VPS13 proteins are widely conserved in eukaryotes and associated with human neurodegenerative and neurodevelopmental diseases. De et al. describe the lipid specificity and structure of yeast Vps13p, providing insight into its role in both TGN late endosome transport and TGN homotypic fusion.

SUMMARY Many neuropeptides and peptide hormones require amidation of their carboxy terminal for full biological activity. The enzyme Peptidyl-α-hydroxyglycine α-amidating lyase (PAL; EC 4.3.2.5) catalyzes the second and last step of this reaction – N-dealkylation of the peptidyl-α-hydroxyglycine to generate the α-amidated peptide and glyoxylate. Here we report the X-ray crystal structure of the PAL catalytic core (PALcc) alone and in complex with the non-peptidic substrate α-hydroxyhippuric acid. The structures show that PAL folds as a six-bladed β-propeller. The active site is formed by a Zn(II) ion coordinated by three histidine residues; the substrate binds to this site with its α-hydroxyl group coordinated to the Zn(II) ion. The structures also reveal a tyrosine residue (Tyr654) at the active site as the catalytic base for hydroxyl deprotonation, an unusual role for tyrosine. A reaction mechanism is proposed based on this structural data and validated by biochemical analysis of site-directed PALcc mutants.

The catalytic core of the peptidyl-␣-hydroxyglycine ␣-amidating lyase (PAL) domain of peptidylglycine ␣-amidating monooxygenase was investigated with respect to its ability to function as a ureidoglycolate lyase and the identity and role of its bound metal ions. The purified PAL catalytic core (PALcc) contains molar equivalents of calcium and zinc along with substoichiometric amounts of iron and functions as a ureidoglycolate lyase. Limiting iron availability in the cells synthesizing PALcc reduces the specific activity of the enzyme produced. Concentrated samples of native PALcc have an absorption maximum at 560 nm, suggestive of a phenolate-Fe(III) charge transfer complex. An essential role for a Tyr residue was confirmed by elimination of PAL activity following site-directed mutagenesis. Purified PALcc in which the only conserved Tyr residue (Tyr 654 ) was mutated to Phe was secreted normally, but was catalytically inactive and lacked bound iron and bound zinc. Our data demonstrate an essential role for Tyr 654 and suggest that it serves as an Fe(III) ligand in an essential iron-zinc bimetallic site.

We explored the effect of copper availability on the synthesis and trafficking of peptidylglycine ␣-amidating monooxygenase (PAM), an essential cuproenzyme whose catalytic domains function in the lumen of peptide-containing secretory granules. Corticotrope tumor cell lines expressing integral membrane and soluble forms of PAM were depleted of copper using bathocuproinedisulfonic acid or loaded with copper by incubation with CuCl 2 . Depleting cellular copper stimulates basal secretion of soluble enzyme produced by endoproteolytic cleavage of PAM in secretory granules and transit of membrane PAM though the endocytic pathway and back into secretory granules. Unlike many cuproenzymes, lack of copper does not lead to instability of PAM. Copper loading decreases cleavage of PAM in secretory granules, secretion of soluble enzyme, and the return of internalized PAM to secretory granules. The trafficking and stability of the soluble, luminal domain of PAM and truncated membrane PAM lacking a cytosolic domain are not affected by copper availability. Taken together, our data demonstrate a role for copper-sensitive cytosolic machinery in directing endocytosed membrane PAM back to secretory granules or to a degradative pathway. The response of PAM to lack of copper suggests that it facilitates copper homeostasis.

Interferon lambda (IFNλ) signaling is a promising therapeutic target against viral infection in murine models, yet little is known about its molecular regulation and its cognate receptor, interferon lambda receptor 1 (IFNLR1) in human lung. We hypothesized that the IFNλ signaling axis was active in human lung macrophages. In human alveolar macrophages (HAMs), we observed increased IFNLR1 expression and robust increase in interferon-stimulated gene (ISG) expression in response to IFNλ ligand. While human monocytes express minimal IFNLR1, differentiation of monocytes into macrophages with macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) increased IFNLR1 mRNA, IFNLR1 protein expression, and cellular response to IFNλ ligation. Conversely, in mice, M-CSF or GM-CSF stimulated macrophages failed to produce ISGs in response to related ligands, IFNL2 or IFNL3, suggesting that IFNLR1 signaling in macrophages is species-specific. We next hypothesized that IFNλ signaling was critical in influenza antiviral responses. In primary human airway epithelial cells and precision-cut human lung slices, influenza infection substantially increased IFNλ levels. Pretreatment of both HAMs and differentiated human monocytes with IFNL1 significantly inhibited influenza infection. IFNLR1 knockout in the myeloid cell line, THP-1, exhibited reduced interferon responses to either direct or indirect exposure to influenza infection suggesting the indispensability of IFNLR1 for antiviral responses. These data demonstrate the presence of IFNλ - IFNLR1 signaling axis in human lung macrophages and a critical role of IFNλ signaling in combating influenza infection.