Friday, May 16, 2025
5/16/2025
Mechanism of CD33 receptor signaling in Alzheimer disease - Project Summary Pursuant to my long-term research interest, understanding the transmembrane (TM) signaling of cell surface receptors, the present proposal aims to elucidate the signaling mechanism of CD33, a key immune modulator of amyloid-β (Aβ) peptide clearance by microglia. The response of microglia, the resident phagocytes of the central nervous system, to Aβ aggregates can be either phagocytotic and neuroprotective or inflammatory and cytotoxic. This influence makes the innate immune response a major determinant of Alzheimer disease (AD) pathogenesis. The activation state and phagocytosis capability of microglia correlate with signaling by the CD33 cell-surface receptor. CD33 inactivation promotes phagocytosis and mitigates Aβ pathology, identifying CD33 inhibition as a promising therapeutic AD avenue. CD33 belongs to the family of sialic acid-binding im- munoglobulin-like receptor (Siglecs) and consists of two extracellular immunoglobulin-like domains (IgV and IgC2), a single-pass transmembrane (TM) domain and a cytosolic (CS) domain. Here, we hypothesize that the IgV-IgC2-TM domain-domain orientations and couplings change upon ligand binding, which transmits a signal (structural change) to the CS domains. Aim 1 lays the structural and biochemical groundwork to test this hy- pothesis. It establishes expression systems to produce isotope-labeled, glycosylated domains, determines their structure, oligomerization state and membrane immersion by NMR spectroscopy. Aim 2 traces the structural changes upon ligand binding from IgV to the CS domain in full-length CD33 reconstituted in phospholipid bicelles to directly test our signaling hypothesis. Specifically, ligand-induced changes in structure, domain- domain orientations and couplings will be established using NMR. The CD33 activity state will be verified in cultured macrophages and structure-based point mutations will be examined to provide target sites for phar- macological CD33 inhibition. Aim 3 examines the link between the CS domain and downstream signaling. The binding motif and affinity of established CD33-binding proteins will be determined by ITC and NMR. This or- ganizes the binding hierarchy of cytosolic ligands, identifies binders whose binding sequences overlap with se- quences that experience structural perturbations upon receptor activation, and identifies additional CD33 can- didate sequences for pharmacological intervention. While fragments of cell surface receptors with extracellular ligand-binding domains have been studied at atomic resolution, the relatively small size of CD33 allows the first study of an entire such receptor in solution. Delineating its complete signaling mechanism at atomic resolution establishes a paradigm for receptor biology and provides novel insight into Siglec biology. The direct targeting of Aβ amyloid as AD therapy has been largely unsuccessful and we provide CD33 target sites verified by cell biology that can be translated to therapeutics to harness the great potential of optimizing the innate immune response to combat AD.
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