3 research outputs found
Identification of the Plasticity-Relevant Fucose-Ī±(1ā2)-Galactose Proteome from the Mouse Olfactory Bulb
Fucose-Ī±(1ā2)-galactose [FucĪ±(1ā2)Gal] sugars have been implicated in the molecular mechanisms that underlie neuronal development, learning, and memory. However, an understanding of their precise roles has been hampered by a lack of information regarding FucĪ±(1ā2)Gal glycoproteins. Here, we report the first proteomic studies of this plasticity-relevant epitope. We identify five classes of putative FucĪ±(1ā2)Gal glycoproteins: cell adhesion molecules, ion channels and solute carriers/transporters, ATP-binding proteins, synaptic vesicle-associated proteins, and mitochondrial proteins. In addition, we show that FucĪ±(1ā2)Gal glycoproteins are enriched in the developing mouse olfactory bulb (OB) and exhibit a distinct spatiotemporal expression that is consistent with the presence of a āglycocodeā to help direct olfactory sensory neuron (OSN) axonal pathfinding. We find that expression of FucĪ±(1ā2)Gal sugars in the OB is regulated by the Ī±(1ā2)fucosyltransferase FUT1. FUT1-deficient mice exhibit developmental defects, including fewer and smaller glomeruli and a thinner olfactory nerve layer, suggesting that fucosylation contributes to OB development. Our findings significantly expand the number of FucĪ±(1ā2)Gal glycoproteins and provide new insights into the molecular mechanisms by which fucosyl sugars contribute to neuronal processes
Development of Sulfonamide Photoaffinity Inhibitors for Probing Cellular Ī³āSecretase
Ī³-Secretase
is a multiprotein complex that catalyzes intramembrane proteolysis
associated with Alzheimerās disease and cancer. Here, we have
developed potent sulfonamide clickable photoaffinity probes that target
Ī³-secretase <i>in vitro</i> and in cells by incorporating
various photoreactive groups and walking the clickable alkyne handle
to different positions around the molecule. We found that benzophenone
is preferred over diazirine as a photoreactive group within the sulfonamide
scaffold for labeling Ī³-secretase. Intriguingly, the placement
of the alkyne at different positions has little effect on probe potency
but has a significant impact on the efficiency of labeling of Ī³-secretase.
Moreover, the optimized clickable photoprobe, 163-BP3, was utilized
as a cellular probe to effectively assess the target engagement of
inhibitors with Ī³-secretase in primary neuronal cells. In addition,
biotinylated 163-BP3 probes were developed and used to capture the
native Ī³-secretase complex in the 3-[(3-cholamidopropyl)Ādimethylammonio]-2-hydroxy-1-propanesulfonate
(CHAPSO) solubilized state. Taken together, these next generation
clickable and biotinylated sulfonamide probes offer new tools to study
Ī³-secretase in biochemical and cellular systems. Finally, the
data provide insights into structural features of the sulfonamide
inhibitor binding site in relation to the active site and into the
design of clickable photoaffinity probes
Systematic Evaluation of Bioorthogonal Reactions in Live Cells with Clickable HaloTag Ligands: Implications for Intracellular Imaging
Bioorthogonal
reactions, including the strain-promoted azideāalkyne
cycloaddition (SPAAC) and inverse electron demand DielsāAlder
(iEDDA) reactions, have become increasingly popular for live-cell
imaging applications. However, the stability and reactivity of reagents
has never been systematically explored in the context of a living
cell. Here we report a universal, organelle-targetable system based
on HaloTag protein technology for directly comparing bioorthogonal
reagent reactivity, specificity, and stability using clickable HaloTag
ligands in various subcellular compartments. This system enabled a
detailed comparison of the bioorthogonal reactions in live cells and
informed the selection of optimal reagents and conditions for live-cell
imaging studies. We found that the reaction of sTCO with monosubstituted
tetrazines is the fastest reaction in cells; however, both reagents
have stability issues. To address this, we introduced a new variant
of sTCO, Ag-sTCO, which has much improved stability and can be used
directly in cells for rapid bioorthogonal reactions with tetrazines.
Utilization of Ag complexes of conformationally strained <i>trans</i>-cyclooctenes should greatly expand their usefulness especially when
paired with less reactive, more stable tetrazines