Dataset Information

Proximity labeling and orthogonal nanobody pulldown (ID-oPD) approaches to map the spinophilin interactome uncover a role for spinophilin in protein regulation.

ABSTRACT: The major synaptic protein phosphatase 1 (PP1) targeting protein, spinophilin can inhibit or target PP1 activity towards diverse substrates. To detail the full panoply of substrates at which spinophilin modulates PP1 activity, we have validated a novel approach that permits proximity labeling and orthogonal nanobody pulldown (ID-oPD) of spinophilin protein complexes. We identified 614 specific and 217 selective spinophilin interacting proteins in a HEK293 overexpression system, many of which are involved in mRNA translation or metabolism. We validated a subset of these interactors using neutravidin and ALFA-tag nanobody pulldowns followed by immunoblotting. Additionally, using wildtype or an F451A mutant spinophilin that limits PP1 binding, we found that the PP1 binding-deficient spinophilin mutant enhanced its own expression, but limited the expression of proteins involved in mRNA translation and metabolism Moreover, in the striatum, spinophilin mRNA is highly neuropil localized and endogenous spinophilin limits its own expression. Overall, our studies demonstrate a novel role for spinophilin in mediating synaptic protein expression and that care must be taken to not over-control when using proximity labeling to detect PP1 targeting protein interactomes.

INSTRUMENT(S): Orbitrap Eclipse, orbitrap QE-plus

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: A.J. Baucum II Amber L. Mosley

PROVIDER: MSV000096575 | MassIVE | Mon Dec 02 12:32:00 GMT 2024

SECONDARY ACCESSION(S): PXD058487

REPOSITORIES: MassIVE

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Proximity labeling and orthogonal nanobody pulldown (ID-oPD) approaches to map the spinophilin interactome uncover a putative role for spinophilin in protein homeostasis.

Claeboe Emily T ET Blake Keyana L KL Shah Nikhil R NR Morris Cameron W CW Hens Basant B Atwood Brady K BK Absalon Sabrina S Mosley Amber L AL Doud Emma H EH Baucum Anthony J AJ

bioRxiv : the preprint server for biology 20250123

Spinophilin is a dendritic spine enriched scaffolding and protein phosphatase 1 targeting protein. To detail spinophilin interacting proteins, we created an Ultra-ID and ALFA-tagged spinophilin encoding construct that permits proximity labeling and orthogonal nanobody pulldown (ID-oPD) of spinophilin-associated protein complexes in heterologous cells. We identified 614 specific, and 312 specific and selective, spinophilin interacting proteins in HEK293 cells and validated a subset of these using ...[more]

PMID: 39896493

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