Lipid Transport Activity Research Articles

Structure and function of Mycobacterium tuberculosis EfpA as a lipid transporter and its inhibition by BRD-8000.3

EfpA, the first major facilitator superfamily (MFS) protein identified in Mycobacterium tuberculosis (Mtb), is an essential efflux pump implicated in resistance to multiple drugs. EfpA-inhibitors have been developed to kill drug-tolerant Mtb. However, the biological function of EfpA has not yet been elucidated. Here, we present the cryo-EM structures of EfpA complexed with lipids or the inhibitor BRD-8000.3 at resolutions of 2.9 Å and 3.4 Å, respectively. Unexpectedly, EfpA forms an antiparallel dimer. Functional studies reveal that EfpA is a lipid transporter and BRD-8000.3 inhibits its lipid transport activity. Intriguingly, the mutation V319F, known to confer resistance to BRD-8000.3, alters the expression level and oligomeric state of EfpA. Based on our results and the observation of other antiparallel dimers in the MFS family, we propose an antiparallel-function model of EfpA. Collectively, our work provides structural and functional insights into EfpA's role in lipid transport and drug resistance, which would accelerate the development of antibiotics against this promising drug target.

Allosteric coupling of substrate binding and proton translocation in MmpL3 transporter from Mycobacterium tuberculosis.

Infections caused by Mycobacterium spp. are very challenging to treat, and multidrug-resistant strains rapidly spread in human populations. Major contributing factors include the unique physiological features of these bacteria, drug efflux, and the low permeability barrier of their outer membrane. Here, we focus on MmpL3 from Mycobacterium tuberculosis, an essential inner membrane transporter of the resistance-nodulation-division superfamily required for the translocation of mycolic acids in the form of trehalose monomycolates (TMM) from the cytoplasm or plasma membrane to the periplasm or outer membrane. The MmpL3-dependent transport of TMM is essential for the growth of M. tuberculosis in vitro, inside macrophages, and in M. tuberculosis-infected mice. MmpL3 is also a validated target for several recently identified anti-mycobacterial agents. In this study, we reconstituted the lipid transport activity of the purified MmpL3 using a two-lipid vesicle system and established the ability of MmpL3 to actively extract phospholipids from the outer leaflet of a lipid bilayer. In contrast, we found that MmpL3 lacks the ability to translocate the same phospholipid substrate across the plasma membrane indicating that it is not an energy-dependent flippase. The lipid extraction activity was modulated by substitutions in critical charged and polar residues of the periplasmic substrate-binding pocket of MmpL3, coupled to the proton transfer activity of MmpL3 and inhibited by a small molecule inhibitor SQ109. Based on the results, we propose a mechanism of allosteric coupling wherein substrate translocation by MmpL3 is coupled to the energy provided by the downhill transfer of protons. The reconstituted activities will facilitate understanding the mechanism of MmpL3-dependent transport of lipids and the discovery of new therapeutic options for Mycobacterium spp. infections.IMPORTANCEMmpL3 from Mycobacterium tuberculosis is an essential transporter involved in the assembly of the mycobacterial outer membrane. It is also an important target in undergoing efforts to discover new anti-tuberculosis drugs effective against multidrug-resistant strains spreading in human populations. The recent breakthrough structural studies uncovered features of MmpL3 that suggested a possible lipid transport mechanism. In this study, we reconstituted and characterized the lipid transport activity of MmpL3 and demonstrated that this activity is blocked by MmpL3 inhibitors and substrate mimics. We further uncovered the mechanism of how the binding of a substrate in the periplasmic domain is communicated to the transmembrane proton relay of MmpL3. The uncovered mechanism and the developed assays provide new opportunities for mechanistic analyses of MmpL3 function and its inhibition.

A comparison of the large-scale gene expression patterns in summer and fall migratory Pantala flavescens (Fabricius) in northern China.

Pantala flavescens (Fabricius) is the most well-known seasonal migratory insect. This research focused on the molecular response of P. flavescens migration in summer and fall. A total of 17,810 assembled unigenes were obtained and 624 differentially expressed genes (DEGs) were identified in summer migration compared to fall migration. A number of DEGs, including cpr49Ae, itm2b, chitinase, cpr11B, laccase2, nd5, vtg2 and so on, had previously been reported to be involved in cold- and high-temperature resistance. Functional enrichment analysis showed three pathways 'that antibacterial humoral response, response to bacterial, and lipid transporter activity' were significantly enriched in summer migration while that six pathways 'structural constituent of cuticle, chitin binding, mitochondrion, propanoate metabolism, citrate cycle, hypertrophic cardiomyopathy' were significantly enriched in fall migration. These results will provide a valuable baseline for further understanding of the molecular mechanisms of insect adaptation to different climate migrations.

Transcriptome analysis of East Asian common octopus, Octopus sinensis, paralarvae.

The genes involved in cephalopod development and their association with hatching and survival during early life stages have been extensively studied. However, few studies have investigated the paralarvae transcriptome of the East Asian common octopus (Octopus sinen sis). This study aimed to identify the genes related to embryonic development and hatching in O. sinensis using RNA sequencing (RNA-seq) and verify the genes most relevant to different embryonic stages. RNA samples from hatched and 25days post-hatching (dph) O. sinensis paralarvae were used to construct cDNA libraries. Clean reads from individual samples were aligned to the reference O. sinensis database to identify the differentially expressed genes (DEGs) between the 0- and 25-dph paralarvae libraries. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to supplement the RNA-seq data for embryogenic developmental stages. A total of 12,597 transcripts were annotated and 5,468 DEGs were identified between the 0- and 25-dph O. sinensis paralarvae, including 2,715 upregulated and 2,753 downregulated transcripts in the 25-dph paralarvae. Several key DEGs were related to transmembrane transport, lipid biosynthesis, monooxygenase activity, lipid transport, neuropeptide signaling, transcription regulation, and protein-cysteine S-palmitoyltransferase activity during the post-hatching development of O. sinensis paralarvae. RT-qPCR analysis further revealed that SLC5A3A, ABCC12, and NPC1 transcripts in 20 and/or 30days post-fertilization (dpf) embryos were significantly higher (p < 0.05) than those in 10-dpf embryos. Transcriptome profiles provide molecular targets to understand the embryonic development, hatching, and survival of O. sinensis paralarvae, and enhance octopus production.

The juvenile hormone analogue, pyriproxifen, alters protein and fat composition of Tenebrio molitor larvae

Abstract Maximising the yield of product from livestock is common practice in the agriculture industry and there is potential to extend this practice to the emerging insect industry, to produce high-quality, sustainable protein. Tenebrio molitor larvae, commonly called yellow mealworms, were fed for 28 days on wheat bran containing the juvenile hormone analogue, pyriproxifen at either 2 mg pyriproxifen/kg wheat bran (JH-PL) or 15 mg pyriproxifen/kg wheat bran (JH-PH). As expected, pupation was inhibited in both pyriproxifen treated groups and significant changes in nutritional composition were observed. Pyriproxifen treated mealworms had a higher protein content per 100 grams of dried material, while fat content was reduced 68% in JH-PH compared to control. These changes were associated with an increase in moisture content and reduction in energy content. The fatty acid profile of extracted fat also displayed significant alterations, with pyriproxifen treated mealworms showing an increase in proportions of saturated fatty acids, reduction in oleic acid but no effect on linoleic acid. The amino acid composition also exhibited a change in composition as a result of pyriproxifen treatment, including an increase in the essential amino acid, lysine, in JH-PH treated mealworms. This change in amino acid profile was associated with a change in the protein composition as observed on SDS-PAGE, with the appearance of a new band identified as the egg-yolk protein, vitellogenin, which has lipid-transporter activity. Hence, pyriproxifen treatment of mealworms has a repartitioning effect, resulting in an increase in the proportion of protein and a decrease in fat on a dry matter basis, demonstrating that mealworm nutrient composition can be manipulated to provide a higher value feed ingredient.

Ligand-dependent interactions between SR-B1 and S1PR1 in macrophages and atherosclerotic plaques

HDLs carry sphingosine-1-phosphate (S1P) and stimulate signaling pathways in different cells including macrophages and endothelial cells, involved in atherosclerotic plaque development. HDL signaling via S1P relies on the HDL receptor scavenger receptor class B, type I (SR-B1) and the sphingosine-1-phosphate receptor 1 (S1PR1), which interact when both are heterologously overexpressed in the HEK293 cell line. In this study, we set out to test if SR-B1 and S1PR1 interacted in primary murine macrophages in culture and atherosclerotic plaques. We used knock-in mice that endogenously expressed S1PR1 tagged with eGFP-(S1pr1eGFP/eGFP mice), combined with proximity ligation analysis to demonstrate that HDL stimulates the physical interaction between SR-B1 and S1PR1 in primary macrophages, that this is dependent on HDL-associated S1P and can be blocked by an inhibitor of SR-B1's lipid transfer activity or an antagonist of S1PR1. We also demonstrate that a synthetic S1PR1-selective agonist, SEW2871, stimulates the interaction between SR-B1 and S1PR1 and that this was also blocked by an inhibitor of SR-B1’s lipid transport activity. Furthermore, we detected abundant SR-B1/S1PR1 complexes in atherosclerotic plaques of S1pr1eGFP/eGFP mice that also lacked apolipoprotein E. Treatment of mice with the S1PR1 antagonist, Ex26, for 12 h disrupted the SR-B1-S1PR1 interaction in atherosclerotic plaques. These findings demonstrate that SR-B1 and S1PR1 form ligand-dependent complexes both in cultured primary macrophages and within atherosclerotic plaques in mice and provide mechanistic insight into how SR-B1 and S1PR1 participate in mediating HDL signaling to activate atheroprotective responses in macrophages.

RNA expression changes driven by altered epigenetics status related to NASH etiology

Non-alcoholic fatty liver disease (NAFLD) is a growing health problem due to the increased obesity rates, among other factors. In its more severe stage (NASH), inflammation, hepatocellular ballooning and fibrosis are present in the liver, which can further evolve to total liver dysfunction or even hepatocarcinoma. As a metabolic disease, is associated to environmental factors such as diet and lifestyle conditions, which in turn can influence the epigenetic landscape of the cells, affecting to the gene expression profile and chromatin organization. In this study we performed ATAC-sequencing and RNA-sequencing to interrogate the chromatin status of liver biopsies in subjects with and without NASH and its effects on RNA transcription and NASH etiology. NASH subjects showed transcriptional downregulation for lipid and glucose metabolic pathways (e.g., ABC transporters, AMPK, FoxO or insulin pathways). A total of 229 genes were differentially enriched (ATAC and mRNA) in NASH, which were mainly related to lipid transport activity, nuclear receptor-binding, dicarboxylic acid transporter, and PPARA lipid regulation. Interpolation of ATAC data with known liver enhancer regions showed differential openness at 8 enhancers, some linked to genes involved in lipid metabolism, (i.e., FASN) and glucose homeostasis (i.e., GCGR). In conclusion, the chromatin landscape is altered in NASH patients compared to patients without this liver condition. This alteration might cause mRNA changes explaining, at least partially, the etiology and pathophysiology of the disease.

Genomic and Proteomic Analyses of Extracellular Products Reveal Major Virulence Factors Likely Accounting for Differences in Pathogenicity to Bivalves between Vibrio mediterranei Strains.

Vibrio mediterranei, a bacterial pathogen of bivalves, has exhibited strain-dependent virulence. The mechanisms behind the variations in bivalve pathogenicity between V. mediterranei strains have remained unclear. However, a preliminary analysis of the extracellular product (ECP) proteomes has revealed differences in protein compositions between low- and high-virulence strains; in addition to 1265 shared proteins, 127 proteins have been identified to be specific to one low-virulence strain and 95 proteins to be specific to two high-virulence strains. We further studied the ECP proteins of the three V. mediterranei strains from functional perspectives using integrated genomics and proteomics approaches. The results showed that lipid metabolism, transporter activity and membrane transporter pathways were more enriched in the ECPs of the two high-virulence strains than in those of the low-virulence strain. Additionally, 73 of the 95 high-virulence strain-specific proteins were found to have coding genes in the genome but were not expressed in the low-virulence strain. Moreover, comparisons with known virulence factors in the Virulence Factor Database (VFDB) and the Pathogen-Host Interactions Database (PHI-base) allowed us to predict more than 10 virulence factors in the categories of antimicrobial activity/competitive advantage, the effector delivery system and immune modulation, and the high-virulence strain-specific ECP proteins consisted of a greater percentage of known virulence factors than the low-virulence strain. Particularly, two virulence factors, MtrC and KatG, were identified in the ECPs of the two high-virulence strains but not in those of the low-virulence strain. Most coding genes of the ECP proteins including known virulence factors were identified on chromosome 1 of V. mediterranei. Our findings indicate that variations in virulence factor composition in the bacterial ECPs may partially account for the differences in the bivalve pathogenicity between V. mediterranei strains.

A single-cell atlas of immune checkpoint molecules in atherosclerosis in ApoE-/- mice

Abstract Introduction Immune checkpoint (IC) targeting is an attractive way to combat atherosclerosis, since ICs regulate the immune response by activating or inhibiting inflammatory pathways. We have shown that ICs exert cell-divergent functions and that cell-specific IC-based treatments can limit immune-related side effects. To design tailored IC-based immunotherapies for atherosclerosis, a detailed single-cell atlas of IC expression during atherogenesis is required. Purpose To obtain cell-type- and atherosclerosis-stage-specific expression profiles of ICs and assess the transcriptome of IC+ cells during atherosclerosis. Methods ApoE-/- mice were fed normal chow diet (NCD) for 10, 20 or 30 weeks, or high-cholesterol diet (HCD) for 6, 10, 14 (study 1) or 20 weeks (study 2). Using mass-cytometry by time of flight (CyTOF), we assessed the cell-type-specific aortic expression of ICs during atheroprogression (study 1). Using cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq), we assessed the transcriptome of aortic IC+ and IC- cells (study 2). Results Using CyTOF, we identified 21 immune cell clusters, including T and B cells, Ly6C- and Ly6C+ monocytes and macrophage (mφ) subsets. On T cell subsets, IC expression remained relatively constant in early plaques independent of the type of diet, apart from an increase in CD40-CD40L and decrease in CD28 and PD-L1 in HCD-fed mice. In advanced plaques from NCD-fed mice, most T cell subsets showed increased PD-L1, GITRL and CD27, while in HCD-fed mice, CD40L was decreased and PD-L1, PD-L2, CD27, CD86-CD28 and GITR-GITRL were induced on these T cell subsets. On myeloid cells, IC expression changed during early atherogenesis. Specifically, in NCD-fed mice, CD11c+MHCII+ mφ upregulated CD40 and PD-L1 and downregulated CD27 and PD-L2, while dendritic cells increased GITR and decreased PD-L2. Additional ICs, such as GITRL, CD40L and CD86-CD28, were induced in advanced plaques from NCD-fed mice. In HCD-fed mice, numerous ICs, i.e. CD40-CD40L, GITR-GITRL, CD86-CD28 and PD-L2, were increased in myeloid subsets in both early and advanced plaques. Using CITE-Seq, we further studied ICs that were strongly regulated during atherogenesis, e.g. GITR. Gene Ontology enrichment analysis of GITR+ and GITR- cells within the myeloid and T cell subsets identified the regulation of various pathways: When compared to their GITR- counterparts, both CD25- and CD25+ CD4+GITR+ T cell subsets showed upregulated pathways of death receptor activity, while CD25+ cells also showed induction of pathways involved in transmembrane lipid transporter activity. In mφ, GITR+ mφ showed upregulated pathways of cell adhesion and cytokine activity compared to GITR- mφ. Conclusion We have identified the expression pattern of numerous ICs on aortic immune cell subsets at various atherosclerosis stages. These data will pave the way towards the development of selective IC-based therapies to combat atherosclerosis.

Molecular signature of the ontogenic development of the prawn Macrobrachium tenellum

The prawn Macrobrachium tenellum shows aquaculture potential due to its well-defined reproductive cycle linked to female nutritional requirements. Significant changes occur in egg composition during the 16 to 17-day embryo development. Understanding the ontogenic proteins is crucial for developmental insights and controlled reproduction. We employed free-label quantitative proteomics to analyze egg peptides at the initial and final stages of wild females. Using the emPAI protocol and Proteome Discoverer 2.0, we identified 89 differentially expressed proteins in M. tenellum eggs. Of these, 27 were exclusive to early-stage development and three to late-stage. Abundant proteins included Vitellogenin, glyceraldehyde-3-phosphate dehydrogenase, histone 4, beta-actin, and hemocyanin. Gene Ontology analysis revealed 518 terms across molecular functions, biological processes, and cellular components using the GoRetriever tool of AgBase and the CateGOrizer tool of the Animal Genome Research Program. Carbohydrate metabolism was significant in early-stage development, with glyceraldehyde-3-phosphate dehydrogenase being the second most abundant protein. Proteins involved in ATP synthesis and cytoplasmic proteins associated with catalytic and binding activities related to primary metabolism were also detected. Our study elucidates the role of Vitellogenin in lipid transport activity and its potential involvement in the juvenile hormone feedback pathway. This pathway includes farnesoic acid O-methyltransferase and juvenile hormone epoxide oxidase, regulating protein biosynthesis, molt cycles (including chitinase activity), and potentially influencing controlled reproduction. Our proteomic analysis provides insights into the molecular mechanisms driving Ontogenic development in Macrobrachium tenellum, with implications for controlled reproduction strategies and advancements in aquaculture practices.

SHMT as a Potential Therapeutic Target for Renal Cell Carcinoma.

Serine hydroxymethyltransferase (SHMT) is a serine-glycine-one-carbon metabolic enzyme in which SHMT1 and SHMT2 encode the cytoplasmic and mitochondrial isoenzymes, respectively. SHMT1 and SHMT2 are key players in cancer metabolic reprogramming, and thus are attractive targets for cancer therapy. However, the role of SHMT in patients with renal cell carcinoma (RCC) has not been fully elucidated. We aimed to systematically analyze the expression, gene regulatory network, prognostic value, and target prediction of SHMT1 and SHMT2 in patients with kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC), and kidney renal papillary cell carcinoma (KIRP); elucidate the association between SHMT expression and RCC; and identify potential new targets for clinical RCC treatment. Several online databases were used for the analysis, including cBioPortal, TRRUST, GeneMANIA, GEPIA, Metascape, UALCAN, LinkedOmics, and TIMER. SHMT1 and SHMT2 transcript levels were significantly down- and upregulated, respectively, in patients with KICH, KIRC, and KIRP, based on sample type, individual cancer stage, sex, and patient age. Compared to men, women with KIRC and KIRP showed significantly up- and downregulated SHMT1 transcript levels, respectively. However, SHMT2 transcript levels were significantly upregulated in the patients mentioned above. KIRC and KIRP patients with high SHMT1 expression had longer survival periods than those with low SHMT1 expression. In patients with KIRC, the findings were similar to those mentioned above. However, in KICH patients, the findings were the opposite regarding SHMT2 expression. SHMT1 versus SHMT2 were altered by 9% versus 3% (n = 66 KICH patients), 4% versus 4% (n = 446 KIRC patients), and 6% versus 7% (n = 280 KIRP patients). SHMT1 versus SHMT2 promoter methylation levels were significantly up- and downregulated in patients with KIRP versus KIRC and KIRP, respectively. SHMT1, SHMT2, and their neighboring genes (NG) formed a complex network of interactions. The molecular functions of SHMT1 and its NG in patients with KICH, KIRC, and KIRP, included clathrin adaptor, metalloendopeptidase, and GTPase regulator activities; lipid binding, active transmembrane transporter activity, and lipid transporter activity; and type I interferon receptor binding, integrin binding, and protein heterodimerization, respectively. Their respective Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were involved in lysosome activity, human immunodeficiency virus 1 infection, and endocytosis; coronavirus disease 2019 and neurodegeneration pathways (multiple diseases); and RIG-I-like receptor signaling pathway, cell cycle, and actin cytoskeleton regulation. The molecular functions of SHMT2 and its NG in patients with KICH, KIRC, and KIRP included cell adhesion molecule binding and phospholipid binding; protein domain-specific binding, enzyme inhibitor activity, and endopeptidase activity; and hormone activity, integrin binding, and protein kinase regulator activity, respectively. For patients with KIRC versus KIRP, the KEGG pathways were involved in cAMP and calcium signaling pathways versus microRNAs (MiRNAs) in cancer cells and neuroactive ligand-receptor interactions, respectively. We identified the key transcription factors of SHMT1 and its NG. SHMT1 and SHMT2 expression levels were different in patients with RCC. SHMT1 and SHMT2 may be potential therapeutic and prognostic biomarkers in these patients. Transcription factor (MYC, STAT1, PPARG, AR, SREBF2, and SP3) and miRNA (miR-17-5P, miR-422, miR-492, miR-137, miR-30A-3P, and miR-493) regulations may be important strategies for RCC treatment.

Proteomic Profiling of Thigh Meat at Different Ages of Chicken for Meat Quality and Development.

Chicken age contributes to the meat characteristics; however, knowledge regarding the pathways and proteins associated with meat quality and muscle development are still scarce, especially in chicken thigh meat. Hence, the objective of this study was to elucidate the intricate relationship between these traits by liquid chromatography mass spectrometry at three different ages. A total of 341 differential expressed proteins (DEPs) were screened out (fold change ≥ 1.50 or ≤0.67 and p < 0.05) among 45 thigh meat samples (15 samples per age) of Beijing-You chicken (BYC), collected at the age of 150, 300, or 450 days (D150, D300, and D450), respectively. Subsequently, based on the protein interaction network and Markov cluster algorithm (MCL) analyses, 91 DEPs were divided into 26 MCL clusters, which were associated with pathways of lipid transporter activity, nutrient reservoir activity, signaling pathways of PPAR and MAPK, focal adhesion, ECM-receptor interaction, the cell cycle, oocyte meiosis, ribosomes, taurine and hypotaurine metabolism, glutathione metabolism, muscle contraction, calcium signaling, nucleic acid binding, and spliceosomes. Overall, our data suggest that the thigh meat of BYC at D450 presents the most desirable nutritional value in the term of free amino acids (FAAs) and intramuscular fat (IMF), and a series of proteins and pathways associated with meat quality and development were identified. These findings also provide comprehensive insight regarding these traits across a wide age spectrum.

Integrated analysis of transcriptome and proteome changes related to the male-sterile mutant MS7-2 in wucai (Brassica Campestris L.)

Male sterility is a complex developmental process involving the differentiation of different tissues and the coordinated expression of genes. It is not only used to produce hybrids with effective utilization of heterosis but also an ideal tool to study plant development networks. Here, we carried out morphological and cytological observations on the flower buds of the previously studied male-sterile mutant MS7-2 and its wild-type fertile plant MF7-2 at different developmental stages and performed transcriptomic and proteomic analyses. Compared with the anthers of MF7-2, the anthers of MS7-2 were obviously atrophied, the anther surface was concave, the tapetum was abnormally expanded at the mononuclear microspore stage and normal pollen grains could not be produced. Proteomic analysis showed that the differentially expressed proteins (DEPs) were mainly involved in lipid transport, lipid binding and lipase activity and were mainly concentrated in carbohydrate metabolism, biosynthesis of other secondary metabolites and lipid metabolism. Transcriptomic results showed that the differentially expressed genes (DEGs) were mainly related to cell membrane components and were significantly enriched in carbohydrate metabolism, lipid metabolism, membrane transport and other pathways. In addition, transcription factors involved in anther and pollen development were also found to be significantly down-regulated in MS7-2, such as AMS, bHLH66, bHLH69 and WRKY 34. Weighted gene coexpression network analysis (WGCNA) screened two key modules of anther abortion in wucai. The DEGs in the two modules were mainly enriched in the “terpenoid backbone biosynthesis,” “pentose and glucuronate interconversions” and “cutin, suberine and wax biosynthesis” pathways, and three hub genes were screened as BraA02g028550.3C (LACS5), BraA01g016670.3C (VHA-G3) and BraA03g043400. 3C (AMS). Through transcriptomic and proteomic analyses, a regulatory network of MS7-2 male sterility of was constructed. It is speculated that the down-regulation of these genes and proteins in MS7-2 anthers inhibits the synthesis of some lipids and endogenous metabolites and accumulates a large amount of reactive oxygen species (ROS) in the tapetum, leading to the failure of normal PCD and the abortion of MS7-2. This study provides new insights into the molecular mechanism of male sterility in wucai.

Alpha lipoic acid treatment in late middle age improves cognitive function: Proteomic analysis of the protective mechanisms in the hippocampus

Alpha lipoic acid (ALA), a powerful antioxidant, has the potential to relieve age-related cognitive impairment and neurodegenerative disease. Clinical randomized controlled studies have demonstrated the cognitive improvement effects of lipoic acid in Alzheimer's disease (AD). In the present study, we examined the effects of ALA on cognitive function in ageing mice and its protective mechanisms. Eighteen-month-old male C57BL6/J mice received ALA or normal saline for 2 months. The Morris water maze test revealed improved cognitive function in animals that received ALA. Furthermore, tandem Mass Tags (TMT) based liquid chromotography with mass spectrometry/mass spectrometry (LC-MS/MS) was established to identify the target proteins. The results showed that 10 proteins were changed significantly. Gene Ontology (GO) analysis indicated that the upregulated proteins were enriched in terminal bouton, synaptic transmission and lipid transporter activity while the down-regulated proteins were involved in nuclear transcription factor-κB binding, apoptosis and mitogen-activated protein kinase binding. Based on the GO results, two upregulated proteins oxysterol-binding protein-related protein 10 (OSBPL10) and oligophrenin 1 (OPHN1), and one downregulated protein, CDK5 regulatory subunit-associated protein 3 (CDK5rap3), were validated through Western blotting. The results were consistent with the proteomic results. Modulation of synaptic transmission, lipid transporter activity and neuroinflammation appears to be the mechanisms of ALA in the aged brain.

ORP8 acts as a lipophagy receptor to mediate lipid droplet turnover.

Lipophagy, the selective engulfment of lipid droplets (LDs) by autophagosomes for lysosomal degradation, is critical to lipid and energy homeostasis. Here we show that the lipid transfer protein ORP8 is located on LDs and mediates the encapsulation of LDs by autophagosomal membranes. This function of ORP8 is independent of its lipid transporter activity and is achieved through direct interaction with phagophore-anchored LC3/GABARAPs. Upon lipophagy induction, ORP8 has increased localization on LDs and is phosphorylated by AMPK, thereby enhancing its affinity for LC3/GABARAPs. Deletion of ORP8 or interruption of ORP8-LC3/GABARAP interaction results in accumulation of LDs and increased intracellular triglyceride. Overexpression of ORP8 alleviates LD and triglyceride deposition in the liver of ob/ob mice, and Osbpl8-/- mice exhibit liver lipid clearance defects. Our results suggest that ORP8 is a lipophagy receptor that plays a key role in cellular lipid metabolism.

Proteomic analysis of zebrafish (Danio rerio) embryos exposed to benzyl benzoate

Benzyl benzoate (BB) is widely used in the food, cosmetics, agriculture, and pharmaceutical industries and is discharged into the aquatic environment via various water sources, including wastewater. Research on the bioaccumulation and possible toxicity of BB has been conducted, but the biochemical responses to BB toxicity are not fully understood, and the specific molecular pathways by which BB causes toxicity remain unknown. In this study, label-free quantitative proteomics based on mass spectrometry was applied to investigate protein profiles in zebrafish (Danio rerio) embryos exposed to BB (1 µg/mL) for 7 days. A total of 83 differentially expressed proteins (DEPs) were identified, including 49 up-regulated and 34 down-regulated proteins. The biological functions of proteins regulated by BB were grouped into functional categories and subcategories, including the biosynthesis of organonitrogen compound biosynthetic process, translation, amide biosynthetic process, lipid transport, stress response, and cytoskeletal activity. The results provide novel insight into the molecular basis of the ecotoxicity of BB in aquatic ecosystems.

Lipid Transport from Endoplasmic Reticulum to Autophagic Membranes.

Autophagy is an intracellular degradation system involving de novo generation of autophagosomes, which function as a transporting vesicle of cytoplasmic components to lysosomes for degradation. Isolation membranes (IMs) or phagophores, the precursor membranes of autophagosomes, require millions of phospholipids to expand and transform into autophagosomes, with the endoplasmic reticulum (ER) being the primary lipid source. Recent advances in structural and biochemical studies of autophagy-related proteins have revealed their lipid transport activities: Atg2 at the interface between IM and ER possesses intermembrane lipid transfer activities, while Atg9 at IM and VMP1 and TMEM41B at ER possess lipid scrambling activities. In this review, we summarize recent advances in the establishment of the lipid transport activities of these proteins and their collaboration mechanisms for lipid transport between the ER and IM, and further discuss how unidirectional lipid transport from the ER to IM occurs during autophagosome formation.

Association between greenness and dyslipidemia in patients with coronary heart disease: A proteomic approach

BackgroundResidential surrounding greenness may be protective of dyslipidemia are often theorized but remain poorly quantified. In particular, the underlying biological mechanisms of blood lipid changes with green spaces remain unclear. MethodsOur observational epidemiology study included a residentially stable sample of 1035 coronary heart disease patients, and proteomics study included 16 participants. Normalized Difference Vegetation Index (NDVI) was used to evaluate residential greenness exposures. Proteomics technology was used to identify plasma greenness-related proteome disturbance, and the pathway analysis was employed to evaluate the potential biological mechanisms of greenness decreasing dyslipidemia risk. ResultHigher residential surrounding greenness in the 500-m area was associated with lower risks of dyslipidemia (odds ratio (OR) = 0.871, 95% confidence interval (CI): 0.763, 0.994 for per one-quartile NDVI increase). Lymphocytes mediated 18.7% of the association between greenness and dyslipidemia. Greenness related proteins (including PLXDC1, IGFBP2 and LY6D) may regulate the biological functions of lipid metabolism and transport-related proteins (including ADIPOQ and CES1) through a series of biological processes. ConclusionPeople in greener surroundings have a lower risk of dyslipidemia, which may be due to their lower inflammation, stronger lipid transporter activity, and normal cholesterol metabolism.

Interleukin 20 receptor A expression in colorectal cancer and its clinical significance.

BackgroundInterleukin 20 receptor A (IL20RA) has been shown to play a role in the establishment and progression of multiple tumors. However, the expression of this protein in colorectal cancer (CRC) and its correlation with the clinicopathological parameters of CRC have remained unclear.MethodsA total of 323 paraffin sections including CRC tissues and adjacent normal tissues after surgery were collected. IL20RA protein expression was detected by immunohistochemical staining. The difference expression of IL20RA mRNA between CRC and normal tissues was also explored in the Oncomine and GEO databases. In addition, the IL20RA-related differentially expressed genes were analyzed in TCGA database and enrichment analysis was conducted to explore the cell functions and pathways related to IL20RA expression.ResultsThere was increased IL20RA expression in CRC compared with that in normal tissues. High IL20RA expression was associated with greater tumor diameter, lymph node metastasis, and poor TNM stage in CRC, while also being suggestive of poor prognosis. The main pathways of IL20RA-related differentially expressed genes in TCGA were protein heterodimerization activity, oxygen binding, oxygen transporter activity, hormone activity, and lipid transporter activity. Meanwhile, IL20RA-related differentially expressed genes were mainly enriched in peroxidase, nucleotide stimulant repair, fatty acid metabolism, basal transcription factor, and RNA degradation.ConclusionsIL20RA might have a role as a biomarker for CRC. Its upregulation might contribute to an aggressive phenotype in CRC. IL20RA’s involvement in the development and progression of CRC might occur through it affecting fatty acid metabolism, oxygen binding, oxygen transport, and hormone activity.

Abstract PO-025: Investigating lipid homeostasis in pancreatic ductal adenocarcinoma under tumor-like stress

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with a 5-year survival rate of only 10%. Nearly 95% of PDAC harbor oncogenic KRAS, which has been largely “undruggable” in the past. One hallmark of PDAC tumor microenvironment is dense desmoplasia, as a result of abnormal accumulation of extracellular matrix and proliferative fibroblasts. During tumorigenesis, pancreatic stellate cells (PSCs) become activated to a myofibroblast-like phenotype and contribute to the fibrotic environment of PDAC. Desmoplasia-induced hypovascularity severely limits the delivery of oxygen and nutrients. Our laboratory revealed that hypoxia is prominent even at the pancreatic intraepithelial neoplasia (PanIN) stage, which is the dominant precursor lesion of PDAC. Hypoxia inhibits oxygen-dependent processes in cancer metabolism, such as unsaturated lipid biosynthesis which requires oxygen as the terminal electron acceptor. Hypoxia also stimulates exogenous lipid uptake. Previous publications demonstrated that RAS-transformed cells preferentially take up unsaturated lysophosphatidylcholines (LPCs) from culture media. However, under limited nutrient conditions, in the desmoplastic environment, the potential lipid source is not well understood. Interestingly, a lipidomic study of a PSC secretome displayed a significant elevation of LPCs, suggesting that PSCs are the potential lipid source in the PDAC microenvironment. Our data indicate that exogenous unsaturated fatty acids sustain PDAC cell viability under hypoxia and nutrient deficiency. We also find PDAC cell survival under tumor-like stress is enhanced in PSC conditioned medium, but not in delipidated conditioned medium. Strikingly, we show that cancer-associated fibroblasts are not experiencing hypoxic stress as much as malignant epithelium cells in vivo by analyzing single-cell RNA-seq data and IHC, which suggests the capability of cancer-associated fibroblasts in providing unsaturated lipids to PDAC cells. Based on our lipidomic mass spectra of PSC conditioned medium, we identify that LPCs are actively imported by PDAC cells and prove that unsaturated LPCs maintain PDAC cell survival under nutritional stress. Our overall hypothesis is that activated PSCs support PDAC cell survival by providing unsaturated lipids, particularly LPCs, and inhibition of unsaturated lipid uptake through impeding lipid transporter activity or LPC synthesis by PSCs could potentially induce PDAC cell death under metabolically challenging conditions. We will hinder possible pathways responsible for LPC uptake in PDAC cells and generate in vivo models to achieve therapeutic benefits of PDAC. In summary, our research aims to demonstrate the metabolic crosstalk between PSCs and PDAC cells under stress conditions. The key advance of this study is that activated PSCs suppress PDAC cell death by supplying tumor cells with unsaturated LPCs for maintaining lipid homeostasis, in a hypoxic and nutrient-poor environment. Our findings will reveal a novel metabolic target for developing combinatorial therapy of PDAC. Citation Format: Xu Han, Michelle Burrows, Celeste Simon, Yanqing Jiang, Brian Keith. Investigating lipid homeostasis in pancreatic ductal adenocarcinoma under tumor-like stress [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-025.