Mouse LRRC8A(BRIL):D in GDN
Ontology highlight
ABSTRACT:
SUBMITTER: Antony Lurie
PROVIDER: EMPIAR-12510 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:G-protein-coupled receptors (GPCRs)-the largest family of cell-surface membrane proteins-mediate the intracellular signal transduction of many external ligands. Thus, GPCRs have become important drug targets. X-ray crystal structures of GPCRs are very useful for structure-based drug design (SBDD). Herein, we produced a new antibody (SRP2070) targeting the thermostabilised apocytochrome b562 from Escherichia coli M7W/H102I/R106L (BRIL). We found that a fragment of this antibody (SRP2070Fab) facilitated the crystallisation of the BRIL-tagged, ligand bound GPCRs, 5HT1B and AT2R. Furthermore, the electron densities of the ligands were resolved, suggesting that SPR2070Fab is versatile and adaptable for GPCR SBDD. We anticipate that this new tool will significantly accelerate structure determination of other GPCRs and the design of small molecular drugs targeting them.
| S-EPMC7363855 | biostudies-literature
Project description:Volume-regulated anion channels (VRACs) participate in the cellular response to osmotic swelling. These membrane proteins consist of heteromeric assemblies of LRRC8 subunits, whose compositions determine permeation properties. Although structures of the obligatory LRRC8A, also referred to as SWELL1, have previously defined the architecture of VRACs, the organization of heteromeric channels has remained elusive. Here we have addressed this question by the structural characterization of murine LRRC8A/C channels. Like LRRC8A, these proteins assemble as hexamers. Despite 12 possible arrangements, we find a predominant organization with an A:C ratio of two. In this assembly, four LRRC8A subunits cluster in their preferred conformation observed in homomers, as pairs of closely interacting proteins that stabilize a closed state of the channel. In contrast, the two interacting LRRC8C subunits show a larger flexibility, underlining their role in the destabilization of the tightly packed A subunits, thereby enhancing the activation properties of the protein.
| S-EPMC9851909 | biostudies-literature
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| EMPIAR-10196 | biostudies-other
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| EMPIAR-10258 | biostudies-other
Project description:BRIL (bone-restricted IFITM-like), is a short transmembrane protein expressed almost exclusively in osteoblasts. Although much is known about its bone-restricted gene expression pattern and protein biochemical and topological features, little information is available for BRIL physiological function. Two autosomal dominant forms of osteogenesis imperfecta (OI) are caused by distinct, but recurrent mutations in the BRIL gene. Yet, the underlying mechanisms by which those mutations lead to OI are still poorly understood. A previous report indicated that BRIL knockout (KO) mice had bone deformities, shortened long bones, and reproductive problems. Here we generated and systematically analyzed the skeletal phenotype of a new global Bril KO/LacZ knockin mouse model. KO mice reproduced and thrived normally up to 12 month of age. The skeletal phenotype of KO and WT littermates was assessed at embryonic (E13.5 to E18.5) and postnatal (2 days, 3 weeks, 3 months and 8 months) time-points. Embryos from E13.5 through to E18.5 showed significant X-Gal staining in all skeletal elements without any apparent patterning anomalies. Although bone deformities were never observed at any postnatal ages, minor and transient differences were noted in terms of bone length and static uCT parameters, but not systematically across all ages and genders. These changes, however, were not accompanied by significant alteration in bone material properties as assessed by a 3-point bending test. In addition, no changes were detected in circulating serum markers of bone turnover (P1NP, CTX-I, and osteocalcin). Gene expression monitoring also revealed no major impact of the loss of BRIL. Further, when mice were challenged with a surgically-induced fracture in tibia, bones repaired equally well in the KO mice as compared to WT. Finally, we showed that BRIL C-terminus is not a bona fide binding site for calcium. In conclusion, our in depth analysis suggest that skeletal patterning, bone mass accrual and remodeling in mice proceeded independent of BRIL.
| S-EPMC5589259 | biostudies-literature
Project description:Rationale: The transformation of fibroblasts into activated myofibroblasts is a critical step that results in cardiac fibrosis upon myocardial infarction (MI). Leucine-rich repeat-containing protein-8A (LRRC8A) is a multi-functional protein involved in cell survival, growth, and proliferation, whereas its role in regulating myofibroblast phenotypes and myocardial fibrosis remains unknown. Methods: Conditional myofibroblast-specific Lrrc8a knockout mouse models were established by crossing the Lrrc8a flox/flox mice with the tamoxifen-inducible periostin-Cre transgenic mice. The involvement of LRRC8A in regulating cardiac fibrosis post-MI and myofibroblast phenotypes induced by transforming growth factor-β1 (TGF-β1) was comprehensively evaluated. The mechanisms underlying LRRC8A regulation of myofibroblast phenotypes were determined by RNA sequencing-driven analysis followed by cause-effect experiments. Results: LRRC8A expression was significantly elevated in the fibrotic tissues and the fibroblasts isolated from the post-MI hearts. Compared with the wild-type (WT) littermates, the specific knockout of LRRC8A in myofibroblasts greatly attenuated myofibroblast transformation, fibrotic remodeling, and ventricular dysfunction after MI. Silencing of LRRC8A expression suppressed, whereas overexpression of LRRC8A enhanced, the pro-fibrotic myofibroblast phenotypes in isolated cardiac fibroblasts upon stimulation with TGF-β1. LRRC8A participated in TGF-β1-induced myofibroblast transformation via activating JAK2-STAT3 signaling. Furthermore, LRRC8A activated the JAK2-STAT3 pathway via its C-terminal leucine-rich repeat-domain (LRRD), directly interacting with growth factor receptor-bound protein 2 (GRB2), an adaptor protein associated with and necessary for tyrosine-phosphorylated JAK2. Conclusions: LRRC8A regulates myofibroblast transformation and cardiac fibrosis following MI. LRRC8A inhibition might be a promising strategy for cardiac fibrosis and heart failure.
| S-EPMC9373806 | biostudies-literature