SYT6: a newly identified protein involved in ER - trans-Golgi network Membrane Contact Sites

Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.SYT6 is a newly identified lipid transport protein from ER - trans-Golgi network Membrane Contact Sites. Our results show that: • SYT6 contacts trans-Golgi network vesicles through its coiled-coil domain. • SYT6 can efectively respond to Ca2+ using its terminal C2C domain. • SYT6-C2 domains preferentially bind to negatively charged membranes (with PI4P and PS) in presence of Ca2+.This research has been funded by AEI (PID2021-127649OB-I00 and PGC-2018-098789-B-I00) and FEDER-Junta de Andalucía (UMA18-FEDERJA-154). The attendance to this meeting was supported by Plan Propio de Investigación, Transferencia y Divulgación Científica de la Universidad de Málaga, Campus de Excelencia Internacional Andalucía Tech

A transgenic Camelina sativa seed oil effectively replaces fish oil as a dietary source of eicosapentaenoic acid in mice

Background: Fish currently supplies only 40% of the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) required to allow all individuals globally to meet the minimum intake recommendation of 500 mg/d. Therefore, alternative sustainable sources are needed. Objective: The main objective was to investigate the ability of genetically engineered Camelina sativa (20% EPA) oil (CO) to enrich tissue EPA and DHA relative to an EPA-rich fish oil (FO) in mammals. Methods: Six-week-old male C57BL/6J mice were fed for 10 wk either a palm oil–containing control (C) diet or diets supplemented with EPA-CO or FO, with the C, low-EPA CO (COL), high-EPA CO (COH), low-EPA FO (FOL), and high-EPA FO (FOH) diets providing 0, 0.4, 3.4, 0.3, and 2.9 g EPA/kg diet, respectively. Liver, muscle, and brain were collected for fatty acid analysis, and blood glucose and serum lipids were quantified. The expression of selected hepatic genes involved in EPA and DHA biosynthesis and in modulating their cellular impact was determined. Results: The oils were well tolerated, with significantly greater weight gain in the COH and FOH groups relative to the C group (P < 0.001). Significantly lower (36–38%) blood glucose concentrations were evident in the FOH and COH mice relative to C mice (P < 0.01). Hepatic EPA concentrations were higher in all EPA groups relative to the C group (P < 0.001), with concentrations of 0.0, 0.4, 2.9, 0.2, and 3.6 g/100 g liver total lipids in the C, COL, COH, FOL, and FOH groups, respectively. Comparable dose-independent enrichments of liver DHA were observed in mice fed CO and FO diets (P < 0.001). Relative to the C group, lower fatty acid desaturase 1 (Fads1) expression (P < 0.005) was observed in the COH and FOH groups. Higher fatty acid desaturase 2 (Fads2), peroxisome proliferator–activated receptor α (Ppara), and peroxisome proliferator–activated receptor γ (Pparg) (P < 0.005) expressions were induced by CO. No impact of treatment on liver X receptor α (Lxra) or sterol regulatory element-binding protein 1c (Srebp1c) was evident. Conclusions: Oil from transgenic Camelina is a bioavailable source of EPA in mice. These data provide support for the future assessment of this oil in a human feeding trial

Modifying the lipid content and composition of plant seeds: engineering the production of LC-PUFA

Omega-3 fatty acids are characterized by a double bond at the third carbon atom from the end of the carbon chain. Latterly, long chain polyunsaturated omega-3 fatty acids such as eicosapentaenoic acid (EPA; 20:5Δ5,8,11,14,17) and docosahexanoic acid (DHA; 22:6 Δ4,7,10,13,16,19), which typically only enter the human diet via the consumption of oily fish, have attracted much attention. The health benefits of the omega-3 LC-PUFAs EPA and DHA are now well established. Given the desire for a sustainable supply of omega-LC-PUFA, efforts have focused on enhancing the composition of vegetable oils to include these important fatty acids. Specifically, EPA and DHA have been the focus of much study, with the ultimate goal of producing a terrestrial plant-based source of these so-called fish oils. Over the last decade, many genes encoding the primary LC-PUFA biosynthetic activities have been identified and characterized. This has allowed the reconstitution of the LC-PUFA biosynthetic pathway in oilseed crops, producing transgenic plants engineered to accumulate omega-3 LC-PUFA to levels similar to that found in fish oil. In this review, we will describe the most recent developments in this field and the challenges of overwriting endogenous seed lipid metabolism to maximize the accumulation of these important fatty acids

Plasma membrane lipid remodeling during cold acclimation is mediated by the ER-PM contact sites-localized synaptotagmins 1 and 3

Cold acclimation is the capacity of certain plants to increase their freezing tolerance in response to a period of low non-freezing temperatures. Cold acclimation involves a series of biochemical and physiological adaptations, including a deep transcriptional reprogramming and drastic changes in the lipid composition of cellular membranes in order to prevent the freeze-induced damage (1). While a profound knowledge has been acquired on the regulation of gene expression triggered by cold-acclimation, very little is known about the mechanisms governing the cold-induced changes in membranes’ lipid composition. In this study we report that in Arabidopsis, the constitutively expressed Synaptotagmin 1 (SYT1) and the cold-induced homolog Synaptotagmin 3 (SYT3) are essential for cold- acclimated freezing tolerance and for the lipid remodelling of the plasma membrane during cold-acclimation. SYT1 and SYT3 are phospholipid-binding proteins located in Endoplasmic Reticulum-Plasma Membrane contact sites (ER-PMcs), conserved structures defined as regions of the cortical ER in close apposition to the PM (2). ER-PMcs facilitate the non-vesicular lipid transport between ER and PM in yeast and mammals, and are essential for lipid homeostasis (3). In contrast to the high and ubiquitous SYT1 expression, SYT3 expression is low and mainly restricted to meristemoids, young stomata, and old primary root. TIRF microscopy analyses show that during cold acclimation there is an increase of SYT1::SYT1:GFP and SYT3::SYT3:GFP signals as spots at the PM. High-resolution lipidome analyses show the over-accumulation of phosphatidylinositols phosphate (PIPs) and glycerolipids in vivo in syt1 and specially syt1/syt3 mutant plants compared to WT in one-week cold-acclimated plants. Interestingly, protein-lipid overlay assays (membrane-strips and PIP-strips) reveal PIPs and glycerolipids as major interactors for both, SYT1 and SYT3. Here we show that 1) Arabidopsis SYT1 and SYT3 are induced by cold, 2) SYT1 and SYT3 localize to ER-PMcs, 3) the specific lipids that directly interact with SYT1 and SYT3 accumulate in syt1/syt3 mutant after cold acclimation, and 4) syt1/syt3 show reduced cold acclimated freezing tolerance. We propose that SYT1 and SYT3 have essential roles in ER-PMcs mediated lipid remodelling during cold acclimation, which in turn leads to freezing tolerance.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

NTMC2T5 protein family: newly identified ER-chloroplast contact site proteins involved in abiotic stress.

Plants are sessile organisms and therefore they have perfected a complex molecular signalling network to detect and respond to the different environmental stresses such as high temperatures, salinity, or drought. In plants, fatty acid synthesis takes place at chloroplasts, and they are assembled into glycerolipids and sphingolipids at the endoplasmic reticulum (ER). Then, the newly synthetized lipids in the ER are delivered to chloroplast via a non-vesicular pathway, likely through lipid transport proteins (LTP). These LTP would be localized in ER-chloroplast membrane contact sites (MCS). Synaptotagmin-like mitochondrial-lipid-binding (SMP) domain proteins are evolutionarily conserved LTP in eukaryotes that localize at MCS. They are involved in tethering of these MCS through interaction with other proteins/membrane lipids and in transferring of glycerolipids between these two membranes. We have studied the occurrence of SMP proteins in A. thaliana and S. lycopersicum by searching remote orthologs of human E-Syt1 (SMP protein). By using transient expression in N. benthamiana leaves and confocal microscopy, we have identified the NTMC2T5 family with two homologs in A. thaliana and only one in S. lycopersicum that are anchored to the chloroplast outer membrane and are interacting with the ER (at ER-chloroplast MCS). Our preliminary data have unequivocally demonstrated that NTMC2T5 proteins are anchored to the chloroplast, and they bind in trans the ER. Additionally, it is predicted that these proteins contain a SMP domain which is a lipid-transfer domain, indicating that these proteins could be responsible for some of the lipid transferring events at ER-chloroplast MCS that are still unknown. Our preliminary phenotypic analyses have shown that these proteins are involved in salt tolerance. Finally, we have observed that clustering of chloroplasts around the nucleus occurred when we overexpressed these proteins in Nicotiana benthamiana leaves.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Calibration of Numerical Modelling of Hydrological and Hydraulic Processes in Arnas Experimental Basin

Source: ICHE Conference Archive - https://mdi-de.baw.de/icheArchiv

Arabidopsis Synaptotagmins 1 and 3 are involved in lipid homeostasis at ER-PM contact sites under cold stress

As sessile organisms, plants must cope with abiotic stress such as soil salinity, drought, and extreme temperatures. This stress signal can activate a phospholipase C (PLC), which hydrolyses PIP2 to generate IP3 and diacylglycerol (DAG). ER-PM contact sites are conserved structures defined as regions of the endoplasmic reticulum (ER) that tightly associate with the plasma membrane (PM). Our recent data suggest that the constitutively expressed Arabidopsis Synaptotagmin 1 (SYT1) and the cold-induced homolog SYT3 are proteins located in these ER-PM contact sites that are essential for freezing tolerance. Additionally, like mammalian Extended Synaptotagmins, membrane tethering is mediated by C2-domains which interact with acidic phospholipids (enhanced by Ca2+). Our experiments of depletion of PM PI(4)P triggers loss of SYT1 and SYT3 at ER-PM CS. Moreover, our analysis in SYT1 and SYT3 proteins predicted a SMP domain like the recently crystalized E-SYT2 which exhibits a hydrophobic groove capable of harbouring phospholipids, suggesting that SYT1 and SYT3 mediate lipid exchange between the ER and the PM. This idea is supported by the over-accumulation of saturated DAG found in SYT1 after a high-resolution lipidome analysis. Additionally, we have identified DGK2 (diacylglycerol kinase 2) as an interactor of SYT1. In summary, our recent studies suggest that SYT1 and SYT3 are ER-PM tether components responsible for the elimination of excess DAG from the PM after its acute generation by PLC in cold conditions.The authors acknowledge the support by the Plan Propio from University of Malaga, Campus de Excelencia Internacional de Andalucía and by the Redes of Excelencia (BIO2014-56153-REDT) and BIO2017-82609-R & BIO2014-55380-R of the Ministerio de Economía, Industria y Competitivida

Differentiating IDH-mutant astrocytomas and 1p19q-codeleted oligodendrogliomas using DSC-PWI:high performance through cerebral blood volume and percentage of signal recovery percentiles

Objective: Presurgical differentiation between astrocytomas and oligodendrogliomas remains an unresolved challenge in neuro-oncology. This research aims to provide a comprehensive understanding of each tumor’s DSC-PWI signatures, evaluate the discriminative capacity of cerebral blood volume (CBV) and percentage of signal recovery (PSR) percentile values, and explore the synergy of CBV and PSR combination for pre-surgical differentiation. Methods: Patients diagnosed with grade 2 and 3 IDH-mutant astrocytomas and IDH-mutant 1p19q-codeleted oligodendrogliomas were retrospectively retrieved (2010–2022). 3D segmentations of each tumor were conducted, and voxel-level CBV and PSR were extracted to compute mean, minimum, maximum, and percentile values. Statistical comparisons were performed using the Mann-Whitney U test and the area under the receiver operating characteristic curve (AUC-ROC). Lastly, the five most discriminative variables were combined for classification with internal cross-validation. Results: The study enrolled 52 patients (mean age 45-year-old, 28 men): 28 astrocytomas and 24 oligodendrogliomas. Oligodendrogliomas exhibited higher CBV and lower PSR than astrocytomas across all metrics (e.g., mean CBV = 2.05 and 1.55, PSR = 0.68 and 0.81 respectively). The highest AUC-ROCs and the smallest p values originated from CBV and PSR percentiles (e.g., PSRp70 AUC-ROC = 0.84 and p value = 0.0005, CBVp75 AUC-ROC = 0.8 and p value = 0.0006). The mean, minimum, and maximum values yielded lower results. Combining the best five variables (PSRp65, CBVp70, PSRp60, CBVp75, and PSRp40) achieved a mean AUC-ROC of 0.87 for differentiation. Conclusions: Oligodendrogliomas exhibit higher CBV and lower PSR than astrocytomas, traits that are emphasized when considering percentiles rather than mean or extreme values. The combination of CBV and PSR percentiles results in promising classification outcomes. Clinical relevance statement: The combination of histogram-derived percentile values of cerebral blood volume and percentage of signal recovery from DSC-PWI enhances the presurgical differentiation between astrocytomas and oligodendrogliomas, suggesting that incorporating these metrics into clinical practice could be beneficial. Key Points: • The unsupervised selection of percentile values for cerebral blood volume and percentage of signal recovery enhances presurgical differentiation of astrocytomas and oligodendrogliomas. • Oligodendrogliomas exhibit higher cerebral blood volume and lower percentage of signal recovery than astrocytomas. • Cerebral blood volume and percentage of signal recovery combined provide a broader perspective on tumor vasculature and yield promising results for this preoperative classification.</p

Differential effects of EPA vs. DHA on postprandial vascular function and the plasma oxylipin profile in men

Our objective was to investigate the impact of EPA versus DHA, on arterial stiffness and reactivity, and underlying mechanisms (with a focus on plasma oxylipins), in the postprandial state. In a 3-arm cross-over acute test meal trial men (n=26, 35-55y) at increased CVD risk, received a high fat (42.4g) test meal providing 4.16 g of EPA or DHA or control oil in random order. At 0 h and 4 h, blood samples were collected to quantify plasma fatty acids, LCn-3PUFAs derived oxylipins, nitrite and hydrogen sulfide and serum lipids and glucose. Vascular function was assessed using blood pressure, Reactive Hyperaemia Index (RHI), Pulse Wave Velocity and Augmentation Index (AIx). The DHA-rich oil significantly reduced AIx by 13% (P=0.047) with the decrease following EPA-rich oil intervention not reaching statistical significance. Both interventions increased EPA and DHA derived oxylipins in the acute postprandial state, with an (1.3 fold) increase in 19,20-DiHDPA evident after DHA intervention (P < 0.001). In conclusion, a single dose of DHA significantly improved postprandial arterial stiffness as assessed by AIx, which if sustained would be associated with a significant decrease in CVD risk. The observed increases in oxylipins provide a mechanistic insight for the AIx effect

IDENTIFICATION OF SYT1 INTERACTORS CONNECTS CALCIUM SIGNALING, ENDOPLASMIC RETICULUM BENDING, PLASMODESMATA AND MEMBRANE CONTACT SITES

Synaptotagmin1 (SYT1) is an Arabidopsis thaliana protein essential for tolerance to several abiotic stresses (Schapire et al., 2008; Pérez-Sancho et al., 2015; Ruiz-Lopez et al., 2020). SYT1 forms endoplasmic reticulum-plasma membrane contact sites (ER-PM CS), microdomains conserved across eukaryotes where protein tethers maintain the membranes of the ER and the PM in close apposition (∼30 nm) without fusing. The short distance between membranes facilitates processes such as ion and lipid transport (Pérez-Sancho et al., 2016). For example, SYT1 transports diacylglycerol (DAG) from the PM to the ER during abiotic stress to assure PM integrity (Ruiz-Lopez et al., 2020). Usually, protein complexes form the core of contact sites. In particular, SYT1 forms dimers with SYT1, SYT3, SYT5 and CLB1; all members of the Arabidopsis SYT family and EPCS tethers (Lee et al., 2020; Ruiz-Lopez et al., 2020). We found that SYT1 interacts with proteins involved in different cellular processes by non-targeted proteomic approaches (IP-MS and TAP-tag). Thus, SYT1 interacts with reticulons (RTN), ER-resident proteins responsible for ER curvature, which is crucial for ER morphology and lipid transport at ER-PM CS (Collado et al., 2019). Additionally, SYT1 interacts with ECAs, Ca2+-ATPases located at the ER membrane. SYT1 also interacts with sterol methyltransferases (SMTs), key enzymes in the route of sitosterol and stigmasterol biosynthesis. The homeostasis of the sitosterol and stigmasterol is crucial for the tolerance to wound, heat and bacterial stress presumably by affecting PM fluidity. MCTPs (multiple C2 domains and transmembrane region proteins), plasmodesmata-exclusive proteins are also SYT1 interactors (Brault et al., 2019). We are now investigating the role of SYT1 in these processes using biochemical, genetic and cellular biology approaches