分类: 生物学 >> 生物物理学 提交时间: 2016-05-12
摘要: Specific arrestin conformations are coupled to distinct downstream effectors, which underlie the functions of many G-protein-coupled receptors (GPCRs). Here, using unnatural amino acid incorporation and fluorine-19 nuclear magnetic resonance (F-19-NMR) spectroscopy, we demonstrate that distinct receptor phospho-barcodes are translated to specific beta-arrestin-1 conformations and direct selective signalling. With its phosphate-binding concave surface, b-arrestin-1 'reads' the message in the receptor phospho-C-tails and distinct phospho-interaction patterns are revealed by F-19-NMR. Whereas all functional phosphopeptides interact with a common phosphate binding site and induce the movements of finger and middle loops, different phospho-interaction patterns induce distinct structural states of b-arrestin-1 that are coupled to distinct arrestin functions. Only clathrin recognizes and stabilizes GRK2-specific b-arrestin-1 conformations. The identified receptor-phosphoselective mechanism for arrestin conformation and the spacing of the multiple phosphatebinding sites in the arrestin enable arrestin to recognize plethora phosphorylation states of numerous GPCRs, contributing to the functional diversity of receptors.
分类: 生物学 >> 生物物理学 >> 细胞生物学 提交时间: 2016-05-12
摘要: Multidrug resistance is a serious threat to public health. Proton motive force-driven antiporters from the major facilitator superfamily (MFS) constitute a major group of multidrug-resistance transporters. Currently, no reports on crystal structures of MFS antiporters in complex with their substrates exist. The E. coli MdfA transporter is a well-studied model system for biochemical analyses of multidrug-resistance MFS antiporters. Here, we report three crystal structures of MdfA-ligand complexes at resolutions up to 2.0 angstrom, all in the inward-facing conformation. The substrate-binding site sits proximal to the conserved acidic residue, D34. Our mutagenesis studies support the structural observations of the substrate-binding mode and the notion that D34 responds to substrate binding by adjusting its protonation status. Taken together, our data unveil the substrate-binding mode of MFS antiporters and suggest a mechanism of transport via this group of transporters.
分类: 生物学 >> 生物物理学 提交时间: 2016-05-11
摘要: Liver cancer stem cells (CSCs) harbour self-renewal and differentiation properties, accounting for chemotherapy resistance and recurrence. However, the molecular mechanisms to sustain liver CSCs remain largely unknown. In this study, based on analysis of several hepatocellular carcinoma (HCC) transcriptome datasets and our experimental data, we find that C8orf4 is weakly expressed in HCC tumours and liver CSCs. C8orf4 attenuates the self-renewal capacity of liver CSCs and tumour propagation. We show that NOTCH2 is activated in liver CSCs. C8orf4 is located in the cytoplasm of HCC tumour cells and associates with the NOTCH2 intracellular domain, which impedes the nuclear translocation of N2ICD. C8orf4 deletion causes the nuclear translocation of N2ICD that triggers the NOTCH2 signalling, which sustains the stemness of liver CSCs. Finally, NOTCH2 activation levels are consistent with clinical severity and prognosis of HCC patients. Altogether, C8orf4 negatively regulates the self-renewal of liver CSCs via suppression of NOTCH2 signalling.