ATP-binding cassette A1 (ABCA1) is a key mediator of cholesterol and phospholipid efflux to apolipoprotein particles. We show that ABCA1 is a constitutively phosphorylated protein in both RAW macrophages and in a human embryonic kidney cell line expressing ABCA1. Furthermore, we demonstrate that phosphorylation of ABCA1 is mediated by protein kinase A (PKA) or a PKAlike kinase in vivo. Through site-directed mutagenesis studies of consensus PKA phosphorylation sites and in vitro PKA kinase assays, we show that Ser-1042 and Ser-2054, located in the nucleotide binding domains of ABCA1, are major phosphorylation sites for PKA. ApoA-I-dependent phospholipid efflux was decreased significantly by mutation of Ser-2054 alone and Ser-1042/Ser-2054 but was not significantly impaired with Ser-1042 alone. The mechanism by which ABCA1 phosphorylation affected ApoA-I-dependent phospholipid efflux did not involve either alterations in ApoA-I binding or changes in ABCA1 protein stability. These studies demonstrate a novel serine (Ser-2054) on the ABCA1 protein crucial for PKA phosphorylation and for regulation of ABCA1 transporter activity.

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease with rising incidence and a remarkable resistance to current therapies. The reasons for this therapeutic failure include the tumor's extensive infiltration by immunosuppressive cells such as myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs). By using light sheet fluorescent microscopy, we identified here direct interactions between these major immunoregulatory cells in PDAC. The in vivo depletion of MDSCs led to a significant reduction in Tregs in the pancreatic tumors. Through videomicroscopy and ex vivo functional assays we have shown that (i) MDSCs are able to induce Treg cells in a cell-cell dependent manner; (ii) Treg cells affect the survival and/or the proliferation of MDSCs. Furthermore, we have observed contacts between MDSCs and Treg cells at different stages of human cancer. Overall our findings suggest that interactions between MDSCs and Treg cells contribute to PDAC immunosuppressive environment.

The ATP Binding cassette transporter, ABCA1, controls the initial steps of reverse cholesterol transport, i.e., the release of cellular phospholipids (PL) to lipid-free apolipoproteins (1, 2). This has been unequivocally demonstrated by the study of Tangier patients (3-6), the naturally occurring loss-of-function model in humans, and by the targeted deletion of the ABCA1 locus in mice (7-9). In both cases, the level of circulating HDL cholesterol is highly reduced as a consequence of impaired cellular release of lipids from the membrane to their plasmatic acceptors (10, 11). This hampers the transformation of nascent lipid-free apolipoproteins into the centripetal cholesterol shuttle, the HDL particle. Molecular events required for the formation of a mature HDL particle include three successive steps (2). Surface docking of lipidpoor apolipoprotein, the core of the future HDL, is followed by the release from the cell surface of PL-enriched particles. These now assume the discoidal conformation permissive for cholesterol loading and maturation to spherical HDL. ABCA1 is considered to be rate limiting in the first two steps and indeed it has been demonstrated that the expression of ABCA1 promotes i ) phosphatidylserine (PS) outward flipping at the plasma membrane, ii ) apolipoprotein A-I (apoA-I) binding to the cell surface, and iii ) cellular release of PL to nascent apolipoproteins (9, 12, 13); however, the temporal and causal relationship among the ABCA1-elicited cellular phenotypes is far from being ascertained. In order to gain insight into this complex phenomenon, we elaborated a structure-function map of the ABCA1 transporter. This analyzes the effect of naturally occurring missense mutations on the function of the transporter and is based on the assessment of ABCA1 membrane topology via a systematic epitope insertion approach. We present evidence here that a spectrum of molecular defects can generate a Tangier phenotype by interfering differently with the activity of the molecule. In particular, we could observe, on appropriately engineered molecules, a dissociation between the outward flipping of PS and the surface binding of apoA-I. Since the loss of either function leads to impaired PL effluxes, this demonstrates that both events are absolute prerequisites for PL loading of apoA-I and thus for HDL formation. In addition, by manipulating structural determinants on the ABCA1