Lipid Transport Proteins Research Articles

The conformational plasticity of structurally unrelated lipid transport proteins correlates with their mode of action.

Lipid transfer proteins (LTPs) are key players in cellular homeostasis and regulation, as they coordinate the exchange of lipids between different cellular organelles. Despite their importance, our mechanistic understanding of how LTPs function at the molecular level is still in its infancy, mostly due to the large number of existing LTPs and to the low degree of conservation at the sequence and structural level. In this work, we use molecular simulations to characterize a representative dataset of lipid transport domains (LTDs) of 12 LTPs that belong to 8 distinct families. We find that despite no sequence homology nor structural conservation, the conformational landscape of LTDs displays common features, characterized by the presence of at least 2 main conformations whose populations are modulated by the presence of the bound lipid. These conformational properties correlate with their mechanistic mode of action, allowing for the interpretation and design of experimental strategies to further dissect their mechanism. Our findings indicate the existence of a conserved, fold-independent mechanism of lipid transfer across LTPs of various families and offer a general framework for understanding their functional mechanism.

Exploring the pathological mechanisms underlying Cohen syndrome.

Cohen Syndrome (CS) is a rare autosomal recessive disorder caused by biallelic mutations in the VPS13B gene. It is characterized by multiple clinical features, including acquired microcephaly, developmental delay, intellectual disability, neutropenia, and retinal degeneration. VPS13B is part of the bridge-like lipid transport (BLTP) protein family, which in mammals also includes VPS13A, -C, and -D. The proteins of this family are peripheral membrane proteins with different sub-cellular localization, but all share similar structural features and have been proposed to act as lipid transport proteins at organellar membrane contact sites. VPS13B is localized at the Golgi apparatus and is essential for the maintenance of organelle architecture. Here we present a review of the experimental data on the function of the protein at the cellular level, discussing the potential link with disease phenotype and review the studies on animal models recapitulating features of the human disease.

Retinol Binding Protein 4 Serves as a Potential Tumor Biomarker and Promotes Malignant Behavior in Gastric Cancer.

Gastric cancer (GC) is a highly phenotypically heterogeneous disease and is caused by a combination of factors. Retinol binding protein 4 (RBP4) is a member of a family of lipid transport proteins that are involved in the transport of substances between cells and play a crucial role in a variety of cancers. However, the expression and role of RBP4 in GC remain unknown. In this study, we explored the expression, prognostic significance, immune microenvironment, drug responsiveness and function of associated signaling pathways of RBP4 in GC using web-based bioinformatics tools. Immunohistochemistry and real-time quantitative PCR were utilized to analyze the tissue and cell expression levels of RBP4. CCK-8, colony formation, EDU incorporation, wound healing and transwell assays were applied to demonstrate the effect of RBP4 on GC cell function. Flow cytometric detection of apoptosis after RBP4 knockdown. Nude mice xenograft model elucidates the role of RBP4 for GC in vivo. Related proteins of the RAS signaling pathway were analyzed by employing Western blot assays. RBP4 is highly expressed in GC. RBP4 is closely associated with patient survival and sensitivity to a wide range of antitumor agents. Knockdown of RBP4 promoted apoptosis and inhibited cell proliferation, invasion and migration. RBP4 promotes GC tumorigenesis in vivo. Finally, RBP4 modulates the RAS/RAF/ERK axis. RBP4 may promote gastric carcinogenesis and development through the RAS/RAF/ERK axis and is expected to be a novel target for GC treatment.

Penthorum chinense Pursh extract ameliorates alcohol-related fatty liver disease in mice via the SIRT1/AMPK signaling axis

Penthorum chinense Pursh (P. chinense), a functional food, has been applied to protect the liver against alcohol-related fatty liver disease (ALD) for a long history in China. This study was designed to evaluate the ameliorative activity of the polyphenolic fraction in P. chinense (PGF) depending on the relief of ALD. The ALD mouse model was established by exposing the mice to a Lieber-DeCarli alcohol liquid diet. We found that PGF administration significantly ameliorated alcohol-induced liver injury, steatosis, oxidative stress, and inflammation in mice. Furthermore, alcohol-increased levels of the critical hepatic lipid synthesis proteins sterol regulatory element binding transcription factor (SREBP-1) and diacylglycerol o-acyltransferase 2 (DGAT2) were attenuated by PGF. Similarly, PGF inhibited the expression of the lipid transport protein very low-density lipoprotein receptor (VLDLR). Interestingly, PGF restored alcohol-inhibited expression of carnitine palmitoyltransferase 1 (CPT1) and peroxisome proliferator-activated receptor alpha (PPARα), essential fatty acid β-oxidation proteins. Mechanistic studies revealed that PGF protects against alcohol-induced hepatocyte injury and lipid deposition via the SIRT1/AMPK signaling pathway. In sum, this research clearly demonstrated the protective effects of PGF against ALD, which was mediated by activating SIRT1/AMPK pathways in hepatocytes. We provide a new theoretical basis for using P. chinense as a functional food in ALD.

A proteome perspective on adaptive responses to physiological challenges in marine mammals

While most mammals decrease energy expenditure while fasting, marine mammals such as northern elephant seals fast for several months while undergoing energy-intensive life history stages (e.g., postnatal development, molting, reproduction). Energy for these functions is provided by mobilization of large energy stores in blubber, which is driven by elevation of stress hormone levels across the fast. We used a proteome approach to examine potential mechanisms regulating fasting metabolism and responses to experimental stress challenges in elephant seals. We compared five proteome datasets and identified proteins that were altered by both fasting and experimental stress challenges across multiple life history stages and tissues in seals. These included lipid and hormone transport proteins (e.g., apolipoproteins, corticosteroid-binding globulin, transthyretin), acute phase proteins (e.g., alpha-2-macroglobulin, haptoglobin), antioxidant enzymes (e.g., glutathione peroxidase 3, catalase), adipokines (e.g., mimecan), and metabolic enzymes (e.g., fatty acid synthase, long chain fatty acid CoA ligase), among others. We suggest that these proteins may function as part of a core adaptive responses to physiological challenges in marine mammals. However, our ability to identify components of these responses has been hindered by several practical and technical challenges, and we will discuss the limitations and considerations for using a proteome approach in comparative physiology studies based on our experience. This research was funded by University of the Pacific and Offce of Naval Research. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The impact of multiple abiotic stresses on ns-LTP2.8 gene transcript and ns-LTP2.8 protein accumulation in germinating barley (Hordeum vulgare L.) embryos.

Abiotic stresses occur more often in combination than alone under regular field conditions limiting in more severe way crop production. Stress recognition in plants primarily occurs in the plasma membrane, modification of which is necessary to maintain homeostasis in response to it. It is known that lipid transport proteins (ns-LTPs) participate in modification of the lipidome of cell membranes. Representative of this group, ns-LTP2.8, may be involved in the reaction to abiotic stress of germinating barley plants by mediating the intracellular transport of hydrophobic particles, such as lipids, helping to maintain homeostasis. The ns-LTP2.8 protein was selected for analysis due to its ability to transport not only linear hydrophobic molecules but also compounds with a more complex spatial structure. Moreover, ns-LTP2.8 has been qualified as a member of pathogenesis-related proteins, which makes it particularly important in relation to its high allergenic potential. This paper demonstrates for the first time the influence of various abiotic stresses acting separately as well as in their combinations on the change in the ns-LTP2.8 transcript, ns-LTP2.8 protein and total soluble protein content in the embryonal axes of germinating spring barley genotypes with different ns-LTP2.8 allelic forms and stress tolerance. Tissue localization of ns-LTP2.8 transcript as well as ns-LTP2.8 protein were also examined. Although the impact of abiotic stresses on the regulation of gene transcription and translation processes remains not fully recognized, in this work we managed to demonstrate different impact on applied stresses on the fundamental cellular processes in very little studied tissue of the embryonal axis of barley.

Investigating a Novel Neurodegenerative Disease Toxic Mechanism Involving Lipid Binding Specificity of Amyloid Oligomers.

Exploring the mechanisms underlying the toxicity of amyloid oligomers (AOs) presents a significant opportunity for discovering cures and developing treatments for neurodegenerative diseases. Recently, using a combination of ion mobility spectrometry-mass spectrometry (IMS-MS) and X-ray crystallography (XRC), we showed that the peptide KVKVLWDVIEV, which is the G95W mutant of αB-Crystallin (90-100) and abbreviated as G6W, self-assembles up to a dodecamer that structurally resembles lipid transport proteins. The glycine to tryptophan mutation promotes not only larger oligomers and enhanced cytotoxicity in brain slices than the wild type but also a narrow hydrophobic cavity suitable for fatty acid or phospholipid binding. Here, we determine the plausibility of a novel cytotoxic mechanism where the G6W's structural motif could perturb lipid homeostasis by determining its lipid binding selectivity and specificity. We show that the G6W oligomers have a strong affinity toward unsaturated phospholipids with a preference toward phospholipids containing 16-C alkyl chains. Molecular dynamics simulations demonstrate how an unsaturated, 16-C phospholipid fits tightly inside and outside G6W's hydrophobic cavity. This binding is exclusive to the G6W peptide, as other amyloid oligomers with different atomic structures, including its wildtype αB-Crystallin (90-100) and several superoxide dismutase 1 (SOD1) peptides that are known to self-assemble into amyloid oligomers (SOD1P28K and SOD1WG-GW), do not experience the same strong binding affinity. While the existing chaperone-lipid hypothesis on amyloid toxicity suggests amyloid-lipid complexes perforate cell membranes, our work provides a new outlook, indicating that soluble amyloid oligomers disrupt lipid homeostasis via selective protein-ligand interactions. The toxic mechanisms may arise from the formation of unique amyloid oligomer structures assisted by lipid ligands or impaired lipid transports.

Andrographolide attenuates sepsis-induced acute kidney injury by inhibiting ferroptosis through the Nrf2/FSP1 pathway

Sepsis is a systemic inflammatory response syndrome caused by infection, which causes renal dysfunction known as sepsis-associated acute kidney injury (S-AKI). Ferroptosis is a form of lipid peroxidation dependent on iron and reactive oxygen species that differs from other forms of programmed cell death at the morphological and biochemical levels. Andrographolide (AG), a natural diterpenoid lactone compound extracted from Andrographis paniculata, has been shown to have therapeutic effects in kidney disease. In this study, we investigated the novel mechanism by which AG attenuates septic acute kidney injury by inhibiting ferroptosis in renal tubular epithelial cells (HK-2) through the Nrf2/FSP1 pathway. Cecum ligation and puncture (CLP)-induced septic rats and lipopolysaccharide (LPS)-induced HK-2 cells were used for in vivo and in vitro experiments. Firstly, in septic rats and HK-2 cells, AG effectively decreased the levels of kidney injury indicators, including blood creatinine, urea nitrogen, and markers of kidney injury such as neutrophil gelatinase-associated lipid transport protein and kidney injury molecule-1 (KIM-1). In addition, AG prevented ferroptotosis, by avoiding the accumulation of iron and lipid peroxidation, and an increase in SLC7A11 and GPX4 in AG-treated HK-2 cells. Furthermore, AG attenuated mitochondrial damage, including mitochondrial swelling, outer membrane rupture, and a reduction in mitochondrial cristae in LPS-treated HK-2 cells. Ferrostatin-1 (Fer-1), a ferroptosis inhibitor, significantly inhibited LPS-induced ferroptosis in HK-2 cells. Importantly, our results confirm that Nrf2/FSP1 is an important pathway for ferroptosis resistance. Nrf2 siRNA hindered the effect of AG in attenuating acute kidney injury and inhibiting ferroptosis. These findings demonstrate that Nrf2/FSP1-mediated HK-2 ferroptosis is associated with AG, alleviates septic acute kidney injury, and indicates a novel avenue for therapeutic interventions in the treatment of acute kidney injury in sepsis.

Abstract 2046 Regulating targeting of bulk lipid transport proteins to membrane contact sites

Abstract 2046 Regulating targeting of bulk lipid transport proteins to membrane contact sites

Uncovering mechanisms of interorganelle lipid transport by enzymatic mass tagging.

Lipid trafficking is critical for the biogenesis and expansion of organelle membranes. Lipid transport proteins (LTPs) have been proposed to facilitate lipid transport at contact sites between organelles. Despite the fundamental importance of LTPs in cell physiology, our knowledge on the mechanisms of interorganelle lipid distribution remains poor due to the scarcity of assays to monitor lipid flux in vivo. In this review, we highlight the recent development of a versatile method named METALIC (Mass tagging-Enabled Tracking of Lipids in Cells), which uses a combination of enzymatic mass tagging and mass spectrometry to track lipid flux between organelles inside living cells. We discuss the methodology, its distinct advantages, limitations as well as its potential to unearth the pipelines of lipid transport and LTP function in vivo.

Defense responses and symbiotic functional initiation in trifoliate orange‒arbuscular mycorrhizal fungi interaction

Arbuscular mycorrhizal fungi (AMF) trigger beneficial effects on their hosts, but it is unknown how plants modulate their defense responses during root colonization of AMF and the symbiotic benefits are initiated. The purpose of this study was to analyze the root mycorrhizal colonization process of trifoliate orange and the responsive patterns of plant growth, root peroxide hydrogen (H2O2), antioxidant enzymes and their encoding gene expression, and sugar, lipid and phosphate transporter protein gene expression at 7‒56 days of inoculation (doi) with Funneliformis mosseae (Fm). Fm developed appressoriums on the root surface at 7 doi, followed by abundant arbuscules in root cortical cells at 28 doi, intracellular vesicles at 42 doi, and root mycorrhizal colonization rate of 41.54% at 56 doi. Plant growth improvement by Fm started at 28 doi. The immune defense response of roots was initiated at 7 doi, as evidenced by the increase of H2O2 levels and superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activity, as well as the up-regulation of PtMn-SOD, PtCu/Zn-SOD, PtPOD, and PtCAT expression, which lasted until 14 doi. Starting at 28 doi, a sugar transporter gene (PtSWEET), a lipid transporter gene (PtSTR), and a phosphate transporter gene (PtPT6) were initiated to be up-regulated, followed by the up-regulation of PtSTR2, PtPT3, and PtPT5 at 42 doi and PtFe-SOD at 56 doi. Arbuscule formation and plant growth improvement together at 28 doi suggested that arbuscules trigger improved growth responses of host plants. This study also reveals the initiation of host immune defense response and function in early root AMF colonization.Graphical

LAMinar Flow: Sterol Transport in a Pathogenic Yeast.

Cryptococcus neoformans is an important fungal pathogen, responsible for over 140,000 deaths per year worldwide. Like other yeasts, C. neoformans relies on ergosterol as its major membrane sterol and carefully regulates its synthesis and distribution. Ergosterol is also targeted by two of the three compound classes currently used to treat cryptococcal infection. We recently reported the discovery and characterization in C. neoformans of a single retrograde ergosterol transporter of the LAM family, Ysp2. Here we review these findings and discuss directions for future research, including the connections between processes that are perturbed by the absence of Ysp2 (which also abrogates cryptococcal virulence) and possible roles for Ysp2 and other, as yet unknown, lipid transport proteins in this organism.

A Noninvasive Circulating Signature of Combined Right Ventricular Pressure and Volume Overload in Tetralogy of Fallot/Pulmonary Atresia/Major Aortopulmonary Collateral Arteries.

Background: Despite surgical advances, children with tetralogy of Fallot/pulmonary atresia/major aortopulmonary collaterals (TOF/PA/MAPCAs) are subject to chronic right ventricular (RV) pressure and volume overload. Current diagnostic tools do not identify adverse myocardial remodeling and cannot predict progression to RV failure. We sought to identify a noninvasive, circulating signature of the systemic response to right heart stress to follow disease progression. Methods: Longitudinal data were collected from patients with TOF/PA/MAPCAs (N = 5) at the time of (1) early RV pressure overload and (2) late RV pressure and volume overload. Plasma protein and microRNA expression were evaluated using high-throughput data-independent mass spectroscopy and Agilent miR Microarray, respectively. Results: At the time of early RV pressure overload, median patient age was 0.34 years (0.02-9.37), with systemic RV pressures, moderate-severe hypertrophy, and preserved systolic function. Late RV pressure and volume overload occurred at a median age of 4.08 years (1.51-10.83), with moderate RV hypertrophy and dilation, and low normal RV function; 277 proteins were significantly dysregulated (log2FC ≥0.6/≤-0.6, FDR≤0.05), predicting downregulation in lipid transport (apolipoproteins), fibrinolytic system, and extracellular matrix structural proteins (talin 1, profilin 1); and upregulation in the respiratory burst. Increasing RV size and decreasing RV function correlated with decreasing structural protein expression. Similarly, miR expression predicted downregulation of extracellular matrix-receptor interactions and upregulation in collagen synthesis. Conclusion: To our knowledge, we show for the first time a noninvasive protein and miR signature reflecting the systemic response to adverse RV myocardial remodeling in TOF/PA/MAPCAs which could be used to follow disease progression.

Extended-Synaptotagmin-1 and -2 control T cell signaling and function

Upon T-cell activation, the levels of the secondary messenger diacylglycerol (DAG) at the plasma membrane need to be controlled to ensure appropriate T-cell receptor signaling and T-cell functions. Extended-Synaptotagmins (E-Syts) are a family of inter-organelle lipid transport proteins that bridge the endoplasmic reticulum and the plasma membrane. In this study, we identify a novel regulatory mechanism of DAG-mediated signaling for T-cell effector functions based on E-Syt proteins. We demonstrate that E-Syts downmodulate T-cell receptor signaling, T-cell-mediated cytotoxicity, degranulation, and cytokine production by reducing plasma membrane levels of DAG. Mechanistically, E-Syt2 predominantly modulates DAG levels at the plasma membrane in resting-state T cells, while E-Syt1 and E-Syt2 negatively control T-cell receptor signaling upon stimulation. These results reveal a previously underappreciated role of E-Syts in regulating DAG dynamics in T-cell signaling.

A population modification gene drive targeting both Saglin and Lipophorin impairs Plasmodium transmission in Anopheles mosquitoes.

Lipophorin is an essential, highly expressed lipid transport protein that is secreted and circulates in insect hemolymph. We hijacked the Anopheles coluzzii Lipophorin gene to make it co-express a single-chain version of antibody 2A10, which binds sporozoites of the malaria parasite Plasmodium falciparum. The resulting transgenic mosquitoes show a markedly decreased ability to transmit Plasmodium berghei expressing the P. falciparum circumsporozoite protein to mice. To force the spread of this antimalarial transgene in a mosquito population, we designed and tested several CRISPR/Cas9-based gene drives. One of these is installed in, and disrupts, the pro-parasitic gene Saglin and also cleaves wild-type Lipophorin, causing the anti-malarial modified Lipophorin version to replace the wild type and hitch-hike together with the Saglin drive. Although generating drive-resistant alleles and showing instability in its gRNA-encoding multiplex array, the Saglin-based gene drive reached high levels in caged mosquito populations and efficiently promoted the simultaneous spread of the antimalarial Lipophorin::Sc2A10 allele. This combination is expected to decrease parasite transmission via two different mechanisms. This work contributes to the design of novel strategies to spread antimalarial transgenes in mosquitoes, and illustrates some expected and unexpected outcomes encountered when establishing a population modification gene drive.

Comparative proximity biotinylation implicates the small GTPase RAB18 in sterol mobilization and biosynthesis

Loss of functional RAB18 causes the autosomal recessive condition Warburg Micro syndrome. To better understand this disease, we used proximity biotinylation to generate an inventory of potential RAB18 effectors. A restricted set of 28 RAB18 interactions were dependent on the binary RAB3GAP1-RAB3GAP2 RAB18-guanine nucleotide exchange factor complex. Twelve of these 28 interactions are supported by prior reports, and we have directly validated novel interactions with SEC22A, TMCO4, and INPP5B. Consistent with a role for RAB18 in regulating membrane contact sites, interactors included groups of microtubule/membrane-remodeling proteins, membrane-tethering and docking proteins, and lipid-modifying/transporting proteins. Two of the putative interactors, EBP and OSBPL2/ORP2, have sterol substrates. EBP is a Δ8-Δ7 sterol isomerase, and ORP2 is a lipid transport protein. This prompted us to investigate a role for RAB18 in cholesterol biosynthesis. We found that the cholesterol precursor and EBP-product lathosterol accumulates in both RAB18-null HeLa cells and RAB3GAP1-null fibroblasts derived from an affected individual. Furthermore, de novo cholesterol biosynthesis is impaired in cells in which RAB18 is absent or dysregulated or in which ORP2 expression is disrupted. Our data demonstrate that guanine nucleotide exchange factor-dependent Rab interactions are highly amenable to interrogation by proximity biotinylation and may suggest that Micro syndrome is a cholesterol biosynthesis disorder.

Legume nodulation and nitrogen fixation require interaction of DnaJ-like protein and lipid transfer protein.

The lipid transport protein (LTP) product of the AsE246 gene of Chinese milk vetch (Astragalus sinicus) contributes to the transport of plant-synthesized lipids to the symbiosome membranes (SMs) that are required for nodule organogenesis in this legume. However, the mechanisms used by nodule-specific LTPs remain unknown. In this study, a functional protein in the DnaJ-like family, designated AsDJL1, was identified and shown to interact with AsE246. Immunofluorescence showed that AsDJL1 was expressed in infection threads (ITs) and in nodule cells and that it co-localized with rhizobium, and an immunoelectron microscopy assay localized the protein to SMs. Via co-transformation into Nicotiana benthamiana cells, AsDJL1 and AsE246 displayed subcellular co-localization in the cells of this heterologous host. Co-immunoprecipitation assays confirmed that AsDJL1 interacted with AsE246 in nodules. The essential interacting region of AsDJL1 was determined to be the zinc finger domain at its C-terminus. Chinese milk vetch plants transfected with AsDJL1-RNAi had significantly decreased numbers of ITs, nodule primordia and nodules as well as reduced (by 83%) nodule nitrogenase activity compared with the controls. By contrast, AsDJL1 overexpression led to increased nodule fresh weight and nitrogenase activity. RNAi-AsDJL1 also significantly affected the abundance of lipids, especially digalactosyldiacylglycerol, in early-infected roots and transgenic nodules. Taken together, the results of this study provide insights into the symbiotic functions of AsDJL1, which may participate in lipid transport to SMs and play an essential role in rhizobial infection and nodule organogenesis.

Apolipoprotein E genotype affects innate susceptibility to Listeria monocytogenes infection in aged male mice.

Apolipoprotein E (ApoE) is a lipid transport protein that is hypothesized to suppress proinflammatory cytokine production, particularly after stimulation with Toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS). Studies using transgenic ApoE human replacement mice (APOE) expressing one of three different allelic variants suggest that there is a hierarchy in terms of responsiveness to proinflammatory stimuli such as APOE4/E4 > APOE3/E3 > APOE2/E2. In this study, we test the hypothesis that APOE genotype can also predict susceptibility to infection with the facultative intracellular gram-positive bacterium Listeria monocytogenes. We found that bone-marrow-derived macrophages isolated from aged APOE4/E4 mice expressed elevated levels of nitric oxide synthase 2 and were highly resistant to in vitro infection with L. monocytogenes compared to APOE3/E3 and APOE2/E2 mice. However, we did not find statistically significant differences in cytokine or chemokine output from either macrophages or whole splenocytes isolated from APOE2/E2, APOE3/E3, or APOE4/E4 mice following L. monocytogenes infection. In vivo, overall susceptibility to foodborne listeriosis also did not differ by APOE genotype in either young (2 mo old) or aged (15 mo old) C57BL/6 mice. However, we observed a sex-dependent susceptibility to infection in aged APOE2/E2 male mice and a sex-dependent resistance to infection in aged APOE4/E4 male mice that was not present in female mice. Thus, these results suggest that APOE genotype does not play an important role in innate resistance to infection with L. monocytogenes but may be linked to sex-dependent changes that occur during immune senescence.

Crystal Structure of the ORP8 Lipid Transport ORD Domain: Model of Lipid Transport.

ORPs are lipid-transport proteins belonging to the oxysterol-binding protein family. They facilitate the transfer of lipids between different intracellular membranes, such as the ER and plasma membrane. We have solved the crystal structure of the ORP8 lipid transport domain (ORD8). The ORD8 exhibited a β-barrel fold composed of anti-parallel β-strands, with three α-helices replacing β-strands on one side. This mixed alpha-beta structure was consistent with previously solved structures of ORP2 and ORP3. A large cavity (≈1860 Å3) within the barrel was identified as the lipid-binding site. Although we were not able to obtain a lipid-bound structure, we used computer simulations based on our crystal structure to dock PS and PI4P molecules into the putative lipid-binding site of the ORD8. Comparative experiments between the short ORD8ΔLid (used for crystallography) and the full-length ORD8 (lid containing) revealed the lid's importance for stable lipid binding. Fluorescence assays revealed different transport efficiencies for PS and PI4P, with the lid slowing down transport and stabilizing cargo. Coarse-grained simulations highlighted surface-exposed regions and hydrophobic interactions facilitating lipid bilayer insertion. These findings enhance our comprehension of ORD8, its structure, and lipid transport mechanisms, as well as provide a structural basis for the design of potential inhibitors.

RBG Motif Bridge-Like Lipid Transport Proteins: Structure, Functions, and Open Questions.

The life of eukaryotic cells requires the transport of lipids between membranes, which are separated by the aqueous environment of the cytosol. Vesicle-mediated traffic along the secretory and endocytic pathways and lipid transfer proteins (LTPs) cooperate in this transport. Until recently, known LTPs were shown to carry one or a few lipids at a time and were thought to mediate transport by shuttle-like mechanisms. Over the last few years, a new family of LTPs has been discovered that is defined by a repeating β-groove (RBG) rod-like structure with a hydrophobic channel running along their entire length. This structure and the localization of these proteins at membrane contact sites suggest a bridge-like mechanism of lipid transport. Mutations in some of these proteins result in neurodegenerative and developmental disorders. Here we review the known properties and well-established or putative physiological roles of these proteins, and we highlight the many questions that remain open about their functions.