Palmitoylation of PD-L1 Regulates Its Membrane Orientation and Immune Evasion

Recently identified palmitoylation of PD-L1 is essential for immune regulation. To elucidate the underlying molecular mechanism, we performed giant plasma membrane vesicle (GPMV) experiments, us-scale all-atom molecular dynamics (MD) simulations and immune killing experiments. GPMV experiments indicated that PD-L1 palmitoylation enhanced its lipid raft affinity. MD simulations revealed dramatically different membrane orientation states of PD-L1 in liquid-ordered (L_o, lipid raft) compared to liquid-disordered (L_d, non-raft) membrane environments. L_d region promoted the lie-down orientation of PD-L1, which could inhibit its association with the PD-1 protein on immune cells and thus promote the immune killing of cancer cells. This hypothesis was supported by immune killing experiments using gamma-delta-T cells as effector cells and NCI-H1299 lung cancer cells as target cells. In short, our study demonstrates that the palmitoylation affects PD-L1 membrane localization and then membrane orientation, which thus regulates its binding with T cell PD-1 and the immune regulation. These observations may guide therapeutic strategies by explicating the regulation of immune checkpoint proteins by post-translational modifications and membrane environments.

Translation initiation factors and active sites of protein synthesis co-localize at the leading edge of migrating fibroblasts

Cell migration is a highly controlled essential cellular process, often dysregulated in tumour cells, dynamically controlled by the architecture of the cell. Studies involving cellular fractionation and microarray profiling have previously identified functionally distinct mRNA populations specific to cellular organelles and architectural compartments. However, the interaction between the translational machinery itself and cellular structures is relatively unexplored. To help understand the role for the compartmentalization and localized protein synthesis in cell migration, we have used scanning confocal microscopy, immunofluorescence and a novel ribopuromycylation method to visualize translating ribosomes. In the present study we show that eIFs (eukaryotic initiation factors) localize to the leading edge of migrating MRC5 fibroblasts in a process dependent on TGN (trans-Golgi network) to plasma membrane vesicle transport. We show that eIF4E and eIF4GI are associated with the Golgi apparatus and membrane microdomains, and that a proportion of these proteins co-localize to sites of active translation at the leading edge of migrating cells.

Stimulation of P2 receptors causes release of IL-1β–loaded microvesicles from human dendritic cells

Dendritic cells (DCs) are professional antigen-presenting cells that initiate the immune response by activating T lymphocytes. DCs express plasma membrane receptors for extracellular nucleotides named P2 receptors (P2Rs). Stimulation of P2Rs in these cells is known to cause chemotaxis, cytokine release, and cell death and to modulate LPS-dependent differentiation. Here we show that stimulation of the P2X7 receptor subtype (P2X7R) causes fast microvesicle shedding from DC plasma membrane. Vesicle release occurs from both immature and mature DCs; however, only vesicles from mature DCs, due to their previous exposure to LPS, contain IL-1β. Microvesicles, whether from immature or mature DCs, also contain caspase-1 and -3 and cathepsin D. They also express the P2X7R in addition to other P2Rs and known markers of immune cells such as major histocompatibility complex II (MHC II) and CD39. Activation of the P2X7R by extracellular ATP causes IL-1β release from the vesicle lumen. Previous studies demonstrated that high extracellular K+ inhibits IL-1β processing and release; here we show that high ionic strength reduces microvesicle shedding when compared with a low ionic strength medium but strongly increases microvesicle IL-1β loading.

Analysis of the Antimalarial Drug Resistance Protein Pfcrt Expressed in Yeast

Mutations in the novel membrane protein Pfcrt were recently found to be essential for chloroquine resistance (CQR) inPlasmodium falciparum, the parasite responsible for most lethal human malaria (Fidock, D. A., Nomura, T., Talley, A. K., Cooper, R. A., Dzekunov, S. M., Ferdig, M. T., Ursos, L. M., Sidhu, A. B., Naude, B., Deitsch, K. W., Su, X. Z., Wootton, J. C., Roepe, P. D., and Wellems, T. E. (2000)Mol. Cell6, 861–871). Pfcrt is localized to the digestive vacuolar membrane of the intraerythrocytic parasite and may function as a transporter. Study of this putative transport function would be greatly assisted by overexpression in yeast followed by characterization of membrane vesicles. Unfortunately, the very high AT content of malarial genes precludes efficient heterologous expression. Thus, we back-translated Pfcrt to design idealized genes with preferred yeast codons, no long poly(A) sequences, and minimal stem-loop structure. We synthesized a designed gene with a two-step PCR method, fused this to N- and C-terminal sequences to aid membrane insertion and purification, and now report efficient expression of wild type and mutant Pfcrt proteins in the plasma membrane ofSaccharomyces cerevisiaeandPichia pastorisyeast. To our knowledge, this is the first successful expression of a full-length malarial parasite integral membrane protein in yeast. Purified membranes and inside-out plasma membrane vesicle preparations were used to analyze wild typeversusCQR-conferring mutant Pfcrt function, which may include effects on H+transport (Dzekunov, S., Ursos, L. M. B., and Roepe, P. D. (2000)Mol. Biochem. Parasitol.110, 107–124), and to perfect a rapid purification of biotinylated Pfcrt. These data expand on the role of Pfcrt in conferring CQR and define a productive route for analysis of importantP. falciparumtransport proteins and membrane associated vaccine candidates.

The oxalate/sulfate antiporter in lobster hepatopancreas: internal and external binding constants

Utilizing a purified basolateral plasma membrane vesicle (BLMV) preparation containing a sulfate/oxalate antiporter, it was demonstrated that sulfate exhibited similar binding characteristics to the transporter whether bound internally or externally. It was also demonstrated that oxalate had similar binding characteristics to the antiporter whether it was bound internally or externally. Oxalate had a greater affinity to the transporter than did sulfate. Several organic anions affected binding and, therefore, overall transport by the antiporter. Most notably, sulfate was the only anion that stimulated oxalate uptake into BLMVs, which suggests a conservative binding specificity for the antiporter. 4-Acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS) and/or 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) inhibited the transport rate, confirming the existence of oxalate/sulfate exchange by the transporter. These results suggest that oxalate, not sulfate, regulates the transport rate because of its greater affinity to the transporter.