Identification and functional characterization of a lipid droplet protein CtLDP1 from an oleaginous yeast Candida tropicalis SY005

Proteins residing in lipid droplets (LDs) of organisms exhibit diverse physiological roles. Since the LD proteins of yeasts are largely unexplored, we have identified a putative LD protein gene, CtLDP1 in the oleaginous yeast Candida tropicalis SY005 and characterized its function. The increased lipid accumulation in SY005 could be correlated with enhanced (~2.67-fold) expression of the CtLDP1 after low-nitrogen stress. The N-terminal transmembrane domain similar to perilipin proteins and the amphipathic α-helices predicted in silico, presumably aid in targeting the CtLDP1 to LD membranes. Heterologous expression of CtLDP1-mCherry fusion in Saccharomyces cerevisiae revealed localization in LDs, yet the expression of CtLDP1 did not show significant effect on LD formation in transformed cells. Molecular docking showed favourable interactions of the protein with sterol class of molecules, but not with triacylglycerol (TAG); and this was further experimentally verified by co-localization of the mCherry-tagged protein in TAG-deficient (but steryl ester containing) LDs. While oleic acid supplementation caused coalescence of LDs into supersized ones (average diameter = 1.19 ± 0.12 μm; n = 160), this effect was suppressed due to CtLDP1 expression, and the cells mostly exhibited numerous smaller LDs (average diameter = 0.46 ± 0.05 μm; n = 160). Moreover, CtLDP1 expression in pet10Δ knockout strain of S. cerevisiae restored multiple LD formation, indicating functional complementation of the protein. Overall, this study documents functional characterization of an LD-stabilizing protein from an oleaginous strain of Candida genus for the first time, and provides insights on the characteristics of LD proteins in oleaginous yeasts for future metabolic engineering.     

Lipid droplets in skeletal muscle during grass snake (Natrix natrix L.) development

Lipid droplets (LDs) are common organelles observed in Eucaryota. They are multifunctional organelles (involved in lipid storage, metabolism, and trafficking) that originate from endoplasmic reticulum (ER). LDs consist of a neutral lipid core, made up of diacyl- and triacylglycerols (DAGs and TAGs) and cholesterol esters (CEs), surrounded by a phospholipid monolayer and proteins, which are necessary for their structure and dynamics.Here, we report the protein and lipid composition as well as characterization and dynamics of grass snake (Natrix natrix) skeletal muscle LDs at different developmental stages. In the present study, we used detailed morphometric, LC-MS, quantitative lipidomic analyses of LDs isolated from the skeletal muscles of the snake embryos, immunofluorescence, and TEM.Our study also provides a valuable insight concerning the LDs' multifunctionality and ability to interact with a variety of organelles. These LD features are reflected in their proteome composition, which contains scaffold proteins, metabolic enzymes signalling polypeptides, proteins necessary for the formation of docking sites, and many others. We also provide insights into the biogenesis and growth of muscle LDs goes beyond the conventional mechanism based on the synthesis and incorporation of TAGs and LD fusion. We assume that the formation and functioning of grass snake muscle LDs are based on additional mechanisms that have not yet been identified, which could be related to the unique features of reptiles that are manifested in the after-hatching period of life, such as a reptile-specific strategy for energy saving during hibernation.     

High confidence proteomic analysis of yeast LDs identifies additional droplet proteins and reveals connections to dolichol synthesis and sterol acetylation

Accurate protein inventories are essential for understanding an organelle’s functions. The lipid droplet (LD) is a ubiquitous intracellular organelle with major functions in lipid storage and metabolism. LDs differ from other organelles because they are bounded by a surface monolayer, presenting unique features for protein targeting to LDs. Many proteins of varied functions have been found in purified LD fractions by proteomics. While these studies have become increasingly sensitive, it is often unclear which of the identified proteins are specific to LDs. Here we used protein correlation profiling to identify 35 proteins that specifically enrich with LD fractions of Saccharomyces cerevisiae. Of these candidates, 30 fluorophore-tagged proteins localize to LDs by microscopy, including six proteins, several with human orthologs linked to diseases, which we newly identify as LD proteins (Cab5, Rer2, Say1, Tsc10, YKL047W, and YPR147C). Two of these proteins, Say1, a sterol deacetylase, and Rer2, a cis-isoprenyl transferase, are enzymes involved in sterol and polyprenol metabolism, respectively, and we show their activities are present in LD fractions. Our results provide a highly specific list of yeast LD proteins and reveal that the vast majority of these proteins are involved in lipid metabolism.     

Identification of the major functional proteins of prokaryotic lipid droplets

Storage of cellular triacylglycerols (TAGs) in lipid droplets (LDs) has been linked to the progression of many metabolic diseases in humans, and to the development of biofuels from plants and microorganisms. However, the biogenesis and dynamics of LDs are poorly understood. Compared with other organisms, bacteria seem to be a better model system for studying LD biology, because they are relatively simple and are highly efficient in converting biomass to TAG. We obtained highly purified LDs from Rhodococcus sp. RHA1, a bacterium that can produce TAG from many carbon sources, and then comprehensively characterized the LD proteome. Of the 228 LD-associated proteins identified, two major proteins, ro02104 and PspA, constituted about 15% of the total LD protein. The structure predicted for ro02104 resembles that of apolipoproteins, the structural proteins of plasma lipoproteins in mammals. Deletion of ro02104 resulted in the formation of supersized LDs, indicating that ro02104 plays a critical role in cellular LD dynamics. The putative α helix of the ro02104 LD-targeting domain (amino acids 83-146) is also similar to that of apolipoproteins. We report the identification of 228 proteins in the proteome of prokaryotic LDs, identify a putative structural protein of this organelle, and suggest that apolipoproteins may have an evolutionarily conserved role in the storage and trafficking of neutral lipids.     

Hydroxysteroid dehydrogenase family proteins on lipid droplets through bacteria, C. elegans, and mammals

Lipid droplets (LDs) are the main fat storing sites in almost all species from bacteria to humans. The perilipin family has been found as LD proteins in mammals, Drosophila, and a couple of slime molds, but no bacterial LD proteins containing sequence conservation were identified. In this study, we reported that the hydroxysteroid dehydrogenase (HSD) family was found on LDs across all organisms by LD proteomic analysis. Imaging experiments confirmed LD targeting of three representative HSD proteins including ro01416 in RHA1, DHS-3 in C. elegans, and 17β-HSD11 in human cells. In C. elegans, 17β-HSD11 family proteins (DHS-3, DHS-4 and DHS-19) were localized on LDs in distinct tissues. In intestinal cells of C. elegans, DHS-3 targeted to cytoplasmic LDs, while DHS-9 labeled nuclear LDs. Furthermore, the N-terminal hydrophobic domains of 17β-HSD11 family were necessary for their targeting to LDs. Last, 17β-HSD11 family proteins induced LD aggregation, and deletion of DHS-3 in C. elegans caused lipid decrease. Independent of their presumptive catalytic sites, 17β-HSD11 family proteins regulated LD dynamics and lipid metabolism through affecting the LD-associated ATGL, which was conserved between C. elegans and humans. Together, these findings for HSDs provide a new insight not only into the mechanistic studies of the dynamics and functions of LDs in multiple organisms, but also into understanding the evolutionary history of the organelle.     

FIT2 organizes lipid droplet biogenesis with ER tubule-forming proteins and septins

Lipid droplets (LDs) are critical for lipid storage and energy metabolism. LDs form in the endoplasmic reticulum (ER). However, the molecular basis for LD biogenesis remains elusive. Here, we show that fat storage–inducing transmembrane protein 2 (FIT2) interacts with ER tubule-forming proteins Rtn4 and REEP5. The association is mainly transmembrane domain based and stimulated by oleic acid. Depletion of ER tubule-forming proteins decreases the number and size of LDs in cells and Caenorhabditis elegans, mimicking loss of FIT2. Through cytosolic loops, FIT2 binds to cytoskeletal protein septin 7, an interaction that is also required for normal LD biogenesis. Depletion of ER tubule-forming proteins or septins delays nascent LD formation. In addition, FIT2-interacting proteins are up-regulated during adipocyte differentiation, and ER tubule-forming proteins, septin 7, and FIT2 are transiently enriched at LD formation sites. Thus, FIT2-mediated nascent LD biogenesis is facilitated by ER tubule-forming proteins and septins.     

Chlamydia trachomatis Infection Leads to Defined Alterations to the Lipid Droplet Proteome in Epithelial Cells

The obligate intracellular bacterium Chlamydia trachomatis is a major human pathogen and a main cause of genital and ocular diseases. During its intracellular cycle, C. trachomatis replicates inside a membrane-bound vacuole termed an “inclusion”. Acquisition of lipids (and other nutrients) from the host cell is a critical step in chlamydial replication. Lipid droplets (LD) are ubiquitous, ER-derived neutral lipid-rich storage organelles surrounded by a phospholipids monolayer and associated proteins. Previous studies have shown that LDs accumulate at the periphery of, and eventually translocate into, the chlamydial inclusion. These observations point out to Chlamydia-mediated manipulation of LDs in infected cells, which may impact the function and thereby the protein composition of these organelles. By means of a label-free quantitative mass spectrometry approach we found that the LD proteome is modified in the context of C. trachomatis infection. We determined that LDs isolated from C. trachomatis-infected cells were enriched in proteins related to lipid metabolism, biosynthesis and LD-specific functions. Interestingly, consistent with the observation that LDs intimately associate with the inclusion, a subset of inclusion membrane proteins co-purified with LD protein extracts. Finally, genetic ablation of LDs negatively affected generation of C. trachomatis infectious progeny, consistent with a role for LD biogenesis in optimal chlamydial growth.     

Proteome of skeletal muscle lipid droplet reveals association with mitochondria and apolipoprotein a-I

The lipid droplet (LD) is a universal organelle governing the storage and turnover of neutral lipids. Mounting evidence indicates that elevated intramuscular triglyceride (IMTG) in skeletal muscle LDs is closely associated with insulin resistance and Type 2 Diabetes Mellitus (T2DM). Therefore, the identification of the skeletal muscle LD proteome will provide some clues to dissect the mechanism connecting IMTG with T2DM. In the present work, we identified 324 LD-associated proteins in mouse skeletal muscle LDs through mass spectrometry analysis. Besides lipid metabolism and membrane traffic proteins, a remarkable number of mitochondrial proteins were observed in the skeletal muscle LD proteome. Furthermore, imaging by fluorescence microscopy and transmission electronic microscopy (TEM) directly demonstrated that mitochondria closely adhere to LDs in vivo. Moreover, our results revealed for the first time that apolipoprotein A-I (apo A-I), the principal apolipoprotein of high density lipoprotein (HDL) particles, was also localized on skeletal muscle LDs. Further studies verified that apo A-I was expressed endogenously by skeletal muscle cells. In conclusion, we report the protein composition and characterization of skeletal muscle LDs and describe a novel LD-associated protein, apo A-I.     

Characterization of a lipid droplet protein from Yarrowia lipolytica that is required for its oleaginous phenotype

Oleaginous microorganisms are characterized by their ability to store high amounts of triacylglycerol (TAG) in intracellular lipid droplets (LDs). In this work, we characterized a protein of the oleaginous yeast Yarrowia lipolytica that is associated with LD and plays a role in the regulation of TAG storage. This protein is required for the oleaginous phenotype of Y. lipolytica because deletion of the coding gene results in a strongly reduced TAG content of the mutant. Therefore, we named it Oleaginicity Inducing LD protein, Oil1. Furthermore, a mutant overexpressing OIL1 accumulates more TAG than the wild type and is delayed in TAG lipolysis when this process is stimulated. We found that Oil1p plays a role in protecting the TAG content of the LD from degradation through lipases under conditions where the cell aims at building up its TAG reserves. Heterologous expression studies showed that Oil1p rescued the phenotype of a Saccharomyces cerevisiae mutant deleted for the perilipin-like protein Pln1p and that its expression in COS-7 cells resulted in increased TAG accumulation, similar to the phenotype of a perilipin 1 expressing control strain. Despite this phenotypical parallels to mammalian perilipins, Oil1p is not a member of this protein family and its activity does not depend on phosphorylation. Rather, our results suggest that ubiquitination might contribute to the function of Oil1p in Y. lipolytica and that a different mechanism evolved in this species to regulate TAG homeostasis.     

Quantitative analysis of the murine lipid droplet-associated proteome during diet-induced hepatic steatosis

Hepatic steatosis is characterized by the accumulation of lipid droplets (LDs), which are composed of a neutral lipid core surrounded by a phospholipid monolayer embedded with many proteins. Although the LD-associated proteome has been investigated in multiple tissues and organisms, the dynamic changes in the murine LD-associated proteome in response to obesity and hepatic steatosis have not been studied. We characterized the hepatic LD-associated proteome of C57BL/6J male mouse livers following high-fat feeding using isobaric tagging for relative and absolute quantification. Of the 1,520 proteins identified with a 5% local false discovery rate, we report a total of 48 proteins that were increased and 52 proteins that were decreased on LDs in response to high-fat feeding. Most notably, ribosomal and endoplasmic reticulum proteins were increased and extracellular and cytosolic proteins were decreased in response to high-fat feeding. Additionally, many proteins involved in fatty acid catabolism or xenobiotic metabolism were enriched in the LD fraction following high-fat feeding. In contrast, proteins involved in glucose metabolism and liver X receptor or retinoid X receptor activation were decreased on LDs of high-fat-fed mice. This study provides insights into unique biological functions of hepatic LDs under normal and steatotic conditions.     

A lipid droplet protein of Nannochloropsis with functions partially analogous to plant oleosins

As our understanding of the dynamics of lipid droplets (LDs) in animal, plant, and fungal cells is rapidly evolving, still little is known about the formation and turnover of these organelles in microalgae. Yet with the growing importance of algal feedstock for the production of biofuels and high-value lipids, there is a need to understand the mechanisms of LD dynamics in microalgae. Thus, we investigated the proteins associated with LDs of the emerging heterokont model alga Nannochloropsis sp. and discovered an abundant hydrophobic lipid droplet surface protein (LDSP) with unique primary sequence but structural similarities to other LD proteins. LDSP abundance in Nannochloropsis cells closely tracked the amount of triacylglycerols during conditions of oil accumulation and degradation. Functional characterization of LDSP in an Arabidopsis (Arabidopsis thaliana) OLEOSIN1-deficient mutant allowed a separation of its physical and structural properties in its interaction with LDs from its physiological or biochemical activities. Although LDSP presence in Arabidopsis predictably affected LD size, it could not reverse the physiological impact of OLEOSIN deficiency on triacylglycerol hydrolysis during germination.     

Dynamics of protein and polar lipid recruitment during lipid droplet assembly in Chlamydomonas reinhardtii

In plants, neutral lipids are frequently synthesized and stored in seed tissues, where the assembly of lipid droplets (LDs) coincides with the accumulation of triacylglycerols (TAGs). In addition, photosynthetic, vegetative cells can form cytosolic LDs and much less information is known about the makeup and biogenesis of these LDs. Here we focus on Chlamydomonas reinhardtii as a reference model for LDs in a photosynthetic cell, because in this unicellular green alga LD dynamics can be readily manipulated by nitrogen availability. Nitrogen deprivation leads to cellular quiescence during which cell divisions cease and TAGs accumulate. The major lipid droplet protein (MLDP) forms a proteinaceous coat surrounding mature LDs. Reducing the amount of MLDP affects LD size and number, TAG breakdown and timely progression out of cellular quiescence following nitrogen resupply. Depending on nitrogen availability, MLDP recruits different proteins to LDs, tubulins in particular. Conversely, depolymerization of microtubules drastically alters the association of MLDP with LDs. LDs also contain select chloroplast envelope membrane proteins hinting at an origin of LDs, at least in part, from chloroplast membranes. Moreover, LD surface lipids are rich in de novo synthesized fatty acids, and are mainly composed of galactolipids which are typical components of chloroplast membranes. The composition of the LD membrane is altered in the absence of MLDP. Collectively, our results suggest a mechanism for LD formation in C. reinhardtii involving chloroplast envelope membranes by which specific proteins are recruited to LDs and a specialized polar lipid monolayer surrounding the LD is formed.     

Differential Roles of Cell Death-inducing DNA Fragmentation Factor-α-like Effector (CIDE) Proteins in Promoting Lipid Droplet Fusion and Growth in Subpopulations of Hepatocytes

Lipid droplets (LDs) are dynamic subcellular organelles whose growth is closely linked to obesity and hepatic steatosis. Cell death-inducing DNA fragmentation factor-α-like effector (CIDE) proteins, including Cidea, Cideb, and Cidec (also called Fsp27), play important roles in lipid metabolism. Cidea and Cidec are LD-associated proteins that promote atypical LD fusion in adipocytes. Here, we find that CIDE proteins are all localized to LD-LD contact sites (LDCSs) and promote lipid transfer, LD fusion, and growth in hepatocytes. We have identified two types of hepatocytes, one with small LDs (small LD-containing hepatocytes, SLHs) and one with large LDs (large LD-containing hepatocytes, LLHs) in the liver. Cideb is localized to LDCSs and promotes lipid exchange and LD fusion in both SLHs and LLHs, whereas Cidea and Cidec are specifically localized to the LDCSs and promote lipid exchange and LD fusion in LLHs. Cideb-deficient SLHs have reduced LD sizes and lower lipid exchange activities. Fasting dramatically induces the expression of Cidea/Cidec and increases the percentage of LLHs in the liver. The majority of the hepatocytes from the liver of obese mice are Cidea/Cidec-positive LLHs. Knocking down Cidea or Cidec significantly reduced lipid storage in the livers of obese animals. Our data reveal that CIDE proteins play differential roles in promoting LD fusion and lipid storage; Cideb promotes lipid storage under normal diet conditions, whereas Cidea and Cidec are responsible for liver steatosis under fasting and obese conditions.     

Whole-genome RNAi screen identifies methylation-related genes influencing lipid metabolism in Caenorhabditis elegans

Lipid droplets (LDs) are highly conserved multifunctional cellular organelles and aberrant lipid storage in LDs can lead to many metabolic diseases. However, the molecular mechanisms governing lipid dynamic changes remain elusive, and the high-throughput screen of genes influencing LD morphology was limited by lacking specific LD marker proteins in the powerful genetic tool Caenorhabditis elegans. In this study, we established a new method to conduct whole-genome RNAi screen using LD resident protein DHS-3 as a LD marker, and identified 78 genes involved in significant LD morphologic changes. Among them, mthf-1, as well as a series of methylation-related genes, was found dramatically influencing lipid metabolism. SREBP-1 and SCD1 homologs in C. elegans were involved in the lipid metabolic change of mthf-1(RNAi) worms, and the regulation of ATGL-1 also contributed to it by decreasing triacylglycerol (TAG) hydrolysis. Overall, this study not only identified important genes involved in LD dynamics, but also provided a new tool for LD study using C. elegans, with implications for the study of lipid metabolic diseases.     

The size and phospholipid composition of lipid droplets can influence their proteome

The proteomic makeup of lipid droplets (LDs) is believed to regulate the function of LDs, which are now recognized as important cellular organelles that are associated with many human metabolic disorders. However, factors that help determine LD proteome remain to be identified and characterized. Here we analyzed the phospholipid and protein composition of LDs isolated from wild type (WT) yeast cells, and also from fld1Δ, cds1, and ino2Δ mutant cells which produce ‘supersized’ LDs. LDs of fld1Δ and WT cells exhibited similar phospholipid profiles, whereas LDs of cds1 and ino2Δ strains had a higher (cds1) or lower (ino2Δ) percentage of phosphatidylcholine than those of WT, respectively. Unexpectedly, the presence of most known LD resident proteins was greatly reduced in the LD fraction isolated from cds1 and ino2Δ, including neutral lipid hydrolases. Consistent with this result, mobilization of neutral lipids was seriously impaired in these two strains. Contrary to the reduction of LD resident proteins, the Hsp90 family molecular chaperones, Hsc82 and Hsp82, were greatly increased in the LD fractions of cds1 and ino2Δ strains without changes at the level of expression. These data demonstrate the impact of LD phopholipids and size on the makeup of LD proteome.     

Abnormal accumulation of lipid droplets in neurons induces the conversion of alpha-Synuclein to proteolytic resistant forms in a Drosophila model of Parkinson’s disease

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by alpha-synuclein (αSyn) aggregation and associated with abnormalities in lipid metabolism. The accumulation of lipids in cytoplasmic organelles called lipid droplets (LDs) was observed in cellular models of PD. To investigate the pathophysiological consequences of interactions between αSyn and proteins that regulate the homeostasis of LDs, we used a transgenic Drosophila model of PD, in which human αSyn is specifically expressed in photoreceptor neurons. We first found that overexpression of the LD-coating proteins Perilipin 1 or 2 (dPlin1/2), which limit the access of lipases to LDs, markedly increased triacylglyclerol (TG) loaded LDs in neurons. However, dPlin-induced-LDs in neurons are independent of lipid anabolic (diacylglycerol acyltransferase 1/midway, fatty acid transport protein/dFatp) and catabolic (brummer TG lipase) enzymes, indicating that alternative mechanisms regulate neuronal LD homeostasis. Interestingly, the accumulation of LDs induced by various LD proteins (dPlin1, dPlin2, CG7900 or Klarsicht^LD-BD) was synergistically amplified by the co-expression of αSyn, which localized to LDs in both Drosophila photoreceptor neurons and in human neuroblastoma cells. Finally, the accumulation of LDs increased the resistance of αSyn to proteolytic digestion, a characteristic of αSyn aggregation in human neurons. We propose that αSyn cooperates with LD proteins to inhibit lipolysis and that binding of αSyn to LDs contributes to the pathogenic misfolding and aggregation of αSyn in neurons.     

Lipid droplets fuel SARS-CoV-2 replication and production of inflammatory mediators

Viruses are obligate intracellular parasites that make use of the host metabolic machineries to meet their biosynthetic needs. Thus, identifying the host pathways essential for the virus replication may lead to potential targets for therapeutic intervention. The mechanisms and pathways explored by SARS-CoV-2 to support its replication within host cells are not fully known. Lipid droplets (LD) are organelles with major functions in lipid metabolism, energy homeostasis and intracellular transport, and have multiple roles in infections and inflammation. Here we described that monocytes from COVID-19 patients have an increased LD accumulation compared to SARS-CoV-2 negative donors. In vitro, SARS-CoV-2 infection were seen to modulate pathways of lipid synthesis and uptake as monitored by testing for CD36, SREBP-1, PPARγ, and DGAT-1 expression in monocytes and triggered LD formation in different human cell lines. LDs were found in close apposition with SARS-CoV-2 proteins and double-stranded (ds)-RNA in infected Vero cells. Electron microscopy (EM) analysis of SARS-CoV-2 infected Vero cells show viral particles colocalizing with LDs, suggestive that LDs might serve as an assembly platform. Pharmacological modulation of LD formation by inhibition of DGAT-1 with A922500 significantly inhibited SARS-CoV-2 replication as well as reduced production of mediators pro-inflammatory response. Taken together, we demonstrate the essential role of lipid metabolic reprograming and LD formation in SARS-CoV-2 replication and pathogenesis, opening new opportunities for therapeutic strategies to COVID-19.     

LDIP cooperates with SEIPIN and LDAP to facilitate lipid droplet biogenesis in Arabidopsis

Cytoplasmic lipid droplets (LDs) are evolutionarily conserved organelles that store neutral lipids and play critical roles in plant growth, development, and stress responses. However, the molecular mechanisms underlying their biogenesis at the endoplasmic reticulum (ER) remain obscure. Here we show that a recently identified protein termed LD-associated protein [LDAP]-interacting protein (LDIP) works together with both endoplasmic reticulum-localized SEIPIN and the LD-coat protein LDAP to facilitate LD formation in Arabidopsis thaliana. Heterologous expression in insect cells demonstrated that LDAP is required for the targeting of LDIP to the LD surface, and both proteins are required for the production of normal numbers and sizes of LDs in plant cells. LDIP also interacts with SEIPIN via a conserved hydrophobic helix in SEIPIN and LDIP functions together with SEIPIN to modulate LD numbers and sizes in plants. Further, the co-expression of both proteins is required to restore normal LD production in SEIPIN-deficient yeast cells. These data, combined with the analogous function of LDIP to a mammalian protein called LD Assembly Factor 1, are discussed in the context of a new model for LD biogenesis in plant cells with evolutionary connections to LD biogenesis in other eukaryotes.

The lipid droplet (LD) proteins LDIP and LDAP cooperate with endoplasmic reticulum-localized SEIPIN to coordinate LD formation in plant cells.