Snx14 proximity labeling reveals a role in saturated fatty acid metabolism and ER homeostasis defective in SCAR20 disease

Abstract

Fatty acids (FAs) are central cellular metabolites that contribute to lipid synthesis, and can be stored or harvested for metabolic energy. Dysregulation in FA processing and storage causes toxic FA accumulation or altered membrane compositions and contributes to metabolic and neurological disorders. Saturated lipids are particularly detrimental to cells, but how lipid saturation levels are maintained remains poorly understood. Here, we identify the cerebellar ataxia SCAR20-associated protein Snx14, an endoplasmic reticulum (ER)-lipid droplet (LD) tethering protein, as a novel factor required to maintain the lipid saturation balance of cellular membranes. We show that Snx14-deficient cells and SCAR20 disease patient-derived cells are hypersensitive to saturated FA (SFA)-mediated lipotoxic cell death that compromises ER integrity. Using APEX2-based proximity labeling, we reveal the protein composition of Snx14-associated ER-LD contacts and define a functional interaction between Snx14 and Δ-9 FA desaturase SCD1. We show that SCD1 is upregulated in SNX14KO cells, and SNX14KO-associated SFA hypersensitivity can be rescued by ectopic SCD1 overexpression. The hydrophobic PXA domain of Snx14 and its interaction with SCD1 are required for Snx14-mediated SFA protection function. Lipidomic profiling reveals that SNX14KO cells exhibit increased membrane saturation, and mimics the lipid profile of SCD1-inhibited cells. Altogether these mechanistic insights reveal a functional interaction between Snx14 and SCD1 in the ER network to maintain FA homeostasis and membrane saturation, defects in which may underlie the neuropathology of SCAR20.Significance StatementSCAR20 disease is an autosomal recessive spinocerebellar ataxia primarily affecting children, and results from loss-of-function mutations in the SNX14 gene. Snx14 is an endoplasmic reticulum (ER)-localized protein that localizes to ER-lipid droplet (LD) contacts and promotes LD biogenesis, but why Snx14 loss causes SCAR20 is unclear. Here, we demonstrate that Snx14-deficient cells and SCAR20 patient-derived fibroblasts have defective ER homeostasis and altered lipid saturation profiles. We reveal a functional interaction between Snx14 and fatty acid desaturase SCD1. Lipidomics shows Snx14-deficient cells contain elevated saturated lipids, closely mirroring SCD1-defective cells. Furthermore, Snx14 and SCD1 interact in the ER, and SCD1 over-expression rescues Snx14 loss. We propose that Snx14 maintains cellular lipid homeostasis, the loss of which underlies the cellular basis for SCAR20 disease.

Snazarus and its human ortholog SNX25 regulate autophagic flux by affecting VAMP8 endocytosis

“…Moreover, recent proteomic data identified PICALM as a high confidence neighbor of SNX14-APEX2 (Datta et al, 2021). We extended this proteomic finding to SNX25, observing close proximity between SNX25 and PICALM by PLA (Fig.…”

“…Given the observed effects on VAMP8 uptake in SNX25-deficient cells, we next focused on PICALM, because of its essential role in small R-SNARE internalization (Miller et al, 2011). Moreover, recent proteomic data identified PICALM as a high confidence neighbor of SNX14- APEX2 (Datta et al, 2021). We extended this proteomic finding to SNX25, observing close proximity between SNX25 and PICALM by PLA (Fig.…”

“…Recent studies have demonstrated important roles for SNX14 in lipid metabolism (Bryant et al, 2018; Datta et al, 2019; Datta et al, 2021; Hariri et al, 2019). SNX14 loss results in saturated fatty acid accumulation and increased sensitivity to lipotoxic stress.…”

CRISPR screens for lipid regulators reveal a role for ER-bound SNX13 in lysosomal cholesterol export

“…Snx14 is an ER- resident protein that accumulates within a subdomain of the ER surrounding lipid droplets in oleic acid-fed cells (cf. Henne et al, 2015; Datta et al, 2019, 2020; Ugrankar et al, 2109). Further studies on the role of SNX13 in cholesterol regulation are presented below.…”

“…As an organelle tether (Henne et al, 2015), SNX13 may coordinate the association of membrane bound compartments to maintain appropriate and tightly regulated cholesterol levels in distinct cellular compartments, particularly in the ER. Further work will be needed to parse the differences in phenotypes observed for the related but distinct SNX13 and SNX14 proteins that both influence cholesterol and neutral lipid accumulation (Bryant et al, 2018;Datta et al, 2019Datta et al, , 2020.…”