Biology subjects

Susa, K. J.

Publications and source records attributed to Susa, K. J..

5 recordsLinked to original sources

bioRxiv2025

Structural basis for regulation of Frizzled-4 signaling by the co-receptor Tetraspanin-12

Norrin is an atypical ligand that regulates retinal angiogenesis through the Wnt/{beta}-catenin pathway. Norrin triggers heterodimerization of Frizzled-4 (FZD4) with Low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6), leading to downstream {beta}-catenin stabilization. Unlike Wnt ligands, Norrin requires the tetraspanin Tspan12 for signaling amplification, but it is not understood why. Here, we report a 3.4-angstrom structure of Tspan12 in complex with FZD4 determined by cryo-electron microscopy. The structure reveals that FZD4 and Tspan12 form a direct complex in the absence of Norrin. The transmembrane (TM) domain of Tspan12 is oriented in a tightly packed four-helix bundle and interacts with TM2 of FZD4 to promote trafficking of Tspan12 to the cell surface. The C-D helices of Tspan12, which mediate binding to Norrin, remain exposed while Tspan12 is in complex with FZD4, facilitating higher-affinity Norrin binding. Cell-based assays reveal that Tspan12 and FZD4 remain associated after Norrin recognition, suggesting that Tspan12 is a core component of the FZD4-Norrin-LRP5/6 signaling complex. Together, these studies reveal the molecular mechanism underlying the assembly of the Tspan12-FZD4 complex and the enhancement of Norrin signaling, suggesting new ways to target the FZD4-Tspan12 complex for ocular diseases characterized by hypo- or hyper-vascularization. SIGNIFICANCE STATEMENTBlood vessel formation in the retina is essential for vision, and its disruption leads to ocular diseases. Norrin regulates retinal angiogenesis by triggering the heterodimerization of Frizzled-4 with co-receptor LRP5/6, mimicking the function of Wnt ligands and leading to downstream {beta}-catenin signaling. Unlike Wnts, Norrin requires a tetraspanin family member, Tspan12, to amplify signaling. We report the structure of the FZD4-Tspan12 co-receptor complex and identify key interacting regions that enable Tspan12 trafficking to the cell surface and higher affinity capture of Norrin. Cell-based assays indicate Tspan12 remains a component of the active Norrin-FZD4-LRP5/6 signaling complex. Our results highlight an underappreciated role of tetraspanins in direct regulation of receptor signaling and suggest new therapeutic avenues for targeting abnormalities in vascular growth.

biochemistry↗

bioRxiv2025

In silico discovery of nanobody binders to a G-protein coupled receptor using AlphaFold-Multimer

Antibodies are central mediators of the adaptive immune response, and they are powerful research tools and therapeutics. Antibody discovery requires substantial experimental effort, such as immunization campaigns or in vitro library screening. Predicting antibody-antigen binding a priori remains challenging. However, recent machine learning methods raise the possibility of in silico antibody discovery, bypassing or reducing initial experimental bottlenecks. Here, we report a virtual screen using AlphaFold-Multimer (AF-M) that prospectively identified nanobody binders to MRGPRX2, a G protein-coupled receptor (GPCR) and therapeutic target for the treatment of pseudoallergic inflammation and itch. Using previously reported nanobody-GPCR structures, we identified a set of AF-M outputs that effectively discriminate between interacting and non-interacting nanobody-GPCR pairs. We used these outputs to perform a prospective in silico screen, identified nanobodies that bind MRGPRX2 with high affinity, and confirmed activity in signaling and functional cellular assays. Our results provide a proof of concept for fully computational antibody discovery pipelines that can circumvent laboratory experiments.

biochemistry↗

bioRxiv2024

Spatiotemporal photocatalytic proximity labeling proteomics reveals ligand-activated extracellular and intracellular EGFR neighborhoods

Photo-proximity labeling proteomics (PLP) methods have recently shown that the cell surface receptors can dynamically form lateral interactome networks. Here, we present a paired set of PLP workflows that simultaneously track neighborhood changes for oncogenic epidermal growth factor receptor (EGFR) with temporal resolution, both outside and inside of cells. We achieved this by augmenting the multiscale PLP workflow we call MultiMap, where three photo-probes with different labeling ranges were photo-activated by one photocatalyst, Eosin Y. By anchoring Eosin Y extracellularly and intracellularly on EGFR, we captured hundreds of proteins on both sides of the cell membrane that change in proximity to EGFR upon EGF activation. Neighbors engaged with EGFR within minutes to over an hour, reflecting dynamic interactomes during early, mid- and late-signaling including phosphorylation, internalization, degradation and transcriptional regulation. This rapid "photographic" labeling approach provides snapshots of signaling neighborhoods, revealing their dynamic nature, and potential for drug targeting.

biochemistry↗

bioRxiv2023

A Spatiotemporal Map of Co-Receptor Signaling Networks Underlying B Cell Activation

The B cell receptor (BCR) signals together with a multi-component co-receptor complex to initiate B cell activation in response to antigen binding. This process underlies nearly every aspect of proper B cell function. Here, we take advantage of peroxidase-catalyzed proximity labeling combined with quantitative mass spectrometry to track B cell co-receptor signaling dynamics from 10 seconds to 2 hours after BCR stimulation. This approach enables tracking of 2,814 proximity-labeled proteins and 1,394 quantified phosphosites and provides an unbiased and quantitative molecular map of proteins recruited to the vicinity of CD19, the key signaling subunit of the co-receptor complex. We detail the recruitment kinetics of essential signaling effectors to CD19 following activation, and then identify new mediators of B cell activation. In particular, we show that the glutamate transporter SLC1A1 is responsible for mediating rapid metabolic reprogramming immediately downstream of BCR stimulation and for maintaining redox homeostasis during B cell activation. This study provides a comprehensive map of the BCR signaling pathway and a rich resource for uncovering the complex signaling networks that regulate B cell activation.

immunology↗

bioRxiv2019

A dynamic interaction between CD19 and the tetraspanin CD81 controls B cell co-receptor trafficking

CD81 and its binding partner CD19 are core subunits of the B cell co-receptor complex. While CD19 is a single-pass transmembrane protein belonging to the extensively studied Ig superfamily, CD81 belongs to a conserved but poorly understood family of four-pass transmembrane proteins called tetraspanins. These functionally diverse proteins play important roles in a wide variety of different organ systems by controlling protein trafficking and other cellular processes. Here, we show that CD81 relies on its ectodomain to control trafficking of CD19 to the cell surface. Moreover, the anti-CD81 antibody 5A6, which binds selectively to activated B cells, recognizes a conformational epitope on CD81 that is masked when CD81 is in complex with CD19. Mutations of CD81 in this contact interface suppress its CD19 surface-export activity. Taken together, these data indicate that the CD81 - CD19 interaction is dynamically regulated upon B cell activation, suggesting that this dynamism can be exploited to regulate B cell function. These results are not only important for understanding B cell biology, but also have important implications for understanding tetraspanin function more generally.

immunology↗