Despite the sequencing of the human and mouse genomes, few genetic mechanisms for protecting against autoimmune disease are currently known. Here we systematically screen the mouse genome for autoimmune regulators to isolate a mouse strain, sanroque, with severe autoimmune disease resulting from a single recessive defect in a previously unknown mechanism for repressing antibody responses to self. The sanroque mutation acts within mature T cells to cause formation of excessive numbers of follicular helper T cells and germinal centres. The mutation disrupts a repressor of ICOS, an essential co-stimulatory receptor for follicular T cells, and results in excessive production of the cytokine interleukin-21. sanroque mice fail to repress diabetes-causing T cells, and develop high titres of autoantibodies and a pattern of pathology consistent with lupus. The causative mutation is in a gene of previously unknown function, roquin (Rc3h1), which encodes a highly conserved member of the RING-type ubiquitin ligase protein family. The Roquin protein is distinguished by the presence of a CCCH zinc-finger found in RNA-binding proteins, and localization to cytosolic RNA granules implicated in regulating messenger RNA translation and stability.

Skeletal growth by endochondral ossification involves tightly coordinated chondrocyte differentiation that creates reserve, proliferating, prehypertrophic, and hypertrophic cartilage zones in the growth plate. Many human skeletal disorders result from mutations in cartilage extracellular matrix (ECM) components that compromise both ECM architecture and chondrocyte function. Understanding normal cartilage development, composition, and structure is therefore vital to unravel these disease mechanisms. To study this intricate process in vivo by proteomics, we analyzed mouse femoral head cartilage at developmental stages enriched in either immature chondrocytes or maturing/hypertrophic chondrocytes (postnatal days 3 and 21, respectively). Using LTQ-Orbitrap tandem mass spectrometry, we identified 703 cartilage proteins. Differentially abundant proteins (q < 0.01) included prototypic markers for both early and late chondrocyte differentiation (epiphycan and collagen X, respectively) and novel ECM and cell adhesion proteins with no previously described roles in cartilage development (tenascin X, vitrin, Urb, emilin-1, and the sushi repeat-containing proteins SRPX and SRPX2). Meta-analysis of cartilage development in vivo and an in vitro chondrocyte culture model (Wilson, R., Diseberg, A. F., Gordon, L., Zivkovic, S., Tatarczuch, L., Mackie, E. J., Gorman, J. J., and Bateman, J. Cartilage is a unique tissue characterized by an abundant extracellular matrix (ECM) 1 and a single cell type, the chondrocyte. However, the permanent hyaline cartilage, which provides the articulating surfaces of long bones and vertebrae, and the transient growth plate cartilage responsible for endochondral bone growth are uniform in neither cellular phenotype nor protein composition. In articular cartilage, the chondrocytes form morphologically distinct regions comprising a superficial region of flattened cells, a sparsely populated middle layer, and a deep zone of hypertrophic chondrocytes embedded in calcified cartilage at the chondro-osseous junc-

Helicoverpa armigera midgut proteins that bind the Bacillus thuringiensis (Bt) δ-endotoxin Cry1Ac were purified by affinity chromatography. SDS-PAGE showed that several proteins were eluted with N-acetylgalactosamine and no further proteins were detected after elution with urea. Tandem mass spectral data for tryptic peptides initially indicated that the proteins resembled aminopeptidases (APNs) from other lepidopterans and cDNA sequences for seven APNs were isolated from H. armigera through a combination of cloning with primers derived from predicted peptide sequences and established EST libraries. Phylogenetic analysis showed lepidopteran APN genes in nine clades of which five were part of a lepidopteran-specific radiation. The Cry1Ac-binding proteins were then identified with four of the seven HaAPN genes. Three of those four APNs are likely orthologs of APNs characterised as Cry1Ac-binding proteins in other lepidopterans. The fourth Cry1Ac-binding APN has orthologs not previously identified as Cry1Ac-binding partners. The HaAPN genes were expressed predominantly in the midgut through larval development. Each showed consistent expression along the length of the midgut but five of the genes were expressed at levels about two orders of magnitude greater than the remaining two. The remaining mass spectral data identified sequences encoding polycalin proteins with multiple lipocalin-like domains. A polycalin has only been previously reported in another lepidopteran, Bombyx mori, but polycalins in both species are now linked with binding of Bt Cry toxins. This is the first report of hybrid, lipocalin-like domains in shorter polycalin sequences that are not present in the longest sequence. We propose that these hybrid domains are generated by alternative splicing of the mRNA.

Summary Objective Cartilage in joints such as the hip and knee experiences repeated phases of heavy loading and low load recovery during the 24-h day/night cycle. Our previous work has shown 24 h rhythmic changes in gene expression at transcript level between night and day in wild type mouse cartilage which is lost in a circadian clock knock-out mouse model. However, it remains unknown to what extent circadian rhythms also regulate protein level gene expression in this matrix rich tissue. Methods We investigated daily changes of protein abundance in mouse femoral head articular cartilage by performing a 48-h time-series LC-MS/MS analysis. Results Out of the 1,177 proteins we identified across all time points, 145 proteins showed rhythmic changes in their abundance within the femoral head cartilage. Among these were molecules that have been implicated in key cartilage functions, including CTGF, MATN1, PAI-1 and PLOD1 & 2. Pathway analysis revealed that protein synthesis, cytoskeleton and glucose metabolism exhibited time-of-day dependent functions. Analysis of published cartilage proteomics datasets revealed that a significant portion of rhythmic proteins were dysregulated in osteoarthritis and/or ageing. Conclusions Our circadian proteomics study reveals that articular cartilage is a much more dynamic tissue than previously thought, with chondrocytes driving circadian rhythms not only in gene transcription but also in protein abundance. Our results clearly call for the consideration of circadian timing mechanisms not only in cartilage biology, but also in the pathogenesis, treatment strategies and biomarker detection in osteoarthritis.