How α-Helical Motifs Form Functionally Diverse Lipid-Binding Compartments.

Lipids are produced site-specifically in cells and then distributed nonrandomly among membranes via vesicular and nonvesicular trafficking mechanisms. The latter involves soluble amphitropic proteins extracting specific lipids from source membranes to function as molecular solubilizers that envelope their insoluble cargo before transporting it to destination sites. Lipid-binding and lipid transfer structural motifs range from multi-ß-strand barrels, to ß-sheet cups and baskets covered by α-helical lids, to multi-α-helical bundles and layers. Here, we focus on how α-helical proteins use amphipathic helical layering and bundling to form modular lipid-binding compartments and discuss the functional consequences. Preformed compartments generally rely on intramolecular disulfide bridging to maintain conformation (e.g., albumins, nonspecific lipid transfer proteins, saposins, nematode polyprotein allergens/antigens). Insights into nonpreformed hydrophobic compartments that expand and adapt to accommodate a lipid occupant are few and provided mostly by the three-layer, α-helical ligand-binding domain of nuclear receptors. The simple but elegant and nearly ubiquitous two-layer, α-helical glycolipid transfer protein (GLTP)-fold now further advances understanding.

Ceramide-1-phosphate transfer protein promotes sphingolipid reorientation needed for binding during membrane interaction.

Lipid transfer proteins acquire and release their lipid cargoes by interacting transiently with source and destination biomembranes. In the GlycoLipid Transfer Protein (GLTP) superfamily, the two-layer all-α-helical GLTP-fold defines proteins that specifically target sphingolipids (SLs) containing either sugar or phosphate headgroups via their conserved but evolutionarily-modified SL recognitions centers. Despite comprehensive structural insights provided by X-ray crystallography, the conformational dynamics associated with membrane interaction and SL uptake/release by GLTP superfamily members have remained unknown. Herein, we report insights gained from molecular dynamics (MD) simulations into the conformational dynamics that enable ceramide-1-phosphate transfer proteins (CPTPs) to acquire and deliver ceramide-1-phosphate (C1P) during interaction with 1-palmitoyl-2-oleoyl phosphatidylcholine bilayers. The focus on CPTP reflects this protein's involvement in regulating pro-inflammatory eicosanoid production and autophagy-dependent inflammasome assembly that drives interleukin (IL-1ß and IL-18) production and release by surveillance cells. We found that membrane penetration by CPTP involved α-6 helix and the α-2 helix N-terminal region, was confined to one bilayer leaflet, and was relatively shallow. Large-scale dynamic conformational changes were minimal for CPTP during membrane interaction or C1P uptake except for the α-3/α-4 helices connecting loop, which is located near the membrane interface and interacts with certain phosphoinositide headgroups. Apart from functioning as a shallow membrane-docking element, α-6 helix was found to adeptly reorient membrane lipids to help guide C1P hydrocarbon chain insertion into the interior hydrophobic pocket of the SL binding site.These findings support a proposed 'hydrocarbon chain-first' mechanism for C1P uptake, in contrast to the 'lipid polar headgroup-first' uptake used by most lipid-transfer proteins.

Ceramide-1-phosphate transfer protein (CPTP) regulation by phosphoinositides.

Ceramide-1-phosphate transfer proteins (CPTPs) are members of the glycolipid transfer protein (GLTP) superfamily that shuttle ceramide-1-phosphate (C1P) between membranes. CPTPs regulate cellular sphingolipid homeostasis in ways that impact programmed cell death and inflammation. CPTP downregulation specifically alters C1P levels in the plasma and trans-Golgi membranes, stimulating proinflammatory eicosanoid production and autophagy-dependent inflammasome-mediated cytokine release. However, the mechanisms used by CPTP to target the trans-Golgi and plasma membrane are not well understood. Here, we monitored C1P intervesicular transfer using fluorescence energy transfer (FRET) and showed that certain phosphoinositides (phosphatidylinositol 4,5 bisphosphate (PI-(4,5)P2) and phosphatidylinositol 4-phosphate (PI-4P)) increased CPTP transfer activity, whereas others (phosphatidylinositol 3-phosphate (PI-3P) and PI) did not. PIPs that stimulated CPTP did not stimulate GLTP, another superfamily member. Short-chain PI-(4,5)P2, which is soluble and does not remain membrane-embedded, failed to activate CPTP. CPTP stimulation by physiologically relevant PI-(4,5)P2 levels surpassed that of phosphatidylserine (PS), the only known non-PIP stimulator of CPTP, despite PI-(4,5)P2 increasing membrane equilibrium binding affinity less effectively than PS. Functional mapping of mutations that led to altered FRET lipid transfer and assessment of CPTP membrane interaction by surface plasmon resonance indicated that di-arginine motifs located in the α-6 helix and the α3-α4 helix regulatory loop of the membrane-interaction region serve as PI-(4,5)P2 headgroup-specific interaction sites. Haddock modeling revealed specific interactions involving the PI-(4,5)P2 headgroup that left the acyl chains oriented favorably for membrane embedding. We propose that PI-(4,5)P2 interaction sites enhance CPTP activity by serving as preferred membrane targeting/docking sites that favorably orient the protein for function.

RNAi-mediated knockdown of gut receptor-like genes prohibitin and α-amylase altered the susceptibility of Galleria mellonella to Cry1AcF toxin.

BACKGROUND: Due to the prolonged usage of Bt-based biopesticides and Bt-transgenic crops worldwide, insects are continually developing resistance against Cry toxins. This resistance may occur if any mechanistic step in the insecticidal process is disrupted possibly because of the alteration in Cry-receptor binding affinity due to mutation in receptor genes. Compared to other lepidopteran insects, Cry receptor-related research has made asymmetric progress in the model insect Galleria mellonella. RESULTS: Present study describes the molecular characterization and functional analysis of five Cry toxin receptor-related genes (prohibitin, GLTP, α-amylase, ADAM and UDP-GT) and a gut repair gene (arylphorin) from the gut tissues of G. mellonella. Protein-protein docking analysis revealed that Cry1AcF putatively binds with all the five candidate proteins, suggesting their receptor-like function. These receptor-like genes were significantly overexpressed in the gut tissues of fourth-instar G. mellonella larvae upon early exposure to a sub-lethal dose of Cry1AcF toxin. However, targeted knockdown (by using bacterially-expressed dsRNAs) of these genes led to variable effect on insect susceptibility to Cry1AcF toxin. Insects pre-treated with prohibitin and α-amylase dsRNA exhibited significant reduction in Cry1AcF-induced mortality, suggesting their probable role as Cry receptor. By contrast, insects pre-treated with GLTP, ADAM and UDP-GT dsRNA exhibited no significant decline in mortality. This maybe explained by the possibility of RNAi feedback regulation (as few of the receptors belong to multigene family) or redundant role of GLTP, ADAM and UDP-GT in Cry intoxication process. CONCLUSION: Since the laboratory culture of G. mellonella develop Bt resistance quite rapidly, findings of the current investigation may provide some useful information for future Cry receptor-related research in the model insect.

miR-196a Upregulation Contributes to Gefitinib Resistance through Inhibiting GLTP Expression.

Tyrosine kinase inhibitor (TKI) therapy has greatly improved lung cancer survival in patients with epidermal growth factor receptor (EGFR) mutations. However, the development of TKI-acquired resistance is the major problem to be overcome. In this study, we found that miR-196a expression was greatly induced in gefitinib-resistant lung cancer cells. To understand the role and mechanism of miR-196a in TKI resistance, we found that miR-196a-forced expression alone increased cell resistance to gefitinib treatment in vitro and in vivo by inducing cell proliferation and inhibiting cell apoptosis. We identified the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) bound to the promoter region of miR-196a and induced miR-196a expression at the transcriptional level. NRF2-forced expression also significantly increased expression levels of miR-196a, and was an upstream inducer of miR-196a to mediate gefitinib resistance. We also found that glycolipid transfer protein (GLTP) was a functional direct target of miR-196a, and downregulation of GLTP by miR-196a was responsible for gefitinib resistance. GLTP overexpression alone was sufficient to increase the sensitivity of lung cancer cells to gefitinib treatment. Our studies identified a new role and mechanism of NRF2/miR-196a/GLTP pathway in TKI resistance and lung tumor development, which may be used as a new biomarker (s) for TKI resistance or as a new therapeutic target in the future.

Functional evaluation of tryptophans in glycolipid binding and membrane interaction by HET-C2, a fungal glycolipid transfer protein.

HET-C2 is a fungal glycolipid transfer protein (GLTP) that uses an evolutionarily-modified GLTP-fold to achieve more focused transfer specificity for simple neutral glycosphingolipids than mammalian GLTPs. Only one of HET-C2's two Trp residues is topologically identical to the three Trp residues of mammalian GLTP. Here, we provide the first assessment of the functional roles of HET-C2 Trp residues in glycolipid binding and membrane interaction. Point mutants HET-C2W208F, HET-C2W208A and HET-C2F149Y all retained >90% activity and 80-90% intrinsic Trp fluorescence intensity; whereas HET-C2F149A transfer activity decreased to ~55% but displayed ~120% intrinsic Trp emission intensity. Thus, neither W208 nor F149 is absolutely essential for activity and most Trp emission intensity (~85-90%) originates from Trp109. This conclusion was supported by HET-C2W109Y/F149Y which displayed ~8% intrinsic Trp intensity and was nearly inactive. Incubation of the HET-C2 mutants with 1-palmitoyl-2-oleoyl-phosphatidylcholine vesicles containing different monoglycosylceramides or presented by lipid ethanol-injection decreased Trp fluorescence intensity and blue-shifted the Trp λmax by differing amounts compared to wtHET-C2. With HET-C2 mutants for Trp208, the emission intensity decreases (~30-40%) and λmax blue-shifts (~12nm) were more dramatic than for wtHET-C2 or F149 mutants and closely resembled human GLTP. When Trp109 was mutated, the glycolipid induced changes in HET-C2 emission intensity and λmax blue-shift were nearly nonexistent. Our findings indicate that the HET-C2 Trp λmax blue-shift is diagnostic for glycolipid binding; whereas the emission intensity decrease reflects higher environmental polarity encountered upon nonspecific interaction with phosphocholine headgroups comprising the membrane interface and specific interaction with the hydrated glycolipid sugar.

Structural insights into lipid-dependent reversible dimerization of human GLTP.

Human glycolipid transfer protein (hsGLTP) forms the prototypical GLTP fold and is characterized by a broad transfer selectivity for glycosphingolipids (GSLs). The GLTP mutation D48V near the `portal entrance' of the glycolipid binding site has recently been shown to enhance selectivity for sulfatides (SFs) containing a long acyl chain. Here, nine novel crystal structures of hsGLTP and the SF-selective mutant complexed with short-acyl-chain monoSF and diSF in different crystal forms are reported in order to elucidate the potential functional roles of lipid-mediated homodimerization. In all crystal forms, the hsGLTP-SF complexes displayed homodimeric structures supported by similarly organized intermolecular interactions. The dimerization interface always involved the lipid sphingosine chain, the protein C-terminus (C-end) and α-helices 6 and 2, but the D48V mutant displayed a `locked' dimer conformation compared with the hinge-like flexibility of wild-type dimers. Differences in contact angles, areas and residues at the dimer interfaces in the `flexible' and `locked' dimers revealed a potentially important role of the dimeric structure in the C-end conformation of hsGLTP and in the precise positioning of the key residue of the glycolipid recognition centre, His140. ΔY207 and ΔC-end deletion mutants, in which the C-end is shifted or truncated, showed an almost complete loss of transfer activity. The new structural insights suggest that ligand-dependent reversible dimerization plays a role in the function of human GLTP.

Role of Gltp in Maturation of Oligodendrocytes Under the Regulation of Nkx2.2.

Myelin, a lipid-enriched multi-layer membrane structure, allows for rapid long-distance saltatory conduction of neuronal impulses. Although glycolipids are the predominant types of lipids in the myelin bilayer, the role of glycolipid transfer protein (GLTP), which selectively mediates the transfer of various glycolipids between phospholipid bilayer, in myelin development and maintenance remains unknown at present. In this study, we identified Gltp as the key lipid metabolism gene in myelin-forming oligodendrocytes (OLs) through integrated omics analysis across independent transcriptomic and single-cell sequencing studies. Gene expression analysis revealed that Gltp is selectively expressed in the differentiated OLs. Functional study demonstrated that its expression is essential for the differentiation of OLs, and promotes the outgrowth of OL membrane. Moreover, we found that the expression of Gltp is regulated by OL-lineage transcriptional factors, such as NKX2.2, OLIG2, SOX10, and MYRF. These findings provide important insights into the unrecognized functions of Gltp in OL differentiation and maturation.

In Silico Docking of Vitamin E Isomers on Transport Proteins.

BACKGROUND: Vitamin E is comprised of α, ß, γ and δ-tocopherols (Ts) and α, ß, γ and δ- tocotrienols (T3s). Vitamin E has neuroprotective antioxidant, anti-cancer, and cholesterol-lowering effects. Intracellular trafficking of these isomers remains largely unknown, except for αT which is selectively transported by αT transfer protein (αTTP). OBJECTIVE: This study aimed to determine the binding of vitamin E isomers on transport proteins using in silico docking. METHODS: Transport proteins were selected using AmiGo Gene Ontology tool based on the same molecular function annotation as αTTP. Protein structures were obtained from the Protein Data Bank. Ligands structures were obtained from ZINC database. In silico docking was performed using SwissDock. RESULTS AND DISCUSSION: A total of 6 transport proteins were found: SEC14-like protein 2, glycolipid transfer protein (GLTP), pleckstrin homology domain-containing family A member 8, collagen type IV alpha-3-binding protein, ceramide-1-phosphate transfer protein and afamin. Compared with other transport proteins, αTTP had the highest affinities for all isomers except ßT3. Binding order of vitamin E isomers toward αTTP was γT > ßT > αT > δT > αT3 > γT3 > δT3 > ßT3. GLTP had a higher affinity for tocotrienols than tocopherols. ßT3 bound stronger to GLTP than αTTP. CONCLUSION: αTTP remained as the most preferred transport protein for most of the isomers. The binding affinity of αT toward αTTP was not the highest than other isomers suggested that other intracellular trafficking mechanisms of these isomers may exist. GLTP may mediate the intracellular transport of tocotrienols, especially ßT3. Improving the bioavailability of these isomers may enhance their beneficial effects to human.

Proteomics of the corpus callosum to identify novel factors involved in hypomyelinated Niemann-Pick Type C disease mice.

Hypomyelination in the central nerves system (CNS) is one of the most obviously pathological features in Niemann-Pick Type C disease (NPC), which is a rare neurodegenerative disorder caused by mutations in the NPC intracellular cholesterol transporter 1 or 2 (Npc1 or Npc2). Npc1 plays key roles in both neurons and oligodendrocytes during myelination, however, the linkage between the disturbed cholesterol transport and inhibited myelination is unrevealed. In this study, mass spectrometry (MS)-based differential quantitative proteomics was applied to compare protein composition in the corpus callosum between wild type (WT) and NPC mice. In total, 3009 proteins from both samples were identified, including myelin structural proteins, neuronal proteins, and astrocyte-specific proteins. In line to hypomyelination, our data revealed downregulation of myelin structural and indispensable proteins in Npc1 mutant mice. Notably, the reduced ceramide synthase 2 (Cers2), UDP glycosyltransferase 8 (Ugt8), and glycolipid transfer protein (Gltp) indicate the altered sphingolipid metabolism in the disease and the involvement of Gltp in myelination. The identification of most reported myelin structural proteins and proteins from other cell types advocates the use of the corpus callosum to investigate proteins in different cell types that regulate myelination.

MicroRNA 196B Regulates HOXA5, HOXB6 and GLTP Expression Levels in Colorectal Cancer Cells.

MiRNAs are non-coding RNAs that play important roles in the pathogenesis of human diseases by regulating target gene expression in specific cells or tissues. Previously we identified colorectal cancer (CRC) associated MIR196B, which was specifically up-regulated in CRC cells and tissue. We also identified 18 putative MIR196B target genes by comparing the mRNAs down-regulated in MIR196B-overexpressed cells with MIR196B target genes predicted by public bioinformatics tools. In this study, we verified the association between MIR196B and three genes, HOXA5, HOXB6 and GLTP. HOXA5, HOXB6 and GLTP transcripts were directly down-regulated by MIR196B. The mRNA and proteins levels of HOXA5, HOXB6 and GLTP were also down-regulated in CRC cells by the up-regulated MIR196B. GLTP protein expression was decreased in CRC tissues compared to adjacent non-tumor tissues. These results suggest that HOXA5, HOXB6, and GLTP were direct target genes of MIR196B in CRC cells, and that the up-regulated MIR196B in CRC tissue regulates the expression levels of HOXA5, HOXB6, and GLTP during colorectal carcinogenesis.

In Vitro Measurement of Sphingolipid Intermembrane Transport Illustrated by GLTP Superfamily Members.

Herein, we describe methodological approaches for measuring in vitro transfer of sphingolipids (SLs) between membranes. The approaches rely on direct tracking of the lipid. Typically, direct tracking involves lipid labeling via attachment of fluorophores or introduction of radioactivity. Members of the GlycoLipid Transfer Protein (GLTP) superfamily are used to illustrate two broadly applicable methods for direct lipid tracking. One method relies on Förster resonance energy transfer (FRET) that enables continuous assessment of fluorophore-labeled SL transfer in real time between lipid donor and acceptor vesicles. The second method relies on tracking of radiolabeled SL transfer by separation of lipid donor and acceptor vesicles at discrete time points. The assays are readily adjustable for assessing lipid transfer (1) between various model membrane assemblies (vesicles, micelles, bicelles, nanodiscs), (2) involving other lipid types by other lipid transfer proteins, (3) with protein preparations that are either crudely or highly purified, and (4) that is spontaneous and occurs in the absence of protein.

Membranes and mammalian glycolipid transferring proteins.

Glycolipids are synthesized in and on various organelles throughout the cell. Their trafficking inside the cell is complex and involves both vesicular and protein-mediated machineries. Most important for the bulk lipid transport is the vesicular system, however, lipids moved by transfer proteins are also becoming more characterized. Here we review the latest advances in the glycolipid transfer protein (GLTP) and the phosphoinositol 4-phosphate adaptor protein-2 (FAPP2) field, from a membrane point of view.