acyl-CoA Binding Protein Research Articles

Phylogeny and subcellular localization analyses reveal distinctions in monocot and eudicot class IV acyl-CoA-binding proteins.

Plant class IV ACBPs diverged with the split of monocots and eudicots. Difference in the subcellular localization supported the functional variation of plant class IV ACBP. Acyl-CoA-binding proteins (ACBPs) are divided into class I-IV in plants. Class IV ACBPs are kelch motif containing proteins that are specific to plants. The currently known subcellular localizations of plant class IV ACBPs are either in the cytosol (Arabidopsis) or in the peroxisomes (rice). However, it is not clear whether peroxisomal localization of class IV ACBP is a shared character that distinguishes eudicots and monocots. Here, the phylogeny of class IV ACBPs from 73 plant species and subcellular localization of class IV ACBPs from six monocots and eudicots were conducted. Phylogenetic analysis of 112 orthologues revealed that monocot class IV ACBPs were basal to the monophyletic clade formed by eudicots and basal angiosperm. Transient expression of GFP fusions in onion epidermal cells demonstrated that monocot maize (Zea mays), wheat (Triticum aestivum), and sorghum (Sorghum bicolor) and eudicot poplar (Populus trichocarpa) all contained at least one peroxisomal localized class IV ACBP, while orthologues from cucumber (Cucumis sativus L.) and soybean (Glycine max) were all cytosolic. Combining the location of Arabidopsis and rice class IV ACBPs, it indicates that maintaining at least one peroxisomal class IV ACBP could be a shared feature within the tested monocots, while cytosolic class IV ACBPs would be preferred in the tested eudicots. Furthermore, the interaction between OsACBP6 and peroxisomal ATP-binding cassette (ABC) transporter provided clues for the functional mechanism of OsACBP6.

ITRAQ-Based Quantitative Proteomics Reveals ChAcb1 as a Novel Virulence Factor in Colletotrichum higginsianum.

Colletotrichum higginsianum is an important hemibiotrophic fungal pathogen that causes anthracnose disease on various cruciferous plants. Discovery of new virulence factors could lead to strategies for effectively controlling anthracnose. Acyl-CoA binding proteins (ACBPs) are mainly involved in binding and trafficking acyl-CoA esters in eukaryotic cells. However, the functions of this important class of proteins in plant fungal pathogens remain unclear. In this study, we performed an isobaric tags for relative and absolute quantification (iTRAQ)-based quantitative proteomic analysis to identify differentially expressed proteins (DEPs) between a nonpathogenic mutant ΔCh-MEL1 and the wild type. Based on iTRAQ data, DEPs in the ΔCh-MEL1 mutant were mainly associated with melanin biosynthesis, carbohydrate and energy metabolism, lipid metabolism, redox processes, and amino acid metabolism. Proteomic analysis revealed that many DEPs might be involved in growth and pathogenesis of C. higginsianum. Among them, an acyl-CoA binding protein, ChAcb1, was selected for further functional studies. Deletion of ChAcb1 caused defects in vegetative growth and conidiation. ChAcb1 is also required for response to hyperosmotic and oxidative stresses, and maintenance of cell wall integrity. Importantly, the ΔChAcb1 mutant exhibited reduced virulence, and microscopic examination revealed that it was defective in appressorial penetration and infectious growth. Furthermore, the ΔChAcb1 mutant was impaired in fatty acid and lipid metabolism. Taken together, ChAcb1 was identified as a new virulence gene in this plant pathogenic fungus.

A Ratiometric Fluorescent Biosensor Reveals Dynamic Regulation of Long-Chain Fatty Acyl-CoA Esters Metabolism.

Despite increasing awareness of the biological impacts of long-chain fatty acyl-CoA esters (LCACoAs), our knowledge about the subcellular distribution and regulatory functions of these acyl-CoA molecules is limited by a lack of methods for detecting LCACoAs in living cells. Here, we report development of a genetically encoded fluorescent sensor that enables ratiometric quantification of LCACoAs in living cells and subcellular compartments. We demonstrate how this FadR-cpYFP fusion "LACSer sensor" undergoes LCACoA-induced conformational changes reflected in easily detectable fluorescence responses, and show proof-of-concept for real-time monitoring of LCACoAs in human cells. Subsequently, we applied LACSer in scientific studies investigating how disruption of ACSL enzymes differentially reduces cytosolic and mitochondrial LCACoA levels, and show how genetic disruption of an acyl-CoA binding protein (ACBP) alters mitochondrial accumulation of LCACoAs.

A Ratiometric Fluorescent Biosensor Reveals Dynamic Regulation of Long‐Chain Fatty Acyl‐CoA Esters Metabolism

AbstractDespite increasing awareness of the biological impacts of long‐chain fatty acyl‐CoA esters (LCACoAs), our knowledge about the subcellular distribution and regulatory functions of these acyl‐CoA molecules is limited by a lack of methods for detecting LCACoAs in living cells. Here, we report development of a genetically encoded fluorescent sensor that enables ratiometric quantification of LCACoAs in living cells and subcellular compartments. We demonstrate how this FadR‐cpYFP fusion “LACSer sensor” undergoes LCACoA‐induced conformational changes reflected in easily detectable fluorescence responses, and show proof‐of‐concept for real‐time monitoring of LCACoAs in human cells. Subsequently, we applied LACSer in scientific studies investigating how disruption of ACSL enzymes differentially reduces cytosolic and mitochondrial LCACoA levels, and show how genetic disruption of an acyl‐CoA binding protein (ACBP) alters mitochondrial accumulation of LCACoAs.

A Functional mTORC1 Containing Raptor Controls Luminal Fat Processing and Epithelium Barrier Function in Transwell Caco‐2 Cultures

As a critical regulatory point of nutrient sensing, growth and metabolism, the mechanistic target of rapamycin complex 1 (mTORC1) is poised to influence intestinal homeostasis under basal conditions and in disease state. We created three stable human colorectal adenocarcinoma Caco-2 cell lines exhibiting normal, low (Raptor knockdown) or high (tuberin knockdown) mTORC1 kinase activity, and grew these cells in Transwell to investigate the contribution of mTORC1 in fat absorption, processing, and barrier permeability. Raptor knockdown impaired lipid and apoB-containing lipoprotein metabolism in Caco-2 cultures provided exogenous oleate in the apical chamber. Raptor-depleted Caco-2 cells secreted less apoB100 (-51%), apoB48 (-66%) and triacylglycerol (-69%) compared with control Caco-2 cells. Genes involved in lipogenesis (FASN, fatty acid synthase), fatty acyl-CoA formation (FATP2, very long-chain acyl-CoA ligase; DBI, diazepam binding inhibitor acyl-CoA binding protein), triacylglycerol synthesis (DGAT1, diacylglycerol O-acyltransferase 1; DGAT2, diacylglycerol O-acyltransferase 2), lipoprotein assembly (APOB, apolipoprotein B; MTTP, microsomal triglyceride transfer protein), and pre-chylomicron transport (VAMP7,vesicle-associated membrane protein 7; SAR1B, secretion associated Ras related GTPase 1B) were downregulated by at least 60% in Caco-2 cells depleted of Raptor versus control Caco-2 cells. The cellular protein abundance of apoB (-89%), MTTP (-63%) and FASN (-80%) were decreased in Raptor-depleted versus control Caco-2 cells. When mTORC1 activity was abrogated, Transwell-type Caco-2 cultures were impermeable to ions (high transepithelial electrical resistance, TEER) and had elevated paracellular transport of 4-kDa FITC dextran. Raptor knockdown likewise rapamycin, a mTORC1 inhibitor, decreased the abundance of small-sized solute pore-forming claudins 2 and 15, and the levels of tight junction proteins occludin and tricellulin. In contrast, resistance against ion diffusion decreased (low TEER) and paracellular transport of 4-kDa FITC dextran increased as mTORC1 activity rose. In conclusion, a functional intestinal mTORC1 is critically important for processing dietary lipids into chylomicrons and establishing the permeability characteristics of the epithelium.

Plant Acyl-CoA-Binding Proteins-Their Lipid and Protein Interactors in Abiotic and Biotic Stresses.

Plants are constantly exposed to environmental stresses during their growth and development. Owing to their immobility, plants possess stress-sensing abilities and adaptive responses to cope with the abiotic and biotic stresses caused by extreme temperatures, drought, flooding, salinity, heavy metals and pathogens. Acyl-CoA-binding proteins (ACBPs), a family of conserved proteins among prokaryotes and eukaryotes, bind to a variety of acyl-CoA esters with different affinities and play a role in the transport and maintenance of subcellular acyl-CoA pools. In plants, studies have revealed ACBP functions in development and stress responses through their interactions with lipids and protein partners. This review summarises the roles of plant ACBPs and their lipid and protein interactors in abiotic and biotic stress responses.

In silico Analysis of Acyl-CoA-Binding Protein Expression in Soybean.

Plant acyl-CoA-binding proteins (ACBPs) form a highly conserved protein family that binds to acyl-CoA esters as well as other lipid and protein interactors to function in developmental and stress responses. This protein family had been extensively studied in non-leguminous species such as Arabidopsis thaliana (thale cress), Oryza sativa (rice), and Brassica napus (oilseed rape). However, the characterization of soybean (Glycine max) ACBPs, designated GmACBPs, has remained unreported although this legume is a globally important crop cultivated for its high oil and protein content, and plays a significant role in the food and chemical industries. In this study, 11 members of the GmACBP family from four classes, comprising Class I (small), Class II (ankyrin repeats), Class III (large), and Class IV (kelch motif), were identified. For each class, more than one copy occurred and their domain architecture including the acyl-CoA-binding domain was compared with Arabidopsis and rice. The expression profile, tertiary structure and subcellular localization of each GmACBP were predicted, and the similarities and differences between GmACBPs and other plant ACBPs were deduced. A potential role for some Class III GmACBPs in nodulation, not previously encountered in non-leguminous ACBPs, has emerged. Interestingly, the sole member of Class III ACBP in each of non-leguminous Arabidopsis and rice had been previously identified in plant-pathogen interactions. As plant ACBPs are known to play important roles in development and responses to abiotic and biotic stresses, the in silico expression profiles on GmACBPs, gathered from data mining of RNA-sequencing and microarray analyses, will lay the foundation for future studies in their applications in biotechnology.

Transcriptomic and proteomic analysis of the underlying mechanisms of digestion of triacylglycerols and phosphatides and absorption and fate of fatty acids along the midgut of Musca domestica

Most dietary lipids are triacylglycerols (TAGs) and phosphatides that are digested by TAG lipases and phospholipases (PLIPs), respectively, originating fatty acids (FA). The genome of Musca domestica has genes coding for phospholipases A1 (1PLIP), A2 (2PLIP), B (BPLIP), and acid lipases (ALIP), as for proteins involved in activation, binding, and metabolism of FA, which expression in the larval midgut was evaluated by RNA-seq. Some of the codified proteins were identified in midgut microvillar-enriched membrane by proteomics. 1PLIPs are the most expressed PLIPs, mainly in anterior midgut whereas 2PLIPs, and BPLIP in middle and posterior midgut, and ALIPs between middle and posterior regions. Absorption of FAs is putatively accomplished by proteins involved in FA activation (acyl-CoA synthetases) found in microvillar-enriched membrane preparations. Furthermore, FA uptake could be enhanced by proteins that bind FAs (FA-binding proteins) and its activated form (acyl-CoA binding proteins) mainly expressed in posterior midgut. Activated FAs could have different fates: synthesis of diacylglycerol (DAG) and TAG through monoacylglycerol and glycerol-3-phosphate pathways; synthesis of phosphatides; energy source by β-oxidation. Most genes coding for enzymes of those routes is expressed mainly at the end of posterior midgut. Data suggest that phosphatides are digested in anterior midgut by Md1PLIPs, releasing lysophosphatides that emulsify fats to be digested by MdALIPs in the middle and posterior midgut. Most resulting FAs is absorbed in the posterior midgut, where they follow the synthesis of DAG, TAG, and phosphatides or are oxidized along the midgut, mainly in highly metabolic middle and posterior midgut regions.

Biosynthesis of the Sex Pheromone Component (E,Z)-7,9-Dodecadienyl Acetate in the European Grapevine Moth, Lobesia botrana, Involving \u220611 Desaturation and an Elusive \u22067 Desaturase

The European grapevine moth, Lobesia botrana, uses (E,Z)-7,9-dodecadienyl acetate as its major sex pheromone component. Through in vivo labeling experiments we demonstrated that the doubly unsaturated pheromone component is produced by ∆11 desaturation of tetradecanoic acid, followed by chain shortening of (Z)-11-tetradecenoic acid to (Z)-9-dodecenoic acid, and subsequently introduction of the second double bond by an unknown ∆7 desaturase, before final reduction and acetylation. By sequencing and analyzing the transcriptome of female pheromone glands of L. botrana, we obtained 41 candidate genes that may be involved in sex pheromone production, including the genes encoding 17 fatty acyl desaturases, 13 fatty acyl reductases, 1 fatty acid synthase, 3 acyl-CoA oxidases, 1 acetyl-CoA carboxylase, 4 fatty acid transport proteins and 2 acyl-CoA binding proteins. A functional assay of desaturase and acyl-CoA oxidase gene candidates in yeast and insect cell (Sf9) heterologous expression systems revealed that Lbo_PPTQ encodes a ∆11 desaturase producing (Z)-11-tetradecenoic acid from tetradecanoic acid. Further, Lbo_31670 and Lbo_49602 encode two acyl-CoA oxidases that may produce (Z)-9-dodecenoic acid by chain shortening (Z)-11-tetradecenoic acid. The gene encoding the enzyme introducing the E7 double bond into (Z)-9-dodecenoic acid remains elusive even though we assayed 17 candidate desaturases in the two heterologous systems.

Investigations of Lipid Binding to Acyl-CoA-Binding Proteins (ACBP) Using Isothermal Titration Calorimetry (ITC).

Isothermal titration calorimetry (ITC) is a quantitative, biophysical method to investigate intermolecular binding between biomolecules by directly measuring the heat exchange in the binding reaction. The assay is carried out in solution when the molecules interact in vitro. This allows to determine values for binding affinity (Kd), binding stoichiometry (n), as well as changes in Gibbs free energy (ΔG), entropy (ΔS), and enthalpy (ΔH). This method also addresses the kinetics of enzymatic reactions for a substrate during conversion to a product. ITC has been used to study the interactions between proteins and ligands such as those of acyl-CoA-binding proteins (ACBPs) and acyl-CoA thioesters or ACBPs with protein partners. ITC has also been used in investigating interactions between antiserum and antigen, as well as those involving RNA and DNA and other macromolecules. We describe the methods used to isolate and purify a recombinant rice ACBP (OsACBP) for ITC. To study OsACBP binding to long-chain acyl-CoA thioesters, a microcalorimeter was used at 30°C, and the ligand (acyl-CoA thioesters or a protein partner in the first cell), was mixed with the ACBP protein solution in a second cell, for more than 40min comprising 20 injections. Subsequently, the binding parameters including the heat-release data were analyzed and various thermodynamic parameters were calculated.

Quantification of intracellular ACBP/DBI levels.

Acyl-CoA binding protein (ACBP), also called diazepam-binding inhibitor (DBI), is a ubiquitous protein that can be secreted from cells by an unconventional pathway. Depending on its levels and on its subcellular localization, ACBP/DBI can regulate lipid metabolism. Several studies have shown that ACBP/DBI is secreted by an autophagy-dependent mechanism, positioning this catabolic pathway as the mechanism that controls lipid metabolism through the intracellular modulation of the levels of this protein. Autophagy is activated, among other stimuli, when cells have increased energy requirements; this causes a drop in the intracellular ACBP/DBI levels due to its release into the extracellular space and triggers an increase in the lipid catabolism. Conversely, when autophagy is inhibited, during pathological (obesity) or physiological (after-meal) situations, the intracellular levels of ACBP/DBI increase resulting in the activation of lipid anabolism, this effect has been demonstrated to be the link between obesity and autophagy inhibition. Here, we detail three different protocols for the detection of the ACBP/DBI levels by immunofluorescence, image flow cytometry or immunoblot techniques, which allow the quantification of ACBP/DBI levels and, indirectly, its autophagy-dependent release.

Epidermal Acyl-CoA-binding protein is indispensable for systemic energy homeostasis

ObjectivesThe skin is the largest sensory organ of the human body and plays a fundamental role in regulating body temperature. However, adaptive alterations in skin functions and morphology have only vaguely been associated with physiological responses to cold stress or sensation of ambient temperatures. We previously found that loss of acyl-CoA-binding protein (ACBP) in keratinocytes upregulates lipolysis in white adipose tissue and alters hepatic lipid metabolism, suggesting a link between epidermal barrier functions and systemic energy metabolism. MethodsTo assess the physiological responses to loss of ACBP in keratinocytes in detail, we used full-body ACBP−/− and skin-specific ACBP−/− knockout mice to clarify how loss of ACBP affects 1) energy expenditure by indirect calorimetry, 2) response to high-fat feeding and a high oral glucose load, and 3) expression of brown-selective gene programs by quantitative PCR in inguinal WAT (iWAT). To further elucidate the role of the epidermal barrier in systemic energy metabolism, we included mice with defects in skin structural proteins (ma/ma Flgft/ft) in these studies. ResultsWe show that the ACBP−/− mice and skin-specific ACBP−/− knockout mice exhibited increased energy expenditure, increased food intake, browning of the iWAT, and resistance to diet-induced obesity. The metabolic phenotype, including browning of the iWAT, was reversed by housing the mice at thermoneutrality (30 °C) or pharmacological β-adrenergic blocking. Interestingly, these findings were phenocopied in flaky tail mice (ma/ma Flgft/ft). Taken together, we demonstrate that a compromised epidermal barrier induces a β-adrenergic response that increases energy expenditure and browning of the white adipose tissue to maintain a normal body temperature. ConclusionsOur findings show that the epidermal barrier plays a key role in maintaining systemic metabolic homeostasis. Thus, regulation of epidermal barrier functions warrants further attention to understand the regulation of systemic metabolism in further detail.

Crystal structure of the rice acyl-CoA-binding protein OsACBP2 in complex with C18:3-CoA reveals a novel pattern of binding to acyl-CoA esters.

Acyl-CoA-binding proteins (ACBPs) are a family of proteins that bind acyl-CoA esters at a conserved acyl-CoA-binding domain. ACBPs maintain intracellular acyl-CoA pools to regulate lipid metabolism. Here, we report on the structure of rice OsACBP2 in complex with C18:3-CoA ester. The residues Y33, K34 and K56 of OsACBP2 play a crucial role in binding the CoA group, while residues N23, L27, K52 and Y55 in one molecule of OsACBP2 cooperate with L27, L28, A59 and A62 from another anchoring the fatty acyl group. Multiangle light scattering assays indicate that OsACBP2 binds C18:3-CoA as a monomer. The first complex structure of a plant ACBP binding with C18:3-CoA is therefore presented, providing a novel model for the interaction between an acyl-CoA ester and the acyl-CoA-binding domain(s).

The overexpression of OsACBP5 protects transgenic rice against necrotrophic, hemibiotrophic and biotrophic pathogens

The most devastating diseases in rice (Oryza sativa) are sheath blight caused by the fungal necrotroph Rhizoctonia solani, rice blast by hemibiotrophic fungus Magnaporthe oryzae, and leaf blight by bacterial biotroph Xanthomonas oryzae (Xoo). It has been reported that the Class III acyl-CoA-binding proteins (ACBPs) such as those from dicots (Arabidopsis and grapevine) play a role in defence against biotrophic pathogens. Of the six Arabidopsis (Arabidopsis thaliana) ACBPs, AtACBP3 conferred protection in transgenic Arabidopsis against Pseudomonas syringae, but not the necrotrophic fungus, Botrytis cinerea. Similar to Arabidopsis, rice possesses six ACBPs, designated OsACBPs. The aims of this study were to test whether OsACBP5, the homologue of AtACBP3, can confer resistance against representative necrotrophic, hemibiotrophic and biotrophic phytopathogens and to understand the mechanisms in protection. Herein, when OsACBP5 was overexpressed in rice, the OsACBP5-overexpressing (OsACBP5-OE) lines exhibited enhanced disease resistance against representative necrotrophic (R. solani & Cercospora oryzae), hemibiotrophic (M. oryzae & Fusarium graminearum) and biotrophic (Xoo) phytopathogens. Progeny from a cross between OsACBP5-OE9 and the jasmonate (JA)-signalling deficient mutant were more susceptible than the wild type to infection by the necrotroph R. solani. In contrast, progeny from a cross between OsACBP5-OE9 and the salicylic acid (SA)-signalling deficient mutant was more susceptible to infection by the hemibiotroph M. oryzae and biotroph Xoo. Hence, enhanced resistance of OsACBP5-OEs against representative necrotrophs appears to be JA-dependent whilst that to (hemi)biotrophs is SA-mediated.

Characterization and function of a sunflower (Helianthus annuus L.) Class II acyl-CoA-binding protein

Acyl-CoA-binding proteins (ACBP) bind to long-chain acyl-CoA esters and phospholipids, enhancing the activity of different acyltransferases in animals and plants. Nevertheless, the role of these proteins in the synthesis of triacylglycerols (TAGs) remains unclear. Here, we cloned a cDNA encoding HaACBP1, a Class II ACBP from sunflower (Helianthus annuus), one of the world’s most important oilseed crop plants. Transcriptome analysis of this gene revealed strong expression in developing seeds from 16 to 30 days after flowering. The recombinant protein (rHaACBP1) was expressed in Escherichia coli and purified to be studied by in vitro isothermal titration calorimetry and for phospholipid binding. Its high affinity for saturated palmitoyl-CoA (16:0-CoA; KD 0.11 μM) and stearoyl-CoA (18:0-CoA; KD 0.13 μM) esters suggests that rHaACBP1 could act in acyl-CoA transfer pathways that involve saturated acyl derivatives. Furthermore, rHaACBP1 also binds to both oleoyl-CoA (18:1-CoA; KD 6.4 μM) and linoleoyl-CoA (18:2-CoA; KD 21.4 μM) esters, the main acyl-CoA substrates used to synthesise the TAGs that accumulate in sunflower seeds. Interestingly, rHaACBP1 also appears to bind to different species of phosphatidylcholines (dioleoyl-PC and dilinoleoyl-PC), glycerolipids that are also involved in TAG synthesis, and while it interacts with dioleoyl-PA, this is less prominent than its binding to the PC derivative. Expression of rHaACBP in yeast alters its fatty acid composition, as well as the composition and size of the host acyl-CoA pool. These results suggest that HaACBP1 may potentially fulfil a role in the transport and trafficking of acyl-CoAs during sunflower seed development.

Pheromone biosynthetic pathway and chemoreception proteins in sex pheromone gland of Eogystia hippophaecolus

The moth Eogystia hippophaecolus (Hua et al.) is a major threat to sea buckthorn plantations in China. Specific and highly efficient artificial sex pheromone traps have been developed and used to control this pest species. However, the biosynthesis of sex pheromones Z7-14: Ac and E3-14:Ac remains poorly understood. We investigated the female pheromone gland transcriptome of E. hippophaecolus and identified two pheromone biosynthesis-activating neuropeptides (PBANs), two pheromone biosynthesis-activating neuropeptide receptors (PBANrs), five acetyl-CoA carboxylases (ACCs), six fatty acid synthases (FASs), 16 Acyl-CoA desaturases (DESs), 26 reductases (REDs), 13 acetyltransferases (ACTs), one fatty acid transport protein (FATP), one acyl-CoA-binding protein (ACBP), and five elongation of very long-chain fatty acid proteins (ELOs) in pheromone biosynthesis pathways. Additionally, we identified 11 odorant-degrading enzymes (ODEs) and 16 odorant-binding proteins (OBPs), 14 chemosensory proteins (CSPs), two sensory neuron membrane proteins (SNMPs), three odorant receptors (ORs), seven ionotropic receptors (IRs), and six gustatory receptors (GRs). 77 unigenes involved in female pheromone biosynthesis, 31 chemoreception proteins and 11 odorant degradation enzymes were identified, which provided insight into the regulation of the pheromone components and pheromone recognition in the sex pheromone gland, and knowledge pertinent to new integrated pest management strategy of interference pheromone biosynthesis and recognition.

Gut microbiota-derived indole 3-propionic acid protects against radiation toxicity via retaining acyl-CoA-binding protein

BackgroundWe have proved fecal microbiota transplantation (FMT) is an efficacious remedy to mitigate acute radiation syndrome (ARS); however, the mechanisms remain incompletely characterized. Here, we aimed to tease apart the gut microbiota-produced metabolites, underpin the therapeutic effects of FMT to radiation injuries, and elucidate the underlying molecular mechanisms.ResultsFMT elevated the level of microbial-derived indole 3-propionic acid (IPA) in fecal pellets from irradiated mice. IPA replenishment via oral route attenuated hematopoietic system and gastrointestinal (GI) tract injuries intertwined with radiation exposure without precipitating tumor growth in male and female mice. Specifically, IPA-treated mice represented a lower system inflammatory level, recuperative hematogenic organs, catabatic myelosuppression, improved GI function, and epithelial integrity following irradiation. 16S rRNA gene sequencing and subsequent analyses showed that irradiated mice harbored a disordered enteric bacterial pattern, which was preserved after IPA administration. Notably, iTRAQ analysis presented that IPA replenishment retained radiation-reprogrammed protein expression profile in the small intestine. Importantly, shRNA interference and hydrodynamic-based gene delivery assays further validated that pregnane X receptor (PXR)/acyl-CoA-binding protein (ACBP) signaling played pivotal roles in IPA-favored radioprotection in vitro and in vivo.ConclusionsThese evidences highlight that IPA is a key intestinal microbiota metabolite corroborating the therapeutic effects of FMT to radiation toxicity. Owing to the potential pitfalls of FMT, IPA might be employed as a safe and effective succedaneum to fight against accidental or iatrogenic ionizing ARS in clinical settings. Our findings also provide a novel insight into microbiome-based remedies toward radioactive diseases.5xbhwSC1V4FsDQpg8AwjrSVideo abstract.

Examination and characterisation of burst spinal cord stimulation on cerebrospinal fluid cellular and protein constituents in patient responders with chronic neuropathic pain - A Pilot Study

IntroductionPatients with neuropathic pain have altered proteomic and neuropeptide constituents in cerebrospinal fluid (CSF) compared to controls. Tonic spinal cord stimulation (SCS) has demonstrated differential expression of neuropeptides in CSF before and after treatment suggesting potential mechanisms of action. Burst-SCS is an evidence-based paraesthesia free waveform utilised for neuropathic pain with a potentially different mechanistic action to tonic SCS. This study examines the dynamic biological changes of CSF at a cellular and proteome level after Burst-SCS. MethodsPatients with neuropathic pain selected for SCS had CSF sampled prior to implant of SCS and following 8 weeks of continuous Burst-SCS. Baseline and 8-week pain scores with demographics were recorded. T cell frequencies were analysed by flow cytometry, proteome analysis was performed using mass spectrometry and secreted cytokines, chemokines and neurotrophins were measured by enzyme-linked immunosorbent assay (ELISA). Results4 patients (2 females, 2 males) with a mean age of 51 years (+/-SEM 2.74, SD 5.48) achieved a reduction in pain of >50% following 8 weeks of Burst-SCS. Analysis of the CSF proteome indicated a significant alteration in protein expression most related to synapse assembly and immune regulators. There was significantly lower expression of the proteins: growth hormone A1 (PRL), somatostatin (SST), nucleobindin-2 (NUCB2), Calbindin (CALB1), acyl-CoA binding protein (DBI), proSAAS (PCSK1N), endothelin-3 (END3) and cholecystokinin (CCK) after Burst-SCS. The concentrations of secreted chemokines and cytokines and the frequencies of T cells were not significantly changed following Burst-SCS. ConclusionThis study characterised the alteration in the CSF proteome in response to burst SCS in vivo. Functional analysis indicated that the alterations in the CSF proteome is predominately linked to synapse assembly and immune effectors. Individual protein analysis also suggests potential supraspinal mechanisms.

The Effects of Histidine Tags on the Energy Landscape on Acyl Co‐A Binding Protein

Protein epitope tags play an important role in protein characterization, allowing for easy detection, tracking and isolation in cellular and biochemical experiments. A polyhistidine tag is an amino acid motif that consists of at least six histidine (His) residues, often located at the N‐ or C‐terminus of the protein. Affinity tags, like His tags, are routinely employed as convenient means of purifying recombinantly expressed proteins. However there have been increasing reports suggesting that His tags interfere with the structure and function of proteins. We hypothesize that His tags affect the energy landscape of proteins. Acyl Co‐A Binding Protein (ACBP) is a single‐domain protein that is thought to fold in a two‐state process To test our hypothesis we studied the stability and folding kinetics of ACBP with differentially located His tags in the presence of buffers with varying pHs with a fluorimeter and a stopped‐flow spectrometer. First, we found that buffer pH differentially affected the charge of the His tag: At a pH of 5.3 Histidine is protonated and at 7 Histidine is deprotonated. Second, we observed a significant difference in the kinetics and thermodynamics of the C‐Terminus His tagged ACBP relative to the taglesss ACBP. In a pH of 5.3, the C‐Term His tagged ACBP was less stable whereas in a pH of 7 it was more stable. On the other hand, there was no significant difference in the thermodynamics or the kinetics of the N‐term ACBP to the tagless ACBP. Taken together our studies suggest that the location of the His tag (and ionic environment) markedly affected the energy landscape of ACBP.Support or Funding InformationDePauw University‐PDF

Subcellular Localization of Rice Acyl-CoA-Binding Proteins ACBP4 and ACBP5 Supports Their Non-redundant Roles in Lipid Metabolism.

Acyl-CoA-binding proteins (ACBPs), conserved at the acyl-CoA-binding domain, can bind acyl-CoA esters as well as transport them intracellularly. Six ACBPs co-exist in each model plant, dicot Arabidopsis thaliana (thale cress) and monocot Oryza sativa (rice). Although Arabidopsis ACBPs have been studied extensively, less is known about the rice ACBPs. OsACBP4 is highly induced by salt treatment, but down-regulated following pathogen infection, while OsACBP5 is up-regulated by both wounding and pathogen treatment. Their differential expression patterns under various stress treatments suggest that they may possess non-redundant functions. When expressed from the CaMV35S promoter, OsACBP4 and OsACBP5 were subcellularly localized to different endoplasmic reticulum (ER) domains in transgenic Arabidopsis. As these plants were not stress-treated, it remains to be determined if OsACBP subcellular localization would change following treatment. Given that the subcellular localization of proteins may not be reliable if not expressed in the native plant, this study addresses OsACBP4:GFP and OsACBP5:DsRED expression from their native promoters to verify their subcellular localization in transgenic rice. The results indicated that OsACBP4:GFP was targeted to the plasma membrane besides the ER, while OsACBP5:DsRED was localized at the apoplast, in contrast to their only localization at the ER in transgenic Arabidopsis. Differences in tagged-protein localization in transgenic Arabidopsis and rice imply that protein subcellular localization studies are best investigated in the native plant. Likely, initial targeting to the ER in a non-native plant could not be followed up properly to the final destination(s) unless it occurred in the native plant. Also, monocot (rice) protein targeting may not be optimally processed in a transgenic dicot (Arabidopsis), perhaps arising from the different processing systems for routing between them. Furthermore, changes in the subcellular localization of OsACBP4:GFP and OsACBP5:DsRED were not detectable following salt and pathogen treatment, respectively. These results suggest that OsACBP4 is likely involved in the intracellular shuttling of acyl-CoA esters and/or other lipids between the plasma membrane and the ER, while OsACBP5 appears to participate in the extracellular transport of acyl-CoA esters and/or other lipids, suggesting that they are non-redundant proteins in lipid trafficking.