Lipid-binding Protein Research Articles

Annexin A6 Polymorphism Is Associated with Pro-atherogenic Lipid Profiles and with the Downregulation of Methotrexate on Anti-Atherogenic Lipid Profiles in Psoriasis.

Annexin A6 (AnxA6) is a lipid-binding protein that regulates cholesterol homeostasis and secretory pathways. However, the correlation of AnxA6 polymorphism with lipometabolism has never been studied in psoriasis. To investigate the impact of AnxA6 polymorphism on lipid profiles and the expression of AnxA6 protein in both peripheral blood mononuclear cells (PBMCs) and lipometabolism in psoriasis. A total of 265 psoriatic patients received methotrexate (MTX) treatment for 12 weeks, after which their lipid profiles were determined by measuring total cholesterol (TC), triglycerides (TGs), lipoprotein (a) [LP(a)], high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein (LDL), apolipoprotein (a)1 (ApoA1), and apolipoprotein B (ApoB). In addition, AnxA6 (rs11960458) was genotyped in 262 patients and the expression of AnxA6 in PBMCs was measured by Western blotting at baseline and week 8 post-MTX treatment. The CC genotype carriers of rs11960458 had a lower expression of AnxA6 and lower levels of the pro-atherogenic lipids TC, LDL, and ApoB compared to TC genotype carriers. MTX significantly downregulated the levels of the anti-atherogenic lipids HDL-C and ApoA1 and the level of AnxA6 in TC genotype carriers, as well as the level of TGs in CC genotype carriers. The polymorphism of AnxA6, rs11960458, was statistically associated with the levels of pro-atherogenic lipids and with the downregulation of MTX on the levels of anti-atherogenic lipids and TGs in psoriasis.

Crystal structure of the OrfX1-OrfX3 complex from the PMP1 neurotoxin gene cluster.

Paraclostridial mosquitocidal protein 1 (PMP1) is a member of the clostridial neurotoxin (CNT) family, which includes botulinum and tetanus neurotoxins. PMP1 has unique selectivity for anopheline mosquitos and is the only known member of the family that targets insects. PMP1 is encoded in an orfX gene cluster, which in addition to the toxin, consists of OrfX1, OrfX2, OrfX3, P47 and NTNH, which have been shown to aid in PMP1 toxicity. We here show that OrfX1 and OrfX3 form a complex and present its structure at 2.7 Å. The OrfX1-OrfX3 complex mimics the structure of full-length OrfX2 and belongs to the lipid-binding TULIP protein superfamily. With this report, the structures of all proteins encoded in the orfX gene cluster of CNTs are now determined.

Exploring Membrane Binding Targets of Disordered Human Tau Aggregates on Lipid Rafts Using Multiscale Molecular Dynamics Simulations.

The self-aggregation of tau, a microtubule-binding protein, has been linked to the onset of Alzheimer's Disease. Recent studies indicate that the disordered tau aggregates, or oligomers, are more toxic than the ordered fibrils found in the intracellular neurofibrillary tangles of tau. At present, details of tau oligomer interactions with lipid rafts, a model of neuronal membranes, are not known. Using molecular dynamics simulations, the lipid-binding events, membrane-damage, and protein folding of tau oligomers on various lipid raft surfaces were investigated. Tau oligomers preferred to bind to the boundary domains (Lod) created by the coexisting liquid-ordered (Lo) and liquid-disordered (Ld) domains in the lipid rafts. Additionally, stronger binding of tau oligomers to the ganglioside (GM1) and phosphatidylserine (PS) domains, and subsequent protein-induced lipid chain order disruption and beta-sheet formation were detected. Our results suggest that GM1 and PS domains, located exclusively in the outer and inner leaflets, respectively, of the neuronal membranes, are specific membrane domain targets, whereas the Lod domains are non-specific targets, of tau oligomers binding to neurons. The molecular details of these specific and non-specific tau bindings to lipid rafts may provide new insights into understanding membrane-associated tauopathies leading to Alzheimer's Disease.

PMON184 Ligand-dependent interaction between phosphatidylcholine transfer protein and PPARδ: therapeutic implications for metabolic syndrome

Abstract Phosphatidylcholine transfer protein (PC-TP; synonym StarD2) is a soluble lipid binding protein that transports phosphatidylcholine (PC) between cellular membranes. PC-TP knockout mice are more sensitive to glucose and insulin, have increased beta oxidation and improved liver health compared to wild-type mice. To better understand the protective metabolic effects associated with PC-TP deletion, we investigated how hepatic PC-TP affects lipid metabolism, sensitivity to glucose and insulin, and energy homeostasis in a mouse model of diet-induced obesity. We generated a liver-specific PC-TP knockout mouse (L-Pctp-/-), which gains less weight and accumulates less liver fat compared to wild-type mice when challenged with a high fat diet. Hepatic deletion of PC-TP also reduces levels of triglycerides and phospholipids in skeletal muscle, suggesting that PC-TP in the liver influences lipid metabolism in the muscle. Gene expression analyses suggest that the observed metabolic changes are related to transcriptional activity of peroxisome proliferative activating receptor (PPAR) family members. An in-cell protein complementation screen between lipid transfer proteins and PPARs uncovered a novel, direct interaction between PC-TP and PPARδ that was not observed for other PPAR family members. We confirmed the PC-TP-PPARδ interaction in Huh7 hepatocytes, where it was found to repress PPARδ-mediated transactivation. Mutations of PC-TP residues implicated in PC binding and transfer combined with a reduction of exogenously supplied lipids through serum starvation relieves PC-TP mediated PPAR repression. Together our data points to a ligand sensitive PC-TP– PPARδ interaction that suppresses PPAR activity. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Exploring the Role of Lipid-Binding Proteins and Oxidative Stress in Neurodegenerative Disorders: A Focus on the Neuroprotective Effects of Nutraceutical Supplementation and Physical Exercise.

The human brain is primarily composed of lipids, and their homeostasis is crucial to carry on normal neuronal functions. In order to provide an adequate amount of lipid transport in and out of the central nervous system, organisms need a set of proteins able to bind them. Therefore, alterations in the structure or function of lipid-binding proteins negatively affect brain homeostasis, as well as increase inflammation and oxidative stress with the consequent risk of neurodegeneration. In this regard, lifestyle changes seem to be protective against neurodegenerative processes. Nutraceutical supplementation with antioxidant molecules has proven to be useful in proving cognitive functions. Additionally, regular physical activity seems to protect neuronal vitality and increases antioxidant defenses. The aim of the present review was to investigate mechanisms that link lipid-binding protein dysfunction and oxidative stress to cognitive decline, also underlining the neuroprotective effects of diet and exercise.

Structure and functionality of a multimeric human COQ7:COQ9 complex

Coenzyme Q (CoQ) is a redox-active lipid essential for core metabolic pathways and antioxidant defense. CoQ is synthesized upon the mitochondrial inner membrane by an ill-defined "complex Q" metabolon. Here, we present structure-function analyses of a lipid-, substrate-, and NADH-bound complex comprising two complex Q subunits: the hydroxylase COQ7 and the lipid-binding protein COQ9. We reveal that COQ7 adopts a ferritin-like fold with a hydrophobic channel whose substrate-binding capacity is enhanced by COQ9. Using molecular dynamics, we further show that two COQ7:COQ9 heterodimers form a curved tetramer that deforms the membrane, potentially opening a pathway for the CoQ intermediates to translocate from the bilayer to the proteins' lipid-binding sites. Two such tetramers assemble into a soluble octamer with a pseudo-bilayer of lipids captured within. Together, these observations indicate that COQ7 and COQ9 cooperate to access hydrophobic precursors within the membrane and coordinate subsequent synthesis steps toward producing CoQ.

Neuronal Ganglioside and Glycosphingolipid (GSL) Metabolism and Disease : Cascades of Secondary Metabolic Errors Can Generate Complex Pathologies (in LSDs).

Glycosphingolipids (GSLs) are a diverse group of membrane components occurring mainly on the surfaces of mammalian cells. They and their metabolites have a role in intercellular communication, serving as versatile biochemical signals (Kaltner et al, Biochem J 476(18):2623-2655, 2019) and in many cellular pathways. Anionic GSLs, the sialic acid containing gangliosides (GGs), are essential constituents of neuronal cell surfaces, whereas anionic sulfatides are key components of myelin and myelin forming oligodendrocytes. The stepwise biosynthetic pathways of GSLs occur at and lead along the membranes of organellar surfaces of the secretory pathway. After formation of the hydrophobic ceramide membrane anchor of GSLs at the ER, membrane-spanning glycosyltransferases (GTs) of the Golgi and Trans-Golgi network generate cell type-specific GSL patterns for cellular surfaces. GSLs of the cellular plasma membrane can reach intra-lysosomal, i.e. luminal, vesicles (ILVs) by endocytic pathways for degradation. Soluble glycoproteins, the glycosidases, lipid binding and transfer proteins and acid ceramidase are needed for the lysosomal catabolism of GSLs at ILV-membrane surfaces. Inherited mutations triggering a functional loss of glycosylated lysosomal hydrolases and lipid binding proteins involved in GSL degradation cause a primary lysosomal accumulation of their non-degradable GSL substrates in lysosomal storage diseases (LSDs). Lipid binding proteins, the SAPs, and the various lipids of the ILV-membranes regulate GSL catabolism, but also primary storage compounds such as sphingomyelin (SM), cholesterol (Chol.), or chondroitin sulfate can effectively inhibit catabolic lysosomal pathways of GSLs. This causes cascades of metabolic errors, accumulating secondary lysosomal GSL- and GG- storage that can trigger a complex pathology (Breiden and Sandhoff, Int J Mol Sci 21(7):2566, 2020).

Inhibition of FABP4 attenuates cardiac fibrosis through inhibition of NLRP3 inflammasome activation.

Objective(s):Cardiac fibrosis is a key biological process of cardiac remodeling and heart failure. Fatty acid-binding protein 4 (FABP4) is a lipid-binding protein that can regulate glucose and lipid homeostasis, and its expression was elevated in heart failure. However, whether FABP4 is involved in cardiac fibrosis remains unknown. Materials and Methods:The cardiac fibrosis model was established in male C57BL/6 mice with subcutaneously infused angiotensin II (Ang-II) (2.8 mg/kg/day) for 4 weeks. DMSO or FABP4 inhibitor BMS309403 (50 mg/kg/day) was intraperitoneally injected for 4 weeks. Ang II-infused mice, FABP4 inhibitor (BMS309403) injected mice, and ventricular tissue were used for morphological studies, and histological and biochemical analyses (FABP4 protein composition and expression).Results:Ang II infusion increased FABP4 mRNA and protein expression in the mouse ventricular tissue. After treatment with FABP4 inhibitor BMS309403 for 4 weeks, mice showed improved cardiac structure and function as detected by echocardiography. BMS309403 suppressed cardiac and systemic inflammatory response, reduced collagen deposition, and mRNA expression of collagen type I (COL1A1) and collagen type III (COL3A1) in Ang II-infused mice. BMS309403 also reduced the number of α-smooth muscle actin (α-SMA)+cells and decreased the mRNA expression of α-SMA, matrix metalloproteinases-2 (MMP-2), MMP-9, and transforming growth factor-β (TGFβ) in ventricular tissue.Conclusion:The inhibitory effect of BMS309403 on cardiac fibrosis might be associated with inhibition of NLRP3 inflammasome activation, which Ang II activated. Thus, our data speculated that inhibition of FABP4 could significantly induce cardiac fibrosis.

Lipid-related FABP5 activation of tumor-associated monocytes fosters immune privilege via PD-L1 expression on Treg cells in hepatocellular carcinoma.

Monocytes/macrophages, a plastic and heterogeneous cell population of the tumor microenvironment (TME), can constitute a major component of most solid tumors. Under the pressure of rapid proliferation of the tumor, monocytes/macrophages can be educated and foster immune tolerance via metabolic reprogramming. Our studies have shown that the activation of FABP5, a lipid-binding protein, decreases the rate of β-oxidation causing the accumulation of lipid droplets in monocytes. We found that hepatocellular carcinoma cells (HCC) increased IL-10 secretion by monocytes, which depended on the expression of FABP5 and suppressing of the PPARα pathway. Moreover, the elevated level of IL-10 promotes PD-L1 expression on Treg cells via the JNK-STAT3 pathway activation. We also observed that elevation of FABP5 in monocytes was negatively related to HCC patients' overall survival time. Thus, FABP5 promotes monocyte/macrophage lipid accumulation, fosters immune tolerance formation, and might represent itself as a therapeutic target in both tumor-associated monocytes (TAMs) and cancer cells.

Therapeutic Implications of FABP4 in Cancer: An Emerging Target to Tackle Cancer.

Metabolic reprogramming is an emerging hallmark of tumor cells. In order to survive in nutrient-deprived environment, tumor cells rewire their metabolic phenotype to provide sufficient energy and build biomass to sustain their transformed state and promote malignant behaviors. Fatty acid uptake and trafficking is an essential part of lipid metabolism within tumor cells. Fatty acid-binding proteins (FABPs), which belongs to a family of intracellular lipid-binding protein, can bind hydrophobic ligands to regulate lipid trafficking and metabolism. In particular, adipocyte fatty acid binding protein (FABP4), one of the most abundant members, has been found to be upregulated in many malignant solid tumors, and correlated with poor prognosis. In multiple tumor types, FABP4 is critical for tumor proliferation, metastasis and drug resistance. More importantly, FABP4 is a crucial driver of malignancy not only by activating the oncogenic signaling pathways, but also rewiring the metabolic phenotypes of tumor cells to satisfy their enhanced energy demand for tumor development. Thus, FABP4 serves as a tumor-promoting molecule in most cancer types, and may be a promising therapeutic target for cancer treatment.

Identification of SEC14 like lipid binding 2(SEC14L2) sequence and expression profiles in the Chinese tree shrew (Tupaia belangeri chinensis).

The product of the SEC14L2 (SEC14 Like Lipid Binding 2) gene belongs to a family of lipid-binding proteins including Sec14p, alpha-tocopherol transfer protein, and cellular retinol-binding protein. SEC14L2 expression enables replication of clinical hepatitis C virus (HCV) isolates in several hepatoma cell lines, and mutations in SEC14L2 may enhance HCV replication in vitro. The Chinese tree shrew (Tupaia belangeri chinensis) is a potential animal model for studying HCV replication, however, the cDNA sequence, protein structure, and expression of the Chinese tree shrew SEC14L2 gene have yet to be characterized. In the present study, we cloned the full-length cDNA sequence of the SEC14L2 in the Chinese tree shrew by using rapid amplification of cDNA ends technology. This led us to determine that, this is 2539 base pairs (bp) in length, the open reading frame sequence is 1212bp, and encodes 403 amino acids. Following this, we constructed a phylogenetic tree based on SEC14L2 molecules from various species and compared SEC14L2 amino acid sequence with other species. This analysis indicated that the Chinese tree shrew SEC14L2 protein (tsSEC14L2) has 96.28% amino acid similarity to the human protein, and is more closely related to the human protein than either mouse or rat protein. The Chinese tree shrew SEC14L2 mRNA was detected in all tissues, and showed highest expression levels in the pancreas, small intestine and trachea, however the tsSEC14L2protein abundance was highest in the liver and small intestine. The Chinese tree shrew SEC14L2 gene was closer in evolutionary relation to humans and non-human primates and expression of the tsSEC14L2protein was highest in the liver and small intestine. These results may provide useful information for tsSEC14L2 function in HCV infection.

Phosphatidic acid binds to and stimulates the activity of ARGAH2 from Arabidopsis

Phosphatidic acid (PA) has emerged as an important lipid signal during abiotic and biotic stress conditions such as drought, salinity, freezing, nutrient starvation, wounding and microbial elicitation. PA acts during stress responses primarily via binding and translocating target proteins or through modulating their activity. Owing to the importance of PA during stress signaling and developmental stages, it is imperative to identify PA interacting proteins and decipher their specific roles. In the present study, we have identified PA binding proteins from the leaves of Arabidopsis thaliana. Mass spectroscopy analysis led to the identification of 21 PA binding proteins with known roles in various cellular processes. One of the PA-binding proteins identified during this study, AtARGAH2, was further studied to unravel the role of PA interaction. Recombinant AtARGAH2 binding with immobilized PA on a solid support validated PA-AtARGAH2 binding invitro. PA binding to AtARGAH2 leads to the enhancement of arginase enzymatic activity in a dose dependent manner. Enzyme kinetics of recombinant AtARGAH2 demonstrated a lower Km value in presence of PA, suggesting role of PA in efficient enzyme-substrate binding. This simple approach could systematically be applied to perform an inclusive study on lipid binding proteins to elucidate their role in physiology of plants.

A Plasma Cuprome Exists With Predictors of Copper Status in Nepalese Children

ObjectivesCopper (Cu) is an essential micronutrient but, due to difficulties of measuring Cu by atomic absorption spectroscopy (AAS), plasma Cu status is rarely assessed in low income countries. A less costly assay is needed. We have explored whether plasma Cu concentration can be predicted by modelling strongly associated plasma proteins, assessed by mass spectrometry (MS), that could be assayed with other nutriproteomic biomarkers on one platform in the future.MethodsIn plasma samples from 500 Nepalese children 6–8 y of age, the mean (SD) plasma Cu concentration measured by AAS was 23.3 ± 5.7 mmol/L, with 13.6% classified as deficient (< 10 mmol/L). We quantified relative abundance of 982 plasma proteins by iTRAQ tandem MS following affinity depletion of six high abundance proteins (Cole J Nutr 2013) and correlated their relative abundance with plasma Cu by linear mixed effects regression. We defined a stringent plasma cuprome comprising proteins associated with Cu at a false discovery rate (q) < 0.01. Missing values were imputed and proteins were fit by forward, stepwise regression analysis, meeting an AIC reduction of ≥ 30, to model plasma Cu status.ResultsThere were 134 (q < 0.01) proteins in the plasma cuprome. Among 62 positive correlates were ceruloplasm (r = 0.65), 9 complement proteins (r = 0.29 to 0.45), 4 serpin protease inhibitors (r = 0.31 to 0.43) with others involved in coagulation, 5 LRR structural motifs as found in toll-like receptors (r = 0.35 to 0.58), signaling enzymes such as CDC42BPA (r = 0.70) and proinflammatory reactants and regulators such as CRP, AGP, amyloids and TNIP1 (r = 0.40 to 0.56). Among 72 negative correlates (r = −0.29 to −0.42) are lipid and sterol binding and transport proteins including apolipoproteins/lipocalins, enzyme regulating proteins, extracellular adhesion, signaling and matrix proteins. Different members of serine peptidase inhibitor members were among + and – correlates. Forward stepwise regression fit 6 proteins (CP, CDC42BPA, LRRC47, TDRD9, LLRIQ1, LRRCC1) that explain 76.5% of the variance (R2) in plasma Cu, sufficient for predicting plasma Cu status of a population.ConclusionsA large, diverse plasma cuprome exists, as revealed in young Nepalese children, from which six proteins may adequately predict plasma Cu status.Funding SourcesBill & Melinda Gates Foundation (OPPGH5241) & Johns Hopkins University.

Transcriptome Analysis of Citrus Dwarfing Viroid Induced Dwarfing Phenotype of Sweet Orange on Trifoliate Orange Rootstock

Dwarfed citrus trees for high-density plantings or mechanized production systems will be key for future sustainable citrus production. Citrus trees consist of two different species of scion and rootstock. Therefore, any observed phenotype results from gene expression in both species. Dwarfed sweet orange trees on trifoliate rootstock have been produced using citrus dwarfing viroid (CDVd). We performed RNA-seq transcriptome analysis of CDVd-infected stems and roots and compared them to non-infected controls. The identified differentially expressed genes validated with RT-qPCR corresponded to various physiological and developmental processes that could be associated with the dwarfing phenotype. For example, the transcription factors MYB13 and MADS-box, which regulate meristem functions and activate stress responses, were upregulated in the stems. Conversely, a calcium-dependent lipid-binding protein that regulates membrane transporters was downregulated in the roots. Most transcriptome reprogramming occurred in the scion rather than in the rootstock; this agrees with previous observations of CDVd affecting the growth of sweet orange stems while not affecting the trifoliate rootstock. Furthermore, the lack of alterations in the pathogen defense transcriptome supports the term “Transmissible small nuclear ribonucleic acid,” which describes CDVd as a modifying agent of tree performance with desirable agronomic traits rather than a disease-causing pathogen.

Systematic analysis and comparison of CaLB genes reveal the functions of GhCaLB42 and GhCaLB123 in fiber development and abiotic stress in cotton

Ca2+-dependent lipid-binding (CaLB) family proteins, containing C2 domain, play an important role in plant growth, development, and abiotic stress response. There has been no systematic identification of CaLB family genes in cotton (Gossypium). In this study, 196 GhCaLBs were identified and divided into five groups by phylogenetic tree analysis in Gossypium hirsutum. Gene structure and conserved protein motif analysis supported the evolutionary conservation of GhCaLBs. Whole-genome or segmental duplications contributed to the expansion of GhCaLBs. 2144 orthologous and paralogous CaLB gene pairs among four cotton species were detected. And Ka/Ks values analysis showed that CaLBs experienced strong purification selection. Numerous hormone-regulated, stress-responsive, and growth and development cis-elements were detected in the 2 kb promoter regions of GhCaLBs. Besides, transcriptome data combined with qRT-PCR analysis showed some GhCaLBs were preferentially expressed in ovule or fiber. Subcellular localization experiment revealed that GhCaLB42-GFP was located in nucleus and cell membrane, while GhCalB123-GFP was in cell membrane. Overexpressing GhCaLB42 significantly promoted the elongation and density of leaf trichomes in transgenic Arabidopsis, while GhCaLB123 overexpression inhibited the elongation of leaf trichomes. Histochemical staining experiments showed that GhCaLB123 overexpression lines could significantly increase the thickness of xylem vessels and interfascicular fibers cell wall of stem compared with WT. GhCaLB42 may positively regulate fiber elongation, and GhCaLB123 inhibited elongation but promoted secondary cell wall (SCW) thickening in fiber. Yeast two-hybrid assays indicated that GhCaLB42 protein had multiple biological functions including fiber development and abiotic stress response, and GhCaLB123 might participate in substance transport for the cellulose synthesis. GhCaLB42 and its two interaction genes GhTHi1–1 and GhCIPK9 were induced to be highly expressed under ABA treatment. These findings provide an important foundation for understanding the functions of specific cotton CaLB genes and for the effective selection of fiber development related genes.

FC065: Combined Proteomic Analyses of Recurrent Idiopathic Nephrotic Syndrome Plasma and Plasma-Treated Human Podocytes

Abstract BACKGROUND AND AIMS Idiopathic nephrotic syndrome (INS) comprises a group of rare glomerulopathies attributed to direct or indirect interaction of putative circulating factors with podocytes. One of the pathological manifestations of INS is the rapid recurrence of focal and segmental glomerulosclerosis (rFSGS) after renal transplant, which occurs in 30–50% of cases. To date, the nature and identity of circulating factors have not been confirmed, while the pathogenic mechanisms in the podocyte are partially understood. Direct analysis of patient plasma proteome has been scarcely addressed, mainly due to methodological difficulties associated with plasma complexity and dynamic range. To better understand the mechanisms involved in FSGS recurrence, the present study had two main objectives: (i) to perform a differential proteomic analysis of soluble plasma proteins and of extracellular vesicles (EV) from rFSGS patients and from controls; and (ii) to study the early proximal signaling events in podocytes in response to plasma from post-transplant rFSGS patients as compared to plasma from controls, by a combination of phosphoproteomics and lipid raft proteomics (raftomics). METHOD We obtained first post-transplant plasma exchange fluid from either rFSGS or non-INS patients (n = 4), the latter used as controls, as well as blood plasma from healthy individuals (n = 4). In a first study, by a combination of immunodepletion and high pH fractionation, we performed in parallel a differential proteomic analysis of soluble plasma proteins and of extracellular vesicles (EV) from the three groups. In a second study, we incubated differentiated human podocytes for 30 min with 10% of plasma exchange fluid from either rFSGS or non-INS patients. Then, we isolated lipid rafts from podocytes by a detergent-free method and performed differential proteomics. In addition, we performed differential phosphoproteomics after phosphopeptide enrichment in a TiO2 column. Proteomics by LC-MS/MS analysis was performed in a TimsTOF Pro mass spectrometer on plasma samples, and in a nanoRSLC-Q Exactive PLUS instrument on podocyte samples. RESULTS The approximate number of proteins identified was 500 in immunodepleted plasma, 1400 in EV, 2500 in podocyte rafts and 4900 podocyte phosphosites. In both soluble and EV protein sets from rFSGS patients, we found a statistically significant increase in a cluster of proteins involved in neutrophil degranulation. A group of lipid binding proteins, generally associated with lipoproteins, was found decreased in the soluble set from rFSGS patients. In addition, several aminoacid transporters involved in mTORC1 activation were found significantly increased in EV from rFSGS individuals. As regards podocyte proteome analyses, the differences found in protein expression and phosphorylation associated with rFSGS plasma incubation suggested alterations in the mTOR pathway, in autophagy, in mitochondrial metabolism and in cytoskeleton organization. In particular, validation experiments indicated an altered phosphorylation pattern of Hsp27. CONCLUSION The technological approaches used in the plasma study show the feasibility of direct proteomic analysis of this material, as well as the potential of some of the identified differential proteins as FSGS biomarkers. The observed changes in podocyte proteome associated with rFSGS plasma incubation highlight some of the potential mechanisms involved in FSGS recurrence and could be used as specific early markers of circulating factor activity on podocytes. Further research will be necessary to confirm and validate these findings.

Abstract 219: Apolipoprotein C3 Induces Inflammasome Activation In Human And Mouse Monocytes Only In De-lipidated Form

Individuals with diabetes suffer from an elevated risk of coronary artery disease (CAD) events. Increased serum levels of apolipoprotein C3 (APOC3) predict incident CAD in individuals with type 1 diabetes (T1D). APOC3 is a small lipid-binding protein carried by lipoproteins, including VLDL, in circulation. Silencing of APOC3 in a mouse model of T1D-accelerated atherosclerosis has been demonstrated to prevent diabetes-accelerated atherosclerosis, strongly suggesting that APOC3 is a causal mediator. However, the exact mechanism whereby APOC3 promotes atherosclerosis in the setting of T1D is unclear. Mice with T1D demonstrated elevated levels of glucose, triglycerides, APOC3 (non-diabetic [ND] mice=442.6 μg/ml, diabetic [D] mice=755.9 μg/ml, p&lt;0.01, N=21 per group), and IL-18 (ND=124.1 pg/ml, D=256.8 pg/ml, p&lt;0.01, N=18-22 per group) in plasma. The elevated levels of plasma APOC3 and IL-18 in diabetic mice and a recent study demonstrating that de-lipidated APOC3 can stimulate inflammasome activation in human monocytes prompted us to investigate whether lipidated APOC3 has similar inflammasome-activating properties. Our results show that de-lipidated APOC3, but not VLDL (which contains APOC3) significantly stimulates IL-1β release from isolated human monocytes (p&lt;0.01 for vehicle vs APOC3, N=3 per group) and mouse monocytes (p&lt;0.01 for vehicle vs APOC3, N=4-7 per group). Mouse monocytes stimulated with de-lipidated APOC3 also had elevated IL-18 release (vehicle=21.2 pg/mg, APOC3=170.2 pg/mg, p&lt;0.001), Il1b mRNA (APOC3=86.7-fold over control), and Nlrp3 mRNA (APOC3=13.8-fold over control), indicating activation of the NLRP3 inflammasome pathway. However, analysis of serum samples from subjects with T1D showed that virtually all APOC3 is bound to lipoproteins (primarily VLDL and HDL). To further confirm the effect of lipidated APOC3, we bound APOC3 to lipid-based small unilamellar vesicles to mimic the physiological state of APOC3 in plasma. The stimulatory effect of APOC3, measured by IL-1β release from mouse monocytes, was lost. Together, our findings suggest that although de-lipidated APOC3 has the ability to induce inflammasome activation in monocytes, virtually no APOC3 circulates in a lipid-free form in subjects with T1D.

Gut Microbiota and Phenotypic Changes Induced by Ablation of Liver- and Intestinal-Type Fatty Acid-Binding Proteins.

Intestinal fatty acid-binding protein (IFABP; FABP2) and liver fatty acid-binding protein (LFABP; FABP1) are small intracellular lipid-binding proteins. Deficiency of either of these proteins in mice leads to differential changes in intestinal lipid transport and metabolism, and to markedly divergent changes in whole-body energy homeostasis. The gut microbiota has been reported to play a pivotal role in metabolic process in the host and can be affected by host genetic factors. Here, we examined the phenotypes of wild-type (WT), LFABP−/−, and IFABP−/− mice before and after high-fat diet (HFD) feeding and applied 16S rRNA gene V4 sequencing to explore guild-level changes in the gut microbiota and their associations with the phenotypes. The results show that, compared with WT and IFABP−/− mice, LFABP−/− mice gained more weight, had longer intestinal transit time, less fecal output, and more guilds containing bacteria associated with obesity, such as members in family Desulfovibrionaceae. By contrast, IFABP−/− mice gained the least weight, had the shortest intestinal transit time, the most fecal output, and the highest abundance of potentially beneficial guilds such as those including members from Akkermansia, Lactobacillus, and Bifidobacterium. Twelve out of the eighteen genotype-related bacterial guilds were associated with body weight. Interestingly, compared with WT mice, the levels of short-chain fatty acids in feces were significantly higher in LFABP−/− and IFABP−/− mice under both diets. Collectively, these studies show that the ablation of LFABP or IFABP induced marked changes in the gut microbiota, and these were associated with HFD-induced phenotypic changes in these mice.

α-Synuclein at the Presynaptic Axon Terminal as a Double-Edged Sword.

α-synuclein (α-syn) is a presynaptic, lipid-binding protein strongly associated with the neuropathology observed in Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and Alzheimer’s Disease (AD). In normal physiology, α-syn plays a pivotal role in facilitating endocytosis and exocytosis. Interestingly, mutations and modifications of precise α-syn domains interfere with α-syn oligomerization and nucleation that negatively affect presynaptic vesicular dynamics, protein expressions, and mitochondrial profiles. Furthermore, the integration of the α-syn oligomers into the presynaptic membrane results in pore formations, ion influx, and excitotoxicity. Targeted therapies against specific domains of α-syn, including the use of small organic molecules, monoclonal antibodies, and synthetic peptides, are being screened and developed. However, the prospect of an effective α-syn targeted therapy is still plagued by low permeability across the blood–brain barrier (BBB), and poor entry into the presynaptic axon terminals. The present review proposes a modification of current strategies, which includes the use of novel encapsulation technology, such as lipid nanoparticles, to bypass the BBB and deliver such agents into the brain.

Serum Protein Biomarkers of Inflammation, Oxidative Stress, and Cerebrovascular and Glial Injury in Concussed Australian Football Players.

Clinical decisions related to sports-related concussion (SRC) are challenging, because of the heterogenous nature of SRC symptoms coupled with the current reliance on subjective self-reported symptom measures. Sensitive and objective methods that can diagnose SRC and determine recovery would aid clinical management, and there is evidence that SRC induces changes in circulating protein biomarkers, indicative of neuroaxonal injury. However, potential blood biomarkers related to other pathobiological responses linked to SRC are still poorly understood. Therefore, here we analyzed blood samples from concussed (male = 30; female = 9) and non-concussed (male = 74; female = 27) amateur Australian rules football players collected during the pre-season (i.e., baseline), and at 2, 6, and 13 days post-SRC to determine time-dependent changes in serum levels of biomarkers related to glial (i.e., brain lipid-binding protein [BLBP]; phosphoprotein enriched in astrocytes 15) and cerebrovascular injury (i.e., von Willebrand factor, claudin-5), inflammation (i.e., fibrinogen, high mobility group box protein 1), and oxidative stress (i.e., 4-hydroxynoneal). In females, BLBP levels were significantly decreased at 2 days post-SRC compared with their pre-season baseline; however, area under the receiver operating characteristic curve (AUROC) analysis found that BLBP was unable to distinguish between SRC and controls. In males, AUROC analysis revealed a statistically significant change at 2 days post-SRC in the serum levels of 4-hydroxynoneal, however the associated AUROC value (0.6373) indicated little clinical utility for this biomarker in distinguishing SRC from controls. There were no other statistically significant findings. These results indicate that the serum biomarkers tested in this study hold little clinical value in the management of SRC at 2, 6, and 13 days post-injury.