Highlights d Upon demyelination, Trem2 -/microglia fail to upregulate lipid metabolism genes d Trem2 -/microglia accumulate cholesteryl ester derived from myelin cholesterol d Accumulation is rescued by ACAT1 inhibitor and LXR agonist d Apoe -/glia defective in cholesterol transport also accumulate cholesteryl ester

SUMMARY Macroautophagy is essential to cell survival during starvation and proceeds by the growth of a double-membraned phagophore, which engulfs cytosol and other substrates. The synthesis and recognition of the lipid phosphatidylinositol 3-phosphate (PI(3)P) is essential for autophagy. The key autophagic PI(3)P sensors, which are conserved from yeast to humans, belong to the PROPPIN family. Here we report the crystal structure of the yeast PROPPIN Hsv2. The structure consists of a seven-bladed β-propeller, and unexpectedly, contains two pseudo-equivalent PI(3)P binding sites on blades 5 and 6. These two sites both contribute to membrane binding in vitro and are collectively required for full autophagic function in yeast. These sites function in concert with membrane binding by a hydrophobic loop in blade 6, explaining the specificity of the PROPPINs for membrane-bound PI(3)P. These observations thus provide a structural and mechanistic framework for one of the conserved central molecular recognition events in autophagy.

Effective delivery of protein therapeutics to the central nervous system (CNS) has been greatly restricted by the blood-brain barrier (BBB). We describe the development of a BBB transport vehicle (TV) comprising an engineered Fc fragment that exploits receptor-mediated transcytosis for CNS delivery of biotherapeutics by binding a highly expressed brain endothelial cell target. TVs were engineered using directed evolution to bind the apical domain of the human transferrin receptor (hTfR) without the use of amino acid insertions, deletions, or unnatural appendages. A crystal structure of the TV-TfR complex revealed the TV binding site to be away from transferrin and FcRn binding sites, which was further confirmed experimentally in vitro and in vivo. Recombinant expression of TVs fused to anti–β-secretase (BACE1) Fabs yielded antibody transport vehicle (ATV) molecules with native immunoglobulin G (IgG) structure and stability. Peripheral administration of anti-BACE1 ATVs to hTfR-engineered mice and cynomolgus monkeys resulted in substantially improved CNS uptake and sustained pharmacodynamic responses. The TV platform readily accommodates numerous additional configurations, including bispecific antibodies and protein fusions, yielding a highly modular CNS delivery platform.

Regulation of the storage of glycogen, one of the major energy reserves, is of utmost metabolic importance. In eukaryotes, this regulation is accomplished through glucose-6-phosphate levels and protein phosphorylation. Glycogen synthase homologs in bacteria and archaea lack regulation, while the eukaryotic enzymes are inhibited by protein kinase mediated phosphorylation and activated by protein phosphatases and glucose-6-phosphate binding. We determined the crystal structures corresponding to the basal activity state and glucose-6-phosphate activated state of yeast glycogen synthase-2. The enzyme is assembled into an unusual tetramer by an insertion unique to the eukaryotic enzymes, and this subunit interface is rearranged by the binding of glucose-6-phosphate, which frees the active site cleft and facilitates catalysis. Using both mutagenesis and intein-mediated phospho-peptide ligation experiments, we demonstrate that the enzyme's response to glucose-6-phosphate is controlled by Arg583 and Arg587, while four additional arginine residues present within the same regulatory helix regulate the response to phosphorylation.allosteric activation | glycosyltransferase G lycogen is a major energy repository in eukaryotes, and the biosynthetic pathway of glycogen synthesis is highly conserved from yeast to humans. The structure of glycogen is characterized by linear chains of glucose linked by α-1,4 glycosidic bonds and branch points occurring every 10 to 13 residues through introduction of α-1,6 linkages. In eukaryotes, glycogen synthase catalyzes the linear polymerization of glucose by transferring glucose residues from UDP-glucose to the 4'-hydroxyl end of a growing glycogen chain and is rate-limiting for synthesis under most circumstances (1).As the first known intracellular target of insulin action (2), the regulation of glycogen synthase (GS) has been a subject of intense investigation for over 50 years. However, only within the past seven years has structural information on any GS enzyme become available. To date, structures for three members of the GS family have been determined-monomeric Escherichia coli enzyme (3), dimeric Agrobacterium enzyme (4), and trimeric Pyrococcus enzyme (5). GS enzymes are members of the GTB fold of glycosyl transferases, which are further divided into two families, GT3 and GT5, based on sequence identity and their responses to posttranslational regulation (6, 7). The eukaryotic GT3 family of enzymes is activated by glucose-6-phosphate, is inhibited by protein phosphorylation, and shares less than 15% pair-wise sequence identity to the GT5 family enzymes (1).Similar to many higher eukaryotes, Saccharomyces cerevisiae has two distinct genes encoding glycogen synthases, GSY1 (708 residues*) and GSY2 (705 residues), of which GSY2 is nutritionally regulated and is the more important isoenzyme for glycogen accumulation (8). In higher eukaryotes the two genes (GYS1 and GYS2) encode distinct isozymes of 738 and 704 residues, respectively, that differ in their tissue expression patterns and t...