Protein glycosylation is an important posttranslational process, which regulates protein folding and functional expression. Studies have shown that abnormal glycosylation in tumor cells affects cancer progression and malignancy. In the current study, we have identified sialylated proteins using an alkynyl sugar probe in two different lung cancer cell lines, CL1-0 and CL1-5 with distinct invasiveness derived from the same parental cell line. Among the identified sialylated proteins, epidermal growth factor receptor (EGFR) was chosen to understand the effect of sialylation on its function. We have determined the differences in glycan sequences of EGFR in both cells and observed higher sialylation and fucosylation of EGFR in CL1-5 than in CL1-0. Further study suggested that overexpression of sialyltransferases in CL1-5 and α1,3-fucosyltransferases (FUT4 or FUT6) in CL1-5 and A549 cells would suppress EGFR dimerization and phosphorylation upon EGF treatment, as compared to the control and CL1-0 cells. Such modulating effects on EGFR dimerization were further confirmed by sialidase or fucosidase treatment. Thus, increasing sialylation and fucosylation could attenuate EGFRmediated invasion of lung cancer cells. However, incorporation of the core fucose by α1,6-fucosylatransferase (FUT8) would promote EGFR dimerization and phosphorylation.sialic acid | glycoproteomics | glycan sequencing | click chemistry | mass spectrometry
G-protein-coupled receptors (GPCRs) are involved in many physiological processes and are therefore key drug targets. Although detailed structural information is available for GPCRs, the effects of lipids on the receptors, and on downstream coupling of GPCRs to G proteins are largely unknown. Here we use native mass spectrometry to identify endogenous lipids bound to three class A GPCRs. We observed preferential binding of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P) over related lipids and confirm that the intracellular surface of the receptors contain hotspots for PtdIns(4,5)P binding. Endogenous lipids were also observed bound directly to the trimeric Gαβγ protein complex of the adenosine A receptor (AR) in the gas phase. Using engineered Gα subunits (mini-Gα mini-Gα and mini-Gα), we demonstrate that the complex of mini-Gα with the β adrenergic receptor (βAR) is stabilized by the binding of two PtdIns(4,5)P molecules. By contrast, PtdIns(4,5)P does not stabilize coupling between βAR and other Gα subunits (mini-Gα or mini-Gα) or a high-affinity nanobody. Other endogenous lipids that bind to these receptors have no effect on coupling, highlighting the specificity of PtdIns(4,5)P. Calculations of potential of mean force and increased GTP turnover by the activated neurotensin receptor when coupled to trimeric Gαβγ complex in the presence of PtdIns(4,5)P provide further evidence for a specific effect of PtdIns(4,5)P on coupling. We identify key residues on cognate Gα subunits through which PtdIns(4,5)P forms bridging interactions with basic residues on class A GPCRs. These modulating effects of lipids on receptors suggest consequences for understanding function, G-protein selectivity and drug targeting of class A GPCRs.
We report a 3.5-angstrom-resolution cryo–electron microscopy structure of a respiratory supercomplex isolated fromMycobacterium smegmatis.It comprises a complex III dimer flanked on either side by individual complex IV subunits. Complex III and IV associate so that electrons can be transferred from quinol in complex III to the oxygen reduction center in complex IV by way of a bridging cytochrome subunit. We observed a superoxide dismutase-like subunit at the periplasmic face, which may be responsible for detoxification of superoxide formed by complex III. The structure reveals features of an established drug target and provides a foundation for the development of treatments for human tuberculosis.
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