Seipin is an endoplasmic reticulum (ER) membrane protein implicated in lipid droplet (LD) biogenesis and mutated in severe congenital lipodystrophy (BSCL2). Here, we show that seipin is stably ...associated with nascent ER–LD contacts in human cells, typically via one mobile focal point per LD. Seipin appears critical for such contacts since ER–LD contacts were completely missing or morphologically aberrant in seipin knockout and BSCL2 patient cells. In parallel, LD mobility was increased and protein delivery from the ER to LDs to promote LD growth was decreased. Moreover, while growing LDs normally acquire lipid and protein constituents from the ER, this process was compromised in seipin‐deficient cells. In the absence of seipin, the initial synthesis of neutral lipids from exogenous fatty acid was normal, but fatty acid incorporation into neutral lipids in cells with pre‐existing LDs was impaired. Together, our data suggest that seipin helps to connect newly formed LDs to the ER and that by stabilizing ER–LD contacts seipin facilitates the incorporation of protein and lipid cargo into growing LDs in human cells.
Synopsis
Seipin, mutated in severe congenital lipodystrophy (BSCL2), is an ER–lipid droplet (LD) contact protein that regulates the extent of ER–LD contacts and facilitates incorporation of lipid and protein cargo into maturing LDs.
Seipin deficiency increases the heterogeneity of ER–LD contacts, resulting in completely missing, rudimentary, or very extensive contacts.
Seipin is required during LD formation for the targeting of ER‐derived fatty acid‐activating enzyme ACSL3 to LDs.
An ER‐to‐LD targeting model peptide and a fluorescent fatty acid analog initially reach newly formed LDs, but their subsequent incorporation into LDs is impaired in the absence of seipin.
In seipin deficiency, the fatty acid flux to neutral lipids becomes compromised when LD formation has been initiated.
Seipin, mutated in severe congenital lipodystrophy (BSCL2), is an ER–lipid droplet (LD) contact protein that regulates the extent of ER–LD contacts and facilitates incorporation of lipid and protein cargo into maturing LDs.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Seipin is an oligomeric integral endoplasmic reticulum (ER) protein involved in lipid droplet (LD) biogenesis. To study the role of seipin in LD formation, we relocalized it to the nuclear envelope ...and found that LDs formed at these new seipin-defined sites. The sites were characterized by uniform seipin-mediated ER-LD necks. At low seipin content, LDs only grew at seipin sites, and tiny, growth-incompetent LDs appeared in a Rab18-dependent manner. When seipin was removed from ER-LD contacts within 1 h, no lipid metabolic defects were observed, but LDs became heterogeneous in size. Studies in seipin-ablated cells and model membranes revealed that this heterogeneity arises via a biophysical ripening process, with triglycerides partitioning from smaller to larger LDs through droplet-bilayer contacts. These results suggest that seipin supports the formation of structurally uniform ER-LD contacts and facilitates the delivery of triglycerides from ER to LDs. This counteracts ripening-induced shrinkage of small LDs.
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•Seipin can determine the site of LD formation•Seipin-mediated ER-LD membrane contacts display a uniform neck-like architecture•Seipin at ER-LD contact facilitates continuous triglyceride transfer to LD•Acute removal of seipin reveals a principle of LD ripening via the ER
Salo et al. show that seipin determines where a lipid droplet (LD) forms and establishes a neck between ER and LD. LDs continuously exchange lipids via ER, and biophysically, larger LDs acquire lipids more readily than smaller ones. Seipin facilitates lipid transfer to droplets, helping small LDs to grow.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Seipin is a disk-like oligomeric endoplasmic reticulum (ER) protein important for lipid droplet (LD) biogenesis and triacylglycerol (TAG) delivery to growing LDs. Here we show through biomolecular ...simulations bridged to experiments that seipin can trap TAGs in the ER bilayer via the luminal hydrophobic helices of the protomers delineating the inner opening of the seipin disk. This promotes the nanoscale sequestration of TAGs at a concentration that by itself is insufficient to induce TAG clustering in a lipid membrane. We identify Ser166 in the α3 helix as a favored TAG occupancy site and show that mutating it compromises the ability of seipin complexes to sequester TAG in silico and to promote TAG transfer to LDs in cells. While the S166D-seipin mutant colocalizes poorly with promethin, the association of nascent wild-type seipin complexes with promethin is promoted by TAGs. Together, these results suggest that seipin traps TAGs via its luminal hydrophobic helices, serving as a catalyst for seeding the TAG cluster from dissolved monomers inside the seipin ring, thereby generating a favorable promethin binding interface.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Microcystins with leucine arginine (MC-LR) is a virulent hepatotoxin, which is commonly present in polluted water with its demethylated derivatives Dha
MC-LR. This study reported a low-cost ...molecularly imprinted polymer network-based electrochemical sensor for detecting MC-LR. The sensor was based on a three-dimensional conductive network composed of multi-walled carbon nanotubes (MWCNTs), graphene quantum dots (GQDs), and gold nanoparticles (AuNPs). The molecularly imprinted polymer was engineered by quantum chemical computation utilizing p-aminothiophenol (p-ATP) and methacrylic acid (MAA) as dual functional monomers and L-arginine as a segment template. The electrochemical reaction mechanism of MC-LR on the sensor was studied for the first time, which is an irreversible electrochemical oxidation reaction involving an electron and two protons, and is controlled by a mixed adsorption-diffusion mechanism. The sensor exhibited a great detection response to MC-LR in the linear range of 0.08-2 μg/L, and the limit of detection (LOD) is 0.0027 μg/L (S/N = 3). In addition, the recoveries of the total amount of MC-LR and Dha
MC-LR in the actual sample by the obtained sensor were in the range from 91.4 to 116.7%, which indicated its great potential for environmental detection.
Magnetic Fe3O4 nanoparticles were successfully functionalized with the low-cost mussel-inspired tannic acid (TA)/polyethylenimine (PEI) under mild conditions and then activated with glutaraldehyde ...(GA) to covalently immobilize laccase for degradation of four model phenolic inhibitors commonly found in lignocellulosic hydrolysate. The immobilized laccase showed the highest relative enzyme activity with the laccase loading of 39.9 mg/g and the enzyme activity recovery of 53.1%. Compared with free laccase, immobilized laccase possessed better relative enzyme activity at acidic pH and wide temperature range, as well as exhibited excellent storage stability and satisfactory reusability. Moreover, it was found that the phenolic degradation products by immobilized laccase did not exhibit obvious inhibition on subsequent enzymatic cellulose hydrolysis. Thus, the low-cost TA/PEI coating for modification of magnetic nanoparticles will provide a promising support for laccase immobilization and the immobilized laccase has great potential in removal of phenolic inhibitors present in lignocellulosic hydrolysate.
•Laccase was immobilized on Fe3O4 nanoparticles modified with TA/PEI coating.•Preparation of TA/PEI modified magnetic carrier was optimized.•Immobilized laccase showed improved stability and catalytic efficiency.•Phenolics degradation products had no effect on enzymatic cellulose hydrolysis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The endoplasmic reticulum (ER) is a large, single-copy, membrane-bound organelle that comprises an elaborate 3D network of diverse structural subdomains, including highly curved tubules, flat sheets, ...and parts that form contacts with nearly every other organelle. The dynamic and complex organization of the ER poses a major challenge on understanding how its functioning – maintenance of the structure, distribution of its functions and communication with other organelles – is orchestrated. In this study, we resolved a unique localization profile within the ER network for several resident ER proteins representing a broad range of functions associated with the ER using immuno-electron microscopy and calculation of a relative labeling index (RLI). Our results demonstrated the effect of changing cellular environment on protein localization and highlighted the importance of correct protein expression level when analyzing its localization at subdomain resolution. We present new software tools for anonymization of images for blind analysis and for quantitative assessment of membrane contact sites (MCSs) from thin section transmission electron microscopy micrographs. The analysis of ER-mitochondria contacts suggested the presence of at least three different types of MCSs that responded differently to changes in cellular lipid loading status.
•Immuno-EM and RLI reveals specific subdomain localization for resident ER proteins.•Protein overexpression can lead to the loss of specific sub-organelle localization.•ER-mitochondria contact site distribution changes pending on lipid loading status.•Lipid loading status affects localization of cholesterol metabolic enzyme SOAT-1.•MCcalc enables quantitative and qualitative analysis of membrane contact sites.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The high recombination rates of photogenerated electron-holes greatly inhibit the catalytic activity of semiconductor photocatalysts. Herein, the heterojunctions of the flower-like g-C
3
N
4
/BiOBr ...composites were synthesized as photocatalysts by a simple hydrothermal process. The X-ray diffraction, scanning electron microscopy, and X-ray photoelectron spectrometer were utilized to characterize the sample's structure and light absorption properties. The results demonstrated that BiOBr-g-C
3
N
4
-4:1 showed excellent photocatalytic properties and 96.6% of bisphenol (BPA) was removed in 120 min with illumination of visible light due to its narrower band gap than that of pure BiOBr. BiOBr offer little electrons during the photocatalytic reaction. Moreover, the heterostructure between BiOBr and g-C
3
N
4
facilitates the separation of photogenerated carriers. Excellent stability was exhibited after five cyclic degradation of methyl orange (MO) with the illumination of visible light. The active species trapping experiment indicated that superoxide radical anions (
O
2
•
-
) and hole (h
+
) have a great effect on the reaction. A possible mechanism was proposed to explain the whole process of photocatalytic reaction.
Recent developments in auxin-inducible degron (AID) technology have increased its popularity for chemogenetic control of proteolysis. However, generation of human AID cell lines is challenging, ...especially in human embryonic stem cells (hESCs). Here, we develop HiHo-AID2, a streamlined procedure for rapid, one-step generation of human cancer and hESC lines with high homozygous degron-tagging efficiency based on an optimized AID2 system and homology-directed repair enhancers. We demonstrate its application for rapid and inducible functional inactivation of twelve endogenous target proteins in five cell lines, including targets with diverse expression levels and functions in hESCs and cells differentiated from hESCs.
Metabolic pathways are reprogrammed in cancer to support cell survival. Here, we report that T-cell acute lymphoblastic leukemia (T-ALL) cells are characterized by increased oxidative phosphorylation ...and robust ATP production. We demonstrate that ORP4L is expressed in T-ALL but not normal T-cells and its abundance is proportional to cellular ATP. ORP4L acts as an adaptor/scaffold assembling CD3ɛ, Gαq/11 and PLCβ3 into a complex that activates PLCβ3. PLCβ3 catalyzes IP3 production in T-ALL as opposed to PLCγ1 in normal T-cells. Up-regulation of ORP4L thus results in a switch in the enzyme responsible for IP3-induced endoplasmic reticulum Ca(2+) release and oxidative phosphorylation. ORP4L knockdown results in suboptimal bioenergetics, cell death and abrogation of T-ALL engraftment in vivo. In summary, we uncovered a signalling pathway operating specifically in T-ALL cells in which ORP4L mediates G protein-coupled ligand-induced PLCβ3 activation, resulting in an increase of mitochondrial respiration for cell survival. Targeting ORP4L might represent a promising approach for T-ALL treatment.
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