New strategies to block the immune evasion activity of programmed death ligand-1 (PD-L1) are urgently needed. When exploring the PD-L1-targeted effects of mechanistically diverse metabolism-targeting drugs, exposure to the dietary polyphenol resveratrol (RSV) revealed its differential capacity to generate a distinct PD-L1 electrophoretic migration pattern. Using biochemical assays, computer-aided docking/molecular dynamics simulations, and fluorescence microscopy, we found that RSV can operate as a direct inhibitor of glyco-PD-L1-processing enzymes (α-glucosidase/α-mannosidase) that modulate N-linked glycan decoration of PD-L1, thereby promoting the endoplasmic reticulum retention of a mannose-rich, abnormally glycosylated form of PD-L1. RSV was also predicted to interact with the inner surface of PD-L1 involved in the interaction with PD-1, almost perfectly occupying the target space of the small compound BMS-202 that binds to and induces dimerization of PD-L1. The ability of RSV to directly target PD-L1 interferes with its stability and trafficking, ultimately impeding its targeting to the cancer cell plasma membrane. Impedance-based real-time cell analysis (xCELLigence) showed that cytotoxic T-lymphocyte activity was notably exacerbated when cancer cells were previously exposed to RSV. This unforeseen immunomodulating mechanism of RSV might illuminate new approaches to restore T-cell function by targeting the PD-1/PD-L1 immunologic checkpoint with natural polyphenols.

Our findings add to the understanding of β2 role in Na 1.5 trafficking and localisation, which must influence cell excitability and electrical coupling in the heart. This study will contribute to knowledge on development of arrhythmias.

The voltage-gated sodium channel is critical for cardiomyocyte function and consists of a protein complex comprising a pore-forming ␣ subunit and two associated ␤ subunits. It has been shown previously that the associated ␤2 subunits promote cell surface expression of the ␣ subunit. The major ␣ isoform in the adult human heart is Na V 1.5, and germline mutations in the Na V 1.5-encoding gene, sodium voltage-gated channel ␣ subunit 5 (SCN5A), often cause inherited arrhythmias. Here, we investigated the mechanisms that regulate ␤2 trafficking and how they may determine proper Na V 1.5 cell surface localization. Using heterologous expression in polarized Madin-Darby canine kidney cells, we show that ␤2 is N-glycosylated in vivo and in vitro at residues 42, 66, and 74, becoming sialylated only at Asn-42. We found that fully nonglycosylated ␤2 was mostly retained in the endoplasmic reticulum, indicating that N-linked glycosylation is required for efficient ␤2 trafficking to the apical plasma membrane. The nonglycosylated variant reached the cell surface by bypassing the Golgi compartment at a rate of only approximately one-third of that of WT ␤2. YFP-tagged, nonglycosylated ␤2 displayed mobility kinetics in the plane of the membrane similar to that of WT ␤2. However, it was defective in promoting surface localization of Na V 1.5. Interestingly, ␤2 with a single intact glycosylation site was as effective as the WT in promoting Na V 1.5 surface localization. In conclusion, our results indicate that N-linked glycosylation of ␤2 is required for surface localization of Na V 1.5, a property that is often defective in inherited cardiac arrhythmias.Genetic alterations leading to channelopathies are frequently found in the voltage-gated sodium (Na V ) 3 channel (1). A well-known ion channel disorder causing ventricular fibrillation is Brugada syndrome (BrS). In this regard, ϳ20% of BrS cases are caused by mutations in SCN5A, the gene encoding Na V 1.5 (i.e. the pore-forming ␣ subunit of the major cardiac Na V channel) (2). The Na V channel allows fast influx of sodium ions, thus generating the rapid upward deflection of the action potential. Therefore, it plays a central role in myocardial cell excitability. The abnormal electrocardiogram observed in BrS is due to Na V channel loss-of-function, often caused by defective Na V 1.5 trafficking and localization to the cell surface (3). Na V 1.5 is localized at the sarcolemma (i.e. the cardiomyocytes' plasma membrane). The differential localization of Na V channel pools at sarcolemma subregions is important for conduction velocity and cardiac impulse propagation (4). Much evidence shows that localization and function of the ␣ subunit are regulated by Na V channel auxiliary ␤ subunits and other associated proteins (5). Analysis of Na V 1.5 trafficking can be envisaged from at least three standpoints: first, to address how Na V 1.5 is targeted to the plasma membrane; second, how Nav1.5 is retained at certain surface domains or subregions; and third, how Na V 1.5 endocytosis and turno...

The voltage-gated sodium channel is vital for cardiomyocyte function, and consists of a protein complex containing a pore-forming α subunit and two associated β subunits. A fundamental, yet unsolved, question is to define the precise function of β subunits. While their location in vivo remains unclear, large evidence shows that they regulate localization of α and the biophysical properties of the channel. The current data support that one of these subunits, β2, promotes cell surface expression of α. The main α isoform in an adult heart is NaV1.5, and mutations in SCN5A, the gene encoding NaV1.5, often lead to hereditary arrhythmias and sudden death. The association of β2 with cardiac arrhythmias has also been described, which could be due to alterations in trafficking, anchoring, and localization of NaV1.5 at the cardiomyocyte surface. Here, we will discuss research dealing with mechanisms that regulate β2 trafficking, and how β2 could be pivotal for the correct localization of NaV1.5, which influences cellular excitability and electrical coupling of the heart. Moreover, β2 may have yet to be discovered roles on cell adhesion and signaling, implying that diverse defects leading to human disease may arise due to β2 mutations.

The voltage-gated sodium channel is critical for cardiomyocyte function. It consists of a protein complex comprising a pore-forming α subunit and associated β subunits. In polarized Madin-Darby canine kidney cells, we show evidence by acyl-biotin exchange that β2 is S-acylated at Cys-182. Interestingly, we found that palmitoylation increases β2 association with detergent-resistant membranes. β2 localizes exclusively to the apical surface. However, depletion of plasma membrane cholesterol, or blocking intracellular cholesterol transport, caused mislocalization of β2, as well as of the non-palmitoylable C182S mutant, to the basolateral domain. Apical β2 did not undergo endocytosis and displayed limited diffusion within the plane of the membrane, in part behaving as cytoskeleton-anchored. Upon acute cholesterol depletion, its mobility was greatly reduced, and a slight reduction was also measured due to lack of palmitoylation, supporting β2 association with cholesterol-rich lipid rafts. Indeed, lipid raft labeling confirmed a partial overlap with apical β2. Although β2 palmitoylation was not found required to promote surface localization of the α subunit, our data suggest that it is likely implicated in lipid raft association and polarized localization of β2.

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