scholarly journals Dissecting the regulatory roles of ORM proteins in the sphingolipid pathway of plants

2021 ◽  
Vol 17 (1) ◽  
pp. e1008284
Author(s):  
Adil Alsiyabi ◽  
Ariadna Gonzalez Solis ◽  
Edgar B. Cahoon ◽  
Rajib Saha
Keyword(s):  
Steady State ◽  
Regulatory Role ◽  
Secretory Proteins ◽  
Ceramide Synthase ◽  
Serine Palmitoyltransferase ◽  
Modeling Framework ◽  
Repressive Effect ◽  
Consistent Manner ◽  
Activation Mechanisms ◽  
Ceramide Synthases

Sphingolipids are a vital component of plant cellular endomembranes and carry out multiple functional and regulatory roles. Different sphingolipid species confer rigidity to the membrane structure, facilitate trafficking of secretory proteins, and initiate programmed cell death. Although the regulation of the sphingolipid pathway is yet to be uncovered, increasing evidence has pointed to orosomucoid proteins (ORMs) playing a major regulatory role and potentially interacting with a number of components in the pathway, including both enzymes and sphingolipids. However, experimental exploration of new regulatory interactions is time consuming and often infeasible. In this work, a computational approach was taken to address this challenge. A metabolic network of the sphingolipid pathway in plants was reconstructed. The steady-state rates of reactions in the network were then determined through measurements of growth and cellular composition of the different sphingolipids in Arabidopsis seedlings. The Ensemble modeling framework was modified to accurately account for activation mechanisms and subsequently used to generate sets of kinetic parameters that converge to the measured steady-state fluxes in a thermodynamically consistent manner. In addition, the framework was appended with an additional module to automate screening the parameters and to output models consistent with previously reported network responses to different perturbations. By analyzing the network’s response in the presence of different combinations of regulatory mechanisms, the model captured the experimentally observed repressive effect of ORMs on serine palmitoyltransferase (SPT). Furthermore, predictions point to a second regulatory role of ORM proteins, namely as an activator of class II (or LOH1 and LOH3) ceramide synthases. This activating role was found to be modulated by the concentration of free ceramides, where an accumulation of these sphingolipid species dampened the activating effect of ORMs on ceramide synthase. The predictions pave the way for future guided experiments and have implications in engineering crops with higher biotic stress tolerance.

scholarly journals Dissecting the regulatory roles of ORM proteins in the sphingolipid pathway of plants

2020 ◽  
Author(s):  
Adil Alsiyabi ◽  
Ariadna Gonzalez Solis ◽  
Edgar B Cahoon ◽  
Rajib Saha
Keyword(s):  
Steady State ◽  
Class Ii ◽  
Regulatory Mechanisms ◽  
Regulatory Role ◽  
Ensemble Modeling ◽  
Regulatory Interactions ◽  
Repressive Effect ◽  
Consistent Manner ◽  
Ceramide Synthases ◽  
The Way

AbstractSphingolipids are a vital component of plant cellular endomembranes and carry out multiple functional and regulatory roles. Different sphingolipid species confer rigidity to the membrane structure, facilitate trafficking of secretory proteins, and initiate programmed cell death. Although the regulation of the sphingolipid pathway is yet to be uncovered, increasing evidence has pointed to orosomucoid proteins (ORMs) playing a major regulatory role and potentially interacting with a number of components in the pathway, including both enzymes and sphingolipids. However, experimental exploration of new regulatory interactions is time consuming and often infeasible. In this work, a computational approach was taken to address this challenge. A metabolic network of the sphingolipid pathway in plants was reconstructed. The steady-state rates of reactions in the network were then determined through measurements of growth and cellular composition of the different sphingolipids in Arabidopsis seedlings. The Ensemble modeling framework was modified to accurately account for activation mechanisms and subsequently used to generate sets of kinetic parameters that converge to the measured steady-state fluxes in a thermodynamically consistent manner. In addition, the framework was appended with an additional module to automate screening the parameters and to output models consistent with previously reported network responses to different perturbations. By analyzing the network’s response in the presence of different combinations of regulatory mechanisms, the model captured the experimentally observed repressive effect of ORMs on SPT. Furthermore, predictions point to a second regulatory role of ORM proteins, namely as an activator of class II (or LOH1 and LOH3) ceramide synthases. This activating role was found to be modulated by the concentration of free ceramides, where an accumulation of these sphingolipid species dampened the activating effect of ORMs on ceramide synthase. The predictions pave the way for future guided experiments and have implications in engineering crops with higher biotic stress tolerance.Author summaryDue to their vital functional and regulatory roles in plant cells, increasing interest has gone into obtaining a complete understanding of the regulatory behavior of the sphingolipid pathway. However, the process of identifying new regulatory interactions is time consuming and often infeasible. To address this issue, ensemble modeling was used as an in silico method to test the ability of different regulatory schemes to predict all known pathway responses in a thermodynamically consistent manner. The analysis resulted in a significant reduction in the number of possible regulatory interactions. Mainly, the model predicts regulatory interactions between ceramides, ORMs, and ceramide synthases (especially class II). This framework can pave the way for biochemists to systematically identify plausible regulatory networks in understudied metabolic networks where knowledge on the underlying regulatory mechanisms is often missing. As future experimental works explore these predictions, an iterative cycle can begin wherein model predictions allow for targeted experiments which in turn generate results that can be reincorporated into the model to further increase prediction accuracy. Such a model-driven approach will significantly reduce the solution space traversed by the experimentalist.

scholarly journals Impact of overproduced heterologous protein characteristics on physiological response in Yarrowia lipolytica steady-state-maintained continuous cultures

2020 ◽  
Vol 104 (22) ◽  
pp. 9785-9800
Author(s):  
Paulina Korpys-Woźniak ◽  
Piotr Kubiak ◽  
Wojciech Białas ◽  
Ewelina Celińska
Keyword(s):  
Steady State ◽  
Yarrowia Lipolytica ◽  
Heterologous Protein ◽  
Target Protein ◽  
Host Cells ◽  
Synthesis Process ◽  
Secretory Proteins ◽  
Heterologous Proteins ◽  
Glycosylation Pattern ◽  
The Impact

Abstract Overproduction of recombinant secretory proteins triggers numerous physiological perturbations. Depending on a given heterologous protein characteristics, the producer cell is faced with different challenges which lead to varying responses in terms of its physiology and the target protein production rate. In the present study, we used steady-state-maintained Yarrowia lipolytica cells to investigate the impact of different heterologous proteins on the physiological behavior of the host cells. Such an approach allowed to uncouple the impact of the overproduction of a particular protein from the phenomena that result from growth phase or are caused by the heterogeneity of the analyzed populations. Altogether, eight variants of recombinant strains, individually overproducing heterologous proteins of varying molecular weight (27–65 kDa) and reporting activity (enzymatic and fluorescent) were subjected to chemostat cultivations. The steady-state-maintained cells were analyzed in terms of the substrate utilization, biomass and metabolites production, as well as the reporter protein synthesis. Simplified distribution of carbon and nitrogen between the respective products, as well as expression analysis of the heterologous genes were conducted. The here-obtained data suggest that using a more transcriptionally active promoter results in channeling more C flux towards the target protein, giving significantly higher specific amounts and production rates of the target polypeptide, at the cost of biomass accumulation, and with no significant impact on the polyols production. The extent of the reporter protein’s post-translational modifications, i.e., the number of disulfide bonds and glycosylation pattern, strongly impacts the synthesis process. Specific responses in terms of the protein formation kinetics, the gene expression levels, and transcript-to-protein linearity were observed. Key Points • Eight expression systems, producing different reporter proteins were analyzed. • The cells were maintained in steady-state by continuous chemostat culturing. • Protein- and promoter-specific effects were observed.

scholarly journals Ceramide Synthases Expression and Role of Ceramide Synthase-2 in the Lung: Insight from Human Lung Cells and Mouse Models

PLoS ONE ◽  
2013 ◽  
Vol 8 (5) ◽  
pp. e62968 ◽  
Author(s):  
Irina Petrache ◽  
Krzysztof Kamocki ◽  
Christophe Poirier ◽  
Yael Pewzner-Jung ◽  
Elad L. Laviad ◽  
...  
Keyword(s):  
Mouse Models ◽  
Human Lung ◽  
Lung Cells ◽  
Ceramide Synthase ◽  
Human Lung Cells ◽  
Ceramide Synthases

Synthesis and Characterization of Fluorescent Probes for the Development of a Ceramide Synthase FRET-Based Assay

2020 ◽  
Author(s):  
Eduardo Izquierdo ◽  
Mireia Casasampere ◽  
Gemma Fabriàs ◽  
José Luis Abad ◽  
Josefina Casas ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Second Messengers ◽  
Sphingolipid Metabolism ◽  
Synthesis And Characterization ◽  
Ceramide Synthase ◽  
Structural Components ◽  
Fluorescent Properties ◽  
Ceramide Synthases ◽  
In Cells

Ceramides are recognized to occupy a pivotal position in sphingolipid metabolism, playing a fundamental role as membrane structural components, as well as second messengers. Ceramide synthases (CerS) are a family of enzymes that catalyze the N-acylation of sphingosine and dihydrosphingosine, thus controlling the levels of intracellular ceramides. In this work, we present the synthesis and the fluorescent properties of new probes for the development of a FRET-based assay for CerS activity. Based on our previous discovery of spisulosine as a suitable probe for CerS activity in cells, the use of a modified NBD-spisulosine with a clickable fatty acid as CerS substrates may allow the ultimate formation of a bichromophoric reporter for a FRET-based analysis of CerS activity in cells.

Perforin Positive Regulatory DCs In Immune Tolerance: Novel Anti-Inflammatory Players In Metabolic Syndrome

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 784-784
Author(s):  
Yael Zlotnikov Klionsky ◽  
Bar Nathansohn ◽  
Chava Rosen ◽  
Anna Aronovich ◽  
Steffen Jung ◽  
...  
Keyword(s):  
Metabolic Syndrome ◽  
T Cells ◽  
Adipose Tissue ◽  
Steady State ◽  
Weight Gain ◽  
Cd8 T Cells ◽  
Regulatory Role ◽  
Cell Populations ◽  
Chimeric Mice

Abstract Immature dendritic cells (imDCs) can have a tolerizing effect in the steady state or following transplantation. However, due to the significant heterogeneity of this cell population it is difficult to study the mechanisms of their tolerance induction. We previously described the generation of a highly defined population of imDCs expressing perforin and granzyme A (Perf-DCs) from hematopoietic progenitors; using TCR transgenic T cells we monitored their ability to delete cognate CD4 and CD8 T cells. While the former are deleted via an MHC-independent mechanism through the nitric oxide system, CD8+ T cell deletion occurs through a unique MHC-dependent perforin-based killing mechanism. This involves activation of Toll-like receptors 7, and signaling through Triggering Receptor Expressed on Myeloid cells -1. Importantly, this novel subpopulation of Perf-DCs was also detected in various lymphoid tissues in normal animals, and its frequency is markedly enhanced upon GM-CSF administration (Zangi et al, Blood 2012). Here, we investigated the potential regulatory role of Perf-DCs in steady state in-vivo by selectively knocking out the expression of perforin in these cells. To this end, we generated BM chimeras using a 1:1 mixture of BM from perforin KO mice and from BM of mice ablated of CD11chigh DCs using diphtheria toxin expression under the CD11c promoter (Birnberg et al, Immunity 2008). In the resulting PKO-DTA chimeras, perforin expression was completely lost in conventional CD11c+ DCs, while 50% of the T and NK cell populations still expressed perforin. At 6 months post transplant, DTA-PKO chimeric mice spontaneously gained more weight than chimeras created using a mixture of normal BM with BM from perforin KO mice (WT-PKO). The increased weight gain observed in DTA-PKO mice prompted us to test whether this phenomenon was accompanied by other metabolic alterations. Indeed, DTA-PKO mice exhibited elevated serum cholesterol and triglyceride levels compared to control WT-PKO chimeras (140±3.5 vs. 115±8.6, 125±31vs. 88±9.8 mg/dl, N≥5). Total body fat percent as measured by body composition MRI was significantly higher in DTA-PKO mice (30.3%±2.2 vs. 14.5%±2.3), along with highly elevated levels of leptin (37±10.5 vs. 9.8±3 ng/ml). In addition, DTA-PKO chimeric mice exhibited glucose intolerance (p=0.034) and reduced insulin sensitivity (p=6.07x10-6). Immunohistological evaluation revealed a significant reduction in the percentage of insulin expressing pancreatic β cell- tissue (2.2%±0.54 vs. 5.75%±1.98). Importantly, the visceral adipose tissue (VAT) of DTA-PKO chimeras contained more crown-like structures typically formed when macrophages within inflamed AT surround dead adipocytes. Based on these characteristics of metabolic syndrome that develop in DTA-PKO chimeras over 6 months, we tested whether high-fat diet (HFD) enhances the rate of disease development. Indeed, DTA-PKO chimeras maintained on HFD displayed more pronounced weight gain compared to their HFD-maintained WT counterparts when tested 6 weeks after HFD initiation. Likewise, cholesterol and triglycerides as well as leptin and IL-1b in the serum, and TNF-α and IL-6 in the AT were elevated in DTA-PKO mice compared to the WT-PKO animals. Importantly, analysis of immune cell populations in collagenase-digested VAT revealed more CD8+ and CD4+ T cells in DTA-PKO mice compared to control chimeras (78.3x103±17.5x103 vs. 24.9x103±3.2x103 and 113x103±21x103 vs. 43x103±4.4x103respectively). Thus, triggering of inflammation in the AT previously shown to be mediated by T cells (Winer et al, Nat.Med 2009; Nishimura et al, Nat.Med 2009), is not effectively regulated in mice lacking Perf-DCs. Interestingly, a similar enhanced rate of metabolic imbalance was found in regular diet-fed DTA-PKO chimeras using RIP-mOVA mice expressing ovalbumin in the thymus, pancreas and kidneys, and known to be more prone to autoimmunity. Moreover, a significantly larger fraction of dividing cells was observed when CD8 T cells, isolated from AT of DTA-PKO chimeric RIP-mOVA mice were stimulated against splenocytes of mice expressing ovalbumin in all tissues (wOVA mice). Taken together, our results demonstrate that Perf-DCs have a unique immune regulatory role under steady state, controlling unwanted inflammatory processes in adipose tissue. Further studies of the role of Perf-DCs in other models of autoimmunity are warranted. Disclosures: No relevant conflicts of interest to declare.

Ceramide synthases in mammalians, worms, and insects: emerging schemes

2010 ◽  
Vol 1 (5-6) ◽  
pp. 411-422 ◽  
Author(s):  
André Voelzmann ◽  
Reinhard Bauer
Keyword(s):  
Biological Function ◽  
Sequence Similarity ◽  
Multiple Roles ◽  
Model Systems ◽  
Lipid Homeostasis ◽  
Biological Processes ◽  
Ceramide Synthase ◽  
Biological Functions ◽  
High Sequence Similarity ◽  
Ceramide Synthases

AbstractThe ceramide synthase (CerS) gene family comprises a group of highly conserved transmembrane proteins, which are found in all studied eukaryotes. The key feature of the CerS proteins is their role in ceramide synthase activity. Therefore, their original name ‘longevity assurance gene (Lass) homologs’, after the founding member, the yeast longevity assurance gene lag1, was altered to ‘CerS’. All CerS have high sequence similarity in a domain called LAG1 motif and a subset of CerS proteins is predicted to contain a Homeobox (Hox) domain. These domains could be the key to the multiple roles CerS have. CerS proteins play a role in diverse biological processes such as proliferation, differentiation, apoptosis, stress response, cancer, and neurodegeneration. In this review, we focus on CerS structure and biological function with emphasis of biological functions in the widely used model systems Caenorhabditis elegans and Drosophila melanogaster. Also, we focus on the accumulating data suggesting a role for CerS in lipid homeostasis.

scholarly journals Inactivation of Ceramide Synthase 6 in Mice Results in an Altered Sphingolipid Metabolism and Behavioral Abnormalities

2013 ◽  
Vol 288 (29) ◽  
pp. 21433-21447 ◽  
Author(s):  
Philipp Ebel ◽  
Katharina vom Dorp ◽  
Elisabeth Petrasch-Parwez ◽  
Armin Zlomuzica ◽  
Kiyoka Kinugawa ◽  
...  
Keyword(s):  
Expression Patterns ◽  
Acyl Chain ◽  
Sphingolipid Metabolism ◽  
Ceramide Synthase ◽  
Acyl Chain Length ◽  
Hind Limbs ◽  
Behavioral Abnormalities ◽  
Ceramide Content ◽  
Ceramide Synthases ◽  
The Brain

The N-acyl chain length of ceramides is determined by the specificity of different ceramide synthases (CerS). The CerS family in mammals consists of six members with different substrate specificities and expression patterns. We have generated and characterized a mouse line harboring an enzymatically inactive ceramide synthase 6 (CerS6KO) gene and lacz reporter cDNA coding for β-galactosidase directed by the CerS6 promoter. These mice display a decrease in C16:0 containing sphingolipids. Relative to wild type tissues the amount of C16:0 containing sphingomyelin in kidney is ∼35%, whereas we find a reduction of C16:0 ceramide content in the small intestine to about 25%. The CerS6KO mice show behavioral abnormalities including a clasping abnormality of their hind limbs and a habituation deficit. LacZ reporter expression in the brain reveals CerS6 expression in hippocampus, cortex, and the Purkinje cell layer of the cerebellum. Using newly developed antibodies that specifically recognize the CerS6 protein we show that the endogenous CerS6 protein is N-glycosylated and expressed in several tissues of mice, mainly kidney, small and large intestine, and brain.

scholarly journals A steady-state modeling framework incorporating the Kuroda–Tvergaard model: demonstrated on single crystal crack growth

2019 ◽  
Vol 89 (10) ◽  
pp. 2133-2145
Author(s):  
K. J. Juul ◽  
S. A. El-Naaman ◽  
K. L. Nielsen ◽  
C. F. Niordson
Keyword(s):  
Steady State ◽  
Single Crystal ◽  
Crack Growth ◽  
Modeling Framework
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