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Trans-2-enoyl-CoA reductase limits Ca²⁺ accumulation in the endoplasmic reticulum by inhibiting the Ca²⁺ pump SERCA2b.

ABSTRACT: The endoplasmic reticulum (ER) contains various enzymes that metabolize fatty acids (FAs). Given that FAs are the components of membranes, FA metabolic enzymes might be associated with regulation of ER membrane functions. However, it remains unclear whether there is the interplay between FA metabolic enzymes and ER membrane proteins. Trans-2-enoyl-CoA reductase (TER) is an FA reductase present in the ER membrane and catalyzes the last step in the FA elongation cycle and sphingosine degradation pathway. Here we identify sarco(endo)plasmic reticulum Ca²⁺-ATPase 2b (SERCA2b), an ER Ca²⁺ pump responsible for Ca²⁺ accumulation in the ER, as a TER-binding protein by affinity purification from HEK293 cell lysates. We show that TER directly binds to SERCA2b by in vitro assays using recombinant proteins. Thapsigargin, a specific SERCA inhibitor, inhibits this binding. TER binds to SERCA2b through its conserved C-terminal region. TER overexpression suppresses SERCA2b ATPase activity in microsomal membranes of HEK293 cells. Depletion of TER increases Ca²⁺ storage in the ER and accelerates SERCA2b-dependent Ca²⁺ uptake to the ER after ligand-induced Ca²⁺ release. Moreover, depletion of TER reduces the Ca²⁺-dependent nuclear translocation of nuclear factor of activated T cells 4. These results demonstrate that TER is a negative regulator of SERCA2b, implying the direct linkage of FA metabolism and Ca²⁺ accumulation in the ER.

SUBMITTER: Uchida Y

PROVIDER: S-EPMC7949109 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Trans-2-enoyl-CoA reductase limits Ca2+ accumulation in the endoplasmic reticulum by inhibiting SERCA2b Ca2+ pump

Project description:The endoplasmic reticulum (ER) contains various enzymes that metabolize fatty acids (FAs). How FA metabolic enzymes cooperate with other ER functions, such as calcium (Ca2+) accumulation and its regulated release to the cytosol, is elusive. Trans-2-enoyl-CoA reductase (TER) is a FA reductase present in the ER membrane, and catalyzes the last step of FA elongation cycle and sphingosine degradation pathway. Here we show that TER directly binds to an ER Ca2+ pump, sarco(endo)plasmic reticulum Ca2+-ATPase 2b (SERCA2b), and regulates its activity. Thapsigargin, a specific SERCA inhibitor, inhibits this binding. TER binds to SERCA2b through its conserved C-terminal region. TER overexpression suppresses SERCA2b ATPase activity in microsomal membranes. Depletion of TER increases the ER Ca2+ storage and accelerates SERCA2b-dependent Ca2+ uptake to the ER after ligand-induced Ca2+ release. These results demonstrate that TER is a negative regulator of SERCA2b, implying the direct linkage of FA metabolism and Ca2+ accumulation in the ER.

2021-01-13 | PXD020792 | Pride

Characterization of two cotton cDNAs encoding trans-2-enoyl-CoA reductase reveals a putative novel NADPH-binding motif.

Project description:Very long chain fatty acids are important components of plant lipids, suberins, and cuticular waxes. Trans-2-enoyl-CoA reductase (ECR) catalyses the fourth reaction of fatty acid elongation, which is NADPH dependent. In the present study, the expression of two cotton ECR (GhECR) genes revealed by quantitative RT-PCR analysis was up-regulated during cotton fibre elongation. GhECR1 and 2 each contain open reading frames of 933 bp in length, both encoding proteins consisting of 310 amino acid residues. GhECRs show 32% identity to Saccharomyces cerevisiae Tsc13p at the deduced amino acid level, and the GhECR genes were able to restore the viability of the S. cerevisiae haploid tsc13-deletion strain. A putative non-classical NADPH-binding site in GhECR was predicted by an empirical approach. Site-directed mutagenesis in combination with gas chromatography-mass spectrometry analysis suggests that G(5X)IPXG presents a putative novel NADPH-binding motif of the plant ECR family. The data suggest that both GhECR genes encode functional enzymes harbouring non-classical NADPH-binding sites at their C-termini, and are involved in fatty acid elongation during cotton fibre development.

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A Novel Cytosolic Isoform of Mitochondrial Trans-2-Enoyl-CoA Reductase Enhances Peroxisome Proliferator-Activated Receptor α Activity.

Project description:BackgroundMitochondrial trans-2-enoyl-CoA reductase (MECR) is involved in mitochondrial synthesis of fatty acids and is highly expressed in mitochondria. MECR is also known as nuclear receptor binding factor-1, which was originally reported with yeast two-hybrid screening as a binding protein of the nuclear hormone receptor peroxisome proliferator-activated receptor α (PPARα). However, MECR and PPARα are localized at different compartment, mitochondria, and the nucleus, respectively. Therefore, the presence of a cytosolic or nuclear isoform of MECR is necessary for functional interaction between MECR and PPARα.MethodsTo identify the expression pattern of MECR and the cytosolic form of MECR (cMECR), we performed reverse transcription polymerase chain reaction (RT-PCR) with various tissue samples from Sprague-Dawley rats. To confirm the interaction between cMECR and PPARα, we performed several binding assays such as yeast two-hybrid, coimmunoprecipitation, and bimolecular fluorescence complementation. To observe subcellular localization of these proteins, immunocytochemistry was performed. A luciferase assay was used to measure PPARα activity.ResultsWe provide evidence of an alternatively spliced variant of the rat MECR gene that yields cMECR. The cMECR lacks the N-terminal 76 amino acids of MECR and shows uniform distribution in the cytoplasm and nucleus of HeLa cells. cMECR directly bound PPARα in the nucleus and increased PPARα-dependent luciferase activity in HeLa cells.ConclusionWe found the cytosolic form of MECR (cMECR) was expressed in the cytosolic and/or nuclear region, directly binds with PPARα, and enhances PPARα activity.

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Carboxylation mechanism and stereochemistry of crotonyl-CoA carboxylase/reductase, a carboxylating enoyl-thioester reductase.

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Luminal transport through intact endoplasmic reticulum limits the magnitude of localized Ca2+ signals.

Project description:The endoplasmic reticulum (ER) forms an interconnected network of tubules stretching throughout the cell. Understanding how ER functionality relies on its structural organization is crucial for elucidating cellular vulnerability to ER perturbations, which have been implicated in several neuronal pathologies. One of the key functions of the ER is enabling Ca[Formula: see text] signaling by storing large quantities of this ion and releasing it into the cytoplasm in a spatiotemporally controlled manner. Through a combination of physical modeling and live-cell imaging, we demonstrate that alterations in ER shape significantly impact its ability to support efficient local Ca[Formula: see text] releases, due to hindered transport of luminal content within the ER. Our model reveals that rapid Ca[Formula: see text] release necessitates mobile luminal buffer proteins with moderate binding strength, moving through a well-connected network of ER tubules. These findings provide insight into the functional advantages of normal ER architecture, emphasizing its importance as a kinetically efficient intracellular Ca[Formula: see text] delivery system.

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Observed crowding effects on Mycobacterium tuberculosis 2-trans-enoyl-ACP (CoA) reductase enzyme activity are not due to excluded volume only.

Project description:The cellular milieu is a complex and crowded aqueous solution. Macromolecular crowding effects are commonly studied in vitro using crowding agents. The aim of the present study was to evaluate the effects, if any, of macromolecular synthetic crowding agents on the apparent steady-state kinetic parameters (K m , k cat , and k cat /K m ) of Mycobacterium tuberculosis 2-trans-enoyl-ACP (CoA) reductase (InhA). Negligible effects on InhA activity were observed for ficoll 70, ficoll 400 and dextran 70. A complex effect was observed for PEG 6000. Glucose and sucrose showed, respectively, no effect on InhA activity and decreased k cat /K m for NADH and k cat for 2-trans-dodecenoyl-CoA. Molecular dynamics results suggest that InhA adopts a more compact conformer in sucrose solution. The effects of the crowding agents on the energy (E a and E η ), enthalpy (∆H # ), entropy (∆S # ), and Gibbs free energy (∆G # ) of activation were determined. The ∆G # values for all crowding agents were similar to buffer, suggesting that excluded volume effects did not facilitate stable activated ES # complex formation. Nonlinear Arrhenius plot for PEG 6000 suggests that "soft" interactions play a role in crowding effects. The results on InhA do not unequivocally meet the criteria for crowding effect due to exclude volume only.

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A major isoform of mitochondrial trans-2-enoyl-CoA reductase is dispensable for wax ester production in Euglena gracilis under anaerobic conditions.

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