This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Myelin is a unique, lipid-rich membrane structure that accelerates neurotransmission and supports neuronal function. Sphingolipids are critical myelin components. Yet sphingolipid content and synthesis has not been well characterized in oligodendrocytes, the myelinproducing cells of the CNS. Here, using quantitative real-time PCR, LC-MS/MS-based lipid analysis, and biochemical assays we examined sphingolipid synthesis during the peak period of myelination in the postnatal rat brain. Importantly, we characterized sphingolipid production in isolated oligodendrocytes. We analyzed sphingolipid distribution and levels of critical enzymes and regulators in the sphingolipid biosynthetic pathway, focusing on the serine palmitoyltransferase (SPT) complex, the rate-limiting step in this pathway. During myelination levels of the major SPT subunits increased and oligodendrocyte maturation was accompanied by extensive alterations in the composition of the SPT complex. These included changes in the relative levels of two alternative catalytic subunits, SPTLC2 and -3, in the relative levels of isoforms of the small subunits ssSPTa and -b, and in the isoform distribution of the SPT regulators, the ORMDLs. Myelination progression was accompanied by distinct changes in both the nature of the sphingoid backbone and the N-acyl chains incorporated into sphingolipids. We conclude that the distribution of these changes among sphingolipid family members is indicative of a selective channeling of the ceramide backbone towards specific downstream metabolic pathways during myelination. Our findings provide insights into myelin production in oligodendrocytes and suggest how dysregulation of the biosynthesis of this highly specialized membrane could contribute to demyelinating diseases.
TDP-43 is an RNA/DNA-binding protein of versatile physiological functions and it is also implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS) disease in addition to several other implicated proteins such as mutant SOD1 and FUS etc.Cytoplasmic mis-localization, liquid-liquid phase separation (LLPS) due to RNA depletion and aggregation of TDP-43 are suggested to be important TDP-43-toxicity causing mechanisms for the ALS manifestation. So far, therapeutic options for ALS are extremely minimal and ineffective therefore, multi-faceted approaches such as treating the oxidative stress and inhibiting the TDP-43's aggregation are being actively pursued. In our recent study, an acridine imidazolium derivative compound, AIM4, has been identified to have anti-TDP-43 aggregation propensity however, its mechanism of inhibition is not deciphered. In this study, we have utilized computational methods to examine binding site(s) of AIM4 in the TDP-43 structure and have also compared its binding efficiency with several other relevant compounds. We find that AIM4 has a binding site in the C-terminal amyloidogenic core region of amino acids aa: 288-319, which coincides with one of the key residue motifs that could potentially mediate liquid-liquid phase separation (LLPS) of TDP-43. Importantly, alike to the previously reported effects exerted by RNA molecules, we found that AIM4 could also inhibit the in vitro LLPS of a recombinantly purified C-terminal fragment TDP-43 2C bearing an A315T familial mutation. Antagonistic effects of AIM4 towards LLPS which is believed as the precursor process to the TDP-43's aggregation and the in silico prediction of a binding site of AIM4 on TDP-43 occurring in the same region, assert that AIM4 could be an important .
The ORM/ORMDL family proteins function as regulatory subunits of the serine palmitoyltransferase (SPT) complex, which is the initiating and rate-limiting enzyme in sphingolipid biosynthesis. This complex is tightly regulated by cellular sphingolipid levels, but the sphingolipid sensing mechanism is unknown. Here we show that purified human SPT-ORMDL complexes are inhibited by the central sphingolipid metabolite ceramide. We have solved the cryo-EM structure of the SPT-ORMDL3 complex in a ceramide-bound state. Structure-guided mutational analyses reveal the essential function of this ceramide binding site for the suppression of SPT activity. Structural studies indicate that ceramide can induce and lock the N-terminus of ORMDL3 into an inhibitory conformation. Furthermore, we demonstrate that childhood amyotrophic lateral sclerosis (ALS) variants in the SPTLC1 subunit cause impaired ceramide sensing in the SPT-ORMDL3 mutants. Our work elucidates the molecular basis of ceramide sensing by the SPT-ORMDL complex for establishing sphingolipid homeostasis and indicates an important role of impaired ceramide sensing in disease development.
TDP-43 is an RNA/DNA-binding protein of versatile physiological functions and it is also implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS) disease in addition to several other implicated proteins such as mutant SOD1 and FUS etc.Cytoplasmic mis-localization, liquid-liquid phase separation (LLPS) due to RNA depletion and aggregation of TDP-43 are suggested to be important TDP-43-toxicity causing mechanisms for the ALS manifestation. So far, therapeutic options for ALS are extremely minimal and ineffective therefore, multi-faceted approaches such as treating the oxidative stress and inhibiting the TDP-43's aggregation are being actively pursued. In our recent study, an acridine imidazolium derivative compound, AIM4, has been identified to have anti-TDP-43 aggregation propensity however, its mechanism of inhibition is not deciphered. In this study, we have utilized computational methods to examine binding site(s) of AIM4 in the TDP-43 structure and have also compared its binding efficiency with several other relevant compounds. We find that AIM4 has a binding site in the C-terminal amyloidogenic core region of amino acids aa: 288-319, which coincides with one of the key residue motifs that could potentially mediate liquid-liquid phase separation (LLPS) of TDP-43. Importantly, alike to the previously reported effects exerted by RNA molecules, we found that AIM4 could also inhibit the in vitro LLPS of a recombinantly purified C-terminal fragment TDP-43 2C bearing an A315T familial mutation. Antagonistic effects of AIM4 towards LLPS which is believed as the precursor process to the TDP-43's aggregation and the in silico prediction of a binding site of AIM4 on TDP-43 occurring in the same region, assert that AIM4 could be an important molecule for further investigations on TDP-43's anti-aggregation effects with relevance to the ALS pathogenesis. two tandem RRMs (RNA recognition motifs) and nuclear localization and export signals whereas its C-terminal region is a glycine-rich low complexity and intrinsically disordered domain [21,[24][25][26]. TDP-43 participates in a variety of cellular processes including RNA splicing, mRNA turnover, RNA trafficking, microRNA biogenesis, translation, apoptosis, neurite outgrowth and embryo development [2,27,28]. Also, TDP-43 undergoes caspase mediated abnormal Cterminal fragmentation which may enhance its aggregation in the ALS patients [29].Notably, TDP-43 has been proposed to form prion-like self-seeding aggregates in vitro especially from its C-terminal glycine-rich region which is highly aggregationprone [30,31]. In fact, a fragment encompassing its RRM2 and the C-terminal region aa: 193-414 (termed: TDP-43 2C ) has been shown to have similar aggregation behavior as that of the full-length TDP-43 [30]. Also, the TDP-43 2C aggregates could induce aggregation of monomeric TDP-43 in cell lines via a prion-like seeding mechanism [30]. Recently, modulation of the in vitro aggregation of TDP-43 2C by post-translational modification and anions has been reported [32]. I...
Myelin is a unique, lipid-rich membrane structure that accelerates neurotransmission and supports neuronal function. Sphingolipids are critical components of myelin. Here we examined sphingolipid synthesis during the peak period of myelination in the postnatal rat brain.Importantly, we made measurements in isolated oligodendrocytes, the myelin-producing cells in the central nervous system. We analyzed sphingolipid distribution and levels of critical enzymes and regulators in the sphingolipid biosynthetic pathway, with a focus on the serine palmitoyltransferase (SPT) complex, the rate-limiting step in this pathway. During myelination levels of the major SPT subunits increased and oligodendrocyte maturation was accompanied by extensive alterations in the composition of the SPT complex. These included changes in the relative levels of alternate catalytic subunits, SPTLC2 and -3, the relative levels of isoforms of the small subunits ssSPTa and -b, and in the isoform distribution of the SPT regulators, the ORMDLs. As myelination progressed there were distinct changes in both the nature of the sphingoid backbone and the N-acyl chains incorporated into sphingolipids. The distribution of these changes among sphingolipid family members indicates that there is selective channeling of the ceramide backbone towards specific downstream metabolic pathways during myelination.
Sphingolipids (SLs) are one of the major lipid types present in eukaryotic cells. SLs not only serve as an important constituent of the plasma membrane but also as bioactive lipids which regulate several signal transduction processes such as cell growth and differentiation. The de novo biosynthesis begins in the endoplasmic reticulum (ER) with the condensation of serine and palmitoyl Co‐A by the rate‐limiting enzyme, serine palmitoyltransferase (SPT). SPT is homeostatically regulated by small ER proteins: ORMDLs in mammalian cells and Orms in yeast. The ORMDLs stereo‐specifically senses D‐erythro ceramide to regulate SPT activity in response to the cellular SL levels. Mammalian cells have three ORMDL isoforms that show high amino acid sequence similarity, but why cells need three ORMDLs is still unknown. Here we are investigating the role of different ORMDL isoforms and the amino‐acid regions of ORMDL involved in SPT regulation. We are also looking at regulation of individual ORMDL isoform protein turnover, as an additional mechanism by which SLs are maintained in the cells. The successful generation of ORMDL2/3, ORMDL1/2, and ORMDL1/3 double‐knockout cell lines by CRISPR‐Cas9 reveals that cells are compensating for the missing ORMDLs by increasing the protein levels of the remaining ORMDL. Surprisingly, we do not see any changes in the mRNA levels of the endogenous ORMDL in double‐knockout cell lines, suggesting that ORMDL protein levels are post‐transcriptionally regulated. We find an important clue to regulation by finding that ORMDLs are short‐lived proteins and that their degradation is regulated by p62 receptor‐mediated selective autophagy. Characterization of the ORMDL isoforms shows that individual ORMDL isoforms are alone sufficient to regulate SPT activity. Lipidomic results show that there is no significant change in total ceramide levels in the ORMDL double‐knockout cell line, confirming that each individual ORMDL isoform is sufficient to control ceramide or SL levels in cells. Based on our findings, we still do not know why cells need three different ORMDL isoforms. We hypothesize that each ORMDL isoforms respond to different ceramide levels in cell. To test this hypothesis, we used a cell‐free system to measure the response of each isoform to different levels of ceramide. We found that ORMDL isoforms response to different ceramide levels, to downregulate the activity of SPT. To investigate which region of ORMDL is important for its function, we have generated three deletion mutant constructs on cytosolic N‐ terminus & C‐terminus and two polyA substitution mutant constructs on the cytosolic loop of mORMDL1 by using site‐directed mutagenesis approach. We have confirmed the co‐localization of mutant constructs in the ER by immunofluorescence staining. Further, we have established a platform to test our mutant constructs and we are in the process of testing the mutant constructs. These data strongly suggest that cells require an optimal amount of ORMDL protein and sense the different ceramide levels to ...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.