Pratap, P. P. | Search bioRxiv

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↗

bioRxiv2024

Immunofocusing on the conserved fusion peptide of HIV envelope glycoprotein in rhesus macaques

SummaryDuring infection, the fusion peptide (FP) of HIV envelope glycoprotein (Env) serves a central role in viral fusion with the host cell. As such, the FP is highly conserved and therefore an attractive epitope for vaccine design. Here, we describe a vaccination study in non-human primates (NHPs) where glycan deletions were made on soluble HIV Env to increase FP epitope exposure. When delivered via implantable osmotic pumps, this immunogen primed immune responses against the FP, which were then boosted with heterologous trimers resulting in a focused immune response targeting the conserved FP epitope. Although autologous immunizations did not elicit high affinity FP-targeting antibodies, the conserved FP epitope on a heterologous trimer further matured the lower affinity, FP-targeting B cells. This study suggests using epitope conservation strategies on distinct Env trimer immunogens can focus humoral responses on desired neutralizing epitopes and suppress immune-distracting antibody responses against non-neutralizing epitopes.

immunology↗

bioRxiv2023

Focusing antibody responses to the fusion peptide in rhesusmacaques

Immunodominance of antibodies targeting non-neutralizing epitopes and the high level of somatic hypermutation within germinal centers (GCs) required for most HIV broadly neutralizing antibodies (bnAbs) are major impediments to the development of an effective HIV vaccine. Rational protein vaccine design and non-conventional immunization strategies are potential avenues to overcome these hurdles. Here, we report using implantable osmotic pumps to continuously deliver a series of epitope-targeted immunogens to rhesus macaques over the course of six months to elicit immune responses against the conserved fusion peptide. Antibody specificities and GC responses were tracked longitudinally using electron microscopy polyclonal epitope mapping (EMPEM) and lymph node fine-needle aspirates, respectively. Application of cryoEMPEM delineated key residues for on-target and off-target responses that can drive the next round of structure-based vaccine design.

immunology↗