Seipin: Biological Overview | References
Gene name - Seipin
Cytological map position - 3A7-3A8
Function - Transmembrane lipid-binding protein
Symbol - Seipin
FlyBase ID: FBgn0040336
Genetic map position - chrX:2,619,799-2,622,140
NCBI classification - Putative adipose-regulatory protein
Cellular location - ER transmembrane
Seipin, the gene that causes Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2), is important for adipocyte differentiation and lipid homeostasis. Previous studies in Drosophila revealed that Seipin promotes ER calcium homeostasis through the Ca(2+)-ATPase SERCA, but little is known about the events downstream of perturbed ER calcium homeostasis that lead to decreased lipid storage in Drosophila dSeipin mutants. This study shows that glycolytic metabolites accumulate and the downstream mitochondrial TCA cycle is impaired in dSeipin mutants. The impaired TCA cycle further leads to a decreased level of citrate, a critical component of lipogenesis. Mechanistically, Seipin/SERCA-mediated ER calcium homeostasis is important for maintaining mitochondrial calcium homeostasis. Reduced mitochondrial calcium in dSeipin mutants affects the TCA cycle and mitochondrial function. The lipid storage defects in dSeipin mutant fat cells can be rescued by replenishing mitochondrial calcium or by restoring the level of citrate through genetic manipulations or supplementation with exogenous metabolites. Together, these results reveal that Seipin promotes adipose tissue lipid storage via calcium-dependent mitochondrial metabolism (Ding, 2018).
Impaired lipid metabolism is associated with an imbalance in energy homeostasis and many other disorders. Excessive lipid storage results in obesity, while a lack of adipose tissue leads to lipodystrophy. Clinical investigations reveal that obesity and lipodystrophy share some common secondary effects, especially non-alcoholic fatty liver disease and severe insulin resistance. Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2/CGL2) is one of the most severe lipodystrophy diseases. Patients with BSCL2 manifest almost total loss of adipose tissue as well as fatty liver, insulin resistance, and myohypertrophy. BSCL2 results from mutation of the Seipin gene, which is highly conserved from yeast to human (Ding, 2018).
To study the function of Seipin, genetic models were established in different organisms, including yeast, fly, and mouse, and in human cells. As a transmembrane protein residing in the endoplasmic reticulum (ER) and in the vicinity of lipid droplet (LD) budding sites, Seipin has been shown to be involved in LD formation, phospholipid metabolism, lipolysis, and ER calcium homeostasis. As a result of the functional studies in these models, several factors that interact with Seipin protein were identified, such as the phosphatidic acid phosphatase lipin, 14-3-3β, and glycerol-3-phosphate acyltransferase (GPAT). Drosophila Seipin (dSeipin) functions tissue autonomously in preventing ectopic lipid accumulation in salivary gland (a non-adipose tissue) and in promoting lipid storage in fat tissue (Tian, 2011). The non-adipose tissue phenotype is likely attributed to the increased level of phosphatidic acid (PA) generated by elevated GPAT activity. In adipose tissue Seipin interacts with the ER Ca2+-ATPase SERCA, whose activity is reduced in dSeipin mutants, leading to reduced ER calcium levels. Further genetic analysis suggested that the perturbed level of intracellular calcium contributes to the lipodystrophy. However, it is not known how the depleted ER calcium pool causes decreased lipid storage (Ding, 2018).
Besides the ER, mitochondria are another important intracellular calcium reservoir. Mitochondrial calcium is mainly derived from the ER through the IP3R channel. IP3R not only releases calcium from the ER into the cytosol, but also provides sufficient Ca2+ at mitochondrion-associated ER membranes (MAMs) for activation of the mitochondrial calcium uniporter. The mitochondrial Ca2+ level varies greatly in different cell types and can be modulated by influx and efflux channel proteins, such as MCU and NCLX, a mitochondrial Na+/Ca2+ exchanger. A proper mitochondrial Ca2+ level is implicated in mitochondrial integrity and function. Mitochondrial calcium is needed to support the activity of the mitochondrial matrix dehydrogenases in the TCA cycle. TCA cycle intermediates are used for the synthesis of important compounds, including glucose, amino acids, and fatty acids. Acetyl-CoA, as the basic building block of fatty acids, is generally derived from glycolysis, the TCA cycle, and fatty acid β-oxidation. In mammalian adipocytes, acetyl-CoA derived from the TCA cycle intermediate citrate is crucial for de novo lipid biosynthesis, which contributes significantly to lipid storage (Ding, 2018 and references therein).
This study used multiple comparative omics to analyze the proteomic, transcriptomic, and metabolic differences between larval fat cells of dSeipin mutants and wild type. The results reveal an impairment in channeling glycolytic metabolites to mitochondrial metabolism in dSeipin mutant fat cells, and scarcity of mitochondrial Ca2+, are the causative factors of this metabolic dysregulation. Evidence is provided showing that dSeipin lipodystrophy is rescued by restoring mitochondrial calcium or replenishing citrate. It is proposed that the low ER Ca2+ level in dSeipin mutants cannot maintain a sufficiently high mitochondrial Ca2+ concentration to support the TCA reactions. This in turn leads to reduced lipogenesis in dSeipin mutants (Ding, 2018).
Seipin promotes fat tissue lipid storage via calcium-dependent mitochondrial metabolism. Defective ER calcium homeostasis in dSeipin mutants is associated with reduced mitochondrial calcium and impaired mitochondrial function, such as low production of TCA cycle metabolites. Restoring mitochondrial calcium levels or replenishing citrate, a key TCA cycle product and also an important precursor of lipogenesis, rescues the lipid storage defects in dSeipin mutant fat cells (Ding, 2018).
This study investigated the underlying causes of Seipin-dependent lipodystrophy by integrating multiple omic analyses, including RNA-seq, quantitative proteomics, and metabolomic analysis. Compared to previous studies based on genetics and traditional cellular phenotypic analysis, these combinatory omic approaches provide an unprecedented spectrum of molecular phenotypes, which not only add new information but also pinpoint logical directions for further investigations (Ding, 2018).
Omics analyses, in particular lipidomic analysis, have been utilized to investigate the underlying mechanisms in several previous Seipin studies and led to the finding that PA is elevated in several Seipin mutant models. In this study, based on genetic rescuing assays and quantitative proteomics analysis, it was initially proposed that downregulated glycolysis is the cause of lipodystrophy. However, both the RNA-seq results and metabolomic data argue against this possibility and suggest a new mechanism. Despite reduced levels of glycolytic enzymes, transcription of the corresponding genes is not affected, and glycolytic metabolites, in particular pyruvate, are increased in dSeipin mutants compared to wild type. Metabolomic data further show that citrate and isocitrate, which are the products of the first two steps of the mitochondrial TCA cycle, are dramatically decreased in dSeipin mutants, suggesting a defective metabolic flow downstream of pyruvate. These results lead to a new possibility that the lipid storage defects in dSeipin mutants are caused by a defective TCA cycle and this is indeed supported by the metabolic flux analysis. These findings further suggest the involvement of mitochondria. In line with this, the previous discovery that fatty acid β-oxidation is elevated in dSeipin mutant fat cells may reflect compensation for the reduced TCA cycle and lipogenesis. This possibility is supported by the results of genetic and citrate-supplement rescue experiments and by citrate measurements (Ding, 2018).
It is known that glycolytic enzymes and metabolites are regulated by a metabolic feedback loop, which may complicate the explanation of genetic interactions. The current findings highlight that although genetic analysis and rescue results provide important clues, multiple lines of evidence are critical for unraveling complex intracellular pathways. In this case, the combination of omic results and genetic analysis led to the finding that mitochondrial metabolism is important in Seipin-associated lipodystrophy (Ding, 2018).
Mitochondria are hubs in key cellular metabolic processes, including the TCA cycle, ATP production, and amino acid catabolism. Mitochondria also play a central role in lipid homeostasis by controlling two seemingly opposite metabolic pathways, lipid biosynthesis, and fatty acid breakdown. Therefore, impairment of mitochondrial function in different tissues may lead to different, even opposite, phenotypes in lipid storage. In tissues where lipid biosynthesis is the major pathway, defective mitochondria might result in reduced lipid storage, whereas in tissues where fatty acid oxidation prevails, the same defect might lead to increased lipid storage. Reduced lipid storage in dSeipin mutants suggests the former case. The reduced level of citrate and other TCA cycle products in dSeipin mutants suggests an impairment of mitochondrial function. The reduction of OCR and ATP production, the decreased Rhod-2 staining, and the aberrant enrichment of mitochondria within autophagosomes all further support this notion. Interestingly, in mouse brown adipose tissue, Seipin mutation increases mitochondrial respiration along with normal MitoTracker labeling (Zhou, 2016). The discrepancies suggest that Seipin may have cell type-specific functions. Unlike white adipose tissue, which favors lipid storage/biosynthesis, brown adipose tissue is prone to fatty acid breakdown (Ding, 2018).
The link between mitochondria and Seipin was concealed in several previous studies. GPATs, which are recently reported Seipin-interacting proteins, participate in many mitochondrial processes. For example, mitochondria from brown adipocytes that are deficient in GPAT4 exhibit high oxidative levels, and mitochondrial GPAT is required for mitochondrial dynamics. PA, which is elevated in Seipin mutants, is required for mitochondrial morphology and function. Similarly, mitochondrial impairments were also observed in various lipodystrophic conditions. Downregulation of mitochondrial transcription and altered mitochondrial function were indicated in type III congenital generalized lipodystrophy. Multiple mitochondrial metabolic processes are altered in mice with lipodystrophy caused by Zmpste24 mutation. HIV patients treated with anti-retroviral therapy manifest partial lipodystrophy and impaired mitochondria in adipocytes. Moreover, mitochondrial dysfunction in adipose tissue triggers lipodystrophy and systemic disorders in mice. Therefore, the contribution of mitochondrial dysfunction to the cause or development of lipodystrophic conditions warrants further examination (Ding, 2018).
It has been previously reported that dSeipin/SERCA-mediated ER calcium homeostasis is critical for lipid storage (Bi, 2014). Consistent with this, transcripts encoding calcium signaling factors are enriched in the genes that are differentially expressed between dSeipin mutants and wild type. Mitochondrial calcium is transported from the ER through the ER-resident channel IP3R. The reduction of mitochondrial calcium in dSeipin mutant fat cells suggests that the decreased ER calcium leads to an insufficient level of mitochondrial calcium. Importantly, RNAi of a putative Drosophila mitochondrial calcium efflux channel (NCLX/CG18660) not only restores the mitochondrial calcium level but also rescues the lipid storage defects in dSeipin mutants, indicating that mitochondrial calcium is key for dSeipin-mediated lipid storage. This explains the previous finding that the lipid storage defects in dSeipin mutants are rescued by RNAi of RyR, which is not required for ER-mitochondrion calcium transport, but not by RNAi of IP3R (Ding, 2018).
Cellular calcium has been linked to lipid storage and related diseases in recent studies. Comprehensive genetic screening in Drosophila showed that ER calcium-related proteins are key regulators of lipid storage. In particular, SERCA, as the sole ER calcium influx channel and an interacting partner of Seipin, has been repeatedly implicated in lipid metabolism. Dysfunctional lipid metabolism can disrupt ER calcium homeostasis by inhibiting SERCA and further disturbing systemic glucose homeostasis. Increased SERCA expression was shown to have dramatic anti-diabetic benefits in mouse models. In a genomewide association study, SERCA was been found to be associated with obesity. In addition, cellular calcium influx is important for transcriptional programming of lipid metabolism, including lipolysis in mice. The current study further elucidates that ER calcium and mitochondrial calcium are important for cellular lipid homeostasis. It also provides a new insight into the pathogenic mechanism of congenital lipodystrophy (Ding, 2018).
Since Seipin mutations lead to opposite effects on lipid storage in adipose tissue (lipodystrophy) and non-adipose tissues (ectopic lipid storage), numerous studies have been carried out to understand the underlying mechanisms. In Seipin mutants, elevated GPAT activity leads to an increased level of PA. This may cause the formation of supersized lipid droplets in non-adipose cells because of the fusogenic property of PA in lipid leaflets, and may also lead to adipogenesis defects due to the potential role of PA as an inhibitor of preadipocyte differentiation. The Seipin-mediated lipid storage phenotype is further complicated by the role of Seipin in lipid droplet formation, which is mainly studied in unicellular eukaryotic yeast or in cultured cells from multicellular eukaryotic organisms. Seipin has been found in the ER-LD contact sites, which are considered as essential subcellular foci for LD formation/maturation. Moreover, in mammalian adipose tissue, the role of Seipin in lipogenesis or lipolysis may also be masked by the defect in early adipogenesis (Ding, 2018).
How can previous findings in different model organisms and different cell types be reconciled? Seipin has been characterized as a tissue-autonomous lipid modulator. It is likely that Seipin participates in lipid metabolism via distinct mechanisms in different tissues. Alternatively, the metabolic processes that involve Seipin may have different outcomes in different tissues. For example, mitochondria have a different impact on lipid metabolism in different tissues: In non-fat cells, mitochondria mainly direct energy mobilization, whereas in fat cells, mitochondria mainly lead anabolism. The molecular role of Seipin and the phenotypic outcomes in Seipin mutants may rely on specific cellular and developmental contexts (Ding, 2018).
Metabolic energy is stored in cells primarily as triacylglycerols in lipid droplets (LDs), and LD dysregulation leads to metabolic diseases. The formation of monolayer-bound LDs from the endoplasmic reticulum (ER) bilayer is poorly understood, but the ER protein seipin is essential to this process. This study reports a cryo-electron microscopy structure and functional characterization of Drosophila melanogaster Seipin. The structure reveals a ring-shaped dodecamer with the luminal domain of each monomer resolved at approximately 4.0 Å. Each luminal domain monomer exhibits two distinctive features: a hydrophobic helix (HH) positioned toward the ER bilayer and a beta-sandwich domain with structural similarity to lipid-binding proteins. This structure and functional testing in cells suggest a model in which Seipin oligomers initially detect forming LDs in the ER via HHs and subsequently act as membrane anchors to enable lipid transfer and LD growth (Sui, 2018).
This study reports a molecular structure for D. melanogaster Seipin and test features of this structure both in vitro and in cells. The data indicate that D. melanogaster Seipin forms a large, ring-shaped dodecamer with N- and C-terminal segments oriented towards the cytoplasm, 24 transmembrane domains, and an assembly of folded domains that are localized in the ER lumen. The structure reveals that the macromolecular complex positions 24 hydrophobic helices of both the luminal and N-terminal domains on either side of the ER membrane, possibly to detect and bind neutral lipid lenses forming in the ER bilayer. The dimensions of this complex are consistent with a barrel-shaped ring that connects the ER membrane with nascent LDs during their formation. The molecular structure of Seipin argues strongly that Seipin performs a structural, and possibly a lipid transfer, role in organizing LD formation, and suggest that other effects of seipin deficiency, such as changes in ER calcium homeostasis found with seipin deficiency, are indirect (Sui, 2018).
Many avenues of evidence indicate that the Seipin luminal domain is crucial for its function. This domain is highly evolutionarily conserved and is the location of numerous lipodystrophy mutations, and this study demonstrates that mutating the Drosophila Seipin luminal domain impairs its function in LD biogenesis. The cryo-EM structure of the D. melanogaster luminal domain now defines structural features of this domain that begin to shed light on how Seipin functions. Most clearly, the structure suggests that Seipin oligomers position multiple hydrophobic helices on either side of the ER membrane, possibly to detect phospholipid packing defects due to forming neutral lipid lenses. This is consistent with previous studies that indicate that Seipin foci and nascent LD foci are initially separate but can interact and subsequently co-localize during LD formation. An advantage of an oligomeric structure of multiple LD-binding helices is that it increases the ability of such helices to detect lipid lenses through increased avidity, a property that is relevant to the binding of amphipathic helices to LD surfaces. Whether a dodecameric oligomer is strictly required for this function is in question. Data from other species suggest the number of monomers can vary between species, suggesting there is flexibility in the numbers of Seipin molecules in a macromolecular structure. Also, this study found that mutations at the luminal interface weakened oligomerization in vitro but could still rescue the seipin-deficiency phenotype in cells. However, the latter finding may indicate that these mutations, while sufficient to weaken oligomerization when analyzed biochemically, do not sufficiently reduce the affinity between monomers to abolish oligomerization in the ER membrane. Conceivably, other regions of the protein, such as interactions between transmembrane domains, may contribute to oligomerization in cells and may be essential for seipin function (Sui, 2018).
The structure also suggests that the luminal domain may have functions that are independent from providing an anchor for the complex on the luminal side of the ER membrane. An intriguing possibility is that the luminal domain functions to mediate lipid transfer to growing LDs. The β-sandwich fold of the luminal domain is structurally similar to NPC2, which binds and solubilizes cholesterol in the lumen of the lysosome to deliver it to membrane-embedded NPC1 for export from this organelle to the cytoplasm. Other distantly related NPC2-type proteins (e.g., in Camponotus japonicus) have a similar flexible β-sheet sandwich structure that allows binding of semiochemicals, including fatty acids, used for chemical communication. The structure, and alignments with sequences from other species, suggest that like NPC2, Seipin may have a binding pocket of sufficient size for accommodating hydrophobic molecules. Perhaps the NPC2-like luminal domain of Seipin participates in transferring lipids from the ER luminal leaflet to the nascent LDs to maintain the proper balance or composition of phospholipids or neutral lipids. The identity of a lipid that binds to Seipin is currently unknown and under investigation (Sui, 2018).
Based on the Seipin structure, a new model is suggested for LD formation. In this model, Seipin forms an oligomeric complex in the ER that moves throughout the reticular network in the absence of LDs. When neutral lipids are synthesized, they are initially dispersed within the ER bilayer. However, once the TG concentration in the ER exceeds a critical concentration, lipid lenses form and locally disrupt phospholipid bilayer packing of the ER membrane, resulting in localized surface defects, similar to those on mature LDs. Seipin complexes may recognize the phospholipid packing defects at these lenses by binding via its many amphipathic and hydrophobic helices located at the cytoplasmic N-terminus and in the ER lumen, respectively, and subsequently a Seipin oligomer becomes localized to a neutral lipid lens. As nascent LDs grow toward the cytosol, Seipin may function as a 'molecular washer', effectively anchoring the nascent LD to the ER (via N- terminal helix binding) and allowing for maintenance of the ER-LD connection. This could stabilize ER-LD connections, enabling LD growth and preventing nascent LDs from premature severing, as found with seipin deficiency. In this model, oligomerization could also serve to restrict the diameter of the neck of the budding LDs (Sui, 2018).
In summary, a structure has been determined for one of the key proteins that is crucial for LD formation, providing an initial biochemical and structural glimpse into this fascinating process. The structure reveals an oligomeric complex, and key features of this complex were identified that participate in LD formation, suggesting a structural and/or lipid-transfer function for Seipin that can be further tested. This structural model provides a new framework for the further molecular dissection of the LD formation pathway (Sui, 2018).
How proteins control the biogenesis of cellular lipid droplets (LDs) is poorly understood. Using Drosophila and human cells, this study shows that Seipin, an ER protein implicated in LD biology, mediates a discrete step in LD formation-the conversion of small, nascent LDs to larger, mature LDs. Seipin forms discrete and dynamic foci in the ER that interact with nascent LDs to enable their growth. In the absence of Seipin, numerous small, nascent LDs accumulate near the ER and most often fail to grow. Those that do grow prematurely acquire lipid synthesis enzymes and undergo expansion, eventually leading to the giant LDs characteristic of Seipin deficiency. These studies identify a discrete step of LD formation, namely the conversion of nascent LDs to mature LDs, and define a molecular role for Seipin in this process, most likely by acting at ER-LD contact sites to enable lipid transfer to nascent LDs (Wang, 2016).
This study identified a discreet step in LD formation—the conversion of small, nascent LDs (<200 nm diameter) to larger, mature initial LDs (typically 300~500 nm diameter); Seipin acts at this step of LD biogenesis. Oligomers of Seipin form highly mobile foci in the ER that interact with small, nascent LDs at ER-LD contact sites and enable their growth to mature initial lipid droplets (iLDs), likely by promoting neutral lipid transport. Without Seipin, the precursors of this step, small nascent LDs, accumulate to large numbers adjacent to the ER but fail to grow. With ongoing TG synthesis, some of these intermediates, or possibly other LDs arising from random coalescence of ER TG collections, appear to prematurely engage the expanding LD (eLD) pathway, and undergo expansion mediated by LD-localized lipid synthesis enzymes (e.g., GPAT4 and CCT1). As a late phenotype of seipin deficiency, giant LDs (>1~2 μm diameter) form eventually from these fewer abnormal eLDs, probably by coalescence due to a relative lack of phosphatidylcholine (PC) on their surfaces. This model explains both aspects of the seipin-deficient phenotype that have been observed (many small LDs and occasional giant LDs) (Wang, 2016).
The data uncover a specific step in LD formation that was previously unrecognized, namely the growth and maturation of nascent LDs to mature iLDs. In wildtype cells, LD formation intermediates at this step are difficult to observe, likely due to their rapid transition through the process. However, with Seipin depletion, progression through this step is blocked, resulting in the apparent accumulation of large numbers of intermediates (small, nascent LDs), found in these studies by electron tomography, that fail to mature. These findings indicate that Seipin functions downstream of initial lens formation and TG budding, at a more distal step in the maturation of nascent LDs (Wang, 2016).
How does seipin function in nascent LD maturation? Studies of the tagged endogenous protein show that Seipin localizes to multimeric foci that are highly mobile within the ER, as if scanning the ER for nascent LDs. Some Seipin foci encountered and associated with LiveDrop puncta (nascent LDs), became relatively less mobile, and at this point iLD growth ensued. These observations are consistent with the model of Seipin engaging nascent LDs and facilitating their growth, likely via transfer of additional neutral lipids, to mature iLDs (Wang, 2016).
How Seipin molecularly interacts with nascent LDs to facilitate LD growth is unknown. The data are consistent with findings in yeast that implicated a role for Seipin oligomers in early stages of LD formation. These studies indicated that purified Seipin forms oligomers in a toroid-structure comprising approximately 8~12 Seipin proteins. However, from live-cell imaging, it is estimated that the Seipin foci include considerably more molecular units in cells. Possibly Seipin oligomers anchored in the ER mediate specific contacts with the surface monolayer of nascent LDs and enable the transfer of lipids (such as TG) to the nascent LDs, allowing them to grow. Precisely how Seipin mediates this transfer is currently unknown (Wang, 2016).
Findings in Drosophila and mammalian cells indicate that Seipin localizes to ER-LD contact sites and suggest that Seipin is part of a protein machinery acting at ER-LD contact sites to promote LD maturation. At later time points in LD biogenesis, it was observed that each LD was associated with at least one Seipin punctum. These findings are consistent with several studies in yeast indicating that Seipin localizes to ER-LD contact sites. This study also found that small nascent LDs almost always associate with the ER, even when Seipin was absent, suggesting these LDs are still connected to the ER via contact sites. Consistent with the notion of ER-LD contact sites, the electron-tomograms revealed an absence of ribosomes between the nascent iLDs and the ER membrane. The close connections of the organelles and possible contact sites suggest that other proteins might be involved in establishing ER-LD contact sites and are still present in seipin deficiency (Wang, 2016).
These findings suggest that Seipin's function in converting nascent LDs to mature iLDs is likely its ancient, primary function. In support of this, seipin deficiency resulted in similar phenotypes of larger numbers of LiveDrop-positive foci in cells from flies, human mammary carcinoma cells, and human fibroblasts from seipin-deficient subjects. Additionally, the evolutionarily conserved transmembrane domains and luminal ER loop were sufficient for Seipin's function in LD formation. The role of the variable N-terminal domain is less clear. Overexpressed N-terminus of Drosophila seipin localized to LDs, suggesting that this part of the protein, though not required for formation, may aid in interaction with LDs. In yeast, the N-terminus was found to be functionally important with respect to the timing of LD formation (Wang, 2016).
Although the findings indicate that Seipin functions in an early step in LD formation, they also potentially explain the phenotype of giant LDs found in nearly all cells with seipin deficiency. In the absence of Seipin, large numbers of nascent iLDs accumulate, and some of these LDs aberrantly entered the LD expansion pathway. In support of this, lipid synthesis enzymes, such as GPAT4 and CCT1, that are normally found only on late-forming eLDs were aberrantly targeted to iLDs at early time points, and this mislocalization of eLD proteins to iLDs was crucial for the development of the giant LD phenotype at later time points. Similar abnormal protein targeting to LDs was recently found in yeast lacking seipin orthologues (Wang, 2016).
Several other models have proposed a primary role for Seipin in maintaining ER homeostasis, with associated indirect effects on LD formation. The current data do not support these models. For example, Seipin was hypothesized to primarily regulate lipid metabolism in the ER, including phospholipid synthesis, and this in turn affects LD formation. This study found no evidence to support this model, either in studies of glycerolipid synthesis rates or in the activities of specific enzymes (such as GPAT). This study also found no evidence of accumulation of PA in membranes of Seipin-deficient S2 cells, a finding that has been reported by several groups for seipin deficiency in yeast. No evidence was found of Seipin primarily affecting ER morphology or ER stress activation, which would normally be associated with defects in calcium homeostasis, as has been suggested. Since seipin appears to act downstream of the budding of nascent LDs, it might be expected that ER functions are largely conserved and unaffected by seipin deficiency (Wang, 2016).
In summary, this study has provided evidence that Seipin functions at a previously unrecognized discrete step in LD biogenesis, enabling nascent LDs to grow to mature iLDs. Cells lacking Seipin can still form LDs, but these LDs have irregular size and abnormal lipid and protein composition, which likely leads to cellular dysfunction with respect to storing and reclaiming lipids for cellular needs. In support of this notion, seipin deficiency in humans leads to severe generalized lipodystrophy, with a near absence of adipose mass. Seipin therefore is a crucial part of the cellular protein machinery that serves to organize oil emulsification for fat storage. The current findings also suggest that Seipin likely works in concert with other proteins that mediate ER-LD contact to enable the growth of nascent LDs (Wang, 2016).
Adipose tissue is central to the regulation of lipid metabolism. Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2), one of the most severe lipodystrophy diseases, is caused by mutation of the Seipin gene. Seipin plays an important role in adipocyte differentiation and lipid homeostasis, but its exact molecular functions are still unknown. This study shows that Seipin physically interacts with the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) in both Drosophila and man. SERCA, an endoplasmic reticulum (ER) calcium pump, is solely responsible for transporting cytosolic calcium into the ER lumen. Like dSeipin, dSERCA cell-autonomously promotes lipid storage in Drosophila fat cells. dSeipin affects dSERCA activity and modulates intracellular calcium homeostasis. Adipose tissue-specific knockdown of the ER-to-cytosol calcium release channel ryanodine receptor (RyR) partially restores fat storage in dSeipin mutants. These results reveal that Seipin promotes adipose tissue fat storage by regulating intracellular calcium homeostasis (Bi, 2014).
Lipids are major cellular energy sources, membrane components, and signal molecules. Adipose tissue is the main storage site for neutral lipids. Proper lipid storage by adipose tissue is important for human health. Excess or impaired lipid storage in adipose tissue leads respectively to obesity and lipodystrophy, which are tightly associated with numerous metabolic syndromes such as diabetes, dyslipidemia, hypertriglyceridemia, and hepatic steatosis (Bi, 2014).
Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2), one of the most severe lipodystrophy diseases in man, is characterized by a near-total loss of adipose tissue from birth or early infancy, severe insulin resistance, fatty liver, and muscular hypertrophy. BSCL2 is caused by mutation of the Seipin gene. Seipin encodes a homo-oligomeric protein that is integral to the endoplasmic reticulum (ER) membrane. Studies in yeast, flies, mice, and various cell lines have shown that loss of Seipin function leads to severe lipodystrophy, suppressed adipocyte differentiation, aberrant lipid droplet formation, and ectopic lipid accumulation. However, the exact molecular functions of Seipin remain unknown (Bi, 2014).
Calcium is an important intracellular signal responsible for regulating multiple cellular processes. The ER is the main intracellular calcium storage site and plays a key role in the maintenance of intracellular calcium homeostasis. The ryanodine receptor (RyR) and the inositol 1,4,5-trisphosphate receptor (IP3R) are two calcium channels that release calcium from the ER to the cytosol in response to cellular stimuli. The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pumps cytosolic calcium into the ER lumen and maintains a high calcium concentration difference between the ER lumen and the cytosol at rest. Several studies indicate that ER calcium homeostasis is critical for adipogenesis and lipid storage. For instance, the SERCA inhibitor thapsigargin inhibits the early stages of adipogenesis in cultured cells. The ER calcium sensor STIM1 was found to negatively regulate the differentiation of 3T3-L1 preadipocytes. Drosophila IP3R mutants exhibit excessive food intake and obesity (Bi, 2014).
This study identified that the ER Ca2+-ATPase SERCA is a Seipin binding protein in both Drosophila and man. It was further shown that dSERCA is required cell autonomously for lipid storage in Drosophila fat cells, and Seipin affects SERCA calcium pump activity and regulates intracellular calcium homeostasis in Drosophila. Reducing ER-to-cytosol calcium release restores the lipid storage function of fat cells in dSeipin mutants. These findings may provide an effective therapy for BSCL2 (Bi, 2014).
SERCA pumps are critical for maintaining ER calcium homeostasis and the activity of SERCA is tightly regulated in different cell types. SERCA has been linked to lipodystrophy; the level of SERCA2 is reduced in cells lacking lipin-1 or its activator protein NEP1-R1. Several lines of evidence favor a specific connection between Seipin and SERCA. First, unlike in lipin-1 mutant cells, the protein level of dSERCA is unchanged in dSeipin mutants. Second, overexpression of dSERCA in wild-type does not cause an obvious fat storage phenotype; however, it partially, but significantly, rescues the fat storage defect in dSeipin mutants. In addition, on its own, IP3R RNAi causes a much stronger phenotype than RyR RNAi, but it cannot rescue the dSeipin mutant phenotype, while RyR RNAi rescues strongly. Third, endogenous hSeipin can be coprecipitated with hSERCA2. Purified hSeipin and hSERCA can interact, but a small deletion of hSeipin totally abolishes the hSeipin/hSERCA interaction without affecting the ER localization of hSeipin. Phospholamban (PLN) and sarcolipin (SLN), the best-studied regulators of SERCA in muscle, inhibit SERCA pumps by reducing their affinity for cytosolic calcium. Neither PLN nor SLN is conserved in fly, suggesting that other factors regulate SERCA activity. This study suggested that Seipin may act as a conserved positive regulator of SERCA in adipose tissue, although the mechanisms for modulation of SERCA activity by Seipin are still unknown (Bi, 2014).
Quantitative proteomic mass spectrometry analysis suggests that the lipodystrophy in dSeipin mutants may be due to impaired lipogenesis and elevated fatty acid β-oxidation. This is supported by a previous finding that elevated lipogenesis, induced by overexpressing SREBP, can rescue the fat storage defect of dSeipin mutants. How calcium homeostasis affects lipogenesis and fatty acid β-oxidation is largely unknown. It was reported that mitochondrial fatty acid oxidation can be activated by calcium in isolated rat liver mitochondria. Moreover, SREBP-2 was found to be activated by the SERCA inhibitor thapsigargin through depletion of Insig-1 in cultured CHO cells. This is in contrast to the current findings and cannot at present help to explain why dSERCA RNAi or dSeipin mutants have reduced lipogenesis in fat cells (Bi, 2014).
The ER is the major intracellular calcium storage site as well as being the organelle responsible for lipid biosynthesis and protein folding. The accumulation of misfolded or unfolded proteins results in ER stress. Similarly, loss of ER lumenal calcium triggers ER stress. Interestingly, ER stress leads to elevated lipid storage in yeast and hepatic cells, but the underlying mechanisms are not fully understood. Moreover, hepatic overexpression of SERCA in obese mice reduced chronic ER stress and improved hepatic steatosis. This study has shown that impaired SERCA activity leads to reduced fat storage in adipose tissue. These seemingly opposite effects of impaired ER calcium homeostasis on fat storage in hepatocytes and adipocytes may reflect the tissue-specific manner in which lipid metabolism is regulated. Similar tissue-specific effects on fat storage have also been observed when autophagy is impaired. Hepatocyte-specific knockout of ATG7 or ATG5 by RNAi results in elevated fat storage, while knockdown of ATG5 or ATG7 in the preadipocyte cell line 3T3-L1 leads to decreased TAG accumulation. In addition, mice with adipocyte-specific knockout of ATG7 are lean and have greatly reduced white adipocyte mass (Bi, 2014).
ER calcium homeostasis and ER stress have previously been linked to transcriptional regulation, chaperone activity, and protein folding. At present, it is unknown whether the effect of ER calcium homeostasis on fat storage, such as lipogenesis and fatty acid β-oxidation, is mediated at the gene transcriptional level, the protein activity level, or both. The possibility cannot be ruled out that the elevated cytosol calcium level, as well as the reduced ER calcium level, may contribute to the lipodystrophy. Indeed, it has been reported that in lymphocytes the activity of DGAT is inhibited by an increase in intracellular calcium induced by the ionophore A23187. In addition, previous work suggests that lipolysis is strongly activated in differentiating adipocytes in Seipin mutant mice. However, the increase in intracellular calcium has antilipolytic effects mainly by decreasing phosphorylation of hormone-sensitive lipase (HSL). Therefore, it remains to be determined whether altered intracellular calcium homeostasis in the fat cells of dSeipin mutants has an effect on lipolysis (Bi, 2014).
These findings may be important in man for the following reasons. First, both Seipin and SERCA are highly conserved from fly to human, and endogenous hSeipin and hSERCA2b physically interact. Second, the ER calcium homeostasis regulators STIM1 and SERCA have previously been reported to affect the early stages of adipocyte differentiation, which is consistent with the role of Seipin in adipocyte differentiation. Third, BSCL2 patients are at much higher risk of hypertrophic cardiomyopathy and mild mental retardation, which may also be caused by disruption of intracellular calcium signaling. The finding that modulating ER calcium homeostasis through RyR significantly rescues the dSeipin mutant phenotype suggests a potential therapeutic approach for treating BSCL2 disease and possibly other lipodystrophies (Bi, 2014).
Obesity is characterized by accumulation of excess body fat, while lipodystrophy is characterized by loss or absence of body fat. Despite their opposite phenotypes, these two conditions both cause ectopic lipid storage in non-adipose tissues, leading to lipotoxicity, which has health-threatening consequences. The exact mechanisms underlying ectopic lipid storage remain elusive. This study reports the analysis of a Drosophila model of the most severe form of human lipodystrophy, Berardinelli-Seip Congenital Lipodystrophy 2, which is caused by mutations in the BSCL2/Seipin gene. In addition to reduced lipid storage in the fat body, dSeipin mutant flies accumulate ectopic lipid droplets in the salivary gland, a non-adipose tissue. This phenotype was suppressed by expressing dSeipin specifically within the salivary gland. dSeipin mutants display synergistic genetic interactions with lipogenic genes in the formation of ectopic lipid droplets. These data suggest that dSeipin may participate in phosphatidic acid metabolism and subsequently down-regulate lipogenesis to prevent ectopic lipid droplet formation. In summary, this study has demonstrated a tissue-autonomous role of dSeipin in ectopic lipid storage in lipodystrophy (Tian, 2011).
Berardinelli-Seip congenital lipodystrophy 2 (BSCL2) is caused by loss-of-function mutations in SEIPIN, a protein implicated in both adipogenesis and lipid droplet expansion but whose molecular function remains obscure. This study identify physical and functional interactions between SEIPIN and microsomal isoforms of glycerol-3-phosphate acyltransferase (GPAT) in multiple organisms. Compared to controls, GPAT activity was elevated in SEIPIN-deficient cells and tissues and GPAT kinetic values were altered. Increased GPAT activity appears to underpin the block in adipogenesis and abnormal lipid droplet morphology associated with SEIPIN loss. Overexpression of Gpat3 blocked adipogenesis, and Gpat3 knockdown in SEIPIN-deficient preadipocytes partially restored differentiation. GPAT overexpression in yeast, preadipocytes, and fly salivary glands also formed supersized lipid droplets. Finally, pharmacological inhibition of GPAT in Seipin-/- mouse preadipocytes partially restored adipogenesis. These data identify SEIPIN as an evolutionarily conserved regulator of microsomal GPAT and suggest that GPAT inhibitors might be useful for the treatment of human BSCL2 patients (Pagac, 2016).
Congenital generalized lipodystrophy (CGL; also known as Berardinelli-Seip congenital lipodystrophy [BSCL]) is an autosomal recessive disorder characterized by a near total loss of adipose tissue, severe hypertriglyceridemia, insulin resistance, and fatty liver. To date, four genes have been linked to CGL/BSCL: 1-acylglycerol-3-phosphate-O-acyltransferase-2 (AGPAT2)/CGL1, SEIPIN/CGL2, CAVEOLIN-1/CGL3, and CAVIN-1/CGL4. The most severe form of human CGL/BSCL is caused by loss-of-function mutations in SEIPIN/BSCL2, which encodes an integral membrane protein of the endoplasmic reticulum (ER) with no recognizable functional domains. Seipin knockout (Bscl2-/-) mice have severe lipodystrophy and insulin resistance, demonstrating an essential role for Seipin in adipogenesis. SEIPIN and its non-mammalian orthologs also control the expansion of lipid droplets (LDs). The most prominent feature of Seipin-deficient cells is the formation of 'supersized' LDs. Thus, SEIPIN has a unique role in regulating both systemic (adipogenesis) and cellular (LD expansion) lipid storage (Pagac, 2016).
Recent studies implicate SEIPIN and its yeast ortholog, Fld1 (also known as Sei1), in regulating phospholipid metabolism such that the amount of phosphatidic acid (PA) is increased in SEIPIN-deficient cells and tissue. It was postulated that in preadipocytes, increased PA acts as a peroxisome proliferator-activated receptor γ (PPARγ) antagonist and thus blocks adipogenesis. In non-preadipocytes and yeast, PA promotes LD expansion, likely because it is fusogenic. However, exactly how the ER-localized SEIPIN might regulate the metabolism of phospholipids remains unknown (Pagac, 2016).
Through affinity isolation and tandem mass spectrometry analyses, this study identified proteins that specifically co-precipitate with Fld1-GFP in the yeast Saccharomyces cerevisiae. The most prominent of these proteins was Gat1, a glycerol-3-phosphate acyltransferase (GPAT). As the rate-limiting step in the synthesis of triacylglycerol and glycerophospholipids, GPAT catalyzes the esterification of glycerol-3-phosphate with a long-chain acyl-coenzyme A (acyl-CoA) to initiate the formation of PA. This study found that mammalian SEIPIN specifically interacted with the corresponding mammalian GPAT orthologs, GPAT3 and GPAT4. SEIPIN deficiency in yeast, mammalian cells, and mouse tissues resulted in increased GPAT activity and changes in GPAT kinetics. These data strongly suggest that SEIPIN is an evolutionarily conserved regulator of GPAT and that targeting GPAT may have therapeutic potential in treating BSCL2 (Pagac, 2016).
SEIPIN's role in mammalian lipid storage is unique, because it regulates both adipocyte differentiation and the expansion of cellular LDs. However, as an integral membrane protein of the ER without known functional domains, SEIPIN's mechanism of action has been difficult to understand. This study demonstrates that SEIPIN and its yeast ortholog, Fld1, can interact specifically with the ER-located GPAT isoforms and that this evolutionarily conserved interaction diminishes the specific activity of both yeast and mammalian GPAT isoforms and alters their substrate affinities. These data provide strong evidence that when SEIPIN is absent, enhanced microsomal GPAT activity results in defective adipogenesis and altered LD morphology (Pagac, 2016).
The acylation of glycerol-3-P, catalyzed by GPAT, is the initial and rate-limiting step in the synthesis of triacylglycerol and the glycerophospholipids. The mitochondrial isoform GPAT1 contributes between 20% and 50% of the total GPAT activity in liver but only ~10% of total GPAT activity in other tissue types. A second mitochondrial GPAT isoform, GPAT2, is expressed primarily in testes. The microsomal GPAT isoforms, GPAT3 and GPAT4, have an 80% amino acid identity and, like the two Gat isoforms in yeast, are integral membrane proteins of the ER but may move to the surface of LDs during LD expansion. Similarly, yeast Gat1 may be present on LDs during the stationary phase of yeast growth. Insulin increases the activities of mammalian GPAT3 and GPAT4 by phosphorylation at Ser and Thr residues, although the functional outcome of these modifications is unclear. Overall, the regulation of the ER GPATs remains largely unexplored, and no GPAT-interacting proteins have previously been identified (Pagac, 2016).
Through a non-biased screen in yeast, this study identified the yeast GPAT, Gat1, as a potential interacting partner of the yeast SEIPIN ortholog, Fld1. Multiple lines of evidence support a specific physical association between orthologs of SEIPIN and ER GPATs. Yeast and mammalian SEIPIN were co-immunoprecipitated with their respective yeast and mammalian GPAT orthologs, and both biotinylation and proximity ligation assays confirmed the close proximity of SEIPIN and GPAT3/4. Interestingly, compared to wild-type SEIPIN, the disease-causing missense mutant T78A had a weakened association with GPAT3/4. These data strongly support a specific physical association between SEIPIN and GPAT3/4. However, because both SEIPIN and the ER GPATs are integral membrane proteins that have resisted purification, it was not possible to determine their stoichiometry in vitro or whether their interaction is direct. Because SEIPIN forms oligomers, it is possible that a single GPAT3/4 molecule may interact with a SEIPIN oligomer. Moreover, whether SEIPIN missense mutants such as T78A cause lipodystrophies through reduced interaction with GPAT remains inconclusive, and future studies will focus on this aspect (Pagac, 2016).
The specific link between orthologs of SEIPIN and the ER GPAT isoforms was strongly and consistently supported by functional assays. SEIPIN deficiency is associated with two striking phenotypes: (1) a near complete block in adipogenesis and (2) the enlargement of LDs in yeast and in those mammalian cells and tissues in which SEIPIN is normally highly expressed, such as testes and mature adipocytes. SEIPIN's association with the ER GPAT isoforms can explain its roles in adipogenesis and LD expansion. These data support the hypothesis that when SEIPIN interacts with the ER GPATs, their enzymatic activity is reduced and the production of PA is diminished. Thus, the normal interaction of SEIPIN and the ER GPAT isoforms results in two major consequences: (1) because PA inhibits PPARγ, low GPAT activity during the first few hours of adipocyte differentiation permits PPARγ to be fully active, so that normal adipogenesis can proceed; and (2) in mature adipocytes, yeast, and other cells, SEIPIN-regulated ER GPAT activity controls the size of LDs, possibly by limiting the amount of fusogenic PA. Conversely, in the absence of SEIPIN, ER GPAT-specific activity is enhanced and PA production increases. The high PA concentration may inhibit PPARγ activity or other signaling pathways in preadipocytes, thereby blocking adipogenesis. In other cells, the absence of SEIPIN also results in high GPAT activity and increased local production of PA at the ER, resulting in aberrant LD budding and growth and the formation of supersized LDs. Several critical experiments support this interpretation, including the altered GPAT kinetics when SEIPIN is absent, the near-normal adipocyte differentiation that occurs when both SEIPIN and GPAT3 are absent, the formation of supersized LDs when the ER GPATs are overexpressed, and the reduction in LD size when SEIPIN and GPAT are co-expressed (Pagac, 2016).
SEIPIN has been reported to interact with two other glycerolipid synthetic enzymes, AGPAT2 and LIPIN1. However, there is a fundamental difference between those studies and ours. SEIPIN was proposed to facilitate AGPAT2 and LIPIN1 function, whereas the current data suggest that SEIPIN inhibits GPATs. Moreover, although the interaction of SEIPIN with AGPAT2 and LIPIN1 is relevant for the metabolism of PA, GPAT is recognized as the rate-limiting enzyme in the pathway. Importantly, the notion that SEIPIN anchors and facilitates LIPIN function overlooks existing physiological evidence. For example, depleting lipin-1 in mouse adipocytes reduces the size of LDs, whereas depleting Seipin in mouse adipocytes increases LD size. Furthermore, a co-immunoprecipitation study in yeast could not detect a SEIPIN-LIPIN1 interaction, and the yeast AGPAT2 homolog, Slc4, did not co-precipitate well with Fld1. Importantly, when overexpressed in the salivary gland of Drosophila under the same promoter, GPAT is the only enzyme that gave rise to supersized LDs (Pagac, 2016).
In Drosophila, SEIPIN has been reported to interact with sarco/endoplasmic reticulum calcium ATPase (SERCA), an ER-specific calcium pump (Bi, 2014). However, alterations in ER fatty acid and lipid composition are known to inhibit SERCA activity. Because the current results support a primary function of SEIPIN in ER phospholipid metabolism, the disrupted calcium homeostasis observed in SEIPIN-deficient cells may be secondary to an altered ER phospholipid composition. Recent data also suggest that SEIPIN is involved in the vectorial export of TAG from the ER or the stabilization of ER-LD contact sites. The current findings do not necessarily contradict with those observations; changes in PA concentration/localization of the ER may also impact the vectorial budding of droplets and/or the phospholipid composition of LDs (Pagac, 2016).
Although both GPAT3 and GPAT4 can interact with SEIPIN, they exhibited clear functional differences. Overexpressing either enzyme altered LD morphology at least in Huh7 cells, but only GPAT3 appeared to play a major role in adipogenesis. Surprisingly, although GPAT3 is highly upregulated in differentiating adipocytes, Gpat3-/- mice had relatively modest phenotypic alterations, and decreased weight gain was detected only when Gpat3-/- mice were fed a high-fat diet. GPAT4 is moderately upregulated during adipogenesis, but it is the major microsomal GPAT activity in brown adipose tissue, where its presence is required to limit the oxidation of exogenous fatty acids. Murine GPAT4 is known to localize to LDs; however, the current data suggest that both ER GPATs can localize to LDs, particularly GPAT3 (Pagac, 2016).
Taken as a whole, these data provide strong evidence that SEIPIN interacts with and regulates ER GPATs in mammalian cells, fly, and S. cerevisiae. This is the first example of an evolutionarily conserved, physiological regulator of the ER GPATs (Pagac, 2016).
Lipid droplets (LDs) are important cellular organelles that govern the storage and turnover of lipids. Little is known about how the size of LDs is controlled, although LDs of diverse sizes have been observed in different tissues and under different (patho)physiological conditions. Recent studies have indicated that the size of LDs may influence adipogenesis, the rate of lipolysis and the oxidation of fatty acids. A genome-wide screen has identified ten yeast mutants producing 'supersized' LDs that are up to 50 times the volume of those in wild-type cells. The mutated genes include: FLD1, which encodes a homologue of mammalian seipin; five genes (CDS1, INO2, INO4, CHO2, and OPI3) that are known to regulate phospholipid metabolism; two genes (CKB1 and CKB2) encoding subunits of the casein kinase 2; and two genes (MRPS35 and RTC2) of unknown function. Biochemical and genetic analyses reveal that a common feature of these mutants is an increase in the level of cellular phosphatidic acid (PA). Results from in vivo and in vitro analyses indicate that PA may facilitate the coalescence of contacting LDs, resulting in the formation of "supersized" LDs. In summary, these results provide important insights into how the size of LDs is determined and identify novel gene products that regulate phospholipid metabolism (Fei, 2011).
Search PubMed for articles about Drosophila Seipin
Bi, J., Wang, W., Liu, Z., Huang, X., Jiang, Q., Liu, G., Wang, Y. and Huang, X. (2014). Seipin promotes adipose tissue fat storage through the ER Ca(2)(+)-ATPase SERCA. Cell Metab 19(5): 861-871. PubMed ID: 24807223
Ding, L., Yang, X., Tian, H., Liang, J., Zhang, F., Wang, G., Wang, Y., Ding, M., Shui, G. and Huang, X. (2018). Seipin regulates lipid homeostasis by ensuring calcium-dependent mitochondrial metabolism. Embo J 37(17). PubMed ID: 30049710
Fei, W., Shui, G., Zhang, Y., Krahmer, N., Ferguson, C., Kapterian, T. S., Lin, R. C., Dawes, I. W., Brown, A. J., Li, P., Huang, X., Parton, R. G., Wenk, M. R., Walther, T. C. and Yang, H. (2011). A role for phosphatidic acid in the formation of "supersized" lipid droplets. PLoS Genet 7(7): e1002201. PubMed ID: 21829381
Pagac, M., Cooper, D. E., Qi, Y., Lukmantara, I. E., Mak, H. Y., Wu, Z., Tian, Y., Liu, Z., Lei, M., Du, X., Ferguson, C., Kotevski, D., Sadowski, P., Chen, W., Boroda, S., Harris, T. E., Liu, G., Parton, R. G., Huang, X., Coleman, R. A. and Yang, H. (2016). SEIPIN regulates lipid droplet expansion and adipocyte development by modulating the activity of glycerol-3-phosphate acyltransferase. Cell Rep 17(6): 1546-1559. PubMed ID: 27806294
Sui, X., Arlt, H., Brock, K. P., Lai, Z. W., DiMaio, F., Marks, D. S., Liao, M., Farese, R. V., Jr. and Walther, T. C. (2018). Cryo-electron microscopy structure of the lipid droplet-formation protein seipin. J Cell Biol 217(12): 4080-4091. PubMed ID: 30327422
Tian, Y., Bi, J., Shui, G., Liu, Z., Xiang, Y., Liu, Y., Wenk, M. R., Yang, H. and Huang, X. (2011). Tissue-autonomous function of Drosophila seipin in preventing ectopic lipid droplet formation. PLoS Genet 7(4): e1001364. PubMed ID: 21533227
Wang, H., Becuwe, M., Housden, B. E., Chitraju, C., Porras, A. J., Graham, M. M., Liu, X. N., Thiam, A. R., Savage, D. B., Agarwal, A. K., Garg, A., Olarte, M. J., Lin, Q., Frohlich, F., Hannibal-Bach, H. K., Upadhyayula, S., Perrimon, N., Kirchhausen, T., Ejsing, C. S., Walther, T. C. and Farese, R. V. (2016). Seipin is required for converting nascent to mature lipid droplets. Elife 5. PubMed ID: 27564575
Zhou, Y., Huang, S., Shen, H., Ma, M., Zhu, B. and Zhang, D. (2017). Detection of glutathione in oral squamous cell carcinoma cells With a fluorescent probe during the course of oxidative stress and apoptosis. J Oral Maxillofac Surg 75(1): 223 e221-223 e210. PubMed ID: 27637779
date revised: 22 February 2019
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