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Xie H, Weinstein H. Recognition of Specific PIP2-subtype Composition Triggers the Allosteric Control Mechanism for Selective Membrane Targeting of Cargo Loading and Release Functions of the Intracellular Sterol Transporter StarD4. J Mol Biol 2025:169157. [PMID: 40246223 DOI: 10.1016/j.jmb.2025.169157] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2024] [Revised: 03/26/2025] [Accepted: 04/10/2025] [Indexed: 04/19/2025]
Abstract
We present a comprehensive, quantitative model of the allosteric molecular mechanisms of selective cholesterol (CHL) uptake and delivery by the StarD4 protein - an intracellular cholesterol trafficking protein that facilitates the crucial non-vesicular sterol transport between the plasma membrane and the endoplasmic reticulum. This sterol-specific transfer protein is essential for maintaining the healthy life of human cells. In its physiological function, StarD4 targets both sterol donor and acceptor membranes via interactions with anionic lipids. Experiments have illuminated the kinetics of this sterol transfer and shown it to be modulated by specific phosphatidylinositol phosphates (PIPs) on the target membrane, but the molecular mechanism of the recognition of the PIP2 subtype by StarD4, and how this affects the direction and kinetics of cholesterol transport remained unclear. By revealing a heretofore unrecognized allosteric mechanism that connects the sterol binding site to the part of the protein embedded in the membrane, we show here how StarD4 can respond with different actions to diverse organelle membranes based on their PIP2-subtype composition, in agreement with physiological and experimental evidence. The trajectories of extensive (millisecond range) molecular dynamics (MD) simulation of the StarD4-membrane interactions we calculated, were analyzed with advanced machine learning and information theory methods. Our findings outline how the specific molecular mechanism for recognizing PIP2-subtypes in membranes by StarD4 couples to the defined allosteric pathway that induces the CHL binding pocket to propagate the signal for either uptake or release of the sterol. The central role determined for allostery in these significant advances in the understanding of intracellular cholesterol trafficking by StarD4, aligns with experimentally determined properties of StarD4 function, and interprets them in experimentally testable atomistic terms that explain function-altering results of mutations.
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Affiliation(s)
- Hengyi Xie
- Department of Physiology and Biophysics, Weill Cornell Medicine, New York, NY 10065, USA
| | - Harel Weinstein
- Department of Physiology and Biophysics, Weill Cornell Medicine, New York, NY 10065, USA; Institute for Computational Biomedicine, Weill Cornell Medicine, New York, NY 10065, USA.
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2
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Yilmaz S, Cizmecioglu O. PI3K Signaling at the Crossroads of Lipid Metabolism and Cancer. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2025; 1479:139-164. [PMID: 39616584 DOI: 10.1007/5584_2024_832] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 04/23/2025]
Abstract
The proto-oncogenic PI3K pathway is crucial for the integration of growth factor signaling and metabolic pathways to facilitate the coordination for cell growth. Since transformed cells have the ability to upregulate their anabolic pathways and selectively modulate a subset of metabolites functioning as anti- or pro-tumorigenic signal mediators, the question of how the levels of these metabolites are regulated has also become the center of attention for cancer researchers. Apart from its well-defined roles in glucose metabolism and peptide anabolism, the PI3K pathway appears to be a significant regulator of lipid metabolism and a potentiator of proto-oncogenic bioactive lipid metabolite signaling. In this review, we aim to describe the crosstalk between the PI3K pathway and bioactive lipid species of the three main lipid classes.
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Affiliation(s)
- Sevval Yilmaz
- Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey
| | - Onur Cizmecioglu
- Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey.
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3
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Chiu PL, Orjuela JD, de Groot BL, Aponte Santamaría C, Walz T. Structure and dynamics of cholesterol-mediated aquaporin-0 arrays and implications for lipid rafts. eLife 2024; 12:RP90851. [PMID: 39222068 PMCID: PMC11368405 DOI: 10.7554/elife.90851] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 09/04/2024] Open
Abstract
Aquaporin-0 (AQP0) tetramers form square arrays in lens membranes through a yet unknown mechanism, but lens membranes are enriched in sphingomyelin and cholesterol. Here, we determined electron crystallographic structures of AQP0 in sphingomyelin/cholesterol membranes and performed molecular dynamics (MD) simulations to establish that the observed cholesterol positions represent those seen around an isolated AQP0 tetramer and that the AQP0 tetramer largely defines the location and orientation of most of its associated cholesterol molecules. At a high concentration, cholesterol increases the hydrophobic thickness of the annular lipid shell around AQP0 tetramers, which may thus cluster to mitigate the resulting hydrophobic mismatch. Moreover, neighboring AQP0 tetramers sandwich a cholesterol deep in the center of the membrane. MD simulations show that the association of two AQP0 tetramers is necessary to maintain the deep cholesterol in its position and that the deep cholesterol increases the force required to laterally detach two AQP0 tetramers, not only due to protein-protein contacts but also due to increased lipid-protein complementarity. Since each tetramer interacts with four such 'glue' cholesterols, avidity effects may stabilize larger arrays. The principles proposed to drive AQP0 array formation could also underlie protein clustering in lipid rafts.
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Affiliation(s)
- Po-Lin Chiu
- Department of Cell Biology, Harvard Medical SchoolBostonUnited States
| | - Juan D Orjuela
- Max Planck Tandem Group in Computational Biophysics, Universidad de los AndesBogotáColombia
- Biomedical Engineering Department, Universidad de los AndesBogotáColombia
| | - Bert L de Groot
- Computational Biomolecular Dynamics Group, Max Planck Institute for Multidisciplinary SciencesGöttingenGermany
| | - Camilo Aponte Santamaría
- Max Planck Tandem Group in Computational Biophysics, Universidad de los AndesBogotáColombia
- Molecular Biomechanics Group, Heidelberg Institute for Theoretical StudiesHeidelbergGermany
| | - Thomas Walz
- Department of Cell Biology, Harvard Medical SchoolBostonUnited States
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4
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Chiu PL, Orjuela JD, de Groot BL, Aponte-Santamaría C, Walz T. Structure and dynamics of cholesterol-mediated aquaporin-0 arrays and implications for lipid rafts. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2023.05.16.540959. [PMID: 37292626 PMCID: PMC10245776 DOI: 10.1101/2023.05.16.540959] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/10/2023]
Abstract
Aquaporin-0 (AQP0) tetramers form square arrays in lens membranes through a yet unknown mechanism, but lens membranes are enriched in sphingomyelin and cholesterol. Here, we determined electron crystallographic structures of AQP0 in sphingomyelin/cholesterol membranes and performed molecular dynamics (MD) simulations to establish that the observed cholesterol positions represent those seen around an isolated AQP0 tetramer and that the AQP0 tetramer largely defines the location and orientation of most of its associated cholesterol molecules. At a high concentration, cholesterol increases the hydrophobic thickness of the annular lipid shell around AQP0 tetramers, which may thus cluster to mitigate the resulting hydrophobic mismatch. Moreover, neighboring AQP0 tetramers sandwich a cholesterol deep in the center of the membrane. MD simulations show that the association of two AQP0 tetramers is necessary to maintain the deep cholesterol in its position and that the deep cholesterol increases the force required to laterally detach two AQP0 tetramers, not only due to protein-protein contacts but also due to increased lipid-protein complementarity. Since each tetramer interacts with four such 'glue' cholesterols, avidity effects may stabilize larger arrays. The principles proposed to drive AQP0 array formation could also underlie protein clustering in lipid rafts.
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5
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Abstract
Cholesterol is an essential lipid species of mammalian cells. Cells acquire it through synthesis in the endoplasmic reticulum (ER) and uptake from lipoprotein particles. Newly synthesized cholesterol is efficiently distributed from the ER to other organelles via lipid-binding/transfer proteins concentrated at membrane contact sites (MCSs) to reach the trans-Golgi network, endosomes, and plasma membrane. Lipoprotein-derived cholesterol is exported from the plasma membrane and endosomal compartments via a combination of vesicle/tubule-mediated membrane transport and transfer through MCSs. In this review, we provide an overview of intracellular cholesterol trafficking pathways, including cholesterol flux from the ER to other membranes, cholesterol uptake from lipoprotein donors and transport from the plasma membrane to the ER, cellular cholesterol efflux to lipoprotein acceptors, as well as lipoprotein cholesterol secretion from enterocytes, hepatocytes, and astrocytes. We also briefly discuss human diseases caused by defects in these processes and therapeutic strategies available in such conditions.
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Affiliation(s)
- Elina Ikonen
- Department of Anatomy and Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, 00100 Helsinki, Finland
- Minerva Foundation Institute for Medical Research, 00290 Helsinki, Finland
| | - Vesa M Olkkonen
- Minerva Foundation Institute for Medical Research, 00290 Helsinki, Finland
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6
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Xie H, Weinstein H. Allosterically coupled conformational dynamics in solution prepare the sterol transfer protein StarD4 to release its cargo upon interaction with target membranes. Front Mol Biosci 2023; 10:1197154. [PMID: 37275961 PMCID: PMC10232897 DOI: 10.3389/fmolb.2023.1197154] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2023] [Accepted: 05/04/2023] [Indexed: 06/07/2023] Open
Abstract
Complex mechanisms regulate the cellular distribution of cholesterol, a critical component of eukaryote membranes involved in regulation of membrane protein functions directly and through the physiochemical properties of membranes. StarD4, a member of the steroidogenic acute regulator-related lipid-transfer (StART) domain (StARD)-containing protein family, is a highly efficient sterol-specific transfer protein involved in cholesterol homeostasis. Its mechanism of cargo loading and release remains unknown despite recent insights into the key role of phosphatidylinositol phosphates in modulating its interactions with target membranes. We have used large-scale atomistic Molecular dynamics (MD) simulations to study how the dynamics of cholesterol bound to the StarD4 protein can affect interaction with target membranes, and cargo delivery. We identify the two major cholesterol (CHL) binding modes in the hydrophobic pocket of StarD4, one near S136&S147 (the Ser-mode), and another closer to the putative release gate located near W171, R92&Y117 (the Trp-mode). We show that conformational changes of StarD4 associated directly with the transition between these binding modes facilitate the opening of the gate. To understand the dynamics of this connection we apply a machine-learning algorithm for the detection of rare events in MD trajectories (RED), which reveals the structural motifs involved in the opening of a front gate and a back corridor in the StarD4 structure occurring together with the spontaneous transition of CHL from the Ser-mode of binding to the Trp-mode. Further analysis of MD trajectory data with the information-theory based NbIT method reveals the allosteric network connecting the CHL binding site to the functionally important structural components of the gate and corridor. Mutations of residues in the allosteric network are shown to affect the performance of the allosteric connection. These findings outline an allosteric mechanism which prepares the CHL-bound StarD4 to release and deliver the cargo when it is bound to the target membrane.
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Affiliation(s)
- Hengyi Xie
- Department of Physiology and Biophysics, Weill Cornell Medicine, New York, NY, United States
| | - Harel Weinstein
- Department of Physiology and Biophysics, Weill Cornell Medicine, New York, NY, United States
- Institute for Computational Biomedicine, Weill Cornell Medicine, New York, NY, United States
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Das O, Masid A, Chakraborty M, Gope A, Dutta S, Bhaumik M. Butyrate driven raft disruption trots off enteric pathogen invasion: possible mechanism of colonization resistance. Gut Pathog 2023; 15:19. [PMID: 37085870 PMCID: PMC10122309 DOI: 10.1186/s13099-023-00545-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/31/2022] [Accepted: 04/09/2023] [Indexed: 04/23/2023] Open
Abstract
The gut microbiome derived short chain fatty acids perform multitude of functions to maintain gut homeostasis. Here we studied how butyrate stymie enteric bacterial invasion in cell using a simplistic binary model. The surface of the mammalian cells is enriched with microdomains rich in cholesterol that are known as rafts and act as entry points for pathogens. We showed that sodium butyrate treated RAW264.7 cells displayed reduced membrane cholesterol and less cholera-toxin B binding coupled with increased membrane fluidity compared to untreated cells indicating that reduced membrane cholesterol caused disruption of lipid rafts. The implication of such cellular biophysical changes on the invasion of enteric pathogenic bacteria was assessed. Our study showed, in comparison to untreated cells, butyrate-treated cells significantly reduced the invasion of Shigella and Salmonella, and these effects were found to be reversed by liposomal cholesterol treatment, increasing the likelihood that the rafts' function against bacterial invasion. The credence of ex vivo studies found to be in concordance in butyrate fed mouse model as evident from the significant drift towards a protective phenotype against virulent enteric pathogen invasion as compared to untreated mice. To produce a cytokine balance towards anti-inflammation, butyrate-treated mice produced more of the gut tissue anti-inflammatory cytokine IL-10 and less of the pro-inflammatory cytokines TNF-α, IL-6, and IFN-γ. In histological studies of Shigella infected gut revealed a startling observation where number of neutrophils infiltration was noted which was correlated with the pathology and was essentially reversed by butyrate treatment. Our results ratchet up a new dimension of our understanding how butyrate imparts resistance to pathogen invasion in the gut.
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Affiliation(s)
- Oishika Das
- ICMR-National Institute of Cholera and Enteric Diseases, P-33 C.I. T Road, Beleghata, Kolkata, West Bengal, 700010, India
| | - Aaheli Masid
- ICMR-National Institute of Cholera and Enteric Diseases, P-33 C.I. T Road, Beleghata, Kolkata, West Bengal, 700010, India
| | - Mainak Chakraborty
- ICMR-National Institute of Cholera and Enteric Diseases, P-33 C.I. T Road, Beleghata, Kolkata, West Bengal, 700010, India
| | - Animesh Gope
- ICMR-National Institute of Cholera and Enteric Diseases, P-33 C.I. T Road, Beleghata, Kolkata, West Bengal, 700010, India
| | - Shanta Dutta
- ICMR-National Institute of Cholera and Enteric Diseases, P-33 C.I. T Road, Beleghata, Kolkata, West Bengal, 700010, India
| | - Moumita Bhaumik
- ICMR-National Institute of Cholera and Enteric Diseases, P-33 C.I. T Road, Beleghata, Kolkata, West Bengal, 700010, India.
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Bruno F, Camuso S, Capuozzo E, Canterini S. The Antifungal Antibiotic Filipin as a Diagnostic Tool of Cholesterol Alterations in Lysosomal Storage Diseases and Neurodegenerative Disorders. Antibiotics (Basel) 2023; 12:antibiotics12010122. [PMID: 36671323 PMCID: PMC9855188 DOI: 10.3390/antibiotics12010122] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2022] [Revised: 01/05/2023] [Accepted: 01/05/2023] [Indexed: 01/10/2023] Open
Abstract
Cholesterol is the most considerable member of a family of polycyclic compounds understood as sterols, and represents an amphipathic molecule, such as phospholipids, with the polar hydroxyl group located in position 3 and the rest of the molecule is completely hydrophobic. In cells, it is usually present as free, unesterified cholesterol, or as esterified cholesterol, in which the hydroxyl group binds to a carboxylic acid and thus generates an apolar molecule. Filipin is a naturally fluorescent antibiotic that exerts a primary antifungal effect with low antibacterial activity, interfering with the sterol stabilization of the phospholipid layers and favoring membrane leakage. This polyene macrolide antibiotic does not bind to esterified sterols, but only to non-esterified cholesterol, and it is commonly used as a marker to label and quantify free cholesterol in cells and tissues. Several lines of evidence have indicated that filipin staining could be a good diagnostic tool for the cholesterol alterations present in neurodegenerative (e.g., Alzheimer's Disease and Huntington Disease) and lysosomal storage diseases (e.g., Niemann Pick type C Disease and GM1 gangliosidosis). Here, we have discussed the uses and applications of this fluorescent molecule in lipid storage diseases and neurodegenerative disorders, exploring not only the diagnostic strength of filipin staining, but also its limitations, which over the years have led to the development of new diagnostic tools to combine with filipin approach.
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Affiliation(s)
- Francesco Bruno
- Regional Neurogenetic Centre (CRN), Department of Primary Care, ASP Catanzaro, 88046 Lamezia Terme, Italy
- Association for Neurogenetic Research (ARN), 88046 Lamezia Terme, Italy
| | - Serena Camuso
- Division of Neuroscience, Department of Psychology, Sapienza University of Rome, 00185 Rome, Italy
| | - Elisabetta Capuozzo
- Department of Biochemical Sciences, Sapienza University of Rome, 00185 Rome, Italy
- Correspondence: (E.C.); (S.C.)
| | - Sonia Canterini
- Division of Neuroscience, Department of Psychology, Sapienza University of Rome, 00185 Rome, Italy
- Correspondence: (E.C.); (S.C.)
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9
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Cologna SM, Pathmasiri KC, Pergande MR, Rosenhouse-Dantsker A. Alterations in Cholesterol and Phosphoinositides Levels in the Intracellular Cholesterol Trafficking Disorder NPC. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2023; 1422:143-165. [PMID: 36988880 DOI: 10.1007/978-3-031-21547-6_5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/30/2023]
Abstract
Lipid mistrafficking is a biochemical hallmark of Niemann-Pick Type C (NPC) disease and is classically characterized with endo/lysosomal accumulation of unesterified cholesterol due to genetic mutations in the cholesterol transporter proteins NPC1 and NPC2. Storage of this essential signaling lipid leads to a sequence of downstream events, including oxidative stress, calcium imbalance, neuroinflammation, and progressive neurodegeneration, another hallmark of NPC disease. These observations have been validated in a growing number of studies ranging from NPC cell cultures and animal models to patient specimens. In recent reports, alterations in the levels of another class of critical signaling lipids, namely phosphoinositides, have been described in NPC disease. Focusing on cholesterol and phosphoinositides, the chapter begins by reviewing the interactions of NPC proteins with cholesterol and their role in cholesterol transport. It then continues to describe the modulation of cholesterol efflux in NPC disease. The chapter concludes with a summary of findings related to the functional consequences of perturbations in phosphoinositides in this fatal disease.
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Affiliation(s)
| | | | - Melissa R Pergande
- Department of Chemistry, University of Illinois Chicago, Chicago, IL, USA
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10
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Rosenhouse-Dantsker A, Gazgalis D, Logothetis DE. PI(4,5)P 2 and Cholesterol: Synthesis, Regulation, and Functions. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2023; 1422:3-59. [PMID: 36988876 DOI: 10.1007/978-3-031-21547-6_1] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Indexed: 03/30/2023]
Abstract
Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is the most abundant membrane phosphoinositide and cholesterol is an essential component of the plasma membrane (PM). Both lipids play key roles in a variety of cellular functions including as signaling molecules and major regulators of protein function. This chapter provides an overview of these two important lipids. Starting from a brief description of their structure, synthesis, and regulation, the chapter continues to describe the primary functions and signaling processes in which PI(4,5)P2 and cholesterol are involved. While PI(4,5)P2 and cholesterol can act independently, they often act in concert or affect each other's impact. The chapters in this volume on "Cholesterol and PI(4,5)P2 in Vital Biological Functions: From Coexistence to Crosstalk" focus on the emerging relationship between cholesterol and PI(4,5)P2 in a variety of biological systems and processes. In this chapter, the next section provides examples from the ion channel field demonstrating that PI(4,5)P2 and cholesterol can act via common mechanisms. The chapter ends with a discussion of future directions.
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Affiliation(s)
| | - Dimitris Gazgalis
- Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, Bouvé College of Health Sciences, Northeastern University, Boston, MA, USA
| | - Diomedes E Logothetis
- Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, Bouvé College of Health Sciences, Northeastern University, Boston, MA, USA
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11
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Role of STAR and SCP2/SCPx in the Transport of Cholesterol and Other Lipids. Int J Mol Sci 2022; 23:ijms232012115. [PMID: 36292972 PMCID: PMC9602805 DOI: 10.3390/ijms232012115] [Citation(s) in RCA: 15] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2022] [Revised: 09/30/2022] [Accepted: 10/08/2022] [Indexed: 11/21/2022] Open
Abstract
Cholesterol is a lipid molecule essential for several key cellular processes including steroidogenesis. As such, the trafficking and distribution of cholesterol is tightly regulated by various pathways that include vesicular and non-vesicular mechanisms. One non-vesicular mechanism is the binding of cholesterol to cholesterol transport proteins, which facilitate the movement of cholesterol between cellular membranes. Classic examples of cholesterol transport proteins are the steroidogenic acute regulatory protein (STAR; STARD1), which facilitates cholesterol transport for acute steroidogenesis in mitochondria, and sterol carrier protein 2/sterol carrier protein-x (SCP2/SCPx), which are non-specific lipid transfer proteins involved in the transport and metabolism of many lipids including cholesterol between several cellular compartments. This review discusses the roles of STAR and SCP2/SCPx in cholesterol transport as model cholesterol transport proteins, as well as more recent findings that support the role of these proteins in the transport and/or metabolism of other lipids.
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12
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Morin EE, Salbato S, Walker BR, Naik JS. Endothelial cell membrane cholesterol content regulates the contribution of TRPV4 channels in ACh-induced vasodilation in rat gracilis arteries. Microcirculation 2022; 29:e12774. [PMID: 35689491 PMCID: PMC10389065 DOI: 10.1111/micc.12774] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2022] [Revised: 05/26/2022] [Accepted: 06/06/2022] [Indexed: 02/06/2023]
Abstract
OBJECTIVE Our previous work demonstrated that endothelial cell (EC) membrane cholesterol is reduced following 48 h of chronic hypoxia (CH). CH couples endothelial transient receptor potential subfamily V member 4 (TRPV4) channels to muscarinic receptor signaling through an endothelium-dependent hyperpolarization (EDH) pathway does not present in control animals. TRVPV4 channel activity has been shown to be regulated by membrane cholesterol. Hence, we hypothesize that acute manipulation of endothelial cell membrane cholesterol inversely determines the contribution of TRPV4 channels to endothelium-dependent vasodilation. METHODS Male Sprague-Dawley rats were exposed to ambient atmospheric (atm.) pressure or 48-h of hypoxia (0.5 atm). Vasodilation to acetylcholine (ACh) was determined using pressure myography in gracilis arteries. EC membrane cholesterol was depleted using methyl-β-cyclodextrin (MβCD) and supplemented with MβCD-cholesterol. RESULTS Inhibiting TRPV4 did not affect ACh-induced vasodilation in normoxic controls. However, TRPV4 inhibition reduced resting diameter in control arteries suggesting basal activity. TRPV4 contributes to ACh-induced vasodilation in these arteries when EC membrane cholesterol is depleted. Inhibiting TRPV4 attenuated ACh-induced vasodilation in arteries from CH animals that exhibit lower EC membrane cholesterol than normoxic controls. EC cholesterol repletion in arteries from CH animals abolished the contribution of TRPV4 to ACh-induced vasodilation. CONCLUSION Endothelial cell membrane cholesterol impedes the contribution of TRPV4 channels in EDH-mediated dilation. These results provide additional evidence for the importance of plasma membrane cholesterol content in regulating intracellular signaling and vascular function.
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Affiliation(s)
- Emily E Morin
- Department of Cell Biology and Physiology, University of New Mexico School of Medicine, Albuquerque, New Mexico, USA
| | - Sophia Salbato
- Department of Cell Biology and Physiology, University of New Mexico School of Medicine, Albuquerque, New Mexico, USA
| | - Benjimen R Walker
- Department of Cell Biology and Physiology, University of New Mexico School of Medicine, Albuquerque, New Mexico, USA
| | - Jay S Naik
- Department of Cell Biology and Physiology, University of New Mexico School of Medicine, Albuquerque, New Mexico, USA
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13
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Pelletier RM, Layeghkhavidaki H, Seidah NG, Prat A, Vitale ML. PCSK9 Contributes to the Cholesterol, Glucose, and Insulin2 Homeostasis in Seminiferous Tubules and Maintenance of Immunotolerance in Testis. Front Cell Dev Biol 2022; 10:889972. [PMID: 35586340 PMCID: PMC9108277 DOI: 10.3389/fcell.2022.889972] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2022] [Accepted: 04/14/2022] [Indexed: 11/25/2022] Open
Abstract
The PCSK9 contribution to cholesterol and immunotolerance homeostasis and response to glucose, and insulin in testis and hypophysis were studied using Pcsk9-deficient (-/-) and transgenic [Tg (PCSK9)] mice, and diabetic, obese ob/ob and db/db mice. The spermatids/spermatozoa acrosome, peritubular vessels, and epididymal adipocytes were PCSK9- and LDL-R-positive. The pro-PCSK9/PCSK9 ratio was high in interstitial tissue-fractions (ITf) and spermatozoa and low in seminiferous tubule-fractions (STf) in normal adult mice. This ratio decreased in ITf in ob/ob and db/db mice but increased in tubules in ob/ob mice. Deleting pcsk9 lowered cholesterol in serum but increased testicular cholesterol. Furthermore, HMGCoA-red, ACAT-2 and LDL-R turnover increased whereas SR-BI decreased in ITf; in tubules, ABCA1 decreased and 160 kDa LDL-R increased in Pcsk9 -/- mice. Excess testicular cholesterol could result from increased cholesterol synthesis and uptake with reduction in SR-BI-mediated efflux in ITf and from the overload of apoptotic cells, lowered ABCA1-mediated efflux and stimulated LDL-R protein synthesis in tubules in Pcsk9 -/- mice. Concomitantly with the cholesterol accumulation, tubules showed infiltrates of immune cells, elevated IL-17A and IL-17RA, and changes in the immunotolerance homeostasis. PCSK9 deficiency decreased glucose in tubules and spermatozoa while increasing insulin2 in ITf and tubules not serum. Moreover, IR-α, and IR-β augmented in tubules but decreased in the anterior pituitary; IR-α increased whereas IR-β decreased in ITf. The histology and cholesterol levels were normal in Tg (PCSK9) mouse testis. The excess cholesterol creates a milieu favorable to the action of high IL-17A and IL-17RA, the development of inflammatory conditions and self-tolerance breakdown in testis.
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Affiliation(s)
- R.-Marc Pelletier
- Department of Pathology and Cell Biology, Université de Montréal, Montreal, QC, Canada
| | - Hamed Layeghkhavidaki
- Department of Pathology and Cell Biology, Université de Montréal, Montreal, QC, Canada
| | - Nabil G. Seidah
- Biochemical Neuroendocrinology Laboratory, Montreal Clinical Research Institute (IRCM), Montreal, QC, Canada
| | - Annik Prat
- Biochemical Neuroendocrinology Laboratory, Montreal Clinical Research Institute (IRCM), Montreal, QC, Canada
| | - María L. Vitale
- Department of Pathology and Cell Biology, Université de Montréal, Montreal, QC, Canada
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14
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Lei XH, Bochner BR. Optimization of cell permeabilization in electron flow based mitochondrial function assays. Free Radic Biol Med 2021; 177:48-57. [PMID: 34656699 DOI: 10.1016/j.freeradbiomed.2021.10.014] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/17/2021] [Revised: 10/05/2021] [Accepted: 10/13/2021] [Indexed: 12/21/2022]
Abstract
Permeable cell models have contributed much to the progress in mitochondrial research. Optimization of permeabilization is required to make the cell's plasma membrane permeable to small molecules while keeping the intracellular organelles and their membranes intact and fully functional. Here we report our assessment and optimization of commonly used permeabilizing agents including different saponin preparations, digitonin, and recombinant perfringolysin O employing a new electron flow based mitochondrial assay technology that utilizes a colorimetric redox dye. The results of this study provide guidance in optimizing the conditions for mitochondrial function assays with permeabilized cells using the novel redox dye-based format.
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Affiliation(s)
- Xiang-He Lei
- Biolog, Inc., 21124 Cabot Blvd., Hayward, CA, 94545, USA
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15
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Goedeke L, Canfrán-Duque A, Rotllan N, Chaube B, Thompson BM, Lee RG, Cline GW, McDonald JG, Shulman GI, Lasunción MA, Suárez Y, Fernández-Hernando C. MMAB promotes negative feedback control of cholesterol homeostasis. Nat Commun 2021; 12:6448. [PMID: 34750386 PMCID: PMC8575900 DOI: 10.1038/s41467-021-26787-7] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2020] [Accepted: 10/14/2021] [Indexed: 12/21/2022] Open
Abstract
Intricate regulatory networks govern the net balance of cholesterol biosynthesis, uptake and efflux; however, the mechanisms surrounding cholesterol homeostasis remain incompletely understood. Here, we develop an integrative genomic strategy to detect regulators of LDLR activity and identify 250 genes whose knockdown affects LDL-cholesterol uptake and whose expression is modulated by intracellular cholesterol levels in human hepatic cells. From these hits, we focus on MMAB, an enzyme which catalyzes the conversion of vitamin B12 to adenosylcobalamin, and whose expression has previously been linked with altered levels of circulating cholesterol in humans. We demonstrate that hepatic levels of MMAB are modulated by dietary and cellular cholesterol levels through SREBP2, the master transcriptional regulator of cholesterol homeostasis. Knockdown of MMAB decreases intracellular cholesterol levels and augments SREBP2-mediated gene expression and LDL-cholesterol uptake in human and mouse hepatic cell lines. Reductions in total sterol content were attributed to increased intracellular levels of propionic and methylmalonic acid and subsequent inhibition of HMGCR activity and cholesterol biosynthesis. Moreover, mice treated with antisense inhibitors of MMAB display a significant reduction in hepatic HMGCR activity, hepatic sterol content and increased expression of SREBP2-mediated genes. Collectively, these findings reveal an unexpected role for the adenosylcobalamin pathway in regulating LDLR expression and identify MMAB as an additional control point by which cholesterol biosynthesis is regulated by its end product.
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Affiliation(s)
- Leigh Goedeke
- Vascular Biology and Therapeutics Program, Yale School of Medicine, New Haven, CT, USA
- Department of Internal Medicine, Yale School of Medicine, New Haven, CT, USA
| | - Alberto Canfrán-Duque
- Vascular Biology and Therapeutics Program, Yale School of Medicine, New Haven, CT, USA
- Integrative Cell Signaling and Neurobiology of Metabolism Program, Department of Comparative Medicine and Pathology, Yale School of Medicine, New Haven, CT, 06520, USA
| | - Noemi Rotllan
- Vascular Biology and Therapeutics Program, Yale School of Medicine, New Haven, CT, USA
- Integrative Cell Signaling and Neurobiology of Metabolism Program, Department of Comparative Medicine and Pathology, Yale School of Medicine, New Haven, CT, 06520, USA
| | - Balkrishna Chaube
- Vascular Biology and Therapeutics Program, Yale School of Medicine, New Haven, CT, USA
- Integrative Cell Signaling and Neurobiology of Metabolism Program, Department of Comparative Medicine and Pathology, Yale School of Medicine, New Haven, CT, 06520, USA
| | - Bonne M Thompson
- Center for Human Nutrition, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA
| | - Richard G Lee
- Cardiovascular Group, Antisense Drug Discovery, Ionis Pharmaceuticals, Carlsbad, CA, 92010, USA
| | - Gary W Cline
- Department of Internal Medicine, Yale School of Medicine, New Haven, CT, USA
| | - Jeffrey G McDonald
- Center for Human Nutrition, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA
| | - Gerald I Shulman
- Department of Internal Medicine, Yale School of Medicine, New Haven, CT, USA
- Department of Cellular & Molecular Physiology, Yale School of Medicine, New Haven, CT, USA
| | - Miguel A Lasunción
- Servicio de Bioquímica-Investigación, Hospital Universitario Ramón y Cajal, Instituto Ramón y Cajal de Investigación Sanitaria (IRyCIS) and CIBER de Fisiopatología de la Obesidad y Nutrición (CIBERobn), Madrid, Spain
| | - Yajaira Suárez
- Vascular Biology and Therapeutics Program, Yale School of Medicine, New Haven, CT, USA
- Integrative Cell Signaling and Neurobiology of Metabolism Program, Department of Comparative Medicine and Pathology, Yale School of Medicine, New Haven, CT, 06520, USA
| | - Carlos Fernández-Hernando
- Vascular Biology and Therapeutics Program, Yale School of Medicine, New Haven, CT, USA.
- Integrative Cell Signaling and Neurobiology of Metabolism Program, Department of Comparative Medicine and Pathology, Yale School of Medicine, New Haven, CT, 06520, USA.
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16
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Schoop V, Martello A, Eden ER, Höglinger D. Cellular cholesterol and how to find it. Biochim Biophys Acta Mol Cell Biol Lipids 2021; 1866:158989. [PMID: 34118431 DOI: 10.1016/j.bbalip.2021.158989] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2021] [Revised: 06/01/2021] [Accepted: 06/02/2021] [Indexed: 01/06/2023]
Abstract
Cholesterol is an essential component of eukaryotic cellular membranes. Information about its subcellular localization and transport pathways inside cells are key for the understanding and treatment of cholesterol-related diseases. In this review we give an overview over the most commonly used methods that contributed to our current understanding of subcellular cholesterol localization and transport routes. First, we discuss methods that provide insights into cholesterol metabolism based on readouts of downstream effects such as esterification. Subsequently, we focus on the use of cholesterol-binding molecules as probes that facilitate visualization and quantification of sterols inside of cells. Finally, we explore different analogues of cholesterol which, when taken up by living cells, are integrated and transported in a similar fashion as endogenous sterols. Taken together, we highlight the challenges and advantages of each method such that researchers studying aspects of cholesterol transport may choose the most pertinent approach for their problem.
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Affiliation(s)
- Valentin Schoop
- Heidelberg University Biochemistry Center (BZH), 69120 Heidelberg, Germany
| | - Andrea Martello
- University College London (UCL), Institute of Ophthalmology, EC1V 9EL London, United Kingdom
| | - Emily R Eden
- University College London (UCL), Institute of Ophthalmology, EC1V 9EL London, United Kingdom
| | - Doris Höglinger
- Heidelberg University Biochemistry Center (BZH), 69120 Heidelberg, Germany.
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17
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Cholesterol metabolism: a new molecular switch to control inflammation. Clin Sci (Lond) 2021; 135:1389-1408. [PMID: 34086048 PMCID: PMC8187928 DOI: 10.1042/cs20201394] [Citation(s) in RCA: 64] [Impact Index Per Article: 16.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2021] [Revised: 05/14/2021] [Accepted: 05/17/2021] [Indexed: 12/30/2022]
Abstract
The immune system protects the body against harm by inducing inflammation. During the immune response, cells of the immune system get activated, divided and differentiated in order to eliminate the danger signal. This process relies on the metabolic reprogramming of both catabolic and anabolic pathways not only to produce energy in the form of ATP but also to generate metabolites that exert key functions in controlling the response. Equally important to mounting an appropriate effector response is the process of immune resolution, as uncontrolled inflammation is implicated in the pathogenesis of many human diseases, including allergy, chronic inflammation and cancer. In this review, we aim to introduce the reader to the field of cholesterol immunometabolism and discuss how both metabolites arising from the pathway and cholesterol homeostasis are able to impact innate and adaptive immune cells, staging cholesterol homeostasis at the centre of an adequate immune response. We also review evidence that demonstrates the clear impact that cholesterol metabolism has in both the induction and the resolution of the inflammatory response. Finally, we propose that emerging data in this field not only increase our understanding of immunometabolism but also provide new tools for monitoring and intervening in human diseases, where controlling and/or modifying inflammation is desirable.
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18
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Zheng Koh DH, Saheki Y. Regulation of Plasma Membrane Sterol Homeostasis by Nonvesicular Lipid Transport. CONTACT (THOUSAND OAKS (VENTURA COUNTY, CALIF.)) 2021; 4:25152564211042451. [PMID: 37366378 PMCID: PMC10259818 DOI: 10.1177/25152564211042451] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 06/28/2023]
Abstract
Sterol contributes to the structural integrity of cellular membranes and plays an important role in the regulation of cell signaling in eukaryotes. It is either produced in the endoplasmic reticulum or taken up from the extracellular environment. In most eukaryotic cells, however, the majority of sterol is enriched in the plasma membrane. Thus, the transport of sterol between the plasma membrane and other organelles, including the endoplasmic reticulum, is crucial for maintaining sterol homeostasis. While vesicular transport that relies on membrane budding and fusion reactions plays an important role in bulk sterol transport, this mode of transport is slow and non-selective. Growing evidence suggests a critical role of nonvesicular transport mediated by evolutionarily conserved families of lipid transfer proteins in more rapid and selective delivery of sterol. Some lipid transfer proteins act primarily at the sites of contacts formed between the endoplasmic reticulum and other organelles or the plasma membrane without membrane fusion. In this review, we describe the similarities and differences of sterol biosynthesis and uptake in mammals and yeast and discuss the role of their lipid transfer proteins in maintaining plasma membrane sterol homeostasis.
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Affiliation(s)
- Dylan Hong Zheng Koh
- Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore 308232, Singapore
| | - Yasunori Saheki
- Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore 308232, Singapore
- Institute of Resource Development and
Analysis, Kumamoto University, Kumamoto 860-0811, Japan
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19
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Shear stress activates mitochondrial oxidative phosphorylation by reducing plasma membrane cholesterol in vascular endothelial cells. Proc Natl Acad Sci U S A 2020; 117:33660-33667. [PMID: 33318210 PMCID: PMC7776821 DOI: 10.1073/pnas.2014029117] [Citation(s) in RCA: 50] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
The mechanotransduction of shear stress in vascular endothelial cells is still not completely understood. We show a pathway of shear stress signal transduction mediated by plasma membrane cholesterol-dependent mitochondrial oxidative phosphorylation. The latest imaging technology using domain 4 mutant-derived cholesterol biosensors and a fluorescence resonance energy transfer-based adenosine triphosphate (ATP) biosensor revealed that shear stress rapidly decreases cholesterol levels in the plasma membrane via both efflux and internalization, and reduction in plasma membrane cholesterol was linked to the activation of mitochondrial ATP production. The addition of cholesterol blocked these shear stress effects. Increased mitochondrial ATP production led to ATP release from the endothelial cells, thereby activating purinoceptors in the plasma membrane and leading to purinergic Ca2+ signaling in response to shear stress. Vascular endothelial cells (ECs) sense and respond to hemodynamic shear stress, which is critical for circulatory homeostasis and the pathophysiology of vascular diseases. The mechanisms of shear stress mechanotransduction, however, remain elusive. We previously demonstrated a direct role of mitochondria in the purinergic signaling of shear stress: shear stress increases mitochondrial adenosine triphosphate (ATP) production, triggering ATP release and Ca2+ signaling via EC purinoceptors. Here, we showed that shear stress rapidly decreases cholesterol in the plasma membrane, thereby activating mitochondrial ATP production. Imaging using domain 4 mutant-derived cholesterol biosensors showed that the application of shear stress to cultured ECs markedly decreased cholesterol levels in both the outer and inner plasma membrane bilayers. Flow cytometry showed that the cholesterol levels in the outer bilayer decreased rapidly after the onset of shear stress, reached a minimum (around 60% of the control level) at 10 min, and plateaued thereafter. After the shear stress ceased, the decreased cholesterol levels returned to those seen in the control. A biochemical analysis showed that shear stress caused both the efflux and the internalization of plasma membrane cholesterol. ATP biosensor imaging demonstrated that shear stress significantly increased mitochondrial ATP production. Similarly, the treatment of cells with methyl-β-cyclodextrin (MβCD), a membrane cholesterol-depleting agent, increased mitochondrial ATP production. The addition of cholesterol to cells inhibited the increasing effects of both shear stress and MβCD on mitochondrial ATP production in a dose-dependent manner. These findings indicate that plasma membrane cholesterol dynamics are closely coupled to mitochondrial oxidative phosphorylation in ECs.
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20
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Balla T, Gulyas G, Kim YJ, Pemberton J. PHOSPHOINOSITIDES AND CALCIUM SIGNALING. A MARRIAGE ARRANGED IN ER-PM CONTACT SITES. CURRENT OPINION IN PHYSIOLOGY 2020; 17:149-157. [PMID: 32944676 DOI: 10.1016/j.cophys.2020.08.007] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Calcium (Ca2+) ions are critically important in orchestrating countless regulatory processes in eukaryotic cells. Consequently, cells tightly control cytoplasmic Ca2+ concentrations using a complex array of Ca2+-selective ion channels, transporters, and signaling effectors. Ca2+ transport through various cellular membranes is highly dependent on the intrinsic properties of specific membrane compartments and conversely, local Ca2+ changes have profound effects on the membrane lipid composition of such membrane sub-domains. In particular, inositol phospholipids are a minor class of phospholipids that play pivotal roles in the control of Ca2+-dependent signaling pathways. In this review, we will highlight some of the recent advances in this field as well as their impact in defining future research directions.
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Affiliation(s)
- Tamas Balla
- Section on Molecular Signal Transduction, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892
| | - Gergo Gulyas
- Section on Molecular Signal Transduction, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892
| | - Yeun Ju Kim
- Section on Molecular Signal Transduction, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892
| | - Joshua Pemberton
- Section on Molecular Signal Transduction, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892
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21
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Zhou Y, Borchelt D, Bauson JC, Fazio S, Miles JR, Tavori H, Notterpek L. Subcellular diversion of cholesterol by gain- and loss-of-function mutations in PMP22. Glia 2020; 68:2300-2315. [PMID: 32511821 DOI: 10.1002/glia.23840] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2019] [Revised: 03/22/2020] [Accepted: 04/24/2020] [Indexed: 12/13/2022]
Abstract
Abnormalities of the peripheral myelin protein 22 (PMP22) gene, including duplication, deletion and point mutations are a major culprit in Type 1 Charcot-Marie-Tooth (CMT) diseases. The complete absence of PMP22 alters cholesterol metabolism in Schwann cells, which likely contributes to myelination deficits. Here, we examined the subcellular trafficking of cholesterol in distinct models of PMP22-linked neuropathies. In Schwann cells from homozygous Trembler J (TrJ) mice carrying a Leu16Pro mutation, cholesterol was retained with TrJ-PMP22 in the Golgi, alongside a corresponding reduction in its plasma membrane level. PMP22 overexpression, which models CMT1A caused by gene duplication, triggered cholesterol sequestration to lysosomes, and reduced ATP-binding cassette transporter-dependent cholesterol efflux. Conversely, lysosomal targeting of cholesterol by U18666A treatment increased wild type (WT)-PMP22 levels in lysosomes. Mutagenesis of a cholesterol recognition motif, or CRAC domain, in human PMP22 lead to increased levels of PMP22 in the ER and Golgi compartments, along with higher cytosolic, and lower membrane-associated cholesterol. Importantly, cholesterol trafficking defects observed in PMP22-deficient Schwann cells were rescued by WT but not CRAC-mutant-PMP22. We also observed that myelination deficits in dorsal root ganglia explants from heterozygous PMP22-deficient mice were improved by cholesterol supplementation. Collectively, these findings indicate that PMP22 is critical in cholesterol metabolism, and this mechanism is likely a contributing factor in PMP22-linked hereditary neuropathies. Our results provide a basis for understanding how altered expression of PMP22 impacts cholesterol metabolism.
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Affiliation(s)
- Ye Zhou
- Department of Neuroscience, College of Medicine, McKnight Brain Institute, University of Florida, Gainesville, Florida, USA
| | - David Borchelt
- Department of Neuroscience, College of Medicine, McKnight Brain Institute, University of Florida, Gainesville, Florida, USA
| | - Jodi C Bauson
- Department of Neuroscience, College of Medicine, McKnight Brain Institute, University of Florida, Gainesville, Florida, USA
| | - Sergio Fazio
- Department of Medicine, Knight Cardiovascular Institute, Center for Preventive Cardiology, Oregon Health and Science University, Oregon, Portland, USA
| | - Joshua R Miles
- Department of Medicine, Knight Cardiovascular Institute, Center for Preventive Cardiology, Oregon Health and Science University, Oregon, Portland, USA
| | - Hagai Tavori
- Department of Medicine, Knight Cardiovascular Institute, Center for Preventive Cardiology, Oregon Health and Science University, Oregon, Portland, USA
| | - Lucia Notterpek
- Department of Neuroscience, College of Medicine, McKnight Brain Institute, University of Florida, Gainesville, Florida, USA.,Department of Neurology, College of Medicine, McKnight Brain Institute, University of Florida, Gainesville, Florida, USA
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22
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Iaea DB, Spahr ZR, Singh RK, Chan RB, Zhou B, Bareja R, Elemento O, Di Paolo G, Zhang X, Maxfield FR. Stable reduction of STARD4 alters cholesterol regulation and lipid homeostasis. Biochim Biophys Acta Mol Cell Biol Lipids 2020; 1865:158609. [PMID: 31917335 DOI: 10.1016/j.bbalip.2020.158609] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2019] [Revised: 12/18/2019] [Accepted: 12/31/2019] [Indexed: 12/20/2022]
Abstract
STARD4, a member of the evolutionarily conserved START gene family, is a soluble sterol transport protein implicated in cholesterol sensing and maintenance of cellular homeostasis. STARD4 is widely expressed and has been shown to transfer sterol between liposomes as well as organelles in cells. However, STARD4 knockout mice lack an obvious phenotype, so the overall role of STARD4 is unclear. To model long term depletion of STARD4 in cells, we use short hairpin RNA technology to stably decrease STARD4 expression in human U2OS osteosarcoma cells (STARD4-KD). We show that STARD4-KD cells display increased total cholesterol, slower cholesterol trafficking between the plasma membrane and the endocytic recycling compartment, and increased plasma membrane fluidity. These effects can all be rescued by transient expression of a short hairpin RNA-resistant STARD4 construct. Some of the cholesterol increase was due to excess storage in late endosomes or lysosomes. To understand the effects of reduced STARD4, we carried out transcriptional and lipidomic profiling of control and STARD4-KD cells. Reduction of STARD4 activates compensatory mechanisms that alter membrane composition and lipid homeostasis. Based on these observations, we propose that STARD4 functions as a critical sterol transport protein involved in sterol sensing and maintaining lipid homeostasis.
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Affiliation(s)
- David B Iaea
- Department of Biochemistry, Weill Cornell Medical College, 1300 York Ave, New York, NY 10065, USA; Weill Cornell Medical College, Rockefeller University, Memorial Sloan-Kettering Cancer Center Tri-Institutional Chemical Biology Program, New York, NY 10065, USA
| | - Zachary R Spahr
- Department of Biochemistry, Weill Cornell Medical College, 1300 York Ave, New York, NY 10065, USA
| | - Rajesh K Singh
- Department of Biochemistry, Weill Cornell Medical College, 1300 York Ave, New York, NY 10065, USA
| | - Robin B Chan
- Pathology and Cell Biology, Taub Institute for Research on Alzheimer's Disease and the Aging Brain, Columbia University Medical Center, New York, NY 10032, USA
| | - Bowen Zhou
- Pathology and Cell Biology, Taub Institute for Research on Alzheimer's Disease and the Aging Brain, Columbia University Medical Center, New York, NY 10032, USA
| | - Rohan Bareja
- Institute for Computational Biomedicine, Department of Physiology and Biophysics, Weill Cornell Medical College, 1300 York Ave, New York, NY 10065, USA
| | - Olivier Elemento
- Institute for Computational Biomedicine, Department of Physiology and Biophysics, Weill Cornell Medical College, 1300 York Ave, New York, NY 10065, USA
| | - Gilbert Di Paolo
- Pathology and Cell Biology, Taub Institute for Research on Alzheimer's Disease and the Aging Brain, Columbia University Medical Center, New York, NY 10032, USA
| | - Xiaoxue Zhang
- Department of Biochemistry, Weill Cornell Medical College, 1300 York Ave, New York, NY 10065, USA
| | - Frederick R Maxfield
- Department of Biochemistry, Weill Cornell Medical College, 1300 York Ave, New York, NY 10065, USA; Weill Cornell Medical College, Rockefeller University, Memorial Sloan-Kettering Cancer Center Tri-Institutional Chemical Biology Program, New York, NY 10065, USA.
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23
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Schrader M, Kamoshita M, Islinger M. Organelle interplay-peroxisome interactions in health and disease. J Inherit Metab Dis 2020; 43:71-89. [PMID: 30864148 PMCID: PMC7041636 DOI: 10.1002/jimd.12083] [Citation(s) in RCA: 87] [Impact Index Per Article: 17.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/25/2019] [Revised: 02/28/2019] [Accepted: 03/06/2019] [Indexed: 01/04/2023]
Abstract
Peroxisomes are multifunctional, dynamic, membrane-bound organelles with important functions in cellular lipid metabolism, rendering them essential for human health and development. Important roles for peroxisomes in signaling and the fine-tuning of cellular processes are emerging, which integrate them in a complex network of interacting cellular compartments. Like many other organelles, peroxisomes communicate through membrane contact sites. For example, peroxisomal growth, positioning, and lipid metabolism involves contacts with the endoplasmic reticulum (ER). Here, we discuss the most recent findings on peroxisome-organelle interactions including peroxisome-ER interplay at membrane contacts sites, and functional interplay with mitochondria, lysosomes, and lipid droplets in mammalian cells. We address tether proteins, metabolic cooperation, and the impact of peroxisome interactions on human health and disease.
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Affiliation(s)
- Michael Schrader
- College of Life and Environmental Sciences, BiosciencesUniversity of ExeterExeterUK
| | - Maki Kamoshita
- College of Life and Environmental Sciences, BiosciencesUniversity of ExeterExeterUK
| | - Markus Islinger
- Institute of Neuroanatomy, Center for Biomedicine and Medical Technology Mannheim, Medical Faculty ManheimUniversity of HeidelbergMannheimGermany
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24
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Mechanisms and regulation of cholesterol homeostasis. Nat Rev Mol Cell Biol 2019; 21:225-245. [DOI: 10.1038/s41580-019-0190-7] [Citation(s) in RCA: 1094] [Impact Index Per Article: 182.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 10/24/2019] [Indexed: 12/14/2022]
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25
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Chen L, Chen XW, Huang X, Song BL, Wang Y, Wang Y. Regulation of glucose and lipid metabolism in health and disease. SCIENCE CHINA-LIFE SCIENCES 2019; 62:1420-1458. [PMID: 31686320 DOI: 10.1007/s11427-019-1563-3] [Citation(s) in RCA: 185] [Impact Index Per Article: 30.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/16/2019] [Accepted: 10/15/2019] [Indexed: 02/08/2023]
Abstract
Glucose and fatty acids are the major sources of energy for human body. Cholesterol, the most abundant sterol in mammals, is a key component of cell membranes although it does not generate ATP. The metabolisms of glucose, fatty acids and cholesterol are often intertwined and regulated. For example, glucose can be converted to fatty acids and cholesterol through de novo lipid biosynthesis pathways. Excessive lipids are secreted in lipoproteins or stored in lipid droplets. The metabolites of glucose and lipids are dynamically transported intercellularly and intracellularly, and then converted to other molecules in specific compartments. The disorders of glucose and lipid metabolism result in severe diseases including cardiovascular disease, diabetes and fatty liver. This review summarizes the major metabolic aspects of glucose and lipid, and their regulations in the context of physiology and diseases.
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Affiliation(s)
- Ligong Chen
- School of Pharmaceutical Sciences, Beijing Advanced Innovation Center for Structural Biology, Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Tsinghua University, Beijing, 100084, China.
| | - Xiao-Wei Chen
- State Key Laboratory of Membrane Biology, Institute of Molecular Medicine, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, 100871, China.
| | - Xun Huang
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China.
| | - Bao-Liang Song
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan, 430072, China.
| | - Yan Wang
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan, 430072, China.
| | - Yiguo Wang
- MOE Key Laboratory of Bioinformatics, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, 100084, China.
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26
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Tan L, Tong J, Chun C, Im YJ. Structural analysis of human sterol transfer protein STARD4. Biochem Biophys Res Commun 2019; 520:466-472. [PMID: 31607485 DOI: 10.1016/j.bbrc.2019.10.054] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2019] [Accepted: 10/04/2019] [Indexed: 10/25/2022]
Abstract
The steroidogenic acute regulatory protein (StAR)-related lipid transfer domain-4 (STARD4) is a sterol-binding protein that is involved in cholesterol homeostasis by intracellular sterol transport. In this work, we determined the crystal structures of human STARD4 and its Ω1-loop mutant in apo forms at 1.95 and 1.7 Å resolutions, respectively. The structure of human STARD4 displays a conserved α-helix/β-grip fold containing a deep hydrophobic pocket. The Ω1-loop which serves as a lid for the hydrophobic pocket has a closed conformation. The shape of the sterol-binding cavity in the closed form is not complementary to accommodate cholesterol, suggesting that a conformational change of the Ω1-loop is essential for sterol binding. The human STARD4 displayed sterol transfer activity between liposomes, and the mutations in the Ω1-loop and the hydrophobic wall abolished the transfer activity. This study confirms the structural conservation of the STARD4 subfamily proteins and the flexibility of the Ω1-loop and helix α4 required for sterol transport.
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Affiliation(s)
- Lingchen Tan
- College of Pharmacy, Chonnam National University, 77 Yongbong-ro, Buk-gu, Gwangju, 61186, South Korea
| | - Junsen Tong
- College of Pharmacy, Chonnam National University, 77 Yongbong-ro, Buk-gu, Gwangju, 61186, South Korea
| | - ChangJu Chun
- College of Pharmacy, Chonnam National University, 77 Yongbong-ro, Buk-gu, Gwangju, 61186, South Korea.
| | - Young Jun Im
- College of Pharmacy, Chonnam National University, 77 Yongbong-ro, Buk-gu, Gwangju, 61186, South Korea.
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27
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Orsó E, Robenek H, Boettcher A, Wolf Z, Liebisch G, Kramer W, Schmitz G. Nonglucuronidated Ezetimibe Disrupts CD13- and CD64-Coassembly in Membrane Microdomains and Decreases Cellular Cholesterol Content in Human Monocytes/Macrophages. Cytometry A 2019; 95:869-884. [PMID: 30994973 DOI: 10.1002/cyto.a.23772] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2019] [Revised: 04/01/2019] [Accepted: 04/02/2019] [Indexed: 12/30/2022]
Abstract
Ezetimibe (EZE) and glucuronidated EZE (EZE-Glu) differentially target Niemann-Pick C1-like 1 (NPC1L1) and CD13 (aminopeptidase-N) to inhibit intestinal cholesterol absorption and cholesterol processing in other cells, although the precise molecular mechanisms are not fully elucidated. Cellular effects of EZE, EZE-Glu, and the low-absorbable EZE-analogue S6130 were investigated on human monocyte-derived macrophages upon loading with atherogenic lipoproteins. EZE and S6130, but not EZE-Glu disturbed the colocalization of CD13 and its coreceptor CD64 (Fcγ receptor I) in membrane microdomains, and decreased the presence of both receptors in detergent-resistant membrane fractions. Biotinylated cholesterol absorption inhibitor C-5 (i.e., derivative of EZE) was rapidly internalized to perinuclear tubular structures of cells, resembling endoplasmic reticulum (ER), but CD13 was detected on extracellular sites of the plasma membrane and endolysosomal vesicles. Administration of EZE, but not of EZE-Glu or S6130, was associated with decreased cellular cholesteryl ester content, indicating the sterol-O acyltransferase 1 (SOAT1)-inhibition by EZE. Furthermore, EZE decreased the expression of molecules involved in cholesterol uptake and synthesis, in parallel with increased apolipoprotein A-I-mediated cholesterol efflux and upregulation of efflux-effectors. However, NPC1L1 the other claimed molecular target of EZE, was not detected in macrophages, thereby excluding this protein as target for EZE in macrophages. Thus, EZE is very likely a CD13-linked microdomain-disruptor and SOAT1-inhibitor in macrophages leading to in vitro anti-atherosclerotic effects through a decrease of net cellular cholesterol content. © 2019 International Society for Advancement of Cytometry.
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Affiliation(s)
- Evelyn Orsó
- Institute for Clinical Chemistry and Laboratory Medicine, University Hospital of Regensburg, 93053 Regensburg, Germany
| | - Horst Robenek
- Leibniz Institute for Arteriosclerosis Research, University of Muenster, 48149 Muenster, Germany
| | - Alfred Boettcher
- Institute for Clinical Chemistry and Laboratory Medicine, University Hospital of Regensburg, 93053 Regensburg, Germany
| | - Zsuzsanna Wolf
- Institute for Clinical Chemistry and Laboratory Medicine, University Hospital of Regensburg, 93053 Regensburg, Germany
| | - Gerhard Liebisch
- Institute for Clinical Chemistry and Laboratory Medicine, University Hospital of Regensburg, 93053 Regensburg, Germany
| | - Werner Kramer
- Biomedical and Scientific Consulting, 55130 Mainz, Germany
| | - Gerd Schmitz
- Institute for Clinical Chemistry and Laboratory Medicine, University Hospital of Regensburg, 93053 Regensburg, Germany
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28
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Luo J, Jiang LY, Yang H, Song BL. Intracellular Cholesterol Transport by Sterol Transfer Proteins at Membrane Contact Sites. Trends Biochem Sci 2019; 44:273-292. [DOI: 10.1016/j.tibs.2018.10.001] [Citation(s) in RCA: 102] [Impact Index Per Article: 17.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2018] [Revised: 10/06/2018] [Accepted: 10/10/2018] [Indexed: 12/20/2022]
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Osuna-Ramos JF, Reyes-Ruiz JM, Del Ángel RM. The Role of Host Cholesterol During Flavivirus Infection. Front Cell Infect Microbiol 2018; 8:388. [PMID: 30450339 PMCID: PMC6224431 DOI: 10.3389/fcimb.2018.00388] [Citation(s) in RCA: 107] [Impact Index Per Article: 15.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2018] [Accepted: 10/15/2018] [Indexed: 12/11/2022] Open
Abstract
In recent years the emergence and resurgence of arboviruses have generated a global health alert. Among arboviruses, Dengue (DENV), Zika (ZIKV), Yellow Fever (YFV), and West Nile (WNV) virus, belong to the genus Flavivirus, cause high viremia and occasionally fatal clinical disease in humans. Given the genetic austerity of the virus, they depend on cellular factors and organelles to complete its replication. One of the cellular components required for flavivirus infection is cholesterol. Cholesterol is an abundant lipid in biomembranes of eukaryotes cells and is necessary to maintain the cellular homeostasis. Recently, it has been reported, that cholesterol is fundamental during flavivirus infection in both mammal and insect vector models. During infection with DENV, ZIKV, YFV, and WNV the modulation of levels of host-cholesterol facilitates viral entry, replicative complexes formation, assembly, egress, and control of the interferon type I response. This modulation involves changes in cholesterol uptake with the concomitant regulation of cholesterol receptors as well as changes in cholesterol synthesis related to important modifications in cellular metabolism pathways. In view of the flavivirus dependence of cholesterol and the lack of an effective anti-flaviviral treatment, this cellular lipid has been proposed as a therapeutic target to treat infection using FDA-approved cholesterol-lowering drugs. This review aims to address the dependence of cholesterol by flaviviruses as well as the basis for anti flaviviral therapy using drugs which target is cholesterol synthesis or uptake.
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Affiliation(s)
- Juan Fidel Osuna-Ramos
- Department of Infectomics and Molecular Pathogenesis, Center for Research and Advanced Studies (CINVESTAV-IPN), Ciudad de Mexico, Mexico
| | - José Manuel Reyes-Ruiz
- Department of Infectomics and Molecular Pathogenesis, Center for Research and Advanced Studies (CINVESTAV-IPN), Ciudad de Mexico, Mexico
| | - Rosa Maria Del Ángel
- Department of Infectomics and Molecular Pathogenesis, Center for Research and Advanced Studies (CINVESTAV-IPN), Ciudad de Mexico, Mexico
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30
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Hasegawa H, Li C, Alba BM, Penny DM, Xia Z, Dayao MR, Li P, Zhang J, Zhou J, Lim D, Murawsky CM, Lim AC. Membrane cholesterol modulates STEAP2 conformation during dynamic intracellular trafficking processes leading to broad subcellular distribution. Exp Cell Res 2018; 370:208-226. [PMID: 29940176 DOI: 10.1016/j.yexcr.2018.06.022] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2018] [Revised: 06/16/2018] [Accepted: 06/21/2018] [Indexed: 11/26/2022]
Abstract
STEAP2 is a member of the Six-Transmembrane Epithelial Antigen of the Prostate (STEAP) protein family that is proposed to function as metalloreductase. While STEAP2 shows a complex subcellular distribution pattern localizing to both secretory and endocytic pathway organelles, how such broad steady-state distribution is maintained is unknown. Similarly, whether STEAP2 undergoes any compartment-specific modulation during intracellular trafficking has not been reported. Leveraging a newly-identified monoclonal antibody that recognizes a conformation-sensitive epitope nested in the second extracellular loop of STEAP2, we demonstrate that the epitope formation was dependent on the cholesterol content of the membrane in which STEAP2 was embedded. Monitoring the STEAP2-dependent internalization of this antibody uncovered STEAP2's rapid internalization from the cell surface and their subsequence trafficking to the Golgi region and endosome-like puncta. Acute inhibition of endocytosis also increased the detectable amount of STEAP2 at the plasma membrane. Collectively, these experiments demonstrate that an intricate balance of membrane flux between the secretory and endocytic pathways underlies the characteristic broad subcellular localization of STEAP2. By using a cell-based assay that detects the metalloreductase functions of cell surface-localizing STEAP4, STEAP2's metalloreductase activities were not detectable, suggesting that its enzymatic function is suppressed at the plasma membrane. The conformational modulation of STEAP2 by the local membrane cholesterol content can therefore serve as a potential mechanism to modulate STEAP2 function in a compartment-restricted manner, by coupling a pre-existing difference in cholesterol content among different cellular membranes to a dynamic trafficking process leading to broad subcellular distribution.
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Affiliation(s)
- Haruki Hasegawa
- Department of Therapeutic Discovery, Amgen Inc., South San Francisco, CA 94080, USA.
| | - Cong Li
- Department of Oncology Research, Amgen Inc., South San Francisco, CA 94080, USA
| | - Benjamin M Alba
- Department of Therapeutic Discovery, Amgen Inc., South San Francisco, CA 94080, USA
| | - David M Penny
- Department of Therapeutic Discovery, Amgen Inc., South San Francisco, CA 94080, USA
| | - Zhen Xia
- Department of Therapeutic Discovery, Amgen Inc., South San Francisco, CA 94080, USA
| | - Maria Rosalyn Dayao
- Department of Therapeutic Discovery, Amgen Inc., South San Francisco, CA 94080, USA
| | - Peng Li
- Department of Therapeutic Discovery, Amgen Inc., South San Francisco, CA 94080, USA
| | - Jue Zhang
- Department of Therapeutic Discovery, Amgen Inc., South San Francisco, CA 94080, USA
| | - Jing Zhou
- Department of Therapeutic Discovery, Amgen Inc., South San Francisco, CA 94080, USA
| | - Desiree Lim
- Department of Therapeutic Discovery, Amgen Inc., Burnaby, British Columbia, Canada
| | | | - Ai Ching Lim
- Department of Therapeutic Discovery, Amgen Inc., South San Francisco, CA 94080, USA
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31
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Intracellular and Plasma Membrane Events in Cholesterol Transport and Homeostasis. J Lipids 2018; 2018:3965054. [PMID: 30174957 PMCID: PMC6106919 DOI: 10.1155/2018/3965054] [Citation(s) in RCA: 62] [Impact Index Per Article: 8.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2018] [Accepted: 07/26/2018] [Indexed: 12/13/2022] Open
Abstract
Cholesterol transport between intracellular compartments proceeds by both energy- and non-energy-dependent processes. Energy-dependent vesicular traffic partly contributes to cholesterol flux between endoplasmic reticulum, plasma membrane, and endocytic vesicles. Membrane contact sites and lipid transfer proteins are involved in nonvesicular lipid traffic. Only “active" cholesterol molecules outside of cholesterol-rich regions and partially exposed in water phase are able to fast transfer. The dissociation of partially exposed cholesterol molecules in water determines the rate of passive aqueous diffusion of cholesterol out of plasma membrane. ATP hydrolysis with concomitant conformational transition is required to cholesterol efflux by ABCA1 and ABCG1 transporters. Besides, scavenger receptor SR-B1 is involved also in cholesterol efflux by facilitated diffusion via hydrophobic tunnel within the molecule. Direct interaction of ABCA1 with apolipoprotein A-I (apoA-I) or apoA-I binding to high capacity binding sites in plasma membrane is important in cholesterol escape to free apoA-I. ABCG1-mediated efflux to fully lipidated apoA-I within high density lipoprotein particle proceeds more likely through the increase of “active” cholesterol level. Putative cholesterol-binding linear motifs within the structure of all three proteins ABCA1, ABCG1, and SR-B1 are suggested to contribute to the binding and transfer of cholesterol molecules from cytoplasmic to outer leaflets of lipid bilayer. Together, plasma membrane events and intracellular cholesterol metabolism and traffic determine the capacity of the cell for cholesterol efflux.
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32
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Two polymorphic cholesterol monohydrate crystal structures form in macrophage culture models of atherosclerosis. Proc Natl Acad Sci U S A 2018; 115:7662-7669. [PMID: 29967179 DOI: 10.1073/pnas.1803119115] [Citation(s) in RCA: 44] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
The formation of atherosclerotic plaques in the blood vessel walls is the result of LDL particle uptake, and consequently of cholesterol accumulation in macrophage cells. Excess cholesterol accumulation eventually results in cholesterol crystal deposition, the hallmark of mature atheromas. We followed the formation of cholesterol crystals in J774A.1 macrophage cells with time, during accumulation of LDL particles, using a previously developed correlative cryosoft X-ray tomography (cryo-SXT) and stochastic optical reconstruction microscopy (STORM) technique. We show, in the initial accumulation stages, formation of small quadrilateral crystal plates associated with the cell plasma membrane, which may subsequently assemble into large aggregates. These plates match crystals of the commonly observed cholesterol monohydrate triclinic structure. Large rod-like cholesterol crystals form at a later stage in intracellular locations. Using cryotransmission electron microscopy (cryo-TEM) and cryoelectron diffraction (cryo-ED), we show that the structure of the large elongated rods corresponds to that of monoclinic cholesterol monohydrate, a recently determined polymorph of the triclinic crystal structure. These monoclinic crystals form with an unusual hollow cylinder or helical architecture, which is preserved in the mature rod-like crystals. The rod-like morphology is akin to that observed in crystals isolated from atheromas. We suggest that the crystals in the atherosclerotic plaques preserve in their morphology the memory of the structure in which they were formed. The identification of the polymorph structure, besides explaining the different crystal morphologies, may serve to elucidate mechanisms of cholesterol segregation and precipitation in atherosclerotic plaques.
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Wang YH, Twu YC, Wang CK, Lin FZ, Lee CY, Liao YJ. Niemann-Pick Type C2 Protein Regulates Free Cholesterol Accumulation and Influences Hepatic Stellate Cell Proliferation and Mitochondrial Respiration Function. Int J Mol Sci 2018; 19:ijms19061678. [PMID: 29874879 PMCID: PMC6032364 DOI: 10.3390/ijms19061678] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2018] [Revised: 06/01/2018] [Accepted: 06/04/2018] [Indexed: 12/23/2022] Open
Abstract
Liver fibrosis is the first step toward the progression to cirrhosis, portal hypertension, and hepatocellular carcinoma. A high-cholesterol diet is associated with liver fibrosis via the accumulation of free cholesterol in hepatic stellate cells (HSCs). Niemann-Pick type C2 (NPC2) plays an important role in the regulation of intracellular free cholesterol homeostasis via direct binding with free cholesterol. Previously, we reported that NPC2 was downregulated in liver cirrhosis tissues. Loss of NPC2 enhanced the accumulation of free cholesterol in HSCs and made them more susceptible to transforming growth factor (TGF)-β1. In this study, we showed that knockdown of NPC2 resulted in marked increases in platelet-derived growth factor BB (PDGF-BB)-induced HSC proliferation through enhanced extracellular signal-regulated kinases (ERK), p38, c-Jun N-terminal kinases (JNK), and protein kinase B (AKT) phosphorylation. In contrast, NPC2 overexpression decreased PDGF-BB-induced cell proliferation by inhibiting p38, JNK, and AKT phosphorylation. Although NPC2 expression did not affect caspase-related apoptosis, the autophagy marker light chain 3β (LC3B) was decreased in NPC2 knockdown, and free cholesterol accumulated in the HSCs. The mitochondrial respiration functions (such as oxygen consumption rate, ATP production, and maximal respiratory capacity) were decreased in NPC2 knockdown, and free cholesterol accumulated in the HSCs, while NPC2-overexpressed cells remained normal. In addition, NPC2 expression did not affect the susceptibility of HSCs to lipopolysaccharides (LPS), and U18666A treatment induced free cholesterol accumulation, which enhanced LPS-induced Toll-like receptor 4 (TLR4), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 phosphorylation, interleukin (IL)-1 and IL-6 expression. Our study demonstrated that NPC2-mediated free cholesterol homeostasis controls HSC proliferation and mitochondrial function.
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Affiliation(s)
- Yuan-Hsi Wang
- School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei 110, Taiwan.
- Department of Biotechnology and Laboratory Science in Medicine, National Yang-Ming University, Taipei 112, Taiwan.
| | - Yuh-Ching Twu
- Department of Biotechnology and Laboratory Science in Medicine, National Yang-Ming University, Taipei 112, Taiwan.
| | - Chung-Kwe Wang
- Department of International Medicine, Taipei City Hospital Ranai Branch, Taipei 106, Taiwan.
| | - Fu-Zhen Lin
- School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei 110, Taiwan.
| | - Chun-Ya Lee
- School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei 110, Taiwan.
| | - Yi-Jen Liao
- School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei 110, Taiwan.
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34
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Wei J, Zhang YY, Luo J, Wang JQ, Zhou YX, Miao HH, Shi XJ, Qu YX, Xu J, Li BL, Song BL. The GARP Complex Is Involved in Intracellular Cholesterol Transport via Targeting NPC2 to Lysosomes. Cell Rep 2018; 19:2823-2835. [PMID: 28658628 DOI: 10.1016/j.celrep.2017.06.012] [Citation(s) in RCA: 34] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2017] [Revised: 05/07/2017] [Accepted: 05/31/2017] [Indexed: 10/19/2022] Open
Abstract
Proper intracellular cholesterol trafficking is critical for cellular function. Two lysosome-resident proteins, NPC1 and NPC2, mediate the egress of low-density lipoprotein-derived cholesterol from lysosomes. However, other proteins involved in this process remain largely unknown. Through amphotericin B-based selection, we isolated two cholesterol transport-defective cell lines. Subsequent whole-transcriptome-sequencing analysis revealed two cell lines bearing the same mutation in the vacuolar protein sorting 53 (Vps53) gene. Depletion of VPS53 or other subunits of the Golgi-associated retrograde protein (GARP) complex impaired NPC2 sorting to lysosomes and caused cholesterol accumulation. GARP deficiency blocked the retrieval of the cation-independent mannose 6-phosphate receptor (CI-MPR) to the trans-Golgi network. Further, Vps54 mutant mice displayed reduced cellular NPC2 protein levels and increased cholesterol accumulation, underscoring the physiological role of the GARP complex in cholesterol transport. We conclude that the GARP complex contributes to intracellular cholesterol transport by targeting NPC2 to lysosomes in a CI-MPR-dependent manner.
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Affiliation(s)
- Jian Wei
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, the Institute for Advanced Studies, Wuhan University, Wuhan 430072, China
| | - Ying-Yu Zhang
- The State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, China
| | - Jie Luo
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, the Institute for Advanced Studies, Wuhan University, Wuhan 430072, China
| | - Ju-Qiong Wang
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, the Institute for Advanced Studies, Wuhan University, Wuhan 430072, China
| | - Yu-Xia Zhou
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, the Institute for Advanced Studies, Wuhan University, Wuhan 430072, China
| | - Hong-Hua Miao
- The State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, China
| | - Xiong-Jie Shi
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, the Institute for Advanced Studies, Wuhan University, Wuhan 430072, China
| | - Yu-Xiu Qu
- The State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, China
| | - Jie Xu
- The State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, China
| | - Bo-Liang Li
- The State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, China
| | - Bao-Liang Song
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, the Institute for Advanced Studies, Wuhan University, Wuhan 430072, China.
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35
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Yamauchi Y, Rogers MA. Sterol Metabolism and Transport in Atherosclerosis and Cancer. Front Endocrinol (Lausanne) 2018; 9:509. [PMID: 30283400 PMCID: PMC6157400 DOI: 10.3389/fendo.2018.00509] [Citation(s) in RCA: 38] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/23/2018] [Accepted: 08/14/2018] [Indexed: 01/22/2023] Open
Abstract
Cholesterol is a vital lipid molecule for mammalian cells, regulating fluidity of biological membranes, and serving as an essential constituent of lipid rafts. Mammalian cells acquire cholesterol from extracellular lipoproteins and from de novo synthesis. Cholesterol biosynthesis generates various precursor sterols. Cholesterol undergoes metabolic conversion into oxygenated sterols (oxysterols), bile acids, and steroid hormones. Cholesterol intermediates and metabolites have diverse and important cellular functions. A network of molecular machineries including transcription factors, protein modifiers, sterol transporters/carriers, and sterol sensors regulate sterol homeostasis in mammalian cells and tissues. Dysfunction in metabolism and transport of cholesterol, sterol intermediates, and oxysterols occurs in various pathophysiological settings such as atherosclerosis, cancers, and neurodegenerative diseases. Here we review the cholesterol, intermediate sterol, and oxysterol regulatory mechanisms and intracellular transport machineries, and discuss the roles of sterols and sterol metabolism in human diseases.
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Affiliation(s)
- Yoshio Yamauchi
- Nutri-Life Science Laboratory, Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, Japan
- AMED-CREST, Japan Agency for Medical Research and Development, Tokyo, Japan
- *Correspondence: Yoshio Yamauchi
| | - Maximillian A. Rogers
- Division of Cardiovascular Medicine, Center for Interdisciplinary Cardiovascular Sciences, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, United States
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36
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Fearon AD, Stokes GY. Thermodynamics of Indomethacin Adsorption to Phospholipid Membranes. J Phys Chem B 2017; 121:10508-10518. [PMID: 29064244 DOI: 10.1021/acs.jpcb.7b08359] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
Using second-harmonic generation, we directly monitored adsorption of indomethacin, a nonsteroidal anti-inflammatory drug, to supported lipid bilayers composed of phospholipids of varying phase, cholesterol content, and head group charge without the use of extrinsic labels at therapeutically relevant aqueous concentrations. Indomethacin adsorbed to gel-phase lipids with a high binding affinity, suggesting that like other arylacetic acid-containing drugs, it preferentially interacts with ordered lipid domains. We discovered that adsorption of indomethacin to gel-phase phospholipids was endothermic and entropically driven, whereas adsorption to fluid-phase phospholipids was exothermic and enthalpically driven. As temperature increased from 19 to 34 °C, binding affinities to gel-phase lipids increased by 7-fold but relative surface concentration decreased to one-fifth of the original value. We also compared our results to the entropies reported for indomethacin adsorbed to surfactant micelles, which are used in drug delivery systems, and assert that adsorbed water molecules in the phospholipid bilayer may be buried deeper into the acyl chains and less accessible for disruption. The thermodynamic studies reported here provide mechanistic insight into indomethacin interactions with mammalian plasma membranes in the gastrointestinal tract and inform studies of drug delivery, where indomethacin is commonly used as a prototypical, hydrophobic small-molecule drug.
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Affiliation(s)
- Amanda D Fearon
- Department of Chemistry and Biochemistry, Santa Clara University , 500 El Camino Real, Santa Clara, California 95053, United States
| | - Grace Y Stokes
- Department of Chemistry and Biochemistry, Santa Clara University , 500 El Camino Real, Santa Clara, California 95053, United States
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37
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Effect of the presence of cholesterol in the interfacial microenvironment on the modulation of the alkaline phosphatase activity during in vitro mineralization. Colloids Surf B Biointerfaces 2017; 155:466-476. [PMID: 28472750 DOI: 10.1016/j.colsurfb.2017.04.051] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2017] [Revised: 04/20/2017] [Accepted: 04/25/2017] [Indexed: 01/17/2023]
Abstract
Mineralization of the skeleton starts within cell-derived matrix vesicles (MVs); then, minerals propagate to the extracellular collagenous matrix. Tissue-nonspecific alkaline phosphatase (TNAP) degrades inorganic pyrophosphate (PPi), a potent inhibitor of mineralization, and contributes Pi (Phosphate) from ATP to initiate mineralization. Compared to the plasma membrane, MVs are rich in Cholesterol (Chol) (∼32%) and TNAP, but how Chol influences TNAP activity remains unclear. We have reconstituted TNAP in liposomes of dipalmitoylphosphatidylcholine (DPPC) or dioleoylphosphatidylcholine (DOPC) combined with Chol or its derivatives Cholestenone (Achol) and Ergosterol (Ergo). DPPC plus 36% sterols in liposome increased the catalytic activity of TNAP toward ATP. The presence of Chol also increased the propagation of minerals by 3.4-fold. The catalytic efficiency of TNAP toward ATP was fourfold lower in DOPC proteoliposomes as compared to DPPC proteoliposomes. DOPC proteoliposomes also increased biomineralization by 2.8-fold as compared to DPPC proteoliposomes. TNAP catalyzed the hydrolysis of ATP more efficiently in the case of the proteoliposome consisting of DOPC with 36% Chol. The same behavior emerged with Achol and Ergo. The organization of the lipid and the structure of the sterol influenced the surface tension (γ), the TNAP phosphohydrolytic activity in the monolayer, and the TNAP catalytic efficiency in the bilayers. Membranes in the Lα phase (Achol) provided better kinetic parameters as compared to membranes in the Lo phase (Chol and Ergo). In conclusion, the physical properties and the lateral organization of lipids in proteoliposomes are crucial to control mineral propagation mediated by TNAP activity during mineralization.
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38
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Luo J, Jiang L, Yang H, Song BL. Routes and mechanisms of post-endosomal cholesterol trafficking: A story that never ends. Traffic 2017; 18:209-217. [DOI: 10.1111/tra.12471] [Citation(s) in RCA: 84] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2017] [Revised: 02/08/2017] [Accepted: 02/08/2017] [Indexed: 12/22/2022]
Affiliation(s)
- Jie Luo
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences; Wuhan University; Wuhan China
| | - Luyi Jiang
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences; Wuhan University; Wuhan China
| | - Hongyuan Yang
- School of Biotechnology and Biomolecular Sciences; The University of New South Wales; Sydney Australia
| | - Bao-Liang Song
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences; Wuhan University; Wuhan China
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39
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Iaea DB, Mao S, Lund FW, Maxfield FR. Role of STARD4 in sterol transport between the endocytic recycling compartment and the plasma membrane. Mol Biol Cell 2017; 28:1111-1122. [PMID: 28209730 PMCID: PMC5391187 DOI: 10.1091/mbc.e16-07-0499] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2016] [Revised: 02/03/2017] [Accepted: 02/08/2017] [Indexed: 11/23/2022] Open
Abstract
The kinetics of sterol transport between the plasma membrane and the endocytic recycling compartment is measured using fluorescence microscopy. STARD4, a small, soluble sterol transport protein, is responsible for 25% of the total transport and 33% of nonvesicular transport. Elevated cholesterol dramatically increases sterol transport rate constants. Cholesterol is an essential constituent of membranes in mammalian cells. The plasma membrane and the endocytic recycling compartment (ERC) are both highly enriched in cholesterol. The abundance and distribution of cholesterol among organelles are tightly controlled by a combination of mechanisms involving vesicular and nonvesicular sterol transport processes. Using the fluorescent cholesterol analogue dehydroergosterol, we examined sterol transport between the plasma membrane and the ERC using fluorescence recovery after photobleaching and a novel sterol efflux assay. We found that sterol transport between these organelles in a U2OS cell line has a t1/2 =12–15 min. Approximately 70% of sterol transport is ATP independent and therefore is nonvesicular. Increasing cellular cholesterol levels dramatically increases bidirectional transport rate constants, but decreases in cholesterol levels have only a modest effect. A soluble sterol transport protein, STARD4, accounts for ∼25% of total sterol transport and ∼33% of nonvesicular sterol transport between the plasma membrane and ERC. This study shows that nonvesicular sterol transport mechanisms and STARD4 in particular account for a large fraction of sterol transport between the plasma membrane and the ERC.
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Affiliation(s)
- David B Iaea
- Department of Biochemistry, Weill Cornell Medical College, New York, NY 10065.,Weill Cornell Medical College, Rockefeller University, and Memorial Sloan-Kettering Cancer Center Tri-Institutional Chemical Biology Program, New York, NY 10065
| | - Shu Mao
- Department of Biochemistry, Weill Cornell Medical College, New York, NY 10065
| | - Frederik W Lund
- Department of Biochemistry, Weill Cornell Medical College, New York, NY 10065
| | - Frederick R Maxfield
- Department of Biochemistry, Weill Cornell Medical College, New York, NY 10065 .,Weill Cornell Medical College, Rockefeller University, and Memorial Sloan-Kettering Cancer Center Tri-Institutional Chemical Biology Program, New York, NY 10065
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40
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Zuo H, Wang R, Jiang D, Fang D. Determining the Composition of Active Cholesterol in the Plasma Membrane of Single Cells by using Electrochemiluminescence. ChemElectroChem 2017. [DOI: 10.1002/celc.201600738] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Affiliation(s)
- Huanzhen Zuo
- School of Pharmacy; Nanjing Medical University; Jiangsu 210029 P.R. China
- NanJing Biopharmaceutical Innovation Platform Co. Ltd; Jiangsu 210000 P.R. China
| | - Rui Wang
- School of Pharmacy; Nanjing Medical University; Jiangsu 210029 P.R. China
| | - Dechen Jiang
- School of Chemistry and Chemical Engineering; Nanjing University; Jiangsu 210092 P.R. China
| | - Danjun Fang
- School of Pharmacy; Nanjing Medical University; Jiangsu 210029 P.R. China
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41
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Xu J, Jiang D, Qin Y, Xia J, Jiang D, Chen HY. C3N4 Nanosheet Modified Microwell Array with Enhanced Electrochemiluminescence for Total Analysis of Cholesterol at Single Cells. Anal Chem 2017; 89:2216-2220. [PMID: 28192948 DOI: 10.1021/acs.analchem.6b04635] [Citation(s) in RCA: 53] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Affiliation(s)
- Jingjing Xu
- State
Key Laboratory of Analytical Chemistry for Life Science, School of
Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China
| | - Depeng Jiang
- Department
of Respiratory Medicine, The Second Affiliated Hospital, Chongqing Medical University, Chongqing 400010, China
| | - Yanling Qin
- Department
of Respiratory Medicine, The Second Affiliated Hospital, Chongqing Medical University, Chongqing 400010, China
| | - Juan Xia
- Department
of Respiratory Medicine, The Second Affiliated Hospital, Chongqing Medical University, Chongqing 400010, China
| | - Dechen Jiang
- State
Key Laboratory of Analytical Chemistry for Life Science, School of
Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China
| | - Hong-Yuan Chen
- State
Key Laboratory of Analytical Chemistry for Life Science, School of
Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China
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Varsano N, Dadosh T, Kapishnikov S, Pereiro E, Shimoni E, Jin X, Kruth HS, Leiserowitz L, Addadi L. Development of Correlative Cryo-soft X-ray Tomography and Stochastic Reconstruction Microscopy. A Study of Cholesterol Crystal Early Formation in Cells. J Am Chem Soc 2016; 138:14931-14940. [PMID: 27934213 DOI: 10.1021/jacs.6b07584] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
We have developed a high resolution correlative method involving cryo-soft X-ray tomography (cryo-SXT) and stochastic optical reconstruction microscopy (STORM), which provides information in three dimensions on large cellular volumes at 70 nm resolution. Cryo-SXT morphologically identified and localized aggregations of carbon-rich materials. STORM identified specific markers on the desired epitopes, enabling colocalization between the identified objects, in this case cholesterol crystals, and the cellular environment. The samples were studied under ambient and cryogenic conditions without dehydration or heavy metal staining. The early events of cholesterol crystal development were investigated in relation to atherosclerosis, using as model macrophage cell cultures enriched with LDL particles. Atherosclerotic plaques build up in arteries in a slow process involving cholesterol crystal accumulation. Cholesterol crystal deposition is a crucial stage in the pathological cascade. Our results show that cholesterol crystals can be identified and imaged at a very early stage on the cell plasma membrane and in intracellular locations. This technique can in principle be applied to other biological samples where specific molecular identification is required in conjunction with high resolution 3D-imaging.
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Affiliation(s)
| | | | - Sergey Kapishnikov
- Soft Matter and Functional Materials, Helmholtz-Zentrum Berlin , Albert-Einstein-Strasse 15, D-12489 Berlin, Germany
| | - Eva Pereiro
- ALBA Synchrotron Light Source, MISTRAL Beamline-Experiments Division, 08290 Cerdanyola del Valles, Barcelona, Spain
| | | | - Xueting Jin
- Experimental Atherosclerosis Section, National Heart, Lung, and Blood Institute, National Institutes of Health , Bethesda, Maryland 20892-1422, United States
| | - Howard S Kruth
- Experimental Atherosclerosis Section, National Heart, Lung, and Blood Institute, National Institutes of Health , Bethesda, Maryland 20892-1422, United States
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43
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Lipid transfer proteins and the tuning of compartmental identity in the Golgi apparatus. Chem Phys Lipids 2016; 200:42-61. [DOI: 10.1016/j.chemphyslip.2016.06.005] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2016] [Revised: 06/21/2016] [Accepted: 06/22/2016] [Indexed: 11/23/2022]
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44
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Seo Y, Kim HS, Kang I, Choi SW, Shin TH, Shin JH, Lee BC, Lee JY, Kim JJ, Kook MG, Kang KS. Cathepsin S contributes to microglia-mediated olfactory dysfunction through the regulation of Cx3cl1-Cx3cr1 axis in a Niemann-Pick disease type C1 model. Glia 2016; 64:2291-2305. [PMID: 27687148 DOI: 10.1002/glia.23077] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2016] [Revised: 09/12/2016] [Accepted: 09/15/2016] [Indexed: 12/28/2022]
Abstract
Microglia can aggravate olfactory dysfunction by mediating neuronal death in the olfactory bulb (OB) of a murine model of Niemann-Pick disease type C1 (NPC1), a fatal neurodegenerative disorder accompanied by lipid trafficking defects. In this study, we focused on the crosstalk between neurons and microglia to elucidate the mechanisms underlying extensive microgliosis in the NPC1-affected brain. Microglia in the OB of NPC1 mice strongly expressed CX3C chemokine receptor 1 (Cx3cr1), a specific receptor for the neural chemokine C-X3-C motif ligand 1 (Cx3cl1). In addition, a high level of Cx3cl1 was detected in NPC1 mouse-derived CSF due to enhanced catalytic activity of Cathepsin S (Ctss), which is responsible for Cx3cl1 secretion. Notably, nasal delivery of Cx3cl1 neutralizing antibody or Ctss inhibitor could inhibit the Cx3cl1-Cx3cr1 interaction and support neuronal survival through the suppression of microglial activation, leading to an improvement in the olfactory function in NPC1 mice. Relevant in vitro experiments revealed that intracellular cholesterol accumulation could act as a strong inducer of abnormal Ctss activation and, in turn, stimulated the Cx3cl1-Cx3cr1 axis in microglia via p38 mitogen-activated protein kinase signaling. Our data address the significance of Cx3cl1-Cx3cr1 interaction in the development of microglial neurotoxicity and suggest that Ctss is a key upstream regulator. Therefore, this study contributes to a better understanding of the crosstalk between neurons and microglia in the development of the neurodegeneration and provides a new perspective for the management of olfactory deficits and other microglia-dependent neuropathies. GLIA 2016;64:2291-2305.
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Affiliation(s)
- Yoojin Seo
- Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul, 08826, South Korea.,School of Medicine, Pusan National University, Busan, 49241, South Korea.,Biomedical Research Institute, Pusan National University Hospital, Busan, 49241, South Korea
| | - Hyung-Sik Kim
- Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul, 08826, South Korea.,School of Medicine, Pusan National University, Busan, 49241, South Korea.,Biomedical Research Institute, Pusan National University Hospital, Busan, 49241, South Korea
| | - Insung Kang
- Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul, 08826, South Korea.,Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul, 08826, South Korea
| | - Soon Won Choi
- Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul, 08826, South Korea.,Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul, 08826, South Korea
| | - Tae-Hoon Shin
- Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul, 08826, South Korea.,Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul, 08826, South Korea
| | - Ji-Hee Shin
- Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul, 08826, South Korea.,Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul, 08826, South Korea
| | - Byung-Chul Lee
- Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul, 08826, South Korea.,Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul, 08826, South Korea
| | - Jin Young Lee
- Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul, 08826, South Korea.,Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul, 08826, South Korea
| | - Jae-Jun Kim
- Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul, 08826, South Korea
| | - Myung Geun Kook
- Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul, 08826, South Korea.,Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul, 08826, South Korea
| | - Kyung-Sun Kang
- Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul, 08826, South Korea. .,Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul, 08826, South Korea.
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45
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Pagliassotti MJ, Kim PY, Estrada AL, Stewart CM, Gentile CL. Endoplasmic reticulum stress in obesity and obesity-related disorders: An expanded view. Metabolism 2016; 65:1238-46. [PMID: 27506731 PMCID: PMC4980576 DOI: 10.1016/j.metabol.2016.05.002] [Citation(s) in RCA: 105] [Impact Index Per Article: 11.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/27/2016] [Revised: 04/01/2016] [Accepted: 05/06/2016] [Indexed: 02/07/2023]
Abstract
The endoplasmic reticulum (ER) is most notable for its central roles in calcium ion storage, lipid biosynthesis, and protein sorting and processing. By virtue of its extensive membrane contact sites that connect the ER to most other organelles and to the plasma membrane, the ER can also regulate diverse cellular processes including inflammatory and insulin signaling, nutrient metabolism, and cell proliferation and death via a signaling pathway called the unfolded protein response (UPR). Chronic UPR activation has been observed in liver and/or adipose tissue of dietary and genetic murine models of obesity, and in human obesity and non-alcoholic fatty liver disease (NAFLD). Activation of the UPR in obesity and obesity-related disorders likely has two origins. One linked to classic ER stress involving the ER lumen and one linked to alterations to the ER membrane environment. This review discusses both of these origins and also considers the role of post-translational protein modifications, such as acetylation and palmitoylation, and ER-mitochondrial interactions to obesity-mediated impairments in the ER and activation of the UPR.
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Affiliation(s)
| | - Paul Y Kim
- Department of Biological Sciences, Grambling State University
| | - Andrea L Estrada
- Department of Food Science and Human Nutrition, Colorado State University
| | - Claire M Stewart
- Department of Food Science and Human Nutrition, Colorado State University
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46
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Tong J, Manik MK, Yang H, Im YJ. Structural insights into nonvesicular lipid transport by the oxysterol binding protein homologue family. Biochim Biophys Acta Mol Cell Biol Lipids 2016; 1861:928-939. [DOI: 10.1016/j.bbalip.2016.01.008] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2015] [Revised: 12/23/2015] [Accepted: 01/14/2016] [Indexed: 10/22/2022]
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47
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Flynn MP, Fiedler SE, Karlsson AB, Carr DW, Maizels ET, Hunzicker-Dunn M. Dephosphorylation of MAP2D enhances its binding to vimentin in preovulatory ovarian granulosa cells. J Cell Sci 2016; 129:2983-96. [PMID: 27335427 DOI: 10.1242/jcs.190397] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2016] [Accepted: 06/10/2016] [Indexed: 12/28/2022] Open
Abstract
Preovulatory granulosa cells express the low-molecular-mass MAP2D variant of microtubule-associated protein 2 (MAP2). Activation of the luteinizing hormone choriogonadotropin receptor by human choriogonadotropin (hCG) promotes dephosphorylation of MAP2D on Thr256 and Thr259. We sought to evaluate the association of MAP2D with the cytoskeleton, and the effect of hCG on this association. MAP2D partially colocalized, as assessed by confocal immunofluorescence microscopy, with the vimentin intermediate filament and microtubule cytoskeletons in naive cells. In vitro binding studies showed that MAP2D bound directly to vimentin and β-tubulin. Phosphorylation of recombinant MAP2D on Thr256 and Thr259, which mimics the phosphorylation status of MAP2D in naive cells, reduces binding of MAP2D to vimentin and tubulin by two- and three-fold, respectively. PKA-dependent phosphorylation of vimentin (Ser32 and Ser38) promoted binding of vimentin to MAP2D and increased contraction of granulosa cells with reorganization of vimentin filaments and MAP2D from the periphery into a thickened layer surrounding the nucleus and into prominent cellular extensions. Chemical disruption of vimentin filament organization increased progesterone production. Taken together, these results suggest that hCG-stimulated dephosphorylation of MAP2D at Thr256 and Thr259, phosphorylation of vimentin at Ser38 and Ser72, and the resulting enhanced binding of MAP2D to vimentin might contribute to the progesterone synthetic response required for ovulation.
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Affiliation(s)
- Maxfield P Flynn
- Department of Cell and Molecular Biology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
| | - Sarah E Fiedler
- Department of Medicine, Oregon Health and Sciences University and VA Portland Health Care System, Portland, OR 97239, USA
| | - Amelia B Karlsson
- Department of Cell and Molecular Biology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA School of Molecular Biosciences, Washington State University, Pullman, WA 99164, USA
| | - Daniel W Carr
- Department of Medicine, Oregon Health and Sciences University and VA Portland Health Care System, Portland, OR 97239, USA
| | - Evelyn T Maizels
- Department of Cell and Molecular Biology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
| | - Mary Hunzicker-Dunn
- Department of Cell and Molecular Biology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA School of Molecular Biosciences, Washington State University, Pullman, WA 99164, USA
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48
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Pycnosomes: Condensed Endosomal Structures Secreted by Dictyostelium Amoebae. PLoS One 2016; 11:e0154875. [PMID: 27187592 PMCID: PMC4871501 DOI: 10.1371/journal.pone.0154875] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2016] [Accepted: 04/20/2016] [Indexed: 11/19/2022] Open
Abstract
Dictyostelium discoideum has been used largely as a model organism to study the organization and function of the endocytic pathway. Here we describe dense structures present in D. discoideum endocytic compartments, which we named pycnosomes. Pycnosomes are constitutively secreted in the extracellular medium, from which they can be recovered by differential centrifugation. We identified the most abundant protein present in secreted pycnosomes, that we designated SctA. SctA defines a new family of proteins with four members in D. discoideum, and homologous proteins in other protists and eumetazoa. We developed a monoclonal antibody specific for SctA and used it to further characterize secreted and intracellular pycnosomes. Within cells, immunofluorescence as well as electron microscopy identified pycnosomes as SctA-enriched dense structures in the lumen of endocytic compartments. Pycnosomes are occasionally seen in continuity with intra-endosomal membranes, particularly in U18666A-treated cells where intraluminal budding is highly enhanced. While the exact nature, origin and cellular function of pycnosomes remain to be established, this study provides a first description of these structures as well as a characterization of reagents that can be used for further studies.
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49
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LaPensee CR, Mann JE, Rainey WE, Crudo V, Hunt SW, Hammer GD. ATR-101, a Selective and Potent Inhibitor of Acyl-CoA Acyltransferase 1, Induces Apoptosis in H295R Adrenocortical Cells and in the Adrenal Cortex of Dogs. Endocrinology 2016; 157:1775-88. [PMID: 26986192 DOI: 10.1210/en.2015-2052] [Citation(s) in RCA: 62] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
ATR-101 is a novel, oral drug candidate currently in development for the treatment of adrenocortical cancer. ATR-101 is a selective and potent inhibitor of acyl-coenzyme A:cholesterol O-acyltransferase 1 (ACAT1), an enzyme located in the endoplasmic reticulum (ER) membrane that catalyzes esterification of intracellular free cholesterol (FC). We aimed to identify mechanisms by which ATR-101 induces adrenocortical cell death. In H295R human adrenocortical carcinoma cells, ATR-101 decreases the formation of cholesteryl esters and increases FC levels, demonstrating potent inhibition of ACAT1 activity. Caspase-3/7 levels and terminal deoxynucleotidyl transferase 2'-deoxyuridine 5'-triphosphate nick end labeled-positive cells are increased by ATR-101 treatment, indicating activation of apoptosis. Exogenous cholesterol markedly potentiates the activity of ATR-101, suggesting that excess FC that cannot be adequately esterified increases caspase-3/7 activation and subsequent cell death. Inhibition of calcium release from the ER or the subsequent uptake of calcium by mitochondria reverses apoptosis induced by ATR-101. ATR-101 also activates multiple components of the unfolded protein response, an indicator of ER stress. Targeted knockdown of ACAT1 in an adrenocortical cell line mimicked the effects of ATR-101, suggesting that ACAT1 mediates the cytotoxic effects of ATR-101. Finally, in vivo treatment of dogs with ATR-101 decreased adrenocortical steroid production and induced cellular apoptosis that was restricted to the adrenal cortex. Together, these studies demonstrate that inhibition of ACAT1 by ATR-101 increases FC, resulting in dysregulation of ER calcium stores that result in ER stress, the unfolded protein response, and ultimately apoptosis.
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Affiliation(s)
- Christopher R LaPensee
- Departments of Internal Medicine (C.R.L., G.D.H.), Pathology (J.E.M.), and Molecular and Integrative Physiology (W.E.R., V.C.), University of Michigan, Ann Arbor, Michigan 48109; and Atterocor, Inc (S.W.H.), Ann Arbor, Michigan 48104
| | - Jacqueline E Mann
- Departments of Internal Medicine (C.R.L., G.D.H.), Pathology (J.E.M.), and Molecular and Integrative Physiology (W.E.R., V.C.), University of Michigan, Ann Arbor, Michigan 48109; and Atterocor, Inc (S.W.H.), Ann Arbor, Michigan 48104
| | - William E Rainey
- Departments of Internal Medicine (C.R.L., G.D.H.), Pathology (J.E.M.), and Molecular and Integrative Physiology (W.E.R., V.C.), University of Michigan, Ann Arbor, Michigan 48109; and Atterocor, Inc (S.W.H.), Ann Arbor, Michigan 48104
| | - Valentina Crudo
- Departments of Internal Medicine (C.R.L., G.D.H.), Pathology (J.E.M.), and Molecular and Integrative Physiology (W.E.R., V.C.), University of Michigan, Ann Arbor, Michigan 48109; and Atterocor, Inc (S.W.H.), Ann Arbor, Michigan 48104
| | - Stephen W Hunt
- Departments of Internal Medicine (C.R.L., G.D.H.), Pathology (J.E.M.), and Molecular and Integrative Physiology (W.E.R., V.C.), University of Michigan, Ann Arbor, Michigan 48109; and Atterocor, Inc (S.W.H.), Ann Arbor, Michigan 48104
| | - Gary D Hammer
- Departments of Internal Medicine (C.R.L., G.D.H.), Pathology (J.E.M.), and Molecular and Integrative Physiology (W.E.R., V.C.), University of Michigan, Ann Arbor, Michigan 48109; and Atterocor, Inc (S.W.H.), Ann Arbor, Michigan 48104
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50
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Vurusaner B, Leonarduzzi G, Gamba P, Poli G, Basaga H. Oxysterols and mechanisms of survival signaling. Mol Aspects Med 2016; 49:8-22. [PMID: 27017897 DOI: 10.1016/j.mam.2016.02.004] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2016] [Revised: 02/27/2016] [Accepted: 02/29/2016] [Indexed: 12/19/2022]
Abstract
Oxysterols, a family of oxidation products of cholesterol, are increasingly drawing attention of scientists to their multifaceted biochemical properties, several of them of clear relevance to human pathophysiology. Taken up by cells through both vesicular and non-vesicular ways or often generated intracellularly, oxysterols contribute to modulate not only the inflammatory and immunological response but also cell viability, metabolism and function by modulating several signaling pathways. Moreover, they have been recognized as elective ligands for the most important nuclear receptors. The outcome of such a complex network of intracellular reactions promoted by these cholesterol oxidation products appears to be largely dependent not only on the type of cells, the dynamic conditions of the cellular and tissue environment but also on the concentration of the oxysterols. Here focus has been given to the cascade of molecular events exerted by relatively low concentrations of certain oxysterols that elicit survival and functional signals in the cells, with the aim to contribute to further expand the knowledge about the biological and physiological potential of the biochemical reactions triggered and modulated by oxysterols.
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Affiliation(s)
- Beyza Vurusaner
- Biological Sciences and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University, Orhanli-Tuzla, 34956 Istanbul, Turkey
| | | | - Paola Gamba
- Department of Clinical and Biological Sciences, University of Torino, Torino, Italy
| | - Giuseppe Poli
- Department of Clinical and Biological Sciences, University of Torino, Torino, Italy.
| | - Huveyda Basaga
- Biological Sciences and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University, Orhanli-Tuzla, 34956 Istanbul, Turkey.
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