Mechanisms Of Lipid Transport Research Articles

Allosteric coupling of substrate binding and proton translocation in MmpL3 transporter from Mycobacterium tuberculosis.

Infections caused by Mycobacterium spp. are very challenging to treat, and multidrug-resistant strains rapidly spread in human populations. Major contributing factors include the unique physiological features of these bacteria, drug efflux, and the low permeability barrier of their outer membrane. Here, we focus on MmpL3 from Mycobacterium tuberculosis, an essential inner membrane transporter of the resistance-nodulation-division superfamily required for the translocation of mycolic acids in the form of trehalose monomycolates (TMM) from the cytoplasm or plasma membrane to the periplasm or outer membrane. The MmpL3-dependent transport of TMM is essential for the growth of M. tuberculosis in vitro, inside macrophages, and in M. tuberculosis-infected mice. MmpL3 is also a validated target for several recently identified anti-mycobacterial agents. In this study, we reconstituted the lipid transport activity of the purified MmpL3 using a two-lipid vesicle system and established the ability of MmpL3 to actively extract phospholipids from the outer leaflet of a lipid bilayer. In contrast, we found that MmpL3 lacks the ability to translocate the same phospholipid substrate across the plasma membrane indicating that it is not an energy-dependent flippase. The lipid extraction activity was modulated by substitutions in critical charged and polar residues of the periplasmic substrate-binding pocket of MmpL3, coupled to the proton transfer activity of MmpL3 and inhibited by a small molecule inhibitor SQ109. Based on the results, we propose a mechanism of allosteric coupling wherein substrate translocation by MmpL3 is coupled to the energy provided by the downhill transfer of protons. The reconstituted activities will facilitate understanding the mechanism of MmpL3-dependent transport of lipids and the discovery of new therapeutic options for Mycobacterium spp. infections.IMPORTANCEMmpL3 from Mycobacterium tuberculosis is an essential transporter involved in the assembly of the mycobacterial outer membrane. It is also an important target in undergoing efforts to discover new anti-tuberculosis drugs effective against multidrug-resistant strains spreading in human populations. The recent breakthrough structural studies uncovered features of MmpL3 that suggested a possible lipid transport mechanism. In this study, we reconstituted and characterized the lipid transport activity of MmpL3 and demonstrated that this activity is blocked by MmpL3 inhibitors and substrate mimics. We further uncovered the mechanism of how the binding of a substrate in the periplasmic domain is communicated to the transmembrane proton relay of MmpL3. The uncovered mechanism and the developed assays provide new opportunities for mechanistic analyses of MmpL3 function and its inhibition.

Engineered Lipids for Intracellular Reactive Oxygen Species Scavenging in Steatotic Hepatocytes

AbstractIntracellular reactive oxygen species (ROS) in steatotic cells pose a problem due to their potential to cause oxidative stress and cellular damage. Delivering engineered phospholipids to intracellular lipid droplets in steatotic hepatic cells, using the cell's inherent intracellular lipid transport mechanisms are investigated. Initially, it is shown that tail‐labeled fluorescent lipids assembled into liposomes are able to be transported to intracellular lipid droplets in steatotic HepG2 cells and HHL‐5 cells. Further, an antioxidant, an EUK salen–manganese derivative, which has superoxide dismutase‐like and catalase‐like activity, is covalently conjugated to the tail of a phospholipid and formulated as liposomes for administration. Steatotic HepG2 cells and HHL‐5 cells incubated with these antioxidant liposomes have lower intracellular ROS levels compared to untreated controls and non‐covalently formulated antioxidants. This first proof‐of‐concept study illustrates an alternative strategy to equip native organelles in mammalian cells with engineered enzyme activity.

Specific cellular microenvironments for spatiotemporal regulation of StAR and steroid synthesis.

For many years, research in the field of steroid synthesis has aimed to understand the regulation of the rate-limiting step of steroid synthesis, i.e. the transport of cholesterol from the outer to the inner mitochondrial membrane, and identify the protein involved in the conversion of cholesterol into pregnenolone. The extraordinary work by B Clark, J Wells, S R King, and D M Stocco eventually identified this protein and named it steroidogenic acute regulatory protein (StAR). The group's finding was also one of the milestones in understanding the mechanism of nonvesicular lipid transport between organelles. A notable feature of StAR is its high degree of phosphorylation. In fact, StAR phosphorylation in the acute phase is required for full steroid biosynthesis. As a contribution to this subject, our work has led to the characterization of StAR as a substrate of kinases and phosphatases and as an integral part of a mitochondrion-associated multiprotein complex, essential for StAR function and cholesterol binding and mitochondrial transport to yield maximum steroid production. Results allow us to postulate the existence of a specific cellular microenvironment where StAR protein synthesis and activation, along with steroid synthesis and secretion, are performed in a compartmentalized manner, at the site of hormone receptor stimulation, and involving the compartmentalized formation of the steroid molecule-synthesizing complex.

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.

Fatty acid translocase (FAT/CD36) in silver pomfret (Pampus argenteus): Molecular cloning and functional characterization

Understanding the mechanisms of lipid transport and metabolism in fish is crucial to enhance dietary lipid utilization. Here, fatty acid translocase (CD36) gene was characterized in silver pomfret (Pampus argenteus). The open reading frame of silver pomfret cd36 gene was 1395 bp, encoding 464 amino acids. The silver pomfret CD36 protein contained typical transmembrane regions and N-glycosylation modification sites, and was localized to the cytomembrane. The cd36 gene was ubiquitously expressed in all tested tissues, with the highest expression observed in brain tissue. In vivo, both fasting and short-term high-fat feeding could increase cd36 expression in intestinal tissue. In vitro, cd36 expression was induced by palmitic acid, oleic acid, linolenic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid treatment in intestinal tissue. Furthermore, dual-luciferase reporter assay results indicated that peroxisome proliferator-activated receptor gamma (PPARγ) could enhance cd36 promoter activity, and the co-expression of cd36 and pparγ was observed in EPA-incubated intestine, suggesting that EPA may regulate the expression of cd36 via PPARγ to maintain the homeostasis of intestinal lipid metabolism in silver pomfret. These results highlighted the crucial role of CD36 in silver pomfret, and suggested that the cd36 expression may be regulated by PPARγ. This study could contribute to a greater understanding of lipid metabolism and the development of effective strategies for nutrient requirements in fish.

CSIG-01. DEFINING THE INVOLVEMENT OF THE PERIVASCULAR NICHE IN BRAIN TUMOR METASTASES

Abstract Medulloblastomas (MB) are the most prevalent malignant pediatric brain tumors, originating in either the cerebellum or the dorsal brainstem. They are classified into four distinct subgroups (WNT, SHH, Group 3, and Group 4), characterized by unique gene expression profiles, metastatic patterns, and recurrence rates. Metastatic progression of MB is frequently associated with poor prognosis, affecting approximately 30% of patients at diagnosis. Despite considerable research on primary tumors, there are currently no approved specific treatments for MB leptomeningeal metastases. Thus, gaining further insights into metastatic MB and developing targeted therapies are imperative. The leptomeningeal niche, where metastatic MB tumor cells reside, comprises a complex network of blood vessels supported by perivascular cells, including pericytes, smooth muscle cells, and fibroblasts. However, the specific adhesion molecules and signaling pathways within the leptomeningeal niche that contribute to tumor cell colonization remain elusive. In this study, we hypothesize that the perivascular niche plays a critical role in the colonization and survival of metastatic cells within the leptomeninges. To investigate this, we employed an innovative sLP-mCherry niche labeling system in conjunction with single-cell RNA sequencing to spatially identify and examine the perivascular cells neighboring metastatic MB tumor cells. Through our analysis, we identified upregulated genes associated with cholesterol export in tumor-associated perivascular cells, suggesting their potential involvement in supporting metastatic cell colonization. To further explore this mechanism, we utilized CRISPR and lentiviral overexpression constructs to modulate cholesterol uptake in MB cell lines, observing subsequent changes in metastatic burden. In summary, our study elucidates the importance of the perivascular niche in metastatic medulloblastomas and provides novel insights into the molecular mechanisms that drive tumor cell colonization within the leptomeninges. These investigations into the lipid transport mechanism in the vascular microenvironment opens doors for the development of innovative treatments targeting MB metastases.

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.

Intracellular Membrane Transport in Vascular Endothelial Cells.

The main component of blood and lymphatic vessels is the endothelium covering their luminal surface. It plays a significant role in many cardiovascular diseases. Tremendous progress has been made in deciphering of molecular mechanisms involved into intracellular transport. However, molecular machines are mostly characterized in vitro. It is important to adapt this knowledge to the situation existing in tissues and organs. Moreover, contradictions have accumulated within the field related to the function of endothelial cells (ECs) and their trans-endothelial pathways. This has induced necessity for the re-evaluation of several mechanisms related to the function of vascular ECs and intracellular transport and transcytosis there. Here, we analyze available data related to intracellular transport within ECs and re-examine several hypotheses about the role of different mechanisms in transcytosis across ECs. We propose a new classification of vascular endothelium and hypotheses related to the functional role of caveolae and mechanisms of lipid transport through ECs.

Elucidating bacterial lipid transport mechanisms by the envelope-spanning LetAB complex.

Gram-negative bacteria are surrounded by an outer membrane (OM) that protects them against environmental stressors and toxic compounds, and therefore contributes to antibiotic resistance. To generate this barrier, membrane lipids, which are poorly soluble in water, must be transported across the hydrophilic periplasmic space between the inner membrane (IM) and OM. Bacteria have evolved various mechanisms to achieve active transport across the cell envelope. The Let (Lipophilic Envelope-spanning Tunnel) system, comprising LetA and LetB, is one system thought to be involved in maintaining OM integrity. LetB forms a hydrophobic tunnel long enough to span the periplasm, potentially facilitating the sheltered movement of substrates directly between the two membranes. However, it is unclear how lipids are extracted from the IM and transported into LetB, or vice versa. The only known binding partner of LetB is the IM protein, LetA, which belongs to an unstudied family of proteins. We have obtained a ∼3.5 Å cryo-EM structure of the LetAB complex, which reveals that LetA is a pseudodimeric integral inner membrane protein with two cytoplasmic zinc finger domains that interact with each other to stabilize the protein. LetA binds at one end of LetB, where it interacts with the central tunnel of LetB to form a substrate translocation pathway between the two subunits. Our structural and biochemical data reveal a possible substrate-binding site in LetA. LetA also contains a putative proton/ion permeation pathway, lined with ionizable residues that are essential for function, which we hypothesize may play a role in energizing transport. We will discuss how the LetA and LetB subunits come together to facilitate lipid transport, and provide a model for how proteins in the LetA protein family may function.

Ca2+ and Annexins - Emerging Players for Sensing and Transferring Cholesterol and Phosphoinositides via Membrane Contact Sites.

Maintaining lipid composition diversity in membranes from different organelles is critical for numerous cellular processes. However, many lipids are synthesized in the endoplasmic reticulum (ER) and require delivery to other organelles. In this scenario, formation of membrane contact sites (MCS) between neighbouring organelles has emerged as a novel non-vesicular lipid transport mechanism. Dissecting the molecular composition of MCS identified phosphoinositides (PIs), cholesterol, scaffolding/tethering proteins as well as Ca2+ and Ca2+-binding proteins contributing to MCS functioning. Compelling evidence now exists for the shuttling of PIs and cholesterol across MCS, affecting their concentrations in distinct membrane domains and diverse roles in membrane trafficking. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) at the plasma membrane (PM) not only controls endo-/exocytic membrane dynamics but is also critical in autophagy. Cholesterol is highly concentrated at the PM and enriched in recycling endosomes and Golgi membranes. MCS-mediated cholesterol transfer is intensely researched, identifying MCS dysfunction or altered MCS partnerships to correlate with de-regulated cellular cholesterol homeostasis and pathologies. Annexins, a conserved family of Ca2+-dependent phospholipid binding proteins, contribute to tethering and untethering events at MCS. In this chapter, we will discuss how Ca2+ homeostasis and annexins in the endocytic compartment affect the sensing and transfer of cholesterol and PIs across MCS.

Maternal obesity: new placental paradigms unfolded.

The prevalence of maternal obesity is increasing at an alarming rate, and is providing a major challenge for obstetric practice. Adverse effects on maternal and fetal health are mediated by complex interactions between metabolic, inflammatory, and oxidative stress signaling in the placenta. Endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) are common downstream pathways of cell stress, and there is evidence that this conserved homeostatic response may be a key mediator in the pathogenesis of placental dysfunction. We summarize the current literature on the placental cellular and molecular changes that occur in obese women. A special focus is cast onto placental ER stress in obese pregnancy, which may provide a novel link for future investigation.

Novel pathways of intracellular membrane lipid transport and neurodegenerative diseases

Membrane lipids move from one compartment to another within cells as part of the membranes of vesicular carriers. However, this mode of lipid traffic is complemented by the action of lipid transport proteins that often act at membrane contact sites. Typically, lipid transport by these proteins is achieved by lipid binding modules that shuttle back and forth between the participating membranes. However, recently, studies of VPS13 family proteins (which also comprise the autophagy factor ATG2) have revealed a new mechanism of lipid transfer mediated by rod‐like proteins that bridge two adjacent bilayers and harbor a hydrophobic groove that runs along their entire length. Most interestingly, loss of function mutations of members of this protein family result in neurodegenerative or neurodevelopmental diseases, including Parkinson’s disease (VPS13C) and a Huntington‐like syndrome called chorea acanthocytosis (VPS13A). The talk will provide an overview of the known properties and functions of mammalian VPS13 family proteins and of potential roles of their dysfunction in disease (Leonzino M, Reinisch KM and De Camilli P. 2021. Insights into VPS13 properties and function reveal a new mechanism of eukaryotic lipid transport. PMID: 34216812).

First person – Yiwei Zhang and Hui Tu

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Yiwei Zhang and Hui Tu are co-first authors on ‘ Oligopeptide transporter Slc15A modulates macropinocytosis in Dictyostelium by maintaining intracellular nutrient status’, published in JCS. Yiwei is a PhD student in the lab of Huaqing Cai at Institute of Biophysics, Chinese Academy of Sciences, Beijing, China, where she is interested in understanding the interrelationship between cellular nutrient status and macropinocytic activity, and lipid transport. Hui is a PhD student in the same lab, investigating the mechanisms of macropinocytosis, cell migration and lipid transport.

Activation of TMEM16F lipid scramblase: transport of ions and lipids

TMEM16F is a ubiquitously expressed bifunctional transmembrane protein for Ca2+-activated passive phospholipid scrambling and nonselective ion transduction. It plays important physiological roles in blood coagulation, bone development, immunity, inflammation and viral infection. Though recent cryo-EM studies have revealed the structures of mammalian TMEM16F in closed and intermediate states, the open, activated state remains elusive and thus leaves questions about the activation mechanism and pathways of ion and lipid transport.

Understanding the mechanism of lipid transport through the Mla system in E. coli

In bacteria, the outer membrane (OM) constitutes a protective barrier against noxious compounds present in the environment and is the cell’s first line of defense against stressors in the environment, including antibiotics. In Gram-negative bacteria, lipids are one of the main components of the inner and outer membranes, and their transport requires multiple lipid transporters. Studying how lipid transport systems function will provide insights into the mechanism of how the OM barrier is built and maintained, which has the potential to inform on novel antibiotic design.

Elucidating bacterial lipid transport mechanisms by the envelope spanning LetAB complex

Gram-negative bacteria are surrounded by an outer membrane (OM) that shields against environmental stressors and contributes to antibiotic resistance. The OM is an asymmetric bilayer composed of phospholipids and lipopolysaccharide. Trafficking lipids and other hydrophobic molecules between the two membranes, across the hydrophilic periplasm poses a significant challenge. Bacteria have evolved various mechanisms to achieve this. The MCE system, Let (Lipophilic Envelope-spanning), is one system that plays a role in maintaining membrane OM integrity.

Placental adaptation to maternal malnutrition.

Maternal malnutrition gives rise to both short- and long-term consequences for the survival and health of the offspring. As the intermediary between mother and fetus, the placenta has the potential to interpret environmental signals, such as nutrient availability, and adapt to support fetal growth and development. While this potential is present, it is clear that at times placental adaptation fails to occur resulting in poor pregnancy outcomes. This review will focus on placental responses to maternal undernutrition related to changes in placental vascularization and hemodynamics and placental nutrient transport systems across species. While much of the available literature describes placental responses that result in poor fetal outcomes, novel models have been developed to utilize the inherent variation in fetal weight when dams are nutrient restricted to identify placental adaptations that result in normal-weight offspring. Detailed analyses of the spectrum of placental responses to maternal malnutrition point to alternations in placental histoarchitectural and vascular development, amino acid and lipid transport mechanisms, and modulation of immune-related factors. Dietary supplementation with selected nutrients, such as arginine, has the potential to improve placental growth and function through a variety of mechanisms including stimulating cell proliferation, protein synthesis, angiogenesis, vasodilation, and gene regulation. Improved understanding of placental responses to environmental cues is necessary to develop diagnostic and intervention strategies to improve pregnancy outcomes.

Structures of the mycobacterial membrane protein MmpL3 reveal its mechanism of lipid transport.

The mycobacterial membrane protein large 3 (MmpL3) transporter is essential and required for shuttling the lipid trehalose monomycolate (TMM), a precursor of mycolic acid (MA)-containing trehalose dimycolate (TDM) and mycolyl arabinogalactan peptidoglycan (mAGP), in Mycobacterium species, including Mycobacterium tuberculosis and Mycobacterium smegmatis. However, the mechanism that MmpL3 uses to facilitate the transport of fatty acids and lipidic elements to the mycobacterial cell wall remains elusive. Here, we report 7 structures of the M. smegmatis MmpL3 transporter in its unbound state and in complex with trehalose 6-decanoate (T6D) or TMM using single-particle cryo-electron microscopy (cryo-EM) and X-ray crystallography. Combined with calculated results from molecular dynamics (MD) and target MD simulations, we reveal a lipid transport mechanism that involves a coupled movement of the periplasmic domain and transmembrane helices of the MmpL3 transporter that facilitates the shuttling of lipids to the mycobacterial cell wall.

Effect of overwintering on body composition, antioxidant enzyme activities, fatty acid composition, glucose and lipid-metabolic related gene expression of grass carp (Ctenopharyngodon idellus)

During overwintering in aquaculture, fish are subjected to the coordinated stress of low temperature and starvation. This study investigates the physiological strategies in response to overwintering in grass carp, including glucose and lipid utilization. Grass carp were sampled at 0, 8, and 16 weeks of overwintering. Then, body composition, antioxidant enzyme activities, fatty acid composition, as well as glucose and lipid metabolic gene expression were determined in hepatopancreas, muscle, and adipose tissue. The results indicated that the three tissues exhibited distinct glucose and lipid metabolisms during overwintering. During overwintering, lipolysis was activated in adipose tissue through influencing ATGL gene expression. In hepatopancreas, triglycerides were also utilized during overwintering; starvation of 8 weeks significantly increased the glycogen content via gluconeogenesis activation, but the glycogen content remained steady from the 8th to the 16th week. The muscle firstly utilized glycogen via glycolysis, followed by triglycerides as energy substrates. Interestingly, starvation of 8 weeks significantly increased the triglycerides content of muscle, which may be dependent on LPL-mediated lipid transport mechanism. Hence, during overwintering, glucose was used mainly during the early stage, and muscle was the main utilization site, while lipids were utilized during the whole stage. According to the results indicating that lipids were continuously utilized as energy substrates, antioxidant enzyme activities demonstrated that adipose tissue is one of the most vulnerable tissues to oxidative stress. In addition, principal component analysis (PCA) of fatty acid composition indicated that SFA was the predominant fatty acid used by grass carp during overwintering. Our results provide critical fundamental information for understanding the physiological responses to overwintering stress and development of nutritional feed before and after overwintering in grass carp.