Structure, Tissue Distribution and Genomic Organization of the Murine RRM-Type RNA Binding Proteins TIA-1 and TIAR

TIA-1 and TIAR are RNA binding proteins of the RNA recognition motif (RRM)/ribonucleoprotein (RNP) family that have been implicated as effectors of apoptotic cell death. We report the structures of murine TIA-1 and TIAR (mTIA-1 and mTIAR) deduced from cDNA cloning, the mRNA and protein tissue distribution of mTIA-1 and mTIAR, and the exon-intron structures of the mTIA-1 and mTIAR genes. Both mTIA-1 and mTIAR are comprised of three ∼100 amino acid N-terminal RRM domains and a ∼90 amino acid C-terminal auxiliary domain. This subfamily of RRM proteins is evolutionarily well conserved; mTIA-1 and mTIAR are 80% similar to each other, and 96 and 99% similar to hTIA-1 and hTIAR, respectively. The overall exon-intron structures of the mTIA-1 and mTIAR genes are also similar to each other, as well as to the human TIA-1 gene structure. While Northern blot analysis reveals that mTIA-1 and mTIAR mRNAs have a broad tissue distribution, mTIA-1 and mTIAR proteins are predominantly expressed in brain, testis and spleen. At least two isoforms of both mTIA-1 and mTIAR are generated by alternative splicing. Murine TIA-1 isoforms including or lacking the exon 5 encoded sequences are expressed at a ratio of ∼ 1:1, whereas mTIAR isoforms including or lacking the 5′-end of exon 3 sequences are expressed in a ∼ 1:6 ratio. Molecular characterization of murine TIA-1 and TIAR RNA binding proteins provides the basis for a genetic analysis of the functional roles of these proteins during mammalian developmen

Skeletal muscle deformity and neuronal disorder in Trio exchange factor-deficient mouse embryos

Dbl-homology guanine nucleotide exchange factors (DH-GEFs) regulate actin cytoskeletal reorganization, cell adhesion, and gene transcription via activation of Rho GTPases. However, little is known about the physiological role of mammalian DH-GEFs during development. The DH-GEF family member Trio is of particular interest because it is a multifunctional protein possessing two GEF domains, as well as a protein serine/threonine kinase domain, and trio-like genes in Caenorhabditis elegans and Drosophila were shown to function in neural migration and axon guidance. To determine the role of Trio during mammalian development, we generated a mouse trio loss-of-function mutation (trio(−/−)). Trio function is essential during late embryonic development as genotype analysis indicated that trio(−/−) embryos died between embryonic day (E)-15.5 and birth, or shortly thereafter. In the trio(−/−) embryos, primary skeletal myofibers were relatively normal at E14.5, but by E18.5 highly unusual spherical myofibers accumulated. Trio deficiency may cause a defect in secondary myogenesis, as the appearance of the abnormal trio(−/−) skeletal myofibers temporally coincided with the onset of secondary myogenesis, and smaller secondary myofibers located adjacent to the primary myofibers were absent. The proliferation of trio(−/−) secondary myoblasts appeared normal, suggesting that Trio may regulate secondary myoblast alignment or fusion. trio(−/−) embryos also displayed aberrant organization in several regions within the brain, including the hippocampal formation and olfactory bulb. We thus conclude that Trio is essential for late embryonic development, and that Trio functions in fetal skeletal muscle formation and in the organization of neural tissues

Differential proteins expression distinguished between patients with infectious and noninfectious uveitis

Uveitis is associated with a number of inflammatory diseases and can be infectious or noninfectious. The diagnosis of uveitis is usually based on typical clinical presentation, but sometimes the signs and symptoms are not clear, and the discovery of new biomarkers may help to distinguish between infectious and noninfectious uveitis. In the current study, we investigate the proteome of aqueous humor and associated blood plasma from patients with confirmed infectious or noninfectious uveitis. Aqueous humor (AH) and plasma were obtained from 28 patients with infectious uveitis (IU), 29 patients with noninfectious uveitis (NIU) and 35 healthy controls undergoing cataract surgery. The protein profiles were analyzed by SomaScan proteomic profiling technology. Our results showed proteins differentially expressed in the AH and plasma from IU and NIU patients in comparison with cataract patients. Importantly, 18 proteins were up-regulated and 1 protein was down-regulated in both AH and plasma from IU patients. Furthermore, in NIU patients, 142 proteins were up-regulated and 7 proteins down-regulated in both AH and blood plasma. The results of pathway enrichment analysis for both IU and NIU groups identified mostly to inflammatory and regulatory processes. In conclusion, SomaScan assay was able to detect novel AH and plasma protein biomarkers in IU and NIU patients. The unique proteins found in both AH and plasma suggests a protein signature that could distinguish between infectious and noninfectious uveitis patient populations

Covalent Inhibitors of Interleukin‑2 Inducible T Cell Kinase (Itk) with Nanomolar Potency in a Whole-Blood Assay

We wish to report a strategy that targets interleukin-2 inducible T cell kinase (Itk) with covalent inhibitors. Thus far, covalent inhibition of Itk has not been disclosed in the literature. Structure-based drug design was utilized to achieve low nanomolar potency of the disclosed series even at high ATP concentrations. Kinetic measurements confirmed an irreversible binding mode with off-rate half-lives exceeding 24 h and moderate on-rates. The analogues are highly potent in a cellular IP1 assay as well as in a human whole-blood (hWB) assay. Despite a half-life of approximately 2 h in resting primary T cells, the covalent inhibition of Itk resulted in functional silencing of the TCR pathway for more than 24 h. This prolonged effect indicates that covalent inhibition is a viable strategy to target the inactivation of Itk