CRISPR/Cas9 deletion of ORMDLs reveals complexity in sphingolipid metabolism

Green, Christopher D.; Weigel, Cynthia; Oyeniran, Clément; James, Briana N.; Davis, Deanna; Mahawar, Usha; Newton, Jason; Wattenberg, Binks W.; Maceyka, Michael; Spiegel, Sarah

doi:10.1016/j.jlr.2021.100082

Cited by 24 publications

(24 citation statements)

References 41 publications

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“…Immunoblotting with a pan ORMDL antibody that detects all isoforms (22) showed that the interaction with ORMDLs was completely abolished by the ex2del variant which lacks the whole TMD (Figure 2A). The other variants affecting the SPTLC1-TMD, L39del and F40S41del, showed a diminished interaction with ORMDLs, while Y23F retained interaction with ORMDLs (Figure 2A).…”

Section: Ormdls Fail To Interact With Sptlc1 Variantsmentioning

confidence: 99%

SPTLC1 variants associated with ALS produce distinct sphingolipid signatures through impaired interaction with ORMDL proteins

Lone¹,

Aaltonen²,

Zidell³

et al. 2022

Journal of Clinical Investigation

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Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting motor neurons. Mutations in the SPTLC1 subunit of serine-palmitoyltransferase (SPT), which catalyzes the first step in the de novo synthesis of sphingolipids cause childhood-onset ALS. SPTLC1-ALS variants map to a transmembrane domain that interacts with ORMDL proteins, negative regulators of SPT activity. We show that ORMDL binding to the holoenzyme complex is impaired in cells expressing pathogenic SPTLC1-ALS alleles, resulting in increased sphingolipid synthesis and a distinct lipid signature. C-terminal SPTLC1 variants cause the peripheral sensory neuropathy HSAN1 due to the synthesis of 1-deoxysphingolipids (1-deoxySLs) that form when SPT metabolizes L-alanine instead of L-serine. Limiting L-serine availability in SPTLC1-ALS expressing cells increased 1-deoxySL and shifted the SL profile from an ALS to an HSAN1-like signature. This effect was corroborated in an SPTLC1-ALS pedigree in which the index patient uniquely presented with an HSAN1 phenotype, increased 1-deoxySL levels, and an L-serine deficiency. These data demonstrate how pathogenic variants in different domains of SPTLC1 give rise to distinct clinical presentations that are nonetheless modifiable by substrate availability.

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Section: Ormdls Fail To Interact With Sptlc1 Variantsmentioning

confidence: 99%

SPTLC1 variants associated with ALS produce distinct sphingolipid signatures through impaired interaction with ORMDL proteins

Lone¹,

Aaltonen²,

Zidell³

et al. 2022

Journal of Clinical Investigation

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show abstract

“…For quantification, total sphingolipids were extracted and quantified by high-resolution mass spectrometry. The interaction of ORMDLs with SPT inhibits SPT activity, as knockdown (Fig 2D), (22) or knockout (23) of all three ORMDLs leads to increased SL synthesis. In contrast, while the expression of SPTLC1 variants L39del and ex2del in SPTLC1-KO cells led to an overall increase in SL synthesis compared to WT-SPTLC1 expressing cells, silencing ORMDLs had little or no effect on the activity of the L39del and ex2del variant (Fig 2E), suggesting that increased SL synthesis by the variants is primarily caused by dysfunctional feedback inhibition rather than by an increased activity of the holoenzyme.…”

Section: Resultsmentioning

confidence: 99%

SPTLC1 variants associated with childhood onset amyotrophic lateral sclerosis produce distinct sphingolipid signatures through impaired interaction with ORMDL proteins

Lone

Aaltonen

Zidell

et al. 2022

Preprint

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SummaryAmyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting motor neurons. Mutations in the SPTLC1 subunit of serine-palmitoyltransferase (SPT), which catalyzes the first step in the de novo synthesis of sphingolipids cause childhood-onset ALS. SPTLC1-ALS variants map to a transmembrane domain that interacts with ORMDL proteins, negative regulators of SPT activity. We show that ORMDL binding to the holoenzyme complex is impaired in cells expressing pathogenic SPTLC1-ALS alleles, resulting in increased sphingolipid synthesis and a distinct lipid signature. C-terminal SPTLC1 variants cause the peripheral sensory neuropathy HSAN1 due to the synthesis of 1-deoxysphingolipids (1-deoxySLs) that form when SPT metabolizes L-alanine instead of L-serine. Limiting L-serine availability in SPTLC1-ALS expressing cells increased 1-deoxySL and shifted the SL profile from an ALS to an HSAN1-like signature. This effect was corroborated in an SPTLC1-ALS pedigree in which the index patient uniquely presented with an HSAN1 phenotype, increased 1-deoxySL levels, and an L-serine deficiency. These data demonstrate how pathogenic variants in different domains of SPTLC1 give rise to distinct clinical presentations that are nonetheless modifiable by substrate availability.

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“…In fact, the SPTLC2 N terminus determines the localization of the protein, as it alone can target both ER and mitochondria ( Fig 1F ). Regulatory subunits ORMDL1-3 and ssSPTa-b control SPT activity and acyl-chain specificity ( Han et al, 2009 ; Breslow et al, 2010 ; Harmon et al, 2013 ; Green et al, 2021 ). Structural studies reveal details of the interaction of the regulatory subunits with the SPT-complex, and these protein interactions may also play a role in determining SPT localization and activity.…”

Section: Resultsmentioning

confidence: 99%

Serine palmitoyltransferase assembles at ER–mitochondria contact sites

et al. 2021

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The accumulation of sphingolipid species in the cell contributes to the development of obesity and neurological disease. However, the subcellular localization of sphingolipid-synthesizing enzymes is unclear, limiting the understanding of where and how these lipids accumulate inside the cell and why they are toxic. Here, we show that SPTLC2, a subunit of the serine palmitoyltransferase (SPT) complex, catalyzing the first step in de novo sphingolipid synthesis, localizes dually to the ER and the outer mitochondrial membrane. We demonstrate that mitochondrial SPTLC2 interacts and forms a complex in trans with the ER-localized SPT subunit SPTLC1. Loss of SPTLC2 prevents the synthesis of mitochondrial sphingolipids and protects from palmitate-induced mitochondrial toxicity, a process dependent on mitochondrial ceramides. Our results reveal the in trans assembly of an enzymatic complex at an organellar membrane contact site, providing novel insight into the localization of sphingolipid synthesis and the composition and function of ER–mitochondria contact sites.

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CRISPR/Cas9 deletion of ORMDLs reveals complexity in sphingolipid metabolism

Cited by 24 publications

References 41 publications

SPTLC1 variants associated with ALS produce distinct sphingolipid signatures through impaired interaction with ORMDL proteins

SPTLC1 variants associated with ALS produce distinct sphingolipid signatures through impaired interaction with ORMDL proteins

SPTLC1 variants associated with childhood onset amyotrophic lateral sclerosis produce distinct sphingolipid signatures through impaired interaction with ORMDL proteins

Serine palmitoyltransferase assembles at ER–mitochondria contact sites

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