Identification of the Plasticity-Relevant Fucose-α(1−2)-Galactose Proteome from the Mouse Olfactory Bulb

Fucose-α(1−2)-galactose [Fucα(1−2)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 Fucα(1−2)Gal glycoproteins. Here, we report the first proteomic studies of this plasticity-relevant epitope. We identify five classes of putative Fucα(1−2)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 Fucα(1−2)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 “glycocode” to help direct olfactory sensory neuron (OSN) axonal pathfinding. We find that expression of Fucα(1−2)Gal sugars in the OB is regulated by the α(1−2)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 Fucα(1−2)Gal glycoproteins and provide new insights into the molecular mechanisms by which fucosyl sugars contribute to neuronal processes

<i>In Vitro</i> and <i>in Vivo</i> Characterization of a Tunable Dual-Reactivity Probe of the Nrf2-ARE Pathway

The cell utilizes the Keap1/Nrf2-ARE signaling pathway to detoxify harmful chemicals in order to protect itself from oxidative stress and to maintain its reducing environment. When exposed to oxidative stress and xenobiotic inducers, the redox sensitive Keap1 is covalently modified at specific cysteine residues. Consequently, the latent transcription factor Nrf2 is stabilized and translocates into the nucleus, where it transactivates the expression of detoxification genes through binding to the antioxidant response element (ARE). In the pursuit of potent and bioavailable activators of the ARE, we validated hits from a pathway-directed high-throughput screening campaign by testing them in cell culture and a reporter strain of a whole animal model, <i>Caenorhabditis elegans</i>. These studies allowed us to identify AI-3 as an ARE activator that induces cytoprotective genes in human cells and in worms, which also translated into <i>in vivo</i> activity in mice. AI-3 is an electrophilic ARE activator with two thiol sensitive sites toward a nucleophilic aromatic substitution, and SAR studies indicated the tunability of the system. Tandem LC–MS analysis revealed that AI-3 alkylates Keap1 primarily at Cys151, while AI-3 is reactive toward additional cysteine residues at higher doses <i>in vitro</i> and <i>in vivo</i>. The immediate effects of such alkylation included the disruption of Keap1-Cul3 (low [AI-3]) and/or Keap1-Nrf2 (high [AI-3]) interactions that both led to the stabilization of Nrf2. This further translated into the downstream Nrf2-ARE regulated cytoprotective gene activation. Collectively, AI-3 may become a valuable biological tool and may even provide therapeutic benefits in oxidative stress related diseases

Optimization of an Enzymatic Antibody–Drug Conjugation Approach Based on Coenzyme A Analogs

Phosphopantetheine transferases (PPTases) can be used to efficiently prepare site-specific antibody–drug conjugates (ADCs) by enzymatically coupling coenzyme A (CoA)-linker payloads to 11–12 amino acid peptide substrates inserted into antibodies. Here, a two-step strategy is established wherein in a first step, CoA analogs with various bioorthogonal reactivities are enzymatically installed on the antibody for chemical conjugation with a cytotoxic payload in a second step. Because of the high structural similarity of these CoA analogs to the natural PPTase substrate CoA-SH, the first step proceeds very efficiently and enables the use of peptide tags as short as 6 amino acids compared to the 11–12 amino acids required for efficient one-step coupling of the payload molecule. Furthermore, two-step conjugation provides access to diverse linker chemistries and spacers of varying lengths. The potency of the ADCs was largely independent of linker architecture. In mice, proteolytic cleavage was observed for some C-terminally linked auristatin payloads. The in vivo stability of these ADCs was significantly improved by reduction of the linker length. In addition, linker stability was found to be modulated by attachment site, and this, together with linker length, provides an opportunity for maximizing ADC stability without sacrificing potency

Small Molecule Mediated Proliferation of Primary Retinal Pigment Epithelial Cells

Retinal pigment epithelial (RPE) cells form a monolayer adjacent to the retina and play a critical role in the visual light cycle. Degeneration of RPE cells results in retinal disorders such as age-related macular degeneration. Cell transplant strategies have potential therapeutic value for such disorders; however, risks associated with an inadequate supply of donor cells limit their therapeutic success. The identification of factors that proliferate RPE cells <i>ex vivo</i> could provide a renewable source of cells for transplantation. Here, we report that a small molecule (WS3) can reversibly proliferate primary RPE cells isolated from fetal and adult human donors. Following withdrawal of WS3, RPE cells differentiate into a functional monolayer, as exhibited by their expression of mature RPE genes and phagocytosis of photoreceptor outer segments. Furthermore, chemically expanded RPE cells preserve vision when transplanted into dystrophic Royal College of Surgeons (RCS) rats, a well-established model of retinal degeneration