1.
Ultralow Background Membrane Editors for Spatiotemporal Control of Phosphatidic Acid Metabolism and Signaling
Abstract:
Phosphatidic acid (PA) is a multifunctional lipid with important metabolic and signaling functions, and efforts to dissect its pleiotropy demand strategies for perturbing its levels with spatiotemporal precision. Previous membrane editing approaches for generating local PA pools used light-mediated induced proximity to recruit a PA-synthesizing enzyme, phospholipase D (PLD), from the cytosol to the target organelle membrane. Whereas these optogenetic PLDs exhibited high activity, their residual activity in the dark led to undesired chronic lipid production. Here, we report ultralow background membrane editors for PA wherein light directly controls PLD catalytic activity, as opposed to localization and access to substrates, exploiting a light–oxygen–voltage (LOV) domain-based conformational photoswitch inserted into the PLD sequence and enabling their stable and nonperturbative targeting to multiple organelle membranes. By coupling organelle-targeted LOVPLD activation to lipidomics analysis, we discovered different rates of metabolism for PA and its downstream products depending on the subcellular location of PA production. We also elucidated signaling roles for PA pools on different membranes in conferring local activation of AMP-activated protein kinase signaling. This work illustrates how membrane editors featuring acute, optogenetic conformational switches can provide new insights into organelle-selective lipid metabolic and signaling pathways.
2.
Gigavalent Display of Proteins on Monodisperse Polyacrylamide Hydrogels as a Versatile Modular Platform for Functional Assays and Protein Engineering.
Abstract:
The assembly of robust, modular biological components into complex functional systems is central to synthetic biology. Here, we apply modular "plug and play" design principles to a solid-phase protein display system that facilitates protein purification and functional assays. Specifically, we capture proteins on polyacrylamide hydrogel display beads (PHD beads) made in microfluidic droplet generators. These monodisperse PHD beads are decorated with predefined amounts of anchors, methacrylate-PEG-benzylguanine (BG) and methacrylate-PEG-chloroalkane (CA), that react covalently with SNAP-/Halo-tag fusion proteins, respectively, in a specific, orthogonal, and stable fashion. Anchors, and thus proteins, are distributed throughout the entire bead volume, allowing attachment of ∼109 protein molecules per bead (⌀ 20 μm) -a higher density than achievable with commercial surface-modified beads. We showcase a diverse array of protein modules that enable the secondary capture of proteins, either noncovalently (IgG and SUMO-tag) or covalently (SpyCatcher, SpyTag, SnpCatcher, and SnpTag), in mono- and multivalent display formats. Solid-phase protein binding and enzymatic assays are carried out, and incorporating the photocleavable protein PhoCl enables the controlled release of modules via visible-light irradiation for functional assays in solution. We utilize photocleavage for valency engineering of an anti-TRAIL-R1 scFv, enhancing its apoptosis-inducing potency ∼50-fold through pentamerization.
