Dynamic regulation of N6,2′-O-dimethyladenosine (m6Am) in obesity

The prevalent m6Am mRNA cap modification was recently identified as a valid target for removal by the human obesity gene FTO along with the previously established m6A mRNA modification. However, the deposition and dynamics of m6Am in regulating obesity are unknown. Here, we investigate the liver m6A/m methylomes in mice fed on a high fat Western-diet and in ob/ob mice. We find that FTO levels are elevated in fat mice, and that genes which lost m6Am marking under obesity are overly downregulated, including the two fatty-acid-binding proteins FABP2, and FABP5. Furthermore, the cellular perturbation of FTO correspondingly affect protein levels of its targets. Notably, generally m6Am- but not m6A-methylated genes, are found to be highly enriched in metabolic processes. Finally, we deplete all m6A background via Mettl3 knockout, and unequivocally uncover the association of m6Am methylation with increased mRNA stability, translation efficiency, and higher protein expression. Together, these results strongly implicate a dynamic role for m6Am in obesity-related translation regulation.

Nieman-Pick Type C2 proteins in Aedes aegypti: Their Structure-Function relationships and Expression in Uninfected versus Virus-infected Mosquitos

Aedes aegypti is a major vector that transmits arboviruses through the saliva injected into the host. Salivary proteins help in uninterrupted blood intake and enhance the transmission of pathogens. We studied Nieman-Pick Type C2 (NPC2) proteins, a superfamily of saliva proteins that play important role in arbovirus infections. In vertebrates, a single conserved gene encodes for the NPC2 protein that functions in cholesterol trafficking. Arthropods, in contrast, have several genes that encode for divergent NPC2 proteins. We compared the sequences of 20 A. aegypti NPC2 proteins to the cholesterol-binding residues of human and bovine, and fatty acid-binding residues of ant NPC2 proteins. We identified four and one mosquito NPC2 proteins as potential sterol- and fatty acid-binding proteins, respectively. From the published data, we analysed the expression of NPC2 genes in various tissues and their differential expression in midgut and salivary gland post-arbovirus infections. NPC2 genes are downregulated rather than upregulated in virus-infected tissues. Interestingly, AAEL012064 is the only gene that is downregulated in both the midgut and salivary gland in all virus infections. In addition, AAEL001650 is downregulated in the salivary gland infected with CHIKV, DENV2 or ZIKV. This gene in the midgut is downregulated infected with DENV1 but upregulated with DENV2. We studied the variation in cis elements in the promoter regions of two groups of closely related NPC2 genes and the expression of relevant transcription factors (TFs). In the midgut infected with DENV1 or DENV2, six TFs (CRE-BP1, AP1, c-Jun, c-Fos, Odd, and NF-kB) appear to play an opposing role in the expression of AAEL006854. Two TFs (RXR-beta/alpha and USF) have a potential role in the downregulation of AAEL009556 in CHIKV-infected midgut and salivary gland. Similarly, two TFs (COUP and Ftz) may have a key role in the downregulation of AAEL009555 and AAEL009556 in DENV2-infected salivary gland.

FABP7 Binds to Fatty Acid Micelles: Implications for Lipid Transport

The transport of hydrophobic molecules, including long-chain fatty acids, within cells is highly dynamic. Hydrophobic molecules are unable to freely diffuse through the aqueous cytoplasm without a transporter. Fatty acid binding proteins (FABP) transport these molecules to different cellular compartments. As part of their transport, FABPs often associate with cell membranes to acquire and deliver their bound cargo. Understanding the nature of this transport is becoming increasingly important because lipid signaling functions are associated with metabolic pathways impacting disease pathologies such as carcinomas, autism and schizophrenia. Herein, we focus on Brain fatty acid binding protein (FABP7), which demonstrates localization to the cytoplasm and nucleus, influencing transcription and fatty acid metabolism. We use a combined biophysical approach to elucidate the interaction between FABP7 and model membranes. Specifically, we use microscale thermophoresis to show that FABP7 can bind oleic acid (OA) and docosahexaenoic acid (DHA) micelles, while differential scanning fluorimetry experiments show binding lowers the melting temperature of FABP7. Structural data from NMR and multiscale molecular dynamics simulations reveals that the interaction between FABP7 and micelles is through FABP7 portal region residues. Our simulations also capture binding events where fatty acids dissociate from the model membrane and bind to FABP7. Overall, our data reveals a novel interaction between FABP7 and OA or DHA micelles and provides key structural insight into the transport of hydrophobic molecules.

Expression Profiles and Prognostic Value of FABPs in Colorectal Adenocarcinomas

Colorectal cancer (CRC) is one of the world’s leading causes of cancer-related deaths; thus, it is important to detect it as early as possible. Obesity is thought to be linked to a large rise in the CRC incidence as a result of bad dietary choices, such as a high intake of animal fats. Fatty acid-binding proteins (FABPs) are a set of molecules that coordinate intracellular lipid responses and are highly associated with metabolism and inflammatory pathways. There are nine types of FABP genes that have been found in mammals, which are FABP1–7, FABP9, and FABP12. Each FABP gene has its own roles in different organs of the body; hence, each one has different expression levels in different cancers. The roles of FABP family genes in the development of CRC are still poorly understood. We used a bioinformatics approach to examine FABP family gene expression profiles using the Oncomine, GEPIA, PrognoScan, STRING, cBioPortal, MetaCore, and TIMER platforms. Results showed that the FABP6 messenger (m)RNA level is overexpressed in CRC cells compared to normal cells. The overexpression of FABP6 was found to be related to poor prognosis in CRC patients’ overall survival. The immunohistochemical results in the Human Protein Atlas showed that FABP1 and FABP6 exhibited strong staining in CRC tissues. An enrichment analysis showed that high expression of FABP6 was significantly correlated with the role of microRNAs in cell proliferation in the development of CRC through the insulin-like growth factor (IGF) signaling pathway. FABP6 functions as an intracellular bile-acid transporter in the ileal epithelium. We looked at FABP6 expression in CRC since bile acids are important in the carcinogenesis of CRC. In conclusion, high FABP6 expression is expected to be a potential biomarker for detecting CRC at the early stage.

PSVI-26 Optimized small tissue specific protein isolation trough complexed diafiltration technique

Physiologically active and pure small tissue specific proteins are in demand in various fundamental and applied areas due to its bioactive properties. It is known that proteins can aggregate, as well as form a highly concentrated layer at the border of the filtration membrane or adhere to it, which significantly interferes with filtration. To eliminate these effects, protein-peptide extracts are recommended to be diluted, as well as using special agents, among them amino acids are more suitable for food industry. The purpose of the study is to develop the isolation and purification of proteins with molecular weight lower 50kDa from porcine hearts and aortas, using different amino acids and its combination. It was previously revealed, that family of fatty acid binding proteins (FABP) is one of the most stable and physiologically active protein in such raw materials. The final technology includes extraction, centrifugation, dilution of extract, diafiltration, lyophilization, dissolving in a small volume of distilled water, dialization and final drying. Concentration of 1.0% glycine and 0.1M proline in both diluted extract and exchange solution was revealed to be most effective to prevent protein aggregation. Dialysates were dried lyophilically and O’Farrell 2DE-electrophoresis with MALDI-TOF MS and MS/MS mass spectrometry identification were used. No proteins of 50kDa and bigger were detected. Proteins of lipid metabolism, peroxiredoxin 2, transgelins etc. with molecular weight lower 50kDa were found. The developed technology allows increasing cardiac and adipocytic isoforms of FABP family content. Cardiac isoform was previously identified in raw material, while adipocytic was newly detected. Adipocytic isoform content is lower than cardiac one; therefore, it was not detected in raw material, but application of complexed diafiltration technique led to increasing its content in dialysate. The developed technology can be used for separation and purification of animal proteins in native and stable forms.

Discovery and Characterization of a Bilirubin Inducible Green Fluorescent Protein From the Moray Eel Gymnothorax zonipectis

Since the initial discovery of Aqueoria victoria’s green fluorescence off the coast of Washington’s Puget Sound, biofluorescent marine organisms have been found across the globe. The variety of colors of biofluorescence as well as the variability in the organisms that exhibit this fluorescence is astounding. The mechanisms of biofluorescence in marine organisms are also variable. To fluoresce, some organisms use fluorescent proteins, while others use small molecules. In eels, green biofluorescence was first identified in Anguilla japonica. The green fluorescence in A. japonica was discovered to be caused by a fatty acid binding protein (UnaG) whose fluorescence is induced by the addition of bilirubin. Members of this class of proteins were later discovered in Kaupichthys eels (Chlopsid FP I and Chlopsid FP II). Here, we report the discovery and characterization of the first member of this class of green fluorescent fatty acid binding proteins from the moray eel Gymnothorax zonipectis. This protein, GymFP, is 15.6 kDa with a fluorescence excitation at 496 nm and an emission maximum at 532 nm upon addition of bilirubin. GymFP is 61% homologous to UnaG and 47% homologous to Chlopsid FP I. Here, we report de novo transcriptome assembly, protein expression, and fluorescence spectroscopic characterization of GymFP. These findings extend the fluorescent fatty acid binding proteins into a third family of true eels (Anguilliformes).

Structural transitions permitting ligand entry and exit in bacterial fatty acid binding proteins

Fatty acid (FA) transfer proteins extract FA from membranes and sequester their ligand to facilitate its movement through the cytosol. While detailed views of soluble protein-FA complexes are available, how FA exchange occurs at the membrane has remained unknown. Staphylococcus aureus FakB1 is a prototypical bacterial FA transfer protein that binds palmitate within a narrow, buried tunnel. Here, we determine the conformational change from this closed state to an open state that engages the phospholipid bilayer. Upon membrane binding, a dynamic loop in FakB1 that covers the FA binding site disengages and folds into an amphipathic helix. This helix inserts below the phosphate plane of the bilayer to create a diffusion channel for the FA to exchange between the protein and the membrane. The structure of the bilayer-associated conformation of FakB1 has local similarities with mammalian FA binding proteins and provides a general conceptual framework for how these proteins interact with the membrane to promote lipid transfer.

Fatty acid-binding proteins and fatty acid synthase influence glial reactivity and promote the formation of Müller glia-derived progenitor cells in the avian retina

The capacity for retinal regeneration varies greatly across vertebrates species. A recent comparative epigenetic and transcriptomic investigation of Müller glial (MG) in the retinas of fish, birds and mice revealed that Fatty Acid Binding Proteins (FABPs) are among the most highly up-regulated genes in activated chick MG (Hoang et al., 2020). Herein we provide an in-depth follow-up investigation to describe patterns of expression and how FABPs and fatty acid synthase (FASN) influence glial cells in the chick retina. During development, FABP7 is highly expressed by embryonic retinal progenitor cells (eRPCs) and maturing MG, whereas FABP5 is gradually up-regulated in maturing MG and remains elevated in mature glial cells. PMP2 (FABP8) is expressed by oligodendrocytes and FABP5 is expressed by non-astrocytic inner retinal glial cells, and both of these FABPs are significantly up-regulated in activated MG in damaged or growth factor-treated retinas. In addition to suppressing the formation of MGPCs, we find that FABP-inhibition suppressed the accumulation of proliferating microglia, although the microglia appeared highly reactive. scRNA-seq analyses of cells treated with FABP-inhibitor revealed distinct changes in patterns of expression suggesting that FABPs are involved in the transitions of MG from a resting state to a reactive state and conversion from reactive MG to MGPCs. Inhibition of FABPs in undamaged retinas had a significant impact upon the transcriptomic profiles of MG, with up-regulation of genes associated with gliogenesis, decreases in genes associated with neurogenesis, and suppression of the ability of MG to become MGPCs. scRNA-seq analyses of microglia indicated that FABP inhibition enhances gene modules related to reactivity, proliferation and cytokine signaling. We find that the proliferation of retinal progenitors in the circumferential marginal zone (CMZ) is unaffected by FABP-inhibitor. Upstream of FABP activity, we inhibited FASN in damaged retinas, which reduced numbers of dying cells, increased the proliferation of microglia, and potently suppressed the formation MGPCs in damaged retinas. We conclude that the activity of FASN and FABPs are required early during the formation of proliferating MGPCs. Fatty acid metabolism and cell signaling involving fatty acids are important in regulating glial homeostasis in the retina, and the dedifferentiation and proliferation of microglia and MGPCs.