Bioscience Reports最新文献

Bimolecular fluorescence complementation (BiFC) methodology uses split fluorescent proteins to detect interactions between proteins in living cells. To date, BiFC has been used to investigate receptor dimerization by splitting the fluorescent protein between the intracellular portions of different receptor components. We reasoned that attaching these split proteins to the extracellular N-terminus instead may improve the flexibility of this methodology and reduce the likelihood of impaired intracellular signal transduction. As a proof-of-concept, we used receptors for calcitonin gene-related peptide, which comprise heterodimers of either the calcitonin or calcitonin receptor-like receptor in complex with an accessory protein (receptor activity-modifying protein 1). We created fusion constructs in which split mVenus fragments were attached to either the C-termini or N-termini of receptor subunits. The resulting constructs were transfected into Cos7 and HEK293S cells, where we measured cAMP production in response to ligand stimulation, cell surface expression of receptor complexes, and BiFC fluorescence. Additionally, we investigated ligand-dependent internalization in HEK293S cells. We found N-terminal fusions were better tolerated with regards to cAMP signaling and receptor internalization. N-terminal fusions also allowed reconstitution of functional fluorescent mVenus proteins; however, fluorescence yields were lower than with C-terminal fusion. Our results suggest that BiFC methodologies can be applied to the receptor N-terminus, thereby increasing the flexibility of this approach, and enabling further insights into receptor dimerization.

Given the failure of high-density lipoprotein (HDL) raising therapies to reduce cardiovascular disease risk, attention has turned towards HDL composition and vascular protective functions. In individuals with insulin resistance, exercise interventions recover HDL function. However, the effect of exercise on HDL in otherwise healthy individuals is unknown. This cross-sectional study aimed to measure HDL composition and antioxidant/endothelial anti-inflammatory function in insulin sensitive endurance athlete and healthy control men. HDL was isolated using density gradient ultracentrifugation. HDL composition was measured using microplate assays for apolipoprotein A-I, total cholesterol content and apolipoprotein M. HDL protein composition was measured using nano-liquid chromatography tandem mass spectrometry. HDL subclass distribution was measured by native gel electrophoresis. HDL in vitro antioxidant function was measured by paraoxonase-1 activity assay and anti-inflammatory function assessed in endothelial cells. Compared with controls, endurance athlete HDL had higher apolipoprotein A-1 (1.65 ± 0.62 mg/ml vs 1.21 ± 0.34 mg/ml, P=0.028) and higher total cholesterol content (2.09 ± 0.44 mmol/L vs 1.54 ± 0.33 mmol/L, P<0.001). Proteomics revealed higher apolipoprotein A-II, A-IV and D and transthyretin in endurance athlete HDL versus controls. There was no difference observed in in vitro HDL antioxidant or anti-inflammatory functions between controls and endurance athletes. Despite a more favourable composition, endurance athlete HDL did not have higher in vitro antioxidant or anti-inflammatory function. It is possible that HDL has a ceiling of function, i.e. that healthy HDL function cannot be enhanced by endurance exercise.

GABARAP is a member of the ATG8 family of ubiquitin-like autophagy related proteins. It was initially discovered as a facilitator of GABA-A receptor translocation to the plasma membrane and has since been shown to promote the intracellular transport of a variety of other proteins under non-autophagic conditions. We and others have shown that GABARAP interacts with the Type II phosphatidylinositol 4-kinase, PI4K2A, and that this interaction is important for autophagosome-lysosome fusion. Here, we identify a 7-amino acid segment within the PI4K2A catalytic domain that contains the GABARAP interaction motif (GIM). This segment resides in an exposed loop that is not conserved in the other mammalian Type II PI 4-kinase, PI4K2B, explaining the specificity of GABARAP binding to the PI4K2A isoform. Mutation of the PI4K2A GIM inhibits GABARAP binding and PI4K2A-mediated recruitment of cytosolic GABARAP to subcellular organelles. We further show that GABARAP binds to mono-phosphorylated phosphoinositides, PI3P, PI4P, and PI5P, raising the possibility that these lipids contribute to the binding energies that drive GABARAP-protein interactions on membranes.

Regulatory RNA elements fulfill functions such as translational regulation, control of transcript levels, and regulation of viral genome replication. Trans-acting factors (i.e., RNA-binding proteins) bind the so-called cis elements and confer functionality to the complex. The specificity during protein-RNA complex (RNP) formation often exploits the structural plasticity of RNA. Functional integrity of cis-trans pairs depends on the availability of properly folded RNA elements, and RNA conformational transitions can cause diseases. Knowledge of RNA structure and the conformational space is needed for understanding complex formation and deducing functional effects. However, structure determination of RNAs under in vivo conditions remains challenging. This review provides an overview of structured eukaryotic and viral RNA cis elements and discusses the effect of RNA structural equilibria on RNP formation. We showcase implications of RNA structural changes for diseases, outline strategies for RNA structure-based drug targeting, and summarize the methodological toolbox for deciphering RNA structures.

Aging is an inevitable and irreversible biological process that gradually heightens the risks of various diseases and death. As a newly discovered endogenous gasotransmitter, hydrogen sulfide (H2S) has been identified to exert multiple beneficial impacts on the regulation of aging and age-related pathologies. This study was aimed at systematically exploring the relationship between asynchronous aging processes and H2S concentrations in various tissues of aging mice. Samples of plasma and 13 tissues were collected from four cross-sectional age groups (3, 6, 12 and 18 months of age) covering the lifespan of male C57BL/6J mice. The H2S concentration was quantified by a reported liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with monobromobimane derivatization. Additionally, the expressions of cystathionine γ-lyase (CSE), cystathionine β-synthase and 3-mercaptopyruvate sulfurtransferase, in those tissues were analyzed by Western blotting. We discovered that the H2S concentrations decreased asynchronously with the aging process in plasma, heart, liver, kidney, spleen, subcutaneous fat and brown fat and increased in brain and lung. At least one of the three H2S-generating enzymes expressions was compensatorily up-regulated with the aging process in most tissues, among which the up-regulation of CSE was the most prominent.

Myelination of axons is a key determinant of fast action potential propagation, axonal health and circuit function. Previously considered a static structure, it is now clear that myelin is dynamically regulated in response to neuronal activity in the central nervous system (CNS). However, how activity-dependent signals are conveyed to oligodendrocytes remains unclear. Here, we review the potential mechanisms by which neurons could communicate changing activity levels to myelin, with a focus on the accumulating body of evidence to support activity-dependent vesicular signalling directly onto myelin sheaths. We discuss recent in vivo findings of activity-dependent fusion of neurotransmitter vesicles from non-synaptic axonal sites, and how modulation of this vesicular fusion regulates the stability and growth of myelin sheaths. We also consider the potential mechanisms by which myelin could sense and respond to axon-derived signals to initiate remodelling, and the relevance of these adaptations for circuit function. We propose that axonal vesicular signalling represents an important and underappreciated mode of communication by which neurons can transmit activity-regulated signals to myelinating oligodendrocytes and, potentially, more broadly to other cell types in the CNS.