Macrocycle-stabilization of its interaction with 14-3-3 increases plasma membrane localization and activity of CFTR

Impaired activity of the chloride channel CFTR is the cause of cystic fibrosis. 14-3-3 proteins have been shown to stabilize CFTR and increase its biogenesis and activity. Here, we report the identification and mechanism of action of a macrocycle stabilizing the 14-3-3/CFTR complex, a first-in-class molecular glue. This molecule rescues plasma membrane localization and chloride transport of F508del-CFTR and works additively with the CFTR pharmacological chaperone corrector lumacaftor (VX-809).

Role of Ezrin/Radixin/Moesin in the Surface Localization of Programmed Cell Death Ligand-1 in Human Colon Adenocarcinoma LS180 Cells

Programmed cell death ligand-1 (PD-L1), an immune checkpoint protein highly expressed on the cell surface in various cancer cell types, binds to programmed cell death-1 (PD-1), leading to T-cell dysfunction and tumor survival. Despite clinical successes of PD-1/PD-L1 blockade therapies, patients with colorectal cancer (CRC) receive little benefit because most cases respond poorly. Because high PD-L1 expression is associated with immune evasion and poor prognosis in CRC patients, identifying potential modulators for the plasma membrane localization of PD-L1 may represent a novel therapeutic strategy for enhancing the efficacy of PD-1/PD-L1 blockade therapies. Here, we investigated whether PD-L1 expression in human colorectal adenocarcinoma cells (LS180) is affected by ezrin/radixin/moesin (ERM), functioning as scaffold proteins that crosslink plasma membrane proteins with the actin cytoskeleton. We observed colocalization of PD-L1 with all three ERM proteins in the plasma membrane and detected interactions involving PD-L1, the three ERM proteins, and the actin cytoskeleton. Furthermore, gene silencing of ezrin and radixin, but not of moesin, substantially decreased the expression of PD-L1 on the cell surface without affecting its mRNA level. Thus, in LS180 cells, ezrin and radixin may function as scaffold proteins mediating the plasma membrane localization of PD-L1, possibly by post-translational modification.

Three conserved hydrophobic residues in the CC domain of Pit contribute to its plasma membrane localization and immune induction

Nucleotide-binding leucine-rich repeat (NLR) proteins work as crucial intracellular immune receptors. N-terminal domains of NLRs fall into two groups, namely coiled-coil (CC) and Toll-interleukin 1 receptor (TIR) domains, which play critical roles in signal transduction and disease resistance. However, the activation mechanisms of NLRs, and how their N-termini are involved in immune induction, remain largely unknown. Here, we revealed that the rice NLR Pit self-associates through its CC domain. The CC domain of Pit possesses three conserved hydrophobic residues that are known to be involved in homodimer formation in two NLRs, barley MLA10 and Arabidopsis RPM1. Interestingly, the function of these residues in Pit is different from that in MLA10 and RPM1. Although the three hydrophobic residues are important for Pit-induced disease resistance against rice blast fungus, they do not participate in self-association or in binding to downstream signaling molecules. Based on homology modeling of Pit using the structure of the Arabidopsis NLR ZAR1, we tried to clarify the role of the three conserved hydrophobic residues and found that they are involved in the plasma membrane localization. Our findings provide novel insights for understanding the mechanisms of NLR activation as well as the relationship between subcellular localization and immune induction.

Rice Non-Specific Phospholipase C6 Is Involved in Mesocotyl Elongation

ABSTRACT Mesocotyl elongation of rice is crucial for seedlings pushing out of deep soil. Underlying mechanisms on the phospholipid signaling in mesocotyl growth of rice are elusive. Here we report that the rice non-specific phospholipase C6 (OsNPC6) is involved in the mesocotyl elongation. Our results indicated that all five rice OsNPCs (OsNPC1, OsNPC2, OsNPC3, OsNPC4 and OsNPC6) hydrolyzed the substrate phosphatidylcholine (PC) to phosphocholine (PCho), and all of them showed plasma membrane localization. Overexpression (OE) of OsNPC6 produced plants with shorter mesocotyls, while comparing to those of Nipponbare (Nip) and npc6 mutants. Although the mesocotyl growth of npc6 mutants was not much affected without GA3, it was obviously elongated by the treatment of GA. Upon GA3 treatment, SLENDER RICE1 (SLR1), the DELLA protein of gibberellin (GA) signaling, was drastically increased in OE plants, in contrary, the level of SLR1 was found decreased in npc6 mutants. The GA-enhanced mesocotyl elongation and the GA-impaired SLR1 level in npc6 mutants were attenuated by the supplementation of PCho. Further analysis indicated that the GA-induced expression of phospho-base N-methyltransferase 1 (PMT1) in npc6 mutants was significantly weakened by the addition of PCho. In summary, our results suggest that OsNPC6 is involved in mesocotyl development via modulation of phosphocholine in rice.

Subcellular distribution of ezrin/radixin/moesin and their roles in the cell surface localization and transport function of P-glycoprotein in human colon adenocarcinoma LS180 cells

The ezrin/radixin/moesin (ERM) family proteins act as linkers between the actin cytoskeleton and P-glycoprotein (P-gp) and regulate the plasma membrane localization and functionality of the latter in various cancer cells. Notably, P-gp overexpression in the plasma membrane of cancer cells is a principal factor responsible for multidrug resistance and drug-induced mutagenesis. However, it remains unknown whether the ERM proteins contribute to the plasma membrane localization and transport function of P-gp in human colorectal cancer cells in which the subcellular localization of ERM has yet to be determined. This study aimed to determine the gene expression patterns and subcellular localization of ERM and P-gp and investigate the role of ERM proteins in the plasma membrane localization and transport function of P-gp using the human colon adenocarcinoma cell line LS180. Using real-time reverse transcription polymerase chain reaction and immunofluorescence analyses, we showed higher levels of ezrin and moesin mRNAs than those of radixin mRNA in these cells and preferential distribution of all three ERM proteins on the plasma membrane. The ERM proteins were highly colocalized with P-gp. Additionally, we show that the knockdown of ezrin, but not of radixin and moesin, by RNA interference significantly decreased the cell surface expression of P-gp in LS180 cells without affecting the mRNA expression of P-gp. Furthermore, gene silencing of ezrin substantially increased the intracellular accumulation of rhodamine123, a typical P-gp substrate, with no alterations in the plasma membrane permeability of Evans blue, a passive transport marker. In conclusion, ezrin may primarily regulate the cell surface localization and transport function of P-gp as a scaffold protein without influencing the transcriptional activity of P-gp in LS180 cells. These findings should be relevant for treating colorectal cancer, which is the second leading cause of cancer-related deaths in males and females combined.

α-Synuclein plasma membrane localization correlates with cellular phosphatidylinositol polyphosphate levels

The Parkinson's disease protein α-synuclein (aSyn) promotes membrane fusion and fission by interacting with various negatively charged phospholipids. Despite postulated roles in endocytosis and exocytosis, plasma membrane (PM) interactions of αSyn are poorly understood. Here, we show that phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3), two highly acidic components of inner PM leaflets, mediate plasma membrane localization of endogenous pools of αSyn in A2780, HeLa, SK-MEL-2 and differentiated and undifferentiated neuronal SH-SY5Y cells. We demonstrate that αSyn binds to reconstituted PIP2-membranes in a helical conformation in vitro and that PIP2 synthesizing kinases and hydrolyzing phosphatases reversibly redistribute αSyn in cells. We further delineate that αSyn-PM targeting follows phosphoinositide-3 kinase (PI3K)-dependent changes of cellular PIP2 and PIP3 levels, which collectively suggests that phosphatidylinositol polyphosphates contribute to αSyn's cellular function(s) at the plasma membrane.