphatidylinositol turnover and inositol trisphosphate-gated Ca2+ release from intracellular organelles. Thus, the perforated vesicle retains signal transduction systems necessary for ion channel modulation. Functional dihydropyridine-sensitive Ca2+ channels (L-type) are maintained in the vesicle, and their gating is inhibited by thyrotropin-releasing hormone. Hence, this new patch clamp configuration has allowed a direct detection of the single-channel basis of transmitter-induced inhibition of L-type Ca2+ channels. The modulation of Ca2(+)-channel gating may be an important mechanism for regulating hormone secretion from pituitary cells.     

Interleukin 21 and its receptor are involved in NK cell expansion and regulation of lymphocyte function

Cytokines are important in the regulation of haematopoiesis and immune responses, and can influence lymphocyte development. Here we have identified a class I cytokine receptor that is selectively expressed in lymphoid tissues and is capable of signal transduction. The full-length receptor was expressed in BaF3 cells, which created a functional assay for ligand detection and cloning. Conditioned media from activated human CD3+ T cells supported proliferation of the assay cell line. We constructed a complementary DNA expression library from activated human CD3+ T cells, and identified a cytokine with a four-helix-bundle structure using functional cloning. This cytokine is most closely related to IL2 and IL15, and has been designated IL21 with the receptor designated IL21 R. In vitro assays suggest that IL21 has a role in the proliferation and maturation of natural killer (NK) cell populations from bone marrow, in the proliferation of mature B-cell populations co-stimulated with anti-CD40, and in the proliferation of T cells co-stimulated with anti-CD3.     

Structural basis for the activation of 20S proteasomes by 11S regulators

Most of the non-lysosomal proteolysis that occurs in eukaryotic cells is performed by a nonspecific and abundant barrel-shaped complex called the 20S proteasome. Substrates access the active sites, which are sequestered in an internal chamber, by traversing a narrow opening (alpha-annulus) that is blocked in the unliganded 20S proteasome by amino-terminal sequences of alpha-subunits. Peptide products probably exit the 20S proteasome through the same opening. 11S regulators (also called PA26 (ref. 4), PA28 (ref. 5) and REG) are heptamers that stimulate 20S proteasome peptidase activity in vitro and may facilitate product release in vivo. Here we report the co-crystal structure of yeast 20S proteasome with the 11S regulator from Trypanosoma brucei (PA26). PA26 carboxy-terminal tails provide binding affinity by inserting into pockets on the 20S proteasome, and PA26 activation loops induce conformational changes in alpha-subunits that open the gate separating the proteasome interior from the intracellular environment. The reduction in processivity expected for an open conformation of the exit gate may explain the role of 11S regulators in the production of ligands for major histocompatibility complex class I molecules.