Adrian Salic

Phosphatidylethanolamine (PE) is the second most abundant class of phospholipids in eukaryotic membranes, as well as a precursor for essential posttranslational protein modifications, such as PE conjugates of ubiquitin and ATG8/LC3 that play key roles in autophagy, and glycosylphosphatidylinositol (GPI) anchors of numerous cell surface proteins. Bioorthogonal chemistry has revolutionized how phospholipid biosynthesis, transport, and turnover are studied, with clickable metabolic precursors now available for several phospholipid classes. Yet no metabolic bioorthogonal probe for labeling endogenous PE and PE‐derived protein modifications has been developed. Here, we introduce an alkyne‐tagged ethanolamine analog (AlkEA) that is incorporated into PE via the Kennedy pathway and can be derivatized by copper‐catalyzed azide–alkyne cycloaddition (CuAAC) for visualization and affinity enrichment. Confocal microscopy revealed the subcellular distribution of AlkEA‐labeled PE in the ER, Golgi, mitochondria, and autophagosomes, while lipidomic analysis demonstrated AlkEA incorporation across diverse PE species. AlkEA labeling also allowed affinity isolation of PE‐conjugated LC3 and ubiquitin, as well as that of a prototypical GPI‐anchored protein. AlkEA is thus a minimally perturbing tool broadly applicable to dissecting PE metabolism and PE‐dependent protein modifications.

Phosphatidylethanolamine (PE) is the second most abundant class of phospholipids in eukaryotic membranes, as well as a precursor for essential posttranslational protein modifications, such as PE conjugates of ubiquitin and ATG8/LC3 that play key roles in autophagy, and glycosylphosphatidylinositol (GPI) anchors of numerous cell surface proteins. Bioorthogonal chemistry has revolutionized how phospholipid biosynthesis, transport, and turnover are studied, with clickable metabolic precursors now available for several phospholipid classes. Yet no metabolic bioorthogonal probe for labeling endogenous PE and PE‐derived protein modifications has been developed. Here, we introduce an alkyne‐tagged ethanolamine analog (AlkEA) that is incorporated into PE via the Kennedy pathway and can be derivatized by copper‐catalyzed azide–alkyne cycloaddition (CuAAC) for visualization and affinity enrichment. Confocal microscopy revealed the subcellular distribution of AlkEA‐labeled PE in the ER, Golgi, mitochondria, and autophagosomes, while lipidomic analysis demonstrated AlkEA incorporation across diverse PE species. AlkEA labeling also allowed affinity isolation of PE‐conjugated LC3 and ubiquitin, as well as that of a prototypical GPI‐anchored protein. AlkEA is thus a minimally perturbing tool broadly applicable to dissecting PE metabolism and PE‐dependent protein modifications.

Wnt proteins are lipid-modified morphogens fundamental in development and disease. During Wnt biogenesis, the G-protein-coupled receptor (GPCR)-like transporter Wntless (WLS) escorts lipidated Wnts from the endoplasmic reticulum to the plasma membrane, then transfers them to extracellular carriers, forming active and soluble morphogen-carrier complexes. To dissect the mechanisms involved, we solve cryo-EM structures of Wnt-bound WLS and unliganded WLS, and perform structure-guided functional experiments. Wnts engage WLS via three conserved hairpins, which are all required for Wnt trafficking to the cell surface and carrier-mediated secretion. Wnt release from cells is driven by dramatic conformational changes in the WLS transmembrane domain, reminiscent of GPCR activation, together with WLS extracellular rearrangements. Unexpectedly, we find that Wnt5a bound to WLS forms dimers, with implications for Wnt signaling. These findings define the mechanism of WLS conformational cycling that governs the intracellular transport and extracellular release of Wnt morphogens, essential steps in the Wnt pathway.

Phosphatidylethanolamine (PE) is the second most abundant class of phospholipids in eukaryotic membranes, as well as a precursor for essential post-translational protein modifications, such as PE conjugates of ubiquitin and ATG8/LC3 that play key roles in autophagy, and glycosylphosphatidylinositol (GPI) anchors of numerous cell surface proteins. Bioorthogonal chemistry has revolutionized how phospholipid biosynthesis, transport, and turnover are studied, with clickable metabolic precursors now available for several phospholipid classes. Yet a probe for labeling PE and PE-derived protein modifications has not yet been developed. Here, we introduce an alkyne-tagged ethanolamine analog (AlkEA) that is incorporated into PE via the Kennedy pathway and can be derivatized by copper-catalyzed azide–alkyne cycloaddition (CuAAC) for visualization and affinity enrichment. Confocal microscopy revealed the subcellular distribution of AlkEA-labeled PE in the ER, Golgi, mitochondria, and autophagosomes, while lipidomic analysis demonstrated AlkEA incorporation across diverse PE species. AlkEA labeling also allowed affinity isolation of PE-conjugated LC3 and ubiquitin, as well as that of a prototypical GPI-anchored protein. AlkEA is thus a minimally perturbing tool broadly applicable to dissecting PE metabolism and PE-dependent protein modifications.

Coronavirus-infected cells contain double-membrane vesicles (DMVs) that are key for viral RNA replication and transcription, perforated by hexameric pores connecting the vesicular lumen to the cytoplasm. How pores form and traverse two membranes, and how DMVs organize RNA synthesis, is unknown. Using structure prediction and functional assays, we show that the nonstructural viral membrane protein nsp4 is the key pore organizer, spanning the double membrane and forming most of the pore lining. Nsp4 interacts with nsp3 on the cytoplasmic side and with the viral replicase inside the DMV. Newly synthesized mRNAs exit the DMV into the cytoplasm, passing through a narrow ring of conserved nsp4 residues. Steric constraints imposed by the ring predict that modified nucleobases block mRNA transit, resulting in broad-spectrum anticoronaviral activity.

The steroid hormone aldosterone, produced by the zona glomerulosa (zG) of the adrenal gland, is a master regulator of plasma electrolytes and blood pressure. While aldosterone control by the renin-angiotensin system is well understood, other key regulatory factors have remained elusive. Here, we replicated a prior association between a non-coding variant in WNT2B and an increased risk of primary aldosteronism, a prevalent and debilitating disease caused by excessive aldosterone production. We further show that in both mice and humans, WNT2B is expressed in the mesenchymal capsule surrounding the adrenal cortex, in close proximity to the zG. Global loss of Wnt2b in the mouse results in a dysmorphic and hypocellular zG, with impaired aldosterone production. Similarly, humans harboring WNT2B loss-of-function mutations develop a novel form of Familial Hyperreninemic Hypoaldosteronism, designated here as Type 4. Additionally, we demonstrate that WNT2B signals by activating the non-canonical Wnt/planar cell polarity pathway. Our findings identify WNT2B as a key regulator of zG function and aldosterone production with important clinical implications. Highlights: WNT2B variant is associated with increased risk for primary aldosteronism Wnt2b knock-out mice show defects in adrenal morphology Wnt2b knock-out mice have hyperreninemic hypoaldosteronism WNT2B activates non-canonical Wnt/planar cell polarity signaling WNT2B deficiency causes a new form of familial hyperreninemic hypoaldosteronism

Coronavirus-infected cells contain double-membrane vesicles (DMVs) that are key for viral RNA replication and transcription, perforated by hexameric pores connecting the vesicular lumen to the cytoplasm. How pores form and traverse two membranes, and how DMVs organize RNA synthesis, is unknown. Using structure prediction and functional assays, we show that the non-structural viral membrane protein nsp4 is the key DMV pore organizer, spanning the double membrane and forming most of the pore lining. Nsp4 interacts with nsp3 on the cytoplasmic side and with the viral replicase inside the DMV. Newly synthesized mRNAs exit the DMV into the cytoplasm, passing through a narrow ring of conserved nsp4 residues. Steric constraints imposed by the ring predict that modified nucleobases block mRNA transit, with broad spectrum anti-coronaviral activity.

Glypicans are a family of cell surface heparan sulfate proteoglycans that play critical roles in multiple cell signaling pathways. Glypicans consist of a globular core, an unstructured stalk modified with sulfated glycosaminoglycan chains, and a glycosylphosphatidylinositol anchor. Though these structural features are conserved, their individual contribution to glypican function remains obscure. Here, we investigate how glypican 3 (GPC3), which is mutated in Simpson–Golabi–Behmel tissue overgrowth syndrome, regulates Hedgehog signaling. We find that GPC3 is necessary for the Hedgehog response, surprisingly controlling a downstream signal transduction step. Purified GPC3 ectodomain rescues signaling when artificially recruited to the surface of GPC3-deficient cells but has dominant-negative activity when unattached. Strikingly, the purified stalk, modified with heparan sulfate but not chondroitin sulfate, is necessary and sufficient for activity. Our results demonstrate a novel function for GPC3-associated heparan sulfate and provide a framework for the functional dissection of glycosaminoglycans by in vivo biochemical complementation. This article has an associated First Person interview with the first author of the paper.