PXD069051-1

PXD069051 is an original dataset announced via ProteomeXchange.

Title	Ion channel inhibition by targeted recruitment of NEDD4-2 with divalent nanobodies
Description	Targeted protein degradation or downregulation (TPD/TPDR) mediated by induced recruitment of an endogenous E3 ubiquitin ligase to a substrate is a disruptive paradigm for developing therapeutics. However, <2% of ~600 E3 ligases have been exploited for this modality, and efficacy for multi-subunit membrane ion channel complexes has not been demonstrated. The HECT E3 ligase NEDD4-2 regulates myriad ion channels, but it’s utility for TPD/TPDR is unexplored and uncertain due to complex intramolecular and posttranslational regulation mechanisms. We identified a nanobody that binds NEDD4-2 HECT domain strongly without disrupting sites critical for catalysis as revealed by a cryo-electron microscopy reconstruction and in vitro ubiquitination assays. Recruiting NEDD4-2 to diverse ion channels with distinct topologies (high voltage-activated CaV2.2 calcium channels; voltage-gated potassium channel, KCNQ1; and the epithelial Na+ channel, ENaC, with a clinically relevant Liddle syndrome channelopathy mutation) using divalent nanobodies (DiVas) strongly suppressed their cell surface density and function, even when there was no frank degradation. Global proteomics indicated DiVa recruitment of endogenous NEDD4-2 to a test ion channel, KCNQ1-YFP, displayed dramatically lower off-target effects compared to NEDD4-2 overexpression. The results establish utility of induced recruitment of NEDD4-2 for TPD/TPDR, validate multi-subunit ion channels as susceptible to this modality, and introduce DiVas as a general method to generate ion channel inhibitors.
HostingRepository	PRIDE
AnnounceDate	2026-01-19
AnnouncementXML	Submission_2026-01-18_16:20:05.731.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Henry Colecraft
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: NEWT:9606;
ModificationList	monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Orbitrap Fusion Lumos

Revision	Datetime	Status	ChangeLog Entry
0	2025-10-01 14:50:40	ID requested
⏵ 1	2026-01-18 16:20:06	announced

10.1038/s41467-025-67068-x;

Darko-Boateng A, Afriyie E, Morgenstern TJ, Shanmugam SK, Zou X, Laloudakis YD, Choudhury P, Desai M, Kass RS, Vallese F, Clarke OB, Colecraft HM, Ion channel inhibition by targeted recruitment of NEDD4-2 with divalent nanobodies. Nat Commun, 17(1):378(2025) [pubmed]

submitter keyword: HEK293 cells, NEDD4-2, divalent nanobody, ion channels

Henry Colecraft
contact affiliation	Department of Physiology and Cellular Biophysics, Columbia University Irving Medical Center, New York, NY
contact email	hc2405@cumc.columbia.edu
lab head
Henry Colecraft
contact affiliation	Columbia University Irving Medical Center
contact email	hc2405@cumc.columbia.edu
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD069051
     1. Label: PRIDE project
     2. Name: Ion channel inhibition by targeted recruitment of NEDD4-2 with divalent nanobodies