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1
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Jacko J, Morvová M, Hervay NT, Eliaš D, Gbelská Y, Waczulíková I, Gášková D, Balážová M, Šikurová L. Impact of ERG6 Gene Deletion on Membrane Composition and Properties in the Pathogenic Yeast Candida glabrata. Cell Biochem Biophys 2025; 83:1909-1918. [PMID: 39477913 PMCID: PMC12089240 DOI: 10.1007/s12013-024-01599-w] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 10/11/2024] [Indexed: 05/20/2025]
Abstract
The ERG6 gene is crucial for the biosynthesis of ergosterol, a key component of yeast cell membranes. Our study examines the impact of ERG6 gene deletion on the membrane composition and physicochemical properties of the pathogenic yeast Candida glabrata. Specifically, we investigated changes in selected sterol content, phospholipid composition, transmembrane potential, and PDR16 gene activity. Sterol levels were measured using high-performance liquid chromatography, the phospholipid profile was analysed via thin-layer chromatography, transmembrane potential was assessed with fluorescence spectroscopy, and gene expression levels were determined by quantitative PCR. Our findings revealed a depletion of ergosterol, increased zymosterol and eburicol content, an increased phosphatidylcholine and a reduced phosphatidylethanolamine content in the Δerg6 strain compared to the wt. Additionally, the Δerg6 strain exhibited membrane hyperpolarization without changes in PDR16 expression. Furthermore, the Δerg6 strain showed increased sensitivity to the antifungals myriocin and aureobasidine A. These results suggest that ERG6 gene deletion leads to significant alterations in membrane composition and may activates an alternative ergosterol synthesis pathway in the C. glabrata Δerg6 deletion mutant.
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Affiliation(s)
- J Jacko
- Department of Nuclear Physics and Biophysics, Faculty of Mathematics, Physics and Informatics, Comenius University Bratislava, Bratislava, Slovakia.
| | - M Morvová
- Department of Nuclear Physics and Biophysics, Faculty of Mathematics, Physics and Informatics, Comenius University Bratislava, Bratislava, Slovakia
| | - N Tóth Hervay
- Department of Microbiology and Virology, Faculty of Natural Sciences, Comenius University Bratislava, Bratislava, Slovakia
| | - D Eliaš
- Department of Microbiology and Virology, Faculty of Natural Sciences, Comenius University Bratislava, Bratislava, Slovakia
| | - Y Gbelská
- Department of Microbiology and Virology, Faculty of Natural Sciences, Comenius University Bratislava, Bratislava, Slovakia
| | - I Waczulíková
- Department of Nuclear Physics and Biophysics, Faculty of Mathematics, Physics and Informatics, Comenius University Bratislava, Bratislava, Slovakia
| | - D Gášková
- Institute of Physics, Charles University, Prague, Czechia
| | - M Balážová
- Centre for Biosciences SAS, Institute of Biochemistry and Genetics of Animals SAS, Bratislava, Slovakia
| | - L Šikurová
- Department of Nuclear Physics and Biophysics, Faculty of Mathematics, Physics and Informatics, Comenius University Bratislava, Bratislava, Slovakia
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2
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Kobalter S, Wriessnegger T, Pichler H. Engineering yeast for tailored fatty acid profiles. Appl Microbiol Biotechnol 2025; 109:101. [PMID: 40263140 PMCID: PMC12014800 DOI: 10.1007/s00253-025-13487-1] [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] [Received: 01/24/2025] [Revised: 04/07/2025] [Accepted: 04/08/2025] [Indexed: 04/24/2025]
Abstract
The demand for sustainable and eco-friendly alternatives to fossil and plant oil-derived chemicals has spurred interest in microbial production of lipids, particularly triacylglycerols, fatty acids, and their derivatives. Yeasts are promising platforms for synthesizing these compounds due to their high lipid accumulation capabilities, robust growth, and generally recognized as safe (GRAS) status. There is vast interest in fatty acid and triacylglycerol products with tailored fatty acid chain lengths and compositions, such as polyunsaturated fatty acids and substitutes for cocoa butter and palm oil. However, microbes naturally produce a limited set of mostly long-chain fatty acids, necessitating the development of microbial cell factories with customized fatty acid profiles. This review explores the capabilities of key enzymes involved in fatty acid and triacylglycerol synthesis, including fatty acid synthases, desaturases, elongases, and acyltransferases. It discusses factors influencing fatty acid composition and presents engineering strategies to enhance fatty acid synthesis. Specifically, we highlight successful engineering approaches to modify fatty acid profiles in triacylglycerols and produce tailored fatty acids, and we offer recommendations for host selection to streamline engineering efforts. KEY POINTS: • Detailed overview on all basic aspects of fatty acid metabolism in yeast • Comprehensive description of fatty acid profile tailoring in yeast • Extensive summary of applying tailored fatty acid profiles in production processes.
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Affiliation(s)
- Simon Kobalter
- Austrian Centre of Industrial Biotechnology (acib) GmbH, Petersgasse 14, 8010, Graz, Austria
| | - Tamara Wriessnegger
- Austrian Centre of Industrial Biotechnology (acib) GmbH, Petersgasse 14, 8010, Graz, Austria
| | - Harald Pichler
- Austrian Centre of Industrial Biotechnology (acib) GmbH, Petersgasse 14, 8010, Graz, Austria.
- Institute of Molecular Biotechnology, Graz University of Technology, NAWI Graz, BioTechMed Graz, Petersgasse 14, 8010, Graz, Austria.
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3
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Sokolov SS, Zyrina AN, Akimov SA, Severin FF. Interrelationship between the Non-Vesicular Transport of Sterols and Their Distribution between the Rafts and the Non-Raft Phase of the Plasma Membrane. BIOCHEMISTRY. BIOKHIMIIA 2025; 90:321-333. [PMID: 40367076 DOI: 10.1134/s0006297924604313] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/28/2024] [Revised: 03/07/2025] [Accepted: 03/12/2025] [Indexed: 05/16/2025]
Abstract
Sterols significantly affect the barrier properties of the membrane, which might explains the fact that their concentration is maximal in the plasma membrane (PM). Together with sphingolipids, sterols form rafts, i.e., bilayer regions whose physicochemical properties differ from those of the surrounding PM. The presence of rafts allows membrane proteins to choose the lipid environment optimal for their functioning (in terms of thickness, rigidity, spontaneous curvature, and lateral pressure profile of the bilayer). The ratio between sterols and sphingolipids in the rafts is close to stoichiometric. Theoretically, excess sterol outside the rafts can critically reduce the degree of order of membrane phospholipids. Sterols are synthesized in the endoplasmic reticulum (ER). The active (against the concentration gradient) transport of sterols from the ER to the PM is driven by proteins of the Osh family, while Lam proteins provide passive reverse transport of sterols from the PM to the ER. Inactivation of Osh proteins does not reduce the total level of sterols in the PM but reduces the rate of their movement inside the PM (the mechanisms underlying this effect remains unclear). Therefore, the vesicular transport of sterols from the ER to the PM is probably more active than the non-vesicular transport carried out by Osh proteins. Since sterols are more rigidly anchored and less sterically accessible in the rafts than outside them, we suggested that Lam proteins transport excess sterols from the non-raft phase of the PM to the ER, and Osh proteins return them back to the PM. In this way, the mutual activity of the Osh and lam proteins provides the rotation of sterols between the non-raft fraction of the PM and rafts, with the enrichment of the latter. It is possible that with a decrease in the sterol concentration in the non-raft fraction of the membrane, the rate of the Lam-dependent transport decreases since the degree of order of phospholipids and, consequently, the strength of retention of sterol molecules in the membrane increases, which might represent a mechanisms maintaining the concentration and distribution of sterols in the PM.
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Affiliation(s)
- Svyatoslav S Sokolov
- Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119991, Russia.
| | - Anna N Zyrina
- Chumakov Federal Scientific Center for Research and Development of Immune and Biological Products (Institute of Poliomyelitis), Russian Academy of Sciences, Moscow, 108819, Russia
| | - Sergey A Akimov
- Frumkin Institute of Physical Chemistry and Electrochemistry, Russian Academy of Sciences, Moscow, 119071, Russia
| | - Fedor F Severin
- Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119991, Russia
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4
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Bonzanini V, Haddad Momeni M, Olofsson K, Olsson L, Geijer C. Impact of glucose and propionic acid on even and odd chain fatty acid profiles of oleaginous yeasts. BMC Microbiol 2025; 25:79. [PMID: 39966733 PMCID: PMC11834278 DOI: 10.1186/s12866-025-03788-w] [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] [Received: 11/29/2024] [Accepted: 01/28/2025] [Indexed: 02/20/2025] Open
Abstract
BACKGROUND Odd chain fatty acids (OCFAs) are gaining attention for their valuable medical and nutritional applications. Microbial fermentation offers a sustainable and environmentally friendly alternative for OCFA production compared to traditional extraction or chemical synthesis methods. To achieve an economically feasible OCFA production process, it is essential to identify and develop microbial cell factories capable of producing OCFAs with high titers and yields. RESULTS We selected 19 yeast species, including both oleaginous yeasts and representatives from the Ascomycota and Basidiomycota phyla, based on their known or potential ability to produce OCFAs. These species were screened under various growth conditions to evaluate their OCFA production potential. In glucose-based, nitrogen-limited media, the strains produced fatty acids to varying extents, with OCFAs comprising 0.5-5% of the total fatty acids. When using the OCFAs precursor propionic acid as the sole carbon source, only eight strains exhibited growth, with tolerance to propionic acid concentrations between 5 and 29 g/L. The strains also displayed varying efficiencies in converting propionic acid into fatty acids, yielding between 0.16 and 1.22 g/L of fatty acids, with OCFAs constituting 37-89% of total fatty acids. Among the top performing strains, Cutaneotrichosporon oleaginosus produced the highest OCFA titers and yields (0.94 g/L, 0.07 g/g), Yarrowia lipolytica demonstrated superior growth rates even at elevated propionic acid concentrations, and Rhodotorula toruloides achieved the highest proportion of OCFAs relative to total fatty acids (89%). CONCLUSIONS Our findings highlight the diverse capacities of the selected yeast species for OCFA production, identifying several promising strains for further optimization as microbial cell factories in sustainable OCFA production processes.
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Affiliation(s)
- Veronica Bonzanini
- Department of Life Sciences, Division of Industrial Biotechnology, Chalmers University of Technology, Chalmersplatsen 4, Gothenburg, 412 96, Sweden
- AAK AB, Pulpetgatan 20, Malmö, 215 37, Sweden
| | | | | | - Lisbeth Olsson
- Department of Life Sciences, Division of Industrial Biotechnology, Chalmers University of Technology, Chalmersplatsen 4, Gothenburg, 412 96, Sweden
| | - Cecilia Geijer
- Department of Life Sciences, Division of Industrial Biotechnology, Chalmers University of Technology, Chalmersplatsen 4, Gothenburg, 412 96, Sweden.
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5
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Kelso C, Maccarone AT, de Kroon AIPM, Mitchell TW, Renne MF. Temperature adaptation of yeast phospholipid molecular species at the acyl chain positional level. FEBS Lett 2025; 599:530-544. [PMID: 39673166 PMCID: PMC11848023 DOI: 10.1002/1873-3468.15060] [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] [Received: 06/20/2024] [Revised: 10/02/2024] [Accepted: 10/22/2024] [Indexed: 12/16/2024]
Abstract
Yeast is a poikilothermic organism and adapts its lipid composition to the environmental temperature to maintain membrane physical properties. Studies addressing temperature-dependent adaptation of the lipidome have described changes in the phospholipid composition at the level of sum composition (e.g. PC 32:1) and molecular composition (e.g. PC 16:0_16:1). However, there is little information at the level of positional isomers (e.g. PC 16:0/16:1 versus PC 16:1/16:0). Here, we used collision- and ozone-induced dissociation (CID/OzID) mass spectrometry to investigate homeoviscous adaptation of PC, PE and PS to determine the phospholipid acyl chains at the sn-1 and sn-2 position. Our data establish the sn-molecular species composition of PC, PE and PS in the lipidome of yeast cultured at different temperatures.
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Affiliation(s)
- Celine Kelso
- School of Chemistry and Molecular BioscienceUniversity of WollongongAustralia
- Molecular Horizons InstituteUniversity of WollongongAustralia
| | - Alan T. Maccarone
- School of Chemistry and Molecular BioscienceUniversity of WollongongAustralia
- Molecular Horizons InstituteUniversity of WollongongAustralia
| | - Anton I. P. M. de Kroon
- Membrane Biochemistry & Biophysics, Department of ChemistryUtrecht UniversityThe Netherlands
| | - Todd W. Mitchell
- Molecular Horizons InstituteUniversity of WollongongAustralia
- School of Medical, Indigenous and Health SciencesUniversity of WollongongAustralia
| | - Mike F. Renne
- Membrane Biochemistry & Biophysics, Department of ChemistryUtrecht UniversityThe Netherlands
- Medical Biochemistry and Molecular Biology, Medical FacultySaarland UniversityHomburgGermany
- Preclinical Center for Molecular Signalling (PZMS), Medical FacultySaarland UniversityHomburgGermany
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6
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Qian W, Tang H, Yao H. Lipidomics and temporal-spatial distribution of organelle lipid. J Biol Methods 2025; 12:e99010049. [PMID: 40200947 PMCID: PMC11973048 DOI: 10.14440/jbm.2025.0094] [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: 10/08/2024] [Revised: 11/17/2024] [Accepted: 12/16/2024] [Indexed: 04/10/2025] Open
Abstract
Background Lipids are crucial signaling molecules or cellular membrane components orchestrating biological processes. To gain insights into lipid functions and the communication between organelles, it is essential to understand the subcellular localization of individual lipids. Advancements in lipid quantification techniques, improvements in chemical and spatial resolution for detecting various lipid species, and enhancements in organelle isolation speed have allowed for profiling of the organelle lipidome, capturing its temporal-spatial distribution. Objective This review examined approaches used to develop organelle lipidome and aimed to gain insights into cellular lipid homeostasis from an organelle perspective. In addition, this review discussed the advancements in lipid-mediated inter-organelle communication within complex physiological and pathological processes. Conclusion With the advancement of lipidomic technologies, more detailed explorations of organelle structures and the specific lipid-mediating functions they perform are feasible.
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Affiliation(s)
- Wenjuan Qian
- State Key Laboratory of Genetic Engineering, School of Life Sciences, Human Phenome Institute, Metabonomics and Systems Biology Laboratory at Shanghai International Centre for Molecular Phenomics, Zhongshan Hospital, Fudan University, Shanghai 200032, China
| | - Huiru Tang
- State Key Laboratory of Genetic Engineering, School of Life Sciences, Human Phenome Institute, Metabonomics and Systems Biology Laboratory at Shanghai International Centre for Molecular Phenomics, Zhongshan Hospital, Fudan University, Shanghai 200032, China
| | - Hongyan Yao
- State Key Laboratory of Genetic Engineering, School of Life Sciences, Human Phenome Institute, Metabonomics and Systems Biology Laboratory at Shanghai International Centre for Molecular Phenomics, Zhongshan Hospital, Fudan University, Shanghai 200032, China
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7
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Delfosse V, Drin G. Determining the Relative Affinity of ORPs for Lipid Ligands Using Fluorescence and Thermal Shift Assays. Methods Mol Biol 2025; 2888:259-280. [PMID: 39699737 DOI: 10.1007/978-1-0716-4318-1_18] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2024]
Abstract
Lipid transfer proteins (LTPs) are specialized proteins that convey specific lipids across the cytosol to regulate the lipid composition of organelles and the plasma membrane. Quantifying to which extent these LTPs recognize and transfer various lipid species and subspecies is of prime interest to define their cellular role(s). Here, we describe how to measure in vitro the relative affinity of Osh6p, a yeast phosphatidylserine (PS)/phosphatidylinositol 4-phosphate (PI(4)P) exchanger belonging to the oxysterol-binding protein(OSBP)-related protein (ORP) family, for PS and phosphoinositide subspecies. First, we detail how to produce and purify Osh6p with high purity. Secondly, we describe how to measure its ability to bind PS, PI(4)P, and PI(4,5)P2 by FRET-based and thermal shift assays using liposomes of defined composition. These protocols can allow further analysis of other ORPs or inspire the design of assays to characterize other LTPs. Notably, they can be helpful in defining how LTPs transfer phospholipids subspecies as a function of their acyl chains' length and unsaturation degree and, therefore, whether they can contribute to regulating the acyl chain composition of cell membranes.
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Affiliation(s)
- Vanessa Delfosse
- Centre de Biologie Structurale (CBS), Université de Montpellier, INSERM, CNRS, Montpellier, France
| | - Guillaume Drin
- Université Côte d'Azur, CNRS, INSERM, Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France.
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8
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Ernst R, Renne MF, Jain A, von der Malsburg A. Endoplasmic Reticulum Membrane Homeostasis and the Unfolded Protein Response. Cold Spring Harb Perspect Biol 2024; 16:a041400. [PMID: 38253414 PMCID: PMC11293554 DOI: 10.1101/cshperspect.a041400] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/24/2024]
Abstract
The endoplasmic reticulum (ER) is the key organelle for membrane biogenesis. Most lipids are synthesized in the ER, and most membrane proteins are first inserted into the ER membrane before they are transported to their target organelle. The composition and properties of the ER membrane must be carefully controlled to provide a suitable environment for the insertion and folding of membrane proteins. The unfolded protein response (UPR) is a powerful signaling pathway that balances protein and lipid production in the ER. Here, we summarize our current knowledge of how aberrant compositions of the ER membrane, referred to as lipid bilayer stress, trigger the UPR.
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Affiliation(s)
- Robert Ernst
- Medical Biochemistry and Molecular Biology, Medical Faculty, Saarland University, 66421 Homburg, Germany
- Preclinical Center for Molecular Signaling (PZMS), Medical Faculty, Saarland University, 66421 Homburg, Germany
| | - Mike F Renne
- Medical Biochemistry and Molecular Biology, Medical Faculty, Saarland University, 66421 Homburg, Germany
- Preclinical Center for Molecular Signaling (PZMS), Medical Faculty, Saarland University, 66421 Homburg, Germany
| | - Aamna Jain
- Medical Biochemistry and Molecular Biology, Medical Faculty, Saarland University, 66421 Homburg, Germany
- Preclinical Center for Molecular Signaling (PZMS), Medical Faculty, Saarland University, 66421 Homburg, Germany
| | - Alexander von der Malsburg
- Medical Biochemistry and Molecular Biology, Medical Faculty, Saarland University, 66421 Homburg, Germany
- Preclinical Center for Molecular Signaling (PZMS), Medical Faculty, Saarland University, 66421 Homburg, Germany
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9
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Prokisch S, Büttner S. Partitioning into ER membrane microdomains impacts autophagic protein turnover during cellular aging. Sci Rep 2024; 14:13653. [PMID: 38871812 DOI: 10.1038/s41598-024-64493-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2023] [Accepted: 06/09/2024] [Indexed: 06/15/2024] Open
Abstract
Eukaryotic membranes are compartmentalized into distinct micro- and nanodomains that rearrange dynamically in response to external and internal cues. This lateral heterogeneity of the lipid bilayer and associated clustering of distinct membrane proteins contribute to the spatial organization of numerous cellular processes. Here, we show that membrane microdomains within the endoplasmic reticulum (ER) of yeast cells are reorganized during metabolic reprogramming and aging. Using biosensors with varying transmembrane domain length to map lipid bilayer thickness, we demonstrate that in young cells, microdomains of increased thickness mainly exist within the nuclear ER, while progressing cellular age drives the formation of numerous microdomains specifically in the cortical ER. Partitioning of biosensors with long transmembrane domains into these microdomains increased protein stability and prevented autophagic removal. In contrast, reporters with short transmembrane domains progressively accumulated at the membrane contact site between the nuclear ER and the vacuole, the so-called nucleus-vacuole junction (NVJ), and were subjected to turnover via selective microautophagy occurring specifically at these sites. Reporters with long transmembrane domains were excluded from the NVJ. Our data reveal age-dependent rearrangement of the lateral organization of the ER and establish transmembrane domain length as a determinant of membrane contact site localization and autophagic degradation.
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Affiliation(s)
- Simon Prokisch
- Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, 10691, Stockholm, Sweden
| | - Sabrina Büttner
- Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, 10691, Stockholm, Sweden.
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10
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Duan M, Plemel RL, Takenaka T, Lin A, Delgado BM, Nattermann U, Nickerson DP, Mima J, Miller EA, Merz AJ. SNARE chaperone Sly1 directly mediates close-range vesicle tethering. J Cell Biol 2024; 223:e202001032. [PMID: 38478018 PMCID: PMC10943277 DOI: 10.1083/jcb.202001032] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2020] [Revised: 12/20/2023] [Accepted: 02/22/2024] [Indexed: 03/17/2024] Open
Abstract
The essential Golgi protein Sly1 is a member of the Sec1/mammalian Unc-18 (SM) family of SNARE chaperones. Sly1 was originally identified through remarkable gain-of-function alleles that bypass requirements for diverse vesicle tethering factors. Employing genetic analyses and chemically defined reconstitutions of ER-Golgi fusion, we discovered that a loop conserved among Sly1 family members is not only autoinhibitory but also acts as a positive effector. An amphipathic lipid packing sensor (ALPS)-like helix within the loop directly binds high-curvature membranes. Membrane binding is required for relief of Sly1 autoinhibition and also allows Sly1 to directly tether incoming vesicles to the Qa-SNARE on the target organelle. The SLY1-20 mutation bypasses requirements for diverse tethering factors but loses this ability if the tethering activity is impaired. We propose that long-range tethers, including Golgins and multisubunit tethering complexes, hand off vesicles to Sly1, which then tethers at close range to initiate trans-SNARE complex assembly and fusion in the early secretory pathway.
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Affiliation(s)
- Mengtong Duan
- Department of Biochemistry, University of Washington, Seattle, WA, USA
| | - Rachael L. Plemel
- Department of Biochemistry, University of Washington, Seattle, WA, USA
| | | | - Ariel Lin
- Department of Biochemistry, University of Washington, Seattle, WA, USA
- Department of Biology, California State University, San Bernardino, CA, USA
| | | | - Una Nattermann
- Department of Biochemistry, University of Washington, Seattle, WA, USA
- Biophysics, Structure, and Design Graduate Program, University of Washington, Seattle, WA, USA
- Institute for Protein Design, University of Washington, Seattle, WA, USA
| | | | - Joji Mima
- Institute for Protein Research, Osaka University, Osaka, Japan
| | | | - Alexey J. Merz
- Department of Biochemistry, University of Washington, Seattle, WA, USA
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11
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Laquel P, Ayciriex S, Doignon F, Camougrand N, Fougère L, Rocher C, Wattelet-Boyer V, Bessoule JJ, Testet E. Mlg1, a yeast acyltransferase located in ER membranes associated with mitochondria (MAMs), is involved in de novo synthesis and remodelling of phospholipids. FEBS J 2024; 291:2683-2702. [PMID: 38297966 DOI: 10.1111/febs.17068] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2023] [Revised: 11/27/2023] [Accepted: 01/17/2024] [Indexed: 02/02/2024]
Abstract
In cells, phospholipids contain acyl chains of variable lengths and saturation, features that affect their functions. Their de novo synthesis in the endoplasmic reticulum takes place via the cytidine diphosphate diacylglycerol (CDP-DAG) and Kennedy pathways, which are conserved in eukaryotes. PA is a key intermediate for all phospholipids (PI, PIPs, PS, PE, PC, PG and CL). The de novo synthesis of PA occurs by acylation of glycerophosphate leading to the synthesis of 1-acyl lysoPA and subsequent acylation of 1-acyl lysoPA at the sn-2 position. Using membranes from Escherichia coli overexpressing MLG1, we showed that the yeast gene MLG1 encodes an acyltransferase, leading specifically to the synthesis of PA from 1-acyl lysoPA. Moreover, after their de novo synthesis, phospholipids can be remodelled by acyl exchange with one and/or two acyl chains exchanged at the sn-1 and/or sn-2 position. Based on shotgun lipidomics of the reference and mlg1Δ strains, as well as biochemical assays for acyltransferase activities, we identified an additional remodelling activity for Mlg1p, namely, incorporation of palmitic acid into the sn-1 position of PS and PE. By using confocal microscopy and subcellular fractionation, we also found that this acyltransferase is located in ER membranes associated with mitochondria, a finding that highlights the importance of these organelles in the global cellular metabolism of lipids.
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Affiliation(s)
- Patricia Laquel
- Univ. Bordeaux, CNRS, LBM, UMR 5200, Villenave d'Ornon, France
| | - Sophie Ayciriex
- Univ. Lyon, CNRS, Université Claude Bernard Lyon 1, ISA, UMR 5280, Villeurbanne, France
| | | | | | - Louise Fougère
- Univ. Bordeaux, CNRS, LBM, UMR 5200, Villenave d'Ornon, France
| | | | | | | | - Eric Testet
- Univ. Bordeaux, CNRS, LBM, UMR 5200, Villenave d'Ornon, France
- Bordeaux INP, LBM, UMR 5200, Villenave d'Ornon, France
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12
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Hachem M, Ahmmed MK, Nacir-Delord H. Phospholipidomics in Clinical Trials for Brain Disorders: Advancing our Understanding and Therapeutic Potentials. Mol Neurobiol 2024; 61:3272-3295. [PMID: 37981628 PMCID: PMC11087356 DOI: 10.1007/s12035-023-03793-y] [Citation(s) in RCA: 4] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2023] [Accepted: 10/31/2023] [Indexed: 11/21/2023]
Abstract
Phospholipidomics is a specialized branch of lipidomics that focuses on the characterization and quantification of phospholipids. By using sensitive analytical techniques, phospholipidomics enables researchers to better understand the metabolism and activities of phospholipids in brain disorders such as Alzheimer's and Parkinson's diseases. In the brain, identifying specific phospholipid biomarkers can offer valuable insights into the underlying molecular features and biochemistry of these diseases through a variety of sensitive analytical techniques. Phospholipidomics has emerged as a promising tool in clinical studies, with immense potential to advance our knowledge of neurological diseases and enhance diagnosis and treatment options for patients. In the present review paper, we discussed numerous applications of phospholipidomics tools in clinical studies, with a particular focus on the neurological field. By exploring phospholipids' functions in neurological diseases and the potential of phospholipidomics in clinical research, we provided valuable insights that could aid researchers and clinicians in harnessing the full prospective of this innovative practice and improve patient outcomes by providing more potent treatments for neurological diseases.
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Affiliation(s)
- Mayssa Hachem
- Department of Chemistry and Healthcare Engineering Innovation Center, Khalifa University of Sciences and Technology, P.O. Box 127788, Abu Dhabi, United Arab Emirates.
| | - Mirja Kaizer Ahmmed
- Department of Fishing and Post-Harvest Technology, Chattogram Veterinary and Animal Sciences University, Chattogram, Bangladesh
- Riddet Institute, Massey University, Palmerston North, New Zealand
| | - Houda Nacir-Delord
- Department of Chemistry, Khalifa University of Sciences and Technology, P.O. Box 127788, Abu Dhabi, United Arab Emirates
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13
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Schuster M, Kilaru S, Steinberg G. Azoles activate type I and type II programmed cell death pathways in crop pathogenic fungi. Nat Commun 2024; 15:4357. [PMID: 38821954 PMCID: PMC11143370 DOI: 10.1038/s41467-024-48157-9] [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] [Received: 09/25/2023] [Accepted: 04/22/2024] [Indexed: 06/02/2024] Open
Abstract
Triazoles are widely used to control pathogenic fungi. They inhibit the ergosterol biosynthetic pathway, but the precise mechanisms leading to fungicidal activities in many fungal pathogens are poorly understood. Here, we elucidate the mode of action of epoxiconazole and metconazole in the wheat pathogen Zymoseptoria tritici and the rice blast fungus Magnaporthe oryzae. We show that both azoles have fungicidal activity and reduce fluidity, but not integrity, of the plasma membrane. This impairs localisation of Cdc15-like F-BAR proteins, resulting in defective actin ring assembly and incomplete septation. However, mutant studies and pharmacological experiments in vitro and in planta show that azole lethality is due to a combination of reactive oxygen species-induced apoptosis and macroautophagy. Simultaneous inhibition of both programmed cell death pathways abolishes azole-induced cell death. Other classes of ergosterol biosynthesis inhibitors also induce apoptosis and macroautophagy, suggesting that activation of these two cell death pathways is a hallmark of ergosterol synthesis-targeting fungicides. This knowledge will inform future crop protection strategies.
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14
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Graff J, Schneiter R. FIT2 proteins and lipid droplet emergence, an interplay between phospholipid synthesis, surface tension, and membrane curvature. Front Cell Dev Biol 2024; 12:1422032. [PMID: 38872930 PMCID: PMC11169642 DOI: 10.3389/fcell.2024.1422032] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2024] [Accepted: 05/06/2024] [Indexed: 06/15/2024] Open
Abstract
Lipid droplets (LDs) serve as intracellular compartments primarily dedicated to the storage of metabolic energy in the form of neutral lipids. The processes that regulate and control LD biogenesis are being studied extensively and are gaining significance due to their implications in major metabolic disorders, including type 2 diabetes and obesity. A protein of particular interest is Fat storage-Inducing Transmembrane 2 (FIT2), which affects the emergence step of LD biogenesis. Instead of properly emerging towards the cytosol, LDs in FIT2-deficient cells remain embedded within the membrane of the endoplasmic reticulum (ER). In vitro studies revealed the ability of FIT2 to bind both di- and triacylglycerol (DAG/TAG), key players in lipid storage, and its activity to cleave acyl-CoA. However, the translation of these in vitro functions to the observed embedding of LDs in FIT2 deficient cells remains to be established. To understand the role of FIT2 in vivo, we discuss the parameters that affect LD emergence. Our focus centers on the role that membrane curvature and surface tension play in LD emergence, as well as the impact that the lipid composition exerts on these key parameters. In addition, we discuss hypotheses on how FIT2 could function locally to modulate lipids at sites of LD emergence.
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Affiliation(s)
| | - Roger Schneiter
- Department of Biology, University of Fribourg, Fribourg, Switzerland
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15
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Aptekarev T, Furman G, Badar F, Sokolovsky V, Xia Y. Study of the collagen tissue nanostructure by analyzing the echo decay obtained using the MRI technique. SOFT MATTER 2024; 20:4282-4290. [PMID: 38757720 PMCID: PMC11211971 DOI: 10.1039/d4sm00312h] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 05/18/2024]
Abstract
The multicomponent relaxation observed in nuclear magnetic resonance experiments in biological tissues makes it difficult to establish a correlation between specific relaxation times and tissue structural parameters. The analysis of a nanostructure (the characteristic size of 10-1000 nm) is usually based on formulas for relaxation times which depend on structural parameters at the atomic or molecular levels in the size range of 0.1-5 nm. We have recently developed an analysis method in which relaxation times' anisotropy in a sample is explicitly related to its structure of nanocavities containing a liquid or gas. However, the method is based on the analysis of experimental data on the anisotropy of relaxation times obtained by using the standard NMR technique and rotating the sample relative to a magnetic field and requires a series of experiments. In the present study, to address this challenge, we develop a new method of analysis of a multi-exponential magnetic resonance signal that does not require determining relaxation times and eliminates the sample rotation and the necessity of a series of experiments. Using the magnetic resonance imaging (MRI) technique, the total signal from the whole sample was obtained as a sum of the signals (echo decays) from all voxels. In contrast to previous research, the volumes of nanocavities and their angular distribution can be obtained by analyzing a single total signal for the entire cartilage. In addition, within the framework of this approach, it is possible to identify the reason for the multicomponent nature of relaxation. The proposed method for analyzing a single multi-exponential signal (transverse relaxation) was implemented on cartilage. Using the signal, three anatomical zones of cartilage were studied, revealing significant structural differences between them. The proposed method not only avoids the need for sample rotation but also enables repeated application of layer-by-layer magnetic resonance imaging with micron resolution. The study results allow us to suggest that water molecules contributing to the echo decay are more likely located in nanocavities formed by the fibrillar structure rather than inside the fibrils.
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Affiliation(s)
- Theodore Aptekarev
- Physics Department, Ben Gurion University of the Negev, Beer Sheva, Israel.
| | - Gregory Furman
- Physics Department, Ben Gurion University of the Negev, Beer Sheva, Israel.
| | - Farid Badar
- Physics Department, Oakland University, Rochester, MI, USA
| | | | - Yang Xia
- Physics Department, Oakland University, Rochester, MI, USA
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16
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Reinhard J, Starke L, Klose C, Haberkant P, Hammarén H, Stein F, Klein O, Berhorst C, Stumpf H, Sáenz JP, Hub J, Schuldiner M, Ernst R. MemPrep, a new technology for isolating organellar membranes provides fingerprints of lipid bilayer stress. EMBO J 2024; 43:1653-1685. [PMID: 38491296 PMCID: PMC11021466 DOI: 10.1038/s44318-024-00063-y] [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] [Received: 09/14/2022] [Revised: 02/16/2024] [Accepted: 02/26/2024] [Indexed: 03/18/2024] Open
Abstract
Biological membranes have a stunning ability to adapt their composition in response to physiological stress and metabolic challenges. Little is known how such perturbations affect individual organelles in eukaryotic cells. Pioneering work has provided insights into the subcellular distribution of lipids in the yeast Saccharomyces cerevisiae, but the composition of the endoplasmic reticulum (ER) membrane, which also crucially regulates lipid metabolism and the unfolded protein response, remains insufficiently characterized. Here, we describe a method for purifying organelle membranes from yeast, MemPrep. We demonstrate the purity of our ER membrane preparations by proteomics, and document the general utility of MemPrep by isolating vacuolar membranes. Quantitative lipidomics establishes the lipid composition of the ER and the vacuolar membrane. Our findings provide a baseline for studying membrane protein biogenesis and have important implications for understanding the role of lipids in regulating the unfolded protein response (UPR). The combined preparative and analytical MemPrep approach uncovers dynamic remodeling of ER membranes in stressed cells and establishes distinct molecular fingerprints of lipid bilayer stress.
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Affiliation(s)
- John Reinhard
- Saarland University, Medical Biochemistry and Molecular Biology, Homburg, Germany
- Saarland University, Preclinical Center for Molecular Signaling (PZMS), Homburg, Germany
| | - Leonhard Starke
- Saarland University, Theoretical Physics and Center for Biophysics, Saarbrücken, Germany
| | | | - Per Haberkant
- EMBL Heidelberg, Proteomics Core Facility, Heidelberg, Germany
| | | | - Frank Stein
- EMBL Heidelberg, Proteomics Core Facility, Heidelberg, Germany
| | - Ofir Klein
- Weizmann Institute of Science, Department of Molecular Genetics, Rehovot, Israel
| | - Charlotte Berhorst
- Saarland University, Medical Biochemistry and Molecular Biology, Homburg, Germany
- Saarland University, Preclinical Center for Molecular Signaling (PZMS), Homburg, Germany
| | - Heike Stumpf
- Saarland University, Medical Biochemistry and Molecular Biology, Homburg, Germany
- Saarland University, Preclinical Center for Molecular Signaling (PZMS), Homburg, Germany
| | - James P Sáenz
- Technische Universität Dresden, B CUBE, Dresden, Germany
| | - Jochen Hub
- Saarland University, Theoretical Physics and Center for Biophysics, Saarbrücken, Germany
| | - Maya Schuldiner
- Weizmann Institute of Science, Department of Molecular Genetics, Rehovot, Israel
| | - Robert Ernst
- Saarland University, Medical Biochemistry and Molecular Biology, Homburg, Germany.
- Saarland University, Preclinical Center for Molecular Signaling (PZMS), Homburg, Germany.
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17
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Bento-Oliveira A, Starosta R, de Almeida RFM. Interaction of the antifungal ketoconazole and its diphenylphosphine derivatives with lipid bilayers: Insights into their antifungal action. Arch Biochem Biophys 2024; 753:109919. [PMID: 38307316 DOI: 10.1016/j.abb.2024.109919] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2023] [Revised: 01/25/2024] [Accepted: 01/30/2024] [Indexed: 02/04/2024]
Abstract
Ketoconazole (Ke) is an important antifungal drug, and two of its diphenylphosphinemethyl derivatives (KeP: Ph2PCH2-Ke and KeOP: Ph2P(O)CH2-Ke) have shown improved antifungal activity, namely against a yeast strain lacking ergosterol, suggesting alternative modes of action for azole compounds. In this context, the interactions of these compounds with a model of the cell membrane were investigated, using POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) large unilamellar vesicles and taking advantage of the intrinsic fluorescence of Ke, KeP and KeOP. Steady-state fluorescence spectra and anisotropy, including partition and aggregation studies, as well as fluorescence lifetime measurements, were carried out. In addition, the ability of the compounds to increase membrane permeability was assessed through carboxyfluorescein leakage. The membrane/water mole fraction partition coefficients (Kp,x): (3.31 ± 0.36) x105, (8.31 ± 1.60) x105 and (4.66 ± 0.72) x106, for Ke, KeP and KeOP, respectively, show that all three compounds have moderate to high affinity for the lipid bilayer. Moreover, KeP, and particularly KeOP interact more efficiently with POPC bilayers than Ke, which correlates well with their in vitro antifungal activity. Furthermore, although the three compounds disturb the lipid bilayer, KeOP is the quickest and most efficient one. Hence, the higher affinity and ability to permeabilize the membrane of KeOP when compared to that of KeP, despite the higher lipophilicity of the latter, points to an important role of Ph2P(O)CH2- oxygen. Overall, this work suggests that membrane interactions are important for the antifungal activity of these azoles and should be considered in the design of new therapeutic agents.
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Affiliation(s)
- Andreia Bento-Oliveira
- Centro de Química Estrutural, Institute of Molecular Sciences, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016, Lisboa, Portugal
| | - Radosław Starosta
- Centro de Química Estrutural, Institute of Molecular Sciences, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016, Lisboa, Portugal; Faculty of Chemistry, University of Wroclaw, F. Joliot-Curie 14, 50-383, Wroclaw, Poland
| | - Rodrigo F M de Almeida
- Centro de Química Estrutural, Institute of Molecular Sciences, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016, Lisboa, Portugal.
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18
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Wang J, Shen J, Chen D, Liao B, Chen X, Zong Y, Wei Y, Shi Y, Liu Y, Gou L, Zhou X, Cheng L, Ren B. Secretory IgA reduced the ergosterol contents of Candida albicans to repress its hyphal growth and virulence. Appl Microbiol Biotechnol 2024; 108:244. [PMID: 38421461 PMCID: PMC10904422 DOI: 10.1007/s00253-024-13063-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2023] [Revised: 01/31/2024] [Accepted: 02/08/2024] [Indexed: 03/02/2024]
Abstract
Candida albicans, one of the most prevalent conditional pathogenic fungi, can cause local superficial infections and lethal systemic infections, especially in the immunocompromised population. Secretory immunoglobulin A (sIgA) is an important immune protein regulating the pathogenicity of C. albicans. However, the actions and mechanisms that sIgA exerts directly against C. albicans are still unclear. Here, we investigated that sIgA directs against C. albicans hyphal growth and virulence to oral epithelial cells. Our results indicated that sIgA significantly inhibited C. albicans hyphal growth, adhesion, and damage to oral epithelial cells compared with IgG. According to the transcriptome and RT-PCR analysis, sIgA significantly affected the ergosterol biosynthesis pathway. Furthermore, sIgA significantly reduced the ergosterol levels, while the addition of exogenous ergosterol restored C. albicans hyphal growth and adhesion to oral epithelial cells, indicating that sIgA suppressed the growth of hyphae and the pathogenicity of C. albicans by reducing its ergosterol levels. By employing the key genes mutants (erg11Δ/Δ, erg3Δ/Δ, and erg3Δ/Δ erg11Δ/Δ) from the ergosterol pathway, sIgA lost the hyphal inhibition on these mutants, while sIgA also reduced the inhibitory effects of erg11Δ/Δ and erg3Δ/Δ and lost the inhibition of erg3Δ/Δ erg11Δ/Δ on the adhesion to oral epithelial cells, further proving the hyphal repression of sIgA through the ergosterol pathway. We demonstrated for the first time that sIgA inhibited C. albicans hyphal development and virulence by affecting ergosterol biosynthesis and suggest that ergosterol is a crucial regulator of C. albicans-host cell interactions. KEY POINTS: • sIgA repressed C. albicans hyphal growth • sIgA inhibited C. albicans virulence to host cells • sIgA affected C. albicans hyphae and virulence by reducing its ergosterol levels.
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Affiliation(s)
- Jiannan Wang
- State Key Laboratory of Oral Diseases & National Center for Stomatology & National Clinical Research Center for Oral Diseases, West China School of Stomatology, Sichuan University, Chengdu, 610041, Sichuan, China
| | - Jiawei Shen
- State Key Laboratory of Oral Diseases & National Center for Stomatology & National Clinical Research Center for Oral Diseases, West China School of Stomatology, Sichuan University, Chengdu, 610041, Sichuan, China
| | - Ding Chen
- State Key Laboratory of Oral Diseases & National Center for Stomatology & National Clinical Research Center for Oral Diseases, West China School of Stomatology, Sichuan University, Chengdu, 610041, Sichuan, China
| | - Binyou Liao
- State Key Laboratory of Oral Diseases & National Center for Stomatology & National Clinical Research Center for Oral Diseases, West China School of Stomatology, Sichuan University, Chengdu, 610041, Sichuan, China
| | - Xi Chen
- State Key Laboratory of Oral Diseases & National Center for Stomatology & National Clinical Research Center for Oral Diseases, West China School of Stomatology, Sichuan University, Chengdu, 610041, Sichuan, China
- Department of Operative Dentistry and Endodontics, West China School of Stomatology, Sichuan University, Chengdu, 610041, Sichuan, China
| | - Yawen Zong
- State Key Laboratory of Oral Diseases & National Center for Stomatology & National Clinical Research Center for Oral Diseases, West China School of Stomatology, Sichuan University, Chengdu, 610041, Sichuan, China
- Department of Operative Dentistry and Endodontics, West China School of Stomatology, Sichuan University, Chengdu, 610041, Sichuan, China
| | - Yu Wei
- State Key Laboratory of Oral Diseases & National Center for Stomatology & National Clinical Research Center for Oral Diseases, West China School of Stomatology, Sichuan University, Chengdu, 610041, Sichuan, China
- Department of Operative Dentistry and Endodontics, West China School of Stomatology, Sichuan University, Chengdu, 610041, Sichuan, China
| | - Yangyang Shi
- State Key Laboratory of Oral Diseases & National Center for Stomatology & National Clinical Research Center for Oral Diseases, West China School of Stomatology, Sichuan University, Chengdu, 610041, Sichuan, China
- Department of Operative Dentistry and Endodontics, West China School of Stomatology, Sichuan University, Chengdu, 610041, Sichuan, China
| | - Yaqi Liu
- State Key Laboratory of Oral Diseases & National Center for Stomatology & National Clinical Research Center for Oral Diseases, West China School of Stomatology, Sichuan University, Chengdu, 610041, Sichuan, China
- Department of Pediatric Dentistry, West China Hospital of Stomatology, Sichuan University, Chengdu, 610041, Sichuan, China
| | - Lichen Gou
- State Key Laboratory of Oral Diseases & National Center for Stomatology & National Clinical Research Center for Oral Diseases, West China School of Stomatology, Sichuan University, Chengdu, 610041, Sichuan, China
| | - Xuedong Zhou
- State Key Laboratory of Oral Diseases & National Center for Stomatology & National Clinical Research Center for Oral Diseases, West China School of Stomatology, Sichuan University, Chengdu, 610041, Sichuan, China
- Department of Operative Dentistry and Endodontics, West China School of Stomatology, Sichuan University, Chengdu, 610041, Sichuan, China
| | - Lei Cheng
- State Key Laboratory of Oral Diseases & National Center for Stomatology & National Clinical Research Center for Oral Diseases, West China School of Stomatology, Sichuan University, Chengdu, 610041, Sichuan, China.
- Department of Operative Dentistry and Endodontics, West China School of Stomatology, Sichuan University, Chengdu, 610041, Sichuan, China.
| | - Biao Ren
- State Key Laboratory of Oral Diseases & National Center for Stomatology & National Clinical Research Center for Oral Diseases, West China School of Stomatology, Sichuan University, Chengdu, 610041, Sichuan, China.
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19
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Agarwal AK, Tunison K, Vale G, McDonald JG, Li X, Horton JD, Garg A. Adipose-specific overexpression of human AGPAT2 in mice causes increased adiposity and mild hepatic dysfunction. iScience 2024; 27:108653. [PMID: 38274405 PMCID: PMC10809107 DOI: 10.1016/j.isci.2023.108653] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2023] [Revised: 07/11/2023] [Accepted: 12/04/2023] [Indexed: 01/27/2024] Open
Abstract
AGPAT2, a critical enzyme involved in the biosynthesis of phospholipids and triacylglycerol (TAG), is highly expressed in adipose tissue (AT). Whether overexpression of AGPAT2 in AT will result in increased TAG synthesis (obesity) and its metabolic complications remains unknown. We overexpressed human AGPAT2 specifically in AT using the adiponectin promoter and report increased mass of subcutaneous, gonadal, and brown AT in wild-type mice. Unexpectedly, overexpression of hAGPAT2 did not change the pattern of phospholipid or TAG concentration of the AT depots. Although there is an increase in liver weight, plasma aspartate aminotransferase, and plasma insulin at various time points of the study, it did not result in significant liver dysfunction. Despite increased adiposity in the Tg-AT-hAGPAT2;mAgpat2+/+ mice, there was no significant increase in TAG concentration of AT. Therefore, this study suggests a role of AGPAT2 in the generation of AT, but not for adipocyte TAG synthesis.
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Affiliation(s)
- Anil K. Agarwal
- Section of Nutrition and Metabolic Diseases, Division of Endocrinology, Department of Internal Medicine, UT Southwestern Medical Center, Dallas, TX 75390, USA
- Center for Human Nutrition, UT Southwestern Medical Center, Dallas, TX 75390, USA
| | - Katie Tunison
- Section of Nutrition and Metabolic Diseases, Division of Endocrinology, Department of Internal Medicine, UT Southwestern Medical Center, Dallas, TX 75390, USA
- Center for Human Nutrition, UT Southwestern Medical Center, Dallas, TX 75390, USA
| | - Goncalo Vale
- Center for Human Nutrition, UT Southwestern Medical Center, Dallas, TX 75390, USA
- Department of Molecular Genetics, UT Southwestern Medical Center, Dallas, TX 75390, USA
| | - Jeffrey G. McDonald
- Center for Human Nutrition, UT Southwestern Medical Center, Dallas, TX 75390, USA
- Department of Molecular Genetics, UT Southwestern Medical Center, Dallas, TX 75390, USA
| | - Xilong Li
- Peter O’Donnell Jr. School of Public Health, UT Southwestern Medical Center, Dallas, TX 75390, USA
| | - Jay D. Horton
- Center for Human Nutrition, UT Southwestern Medical Center, Dallas, TX 75390, USA
- Department of Molecular Genetics, UT Southwestern Medical Center, Dallas, TX 75390, USA
| | - Abhimanyu Garg
- Section of Nutrition and Metabolic Diseases, Division of Endocrinology, Department of Internal Medicine, UT Southwestern Medical Center, Dallas, TX 75390, USA
- Center for Human Nutrition, UT Southwestern Medical Center, Dallas, TX 75390, USA
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20
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Ding S, von Meijenfeldt FAB, Bale NJ, Sinninghe Damsté JS, Villanueva L. Production of structurally diverse sphingolipids by anaerobic marine bacteria in the euxinic Black Sea water column. THE ISME JOURNAL 2024; 18:wrae153. [PMID: 39113610 PMCID: PMC11334938 DOI: 10.1093/ismejo/wrae153] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/26/2024] [Revised: 06/13/2024] [Accepted: 08/07/2024] [Indexed: 08/22/2024]
Abstract
Microbial lipids, used as taxonomic markers and physiological indicators, have mainly been studied through cultivation. However, this approach is limited due to the scarcity of cultures of environmental microbes, thereby restricting insights into the diversity of lipids and their ecological roles. Addressing this limitation, here we apply metalipidomics combined with metagenomics in the Black Sea, classifying and tentatively identifying 1623 lipid-like species across 18 lipid classes. We discovered over 200 novel, abundant, and structurally diverse sphingolipids in euxinic waters, including unique 1-deoxysphingolipids with long-chain fatty acids and sulfur-containing groups. Sphingolipids were thought to be rare in bacteria and their molecular and ecological functions in bacterial membranes remain elusive. However, genomic analysis focused on sphingolipid biosynthesis genes revealed that members of 38 bacterial phyla in the Black Sea can synthesize sphingolipids, representing a 4-fold increase from previously known capabilities and accounting for up to 25% of the microbial community. These sphingolipids appear to be involved in oxidative stress response, cell wall remodeling, and are associated with the metabolism of nitrogen-containing molecules. Our findings underscore the effectiveness of multi-omics approaches in exploring microbial chemical ecology.
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Affiliation(s)
- Su Ding
- Department of Marine Microbiology and Biogeochemistry, NIOZ Royal Netherlands Institute for Sea Research, 1797 SZ 't Horntje, Texel, The Netherlands
| | - F A Bastiaan von Meijenfeldt
- Department of Marine Microbiology and Biogeochemistry, NIOZ Royal Netherlands Institute for Sea Research, 1797 SZ 't Horntje, Texel, The Netherlands
| | - Nicole J Bale
- Department of Marine Microbiology and Biogeochemistry, NIOZ Royal Netherlands Institute for Sea Research, 1797 SZ 't Horntje, Texel, The Netherlands
| | - Jaap S Sinninghe Damsté
- Department of Marine Microbiology and Biogeochemistry, NIOZ Royal Netherlands Institute for Sea Research, 1797 SZ 't Horntje, Texel, The Netherlands
- Department of Earth Sciences, Faculty of Geosciences, Utrecht University, 3584 CS Utrecht, The Netherlands
| | - Laura Villanueva
- Department of Marine Microbiology and Biogeochemistry, NIOZ Royal Netherlands Institute for Sea Research, 1797 SZ 't Horntje, Texel, The Netherlands
- Department of Biology, Faculty of Sciences, Utrecht University, 3584 CS Utrecht, The Netherlands
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21
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Renne MF, Ernst R. Membrane homeostasis beyond fluidity: control of membrane compressibility. Trends Biochem Sci 2023; 48:963-977. [PMID: 37652754 PMCID: PMC10580326 DOI: 10.1016/j.tibs.2023.08.004] [Citation(s) in RCA: 27] [Impact Index Per Article: 13.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2023] [Revised: 08/03/2023] [Accepted: 08/04/2023] [Indexed: 09/02/2023]
Abstract
Biomembranes are complex materials composed of lipids and proteins that compartmentalize biochemistry. They are actively remodeled in response to physical and metabolic cues, as well as during cell differentiation and stress. The concept of homeoviscous adaptation has become a textbook example of membrane responsiveness. Here, we discuss limitations and common misconceptions revolving around it. By highlighting key moments in the life cycle of a transmembrane protein, we illustrate that membrane thickness and a finely regulated membrane compressibility are crucial to facilitate proper membrane protein insertion, function, sorting, and inheritance. We propose that the unfolded protein response (UPR) provides a mechanism for endoplasmic reticulum (ER) membrane homeostasis by sensing aberrant transverse membrane stiffening and triggering adaptive responses that re-establish membrane compressibility.
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Affiliation(s)
- Mike F Renne
- Medical Biochemistry and Molecular Biology, Medical Faculty, Saarland University, Homburg, Germany; PZMS, Center for Molecular Signaling, Medical Faculty, Saarland University, Homburg, Germany.
| | - Robert Ernst
- Medical Biochemistry and Molecular Biology, Medical Faculty, Saarland University, Homburg, Germany; PZMS, Center for Molecular Signaling, Medical Faculty, Saarland University, Homburg, Germany.
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22
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Sim SI, Chen Y, Lynch DL, Gumbart JC, Park E. Structural basis of mitochondrial protein import by the TIM23 complex. Nature 2023; 621:620-626. [PMID: 37344598 PMCID: PMC11495887 DOI: 10.1038/s41586-023-06239-6] [Citation(s) in RCA: 43] [Impact Index Per Article: 21.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2021] [Accepted: 05/19/2023] [Indexed: 06/23/2023]
Abstract
Mitochondria import nearly all of their approximately 1,000-2,000 constituent proteins from the cytosol across their double-membrane envelope1-5. Genetic and biochemical studies have shown that the conserved protein translocase, termed the TIM23 complex, mediates import of presequence-containing proteins (preproteins) into the mitochondrial matrix and inner membrane. Among about ten different subunits of the TIM23 complex, the essential multipass membrane protein Tim23, together with the evolutionarily related protein Tim17, has long been postulated to form a protein-conducting channel6-11. However, the mechanism by which these subunits form a translocation path in the membrane and enable the import process remains unclear due to a lack of structural information. Here we determined the cryo-electron microscopy structure of the core TIM23 complex (heterotrimeric Tim17-Tim23-Tim44) from Saccharomyces cerevisiae. Contrary to the prevailing model, Tim23 and Tim17 themselves do not form a water-filled channel, but instead have separate, lipid-exposed concave cavities that face in opposite directions. Our structural and biochemical analyses show that the cavity of Tim17, but not Tim23, forms the protein translocation path, whereas Tim23 probably has a structural role. The results further suggest that, during translocation of substrate polypeptides, the nonessential subunit Mgr2 seals the lateral opening of the Tim17 cavity to facilitate the translocation process. We propose a new model for the TIM23-mediated protein import and sorting mechanism, a central pathway in mitochondrial biogenesis.
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Affiliation(s)
- Sue Im Sim
- Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA
| | - Yuanyuan Chen
- Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA
- California Institute for Quantitative Biosciences, University of California, Berkeley, CA, USA
| | - Diane L Lynch
- School of Physics, Georgia Institute of Technology, Atlanta, GA, USA
- School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA, USA
| | - James C Gumbart
- School of Physics, Georgia Institute of Technology, Atlanta, GA, USA
- School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA, USA
| | - Eunyong Park
- Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.
- California Institute for Quantitative Biosciences, University of California, Berkeley, CA, USA.
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23
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Fait ME, Grillo PD, Garrote GL, Prieto ED, Vázquez RF, Saparrat MCN, Morcelle SR. Biocidal and antibiofilm activities of arginine-based surfactants against Candida isolates. Amino Acids 2023; 55:1083-1102. [PMID: 37382761 DOI: 10.1007/s00726-023-03296-z] [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] [Received: 03/27/2023] [Accepted: 06/19/2023] [Indexed: 06/30/2023]
Abstract
Amino-acid-based surfactants are a group of compounds that resemble natural amphiphiles and thus are expected to have a low impact on the environment, owing to either the mode of surfactant production or its means of disposal. Within this context, arginine-based tensioactives have gained particular interest, since their cationic nature-in combination with their amphiphilic character-enables them to act as broad-spectrum biocides. This capability is based mainly on their interactive affinity for the microbial envelope that alters the latter's structure and ultimately its function. In the work reported here, we investigated the efficiency of Nα-benzoyl arginine decyl- and dodecylamide against Candida spp. to further our understanding of the antifungal mechanism involved. For the assays, both a Candida albicans and a Candida tropicalis clinical isolates along with a C. albicans-collection strain were used as references. As expected, both arginine-based compounds proved to be effective against the strains tested through inhibiting both the planktonic and the sessile growth. Furthermore, atomic force microscopy techniques and lipid monolayer experiments enabled us to gain insight into the effect of the surfactant on the cellular envelope. The results demonstrated that all the yeasts treated exhibited changes in their exomorphologic structure, with respect to alterations in both roughness and stiffness, relative to the nontreated ones. This finding-in addition to the amphiphiles' proven ability to insert themselves within this model fungal membrane-could explain the changes in the yeast-membrane permeability that could be linked to viability loss and mixed-vesicle release.
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Affiliation(s)
- M Elisa Fait
- Centro de Investigación de Proteínas Vegetales (CIProVe-UNLP-Centro Asociado CICPBA), Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Centro Asociado CICPBA, Universidad Nacional de La Plata (UNLP), La Plata, Argentina.
- Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina.
| | - Patricia D Grillo
- Centro de Investigación de Proteínas Vegetales (CIProVe-UNLP-Centro Asociado CICPBA), Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Centro Asociado CICPBA, Universidad Nacional de La Plata (UNLP), La Plata, Argentina
- Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT), Buenos Aires, Argentina
| | - Graciela L Garrote
- Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina
- Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA, CONICET-UNLP-CICPBA), La Plata, Argentina
| | - Eduardo D Prieto
- Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina
- Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas (INIFTA), CONICET, UNLP, CCT-La Plata, La Plata, Argentina
- Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina
- Instituto Ciencias de la Salud, Universidad Nacional Arturo Jauretche, Buenos Aires, Argentina
| | - Romina F Vázquez
- Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina
- Instituto de Investigaciones Bioquímicas de La Plata (INIBIOLP), CCT-La Plata, CONICET, UNLP, La Plata, Argentina
| | - Mario C N Saparrat
- Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina
- Instituto de Fisiología Vegetal (INFIVE-CONICET-UNLP) and Cátedra de Microbiología Agrícola, Facultad de Ciencias Agrarias y Forestales, UNLP, La Plata, Argentina
| | - Susana R Morcelle
- Centro de Investigación de Proteínas Vegetales (CIProVe-UNLP-Centro Asociado CICPBA), Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Centro Asociado CICPBA, Universidad Nacional de La Plata (UNLP), La Plata, Argentina.
- Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina.
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24
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Fuggetta N, Rigolli N, Magdeleine M, Seminara A, Drin G. Reconstitution of ORP-mediated lipid exchange process coupled to PI(4)P metabolism. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.08.04.551917. [PMID: 37577629 PMCID: PMC10418177 DOI: 10.1101/2023.08.04.551917] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 08/15/2023]
Abstract
Lipid distribution in the eukaryotic cells depends on tight couplings between lipid transfer and lipid metabolism. Yet these couplings remain poorly described. Notably, it is unclear to what extent lipid exchangers of the OSBP-related proteins (ORPs) family, coupled to PI(4)P metabolism, contribute to the formation of sterol and phosphatidylserine gradient between the endoplasmic reticulum (ER) and other cell regions. To address this question, we have examined in vitro the activity of Osh4p, a representative ORP, between Golgi mimetic membranes in which PI(4)P is produced by a PI 4-kinase and ER mimetic membranes in which PI(4)P is hydrolyzed by the phosphatase Sac1p. Using quantitative, real-time assays, we demonstrate that Osh4p creates a sterol gradient between the two membranes by sterol/PI(4)P exchange as soon as a PI(4)P gradient is generated at this interface following ATP addition, and define how much PI(4)P must be synthesized for this process. Then, using a kinetic model supported by our in vitro data, we estimate to what extent PI(4)P metabolism can drive lipid transfer in cells. Finally, we show that Sec14p, by transferring phosphatidylinositol between membranes, can support the synthesis of PI(4)P and the creation of a sterol gradient by Osh4p. These results indicate to what extent ORPs, under the control of PI(4)P metabolism, can distribute lipids in the cell.
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Affiliation(s)
- Nicolas Fuggetta
- Université Côte d'Azur, Centre National de la Recherche Scientifique, Institut de Pharmacologie Moléculaire et Cellulaire, 660 route des lucioles, 06560 Valbonne, France
| | - Nicola Rigolli
- Laboratoire de Physique, École Normale Supérieure (LPENS), 75005 Paris, France
| | - Maud Magdeleine
- Université Côte d'Azur, Centre National de la Recherche Scientifique, Institut de Pharmacologie Moléculaire et Cellulaire, 660 route des lucioles, 06560 Valbonne, France
| | - Agnese Seminara
- Malga, Department of Civil, Chemical and Environmental Engineering, University of Genoa, Villa Cambiaso 1, 16145 Genoa, Italy
| | - Guillaume Drin
- Université Côte d'Azur, Centre National de la Recherche Scientifique, Institut de Pharmacologie Moléculaire et Cellulaire, 660 route des lucioles, 06560 Valbonne, France
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25
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Skotland T, Llorente A, Sandvig K. Lipids in Extracellular Vesicles: What Can Be Learned about Membrane Structure and Function? Cold Spring Harb Perspect Biol 2023; 15:a041415. [PMID: 37277192 PMCID: PMC10411865 DOI: 10.1101/cshperspect.a041415] [Citation(s) in RCA: 14] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/07/2023]
Abstract
Extracellular vesicles, such as exosomes, can be used as interesting models to study the structure and function of biological membranes as these vesicles contain only one membrane (i.e., one lipid bilayer). In addition to lipids, they contain proteins, nucleic acids, and various other molecules. The lipid composition of exosomes is here compared to HIV particles and detergent-resistant membranes, which also have a high content of sphingolipids, cholesterol, and phosphatidylserine (PS). We discuss interactions between the lipids in the two bilayers, and especially those between PS 18:0/18:1 in the inner leaflet and the very-long-chain sphingolipids in the outer leaflet, and the importance of cholesterol for these interactions. We also briefly discuss the involvement of ether-linked phospholipids (PLs) in such lipid raft-like structures, and the possible involvement of these and other lipid classes in the formation of exosomes. The urgent need to improve the quality of quantitative lipidomic studies is highlighted.
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Affiliation(s)
- Tore Skotland
- Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital-The Norwegian Radium Hospital, 0379 Oslo, Norway
- Centre for Cancer Cell Reprogramming, Faculty of Medicine, University of Oslo, 0379 Oslo, Norway
| | - Alicia Llorente
- Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital-The Norwegian Radium Hospital, 0379 Oslo, Norway
- Centre for Cancer Cell Reprogramming, Faculty of Medicine, University of Oslo, 0379 Oslo, Norway
- Department of Mechanical, Electronics and Chemical Engineering, Oslo Metropolitan University, 0167 Oslo, Norway
| | - Kirsten Sandvig
- Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital-The Norwegian Radium Hospital, 0379 Oslo, Norway
- Centre for Cancer Cell Reprogramming, Faculty of Medicine, University of Oslo, 0379 Oslo, Norway
- Department of Molecular Biosciences, University of Oslo, 0316 Oslo, Norway
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26
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Reinhard J, Leveille CL, Cornell CE, Merz AJ, Klose C, Ernst R, Keller SL. Remodeling of yeast vacuole membrane lipidomes from the log (one phase) to stationary stage (two phases). Biophys J 2023; 122:1043-1057. [PMID: 36635960 PMCID: PMC10111276 DOI: 10.1016/j.bpj.2023.01.009] [Citation(s) in RCA: 12] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2022] [Revised: 01/03/2023] [Accepted: 01/09/2023] [Indexed: 01/13/2023] Open
Abstract
Upon nutrient limitation, budding yeast of Saccharomyces cerevisiae shift from fast growth (the log stage) to quiescence (the stationary stage). This shift is accompanied by liquid-liquid phase separation in the membrane of the vacuole, an endosomal organelle. Recent work indicates that the resulting micrometer-scale domains in vacuole membranes enable yeast to survive periods of stress. An outstanding question is which molecular changes might cause this membrane phase separation. Here, we conduct lipidomics of vacuole membranes in both the log and stationary stages. Isolation of pure vacuole membranes is challenging in the stationary stage, when lipid droplets are in close contact with vacuoles. Immuno-isolation has previously been shown to successfully purify log-stage vacuole membranes with high organelle specificity, but it was not previously possible to immuno-isolate stationary-stage vacuole membranes. Here, we develop Mam3 as a bait protein for vacuole immuno-isolation, and demonstrate low contamination by non-vacuolar membranes. We find that stationary-stage vacuole membranes contain surprisingly high fractions of phosphatidylcholine lipids (∼40%), roughly twice as much as log-stage membranes. Moreover, in the stationary stage, these lipids have higher melting temperatures, due to longer and more saturated acyl chains. Another surprise is that no significant change in sterol content is observed. These lipidomic changes, which are largely reflected on the whole-cell level, fit within the predominant view that phase separation in membranes requires at least three types of molecules to be present: lipids with high melting temperatures, lipids with low melting temperatures, and sterols.
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Affiliation(s)
- John Reinhard
- Medical Biochemistry and Molecular Biology, Medical Faculty, Saarland University, Homburg, Germany; PZMS, Center for Molecular Signaling, Medical Faculty, Saarland University, Homburg, Germany
| | | | | | - Alexey J Merz
- Department of Biochemistry, University of Washington, Seattle, WA
| | | | - Robert Ernst
- Medical Biochemistry and Molecular Biology, Medical Faculty, Saarland University, Homburg, Germany; PZMS, Center for Molecular Signaling, Medical Faculty, Saarland University, Homburg, Germany.
| | - Sarah L Keller
- Department of Chemistry, University of Washington, Seattle, WA.
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Tsuchiya M, Tachibana N, Nagao K, Tamura T, Hamachi I. Organelle-selective click labeling coupled with flow cytometry allows pooled CRISPR screening of genes involved in phosphatidylcholine metabolism. Cell Metab 2023:S1550-4131(23)00050-5. [PMID: 36917984 DOI: 10.1016/j.cmet.2023.02.014] [Citation(s) in RCA: 31] [Impact Index Per Article: 15.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/02/2023] [Revised: 02/13/2023] [Accepted: 02/17/2023] [Indexed: 03/14/2023]
Abstract
Cellular lipid synthesis and transport are governed by intricate protein networks. Although genetic screening should contribute to deciphering the regulatory networks of lipid metabolism, technical challenges remain-especially for high-throughput readouts of lipid phenotypes. Here, we coupled organelle-selective click labeling of phosphatidylcholine (PC) with flow cytometry-based CRISPR screening technologies to convert organellar PC phenotypes into a simple fluorescence readout for genome-wide screening. This technique, named O-ClickFC, was successfully applied in genome-scale CRISPR-knockout screens to identify previously reported genes associated with PC synthesis (PCYT1A, ACACA), vesicular membrane trafficking (SEC23B, RAB5C), and non-vesicular transport (PITPNB, STARD7). Moreover, we revealed previously uncharacterized roles of FLVCR1 as a choline uptake facilitator, CHEK1 as a post-translational regulator of the PC-synthetic pathway, and CDC50A as responsible for the translocation of PC to the outside of the plasma membrane bilayer. These findings demonstrate the versatility of O-ClickFC as an unprecedented platform for genetic dissection of cellular lipid metabolism.
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Affiliation(s)
- Masaki Tsuchiya
- Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan; PRESTO (Precursory Research for Embryonic Science and Technology), JST, Sanbancho, Chiyodaku, Tokyo 102-0075, Japan
| | - Nobuhiko Tachibana
- Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan; PRESTO (Precursory Research for Embryonic Science and Technology), JST, Sanbancho, Chiyodaku, Tokyo 102-0075, Japan
| | - Kohjiro Nagao
- Department of Biophysical Chemistry, Kyoto Pharmaceutical University, 5 Misasaginakauchi-cho, Yamashina-ku, Kyoto 607-8414, Japan
| | - Tomonori Tamura
- Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan.
| | - Itaru Hamachi
- Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan; ERATO (Exploratory Research for Advanced Technology), JST, Sanbancho, Chiyodaku, Tokyo 102-0075, Japan.
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28
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Orr A, Wickner W. PI3P regulates multiple stages of membrane fusion. Mol Biol Cell 2023; 34:ar17. [PMID: 36735517 PMCID: PMC10011722 DOI: 10.1091/mbc.e22-10-0486] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023] Open
Abstract
The conserved catalysts of intracellular membrane fusion are Rab-family GTPases, effector complexes that bind Rabs for membrane tethering, SNARE proteins of the R, Qa, Qb, and Qc families, and SNARE chaperones of the SM, Sec17/SNAP, and Sec18/NSF families. Yeast vacuole fusion is regulated by phosphatidylinositol-3-phosphate (PI3P). PI3P binds directly to the vacuolar Qc-SNARE and to HOPS, the vacuolar tethering/SM complex. We now report several distinct functions of PI3P in fusion. PI3P binds the N-terminal PX domain of the Qc-SNARE to enhance its engagement for fusion. Even when Qc has been preassembled with the Qa- and Qb-SNAREs, PI3P still promotes trans-SNARE assembly and fusion between these 3Q proteoliposomes and those with R-SNARE, whether with the natural HOPS tether or with a synthetic tether. With HOPS, efficient trans-SNARE complex formation needs PI3P on the 3Q-SNARE proteoliposomes, in cis to the Qc. PI3P is also needed for HOPS to confer resistance to Sec17/Sec18. With a synthetic tether, fusion is supported by PI3P on either fusion partner membrane, but this fusion is blocked by Sec17/Sec18. PI3P thus supports multiple stages of fusion: the engagement of the Qc-SNARE, trans-SNARE complex formation with preassembled Q-SNAREs, HOPS protection of SNARE complexes from Sec17/Sec18, and fusion per se after tethering and Q-SNARE assembly.
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Affiliation(s)
- Amy Orr
- Department of Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, NH 03755-3844
| | - William Wickner
- Department of Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, NH 03755-3844
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29
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Renne MF, Bao X, Hokken MWJ, Bierhuizen AS, Hermansson M, Sprenger RR, Ewing TA, Ma X, Cox RC, Brouwers JF, De Smet CH, Ejsing CS, de Kroon AIPM. Molecular species selectivity of lipid transport creates a mitochondrial sink for di-unsaturated phospholipids. EMBO J 2022; 41:e106837. [PMID: 34873731 PMCID: PMC8762554 DOI: 10.15252/embj.2020106837] [Citation(s) in RCA: 16] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2020] [Revised: 10/22/2021] [Accepted: 10/26/2021] [Indexed: 11/09/2022] Open
Abstract
Mitochondria depend on the import of phospholipid precursors for the biosynthesis of phosphatidylethanolamine (PE) and cardiolipin, yet the mechanism of their transport remains elusive. A dynamic lipidomics approach revealed that mitochondria preferentially import di-unsaturated phosphatidylserine (PS) for subsequent conversion to PE by the mitochondrial PS decarboxylase Psd1p. Several protein complexes tethering mitochondria to the endomembrane system have been implicated in lipid transport in yeast, including the endoplasmic reticulum (ER)-mitochondrial encounter structure (ERMES), ER-membrane complex (EMC), and the vacuole and mitochondria patch (vCLAMP). By limiting the availability of unsaturated phospholipids, we created conditions to investigate the mechanism of lipid transfer and the contributions of the tethering complexes in vivo. Under these conditions, inactivation of ERMES components or of the vCLAMP component Vps39p exacerbated accumulation of saturated lipid acyl chains, indicating that ERMES and Vps39p contribute to the mitochondrial sink for unsaturated acyl chains by mediating transfer of di-unsaturated phospholipids. These results support the concept that intermembrane lipid flow is rate-limited by molecular species-dependent lipid efflux from the donor membrane and driven by the lipid species' concentration gradient between donor and acceptor membrane.
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Affiliation(s)
- Mike F Renne
- Membrane Biochemistry & BiophysicsDepartment of ChemistryUtrecht UniversityUtrechtThe Netherlands
- Present address:
Sir William Dunn School of PathologyUniversity of OxfordOxfordUK
| | - Xue Bao
- Membrane Biochemistry & BiophysicsDepartment of ChemistryUtrecht UniversityUtrechtThe Netherlands
| | - Margriet WJ Hokken
- Membrane Biochemistry & BiophysicsDepartment of ChemistryUtrecht UniversityUtrechtThe Netherlands
- Present address:
Department of Medical MicrobiologyRadboud University Medical CenterRadboud Institute for Molecular Life SciencesNijmegenThe Netherlands
| | - Adolf S Bierhuizen
- Membrane Biochemistry & BiophysicsDepartment of ChemistryUtrecht UniversityUtrechtThe Netherlands
| | - Martin Hermansson
- Department of Biochemistry and Molecular BiologyVILLUM Center for Bioanalytical SciencesUniversity of Southern DenmarkOdenseDenmark
| | - Richard R Sprenger
- Department of Biochemistry and Molecular BiologyVILLUM Center for Bioanalytical SciencesUniversity of Southern DenmarkOdenseDenmark
| | - Tom A Ewing
- Membrane Biochemistry & BiophysicsDepartment of ChemistryUtrecht UniversityUtrechtThe Netherlands
- Present address:
Wageningen Food & Biobased ResearchWageningen University & ResearchWageningenThe Netherlands
| | - Xiao Ma
- Membrane Biochemistry & BiophysicsDepartment of ChemistryUtrecht UniversityUtrechtThe Netherlands
| | - Ruud C Cox
- Membrane Biochemistry & BiophysicsDepartment of ChemistryUtrecht UniversityUtrechtThe Netherlands
| | - Jos F Brouwers
- Biochemistry and Cell BiologyDepartment of Veterinary MedicineUtrecht UniversityUtrechtThe Netherlands
- Present address:
Center for Molecular MedicineUniversity Medical Center UtrechtUtrechtThe Netherlands
| | - Cedric H De Smet
- Membrane Biochemistry & BiophysicsDepartment of ChemistryUtrecht UniversityUtrechtThe Netherlands
| | - Christer S Ejsing
- Department of Biochemistry and Molecular BiologyVILLUM Center for Bioanalytical SciencesUniversity of Southern DenmarkOdenseDenmark
- Cell Biology and Biophysics UnitEuropean Molecular Biology LaboratoryHeidelbergGermany
| | - Anton IPM de Kroon
- Membrane Biochemistry & BiophysicsDepartment of ChemistryUtrecht UniversityUtrechtThe Netherlands
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30
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Creating and sensing asymmetric lipid distributions throughout the cell. Emerg Top Life Sci 2022; 7:7-19. [PMID: 36373850 DOI: 10.1042/etls20220028] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2022] [Revised: 10/24/2022] [Accepted: 11/01/2022] [Indexed: 11/16/2022]
Abstract
A key feature of eukaryotic cells is the asymmetric distribution of lipids along their secretory pathway. Because of the biological significance of these asymmetries, it is crucial to define the mechanisms which create them. Extensive studies have led to the identification of lipid transfer proteins (LTPs) that work with lipid-synthesizing enzymes to carry lipids between two distinct membranes in a directional manner, and are thus able to create asymmetries in lipid distribution throughout the cell. These networks are often in contact sites where two organelle membranes are in close proximity for reasons we have only recently started to understand. A question is whether these networks transfer lipids en masse within the cells or adjust the lipid composition of organelle membranes. Finally, recent data have confirmed that some networks organized around LTPs do not generate lipid asymmetries between membranes but sense them and rectify the lipid content of the cell.
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31
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Kurakin S, Ivankov O, Skoi V, Kuklin A, Uhríková D, Kučerka N. Cations Do Not Alter the Membrane Structure of POPC—A Lipid With an Intermediate Area. Front Mol Biosci 2022; 9:926591. [PMID: 35898308 PMCID: PMC9312375 DOI: 10.3389/fmolb.2022.926591] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2022] [Accepted: 06/07/2022] [Indexed: 12/02/2022] Open
Abstract
Combining small-angle neutron scattering (SANS), small-angle X-ray scattering (SAXS), and densitometric measurements, we have studied the interactions of the divalent cations Ca2+ and Mg2+ with the lipid vesicles prepared of a mixed-chain palmitoyl-oleoyl-phosphatidylcholine (POPC) at 25°C. The structural parameters of the POPC bilayer, such as the bilayer thickness, lateral area, and volume per lipid, displayed no changes upon the ion addition at concentrations up to 30 mM and minor changes at > 30 mM Ca2+ and Mg2+, while some decrease in the vesicle radius was observed over the entire concentration range studied. This examination allows us to validate the concept of lipid–ion interactions governed by the area per lipid suggested previously and to propose the mixed mode of those interactions that emerge in the POPC vesicles. We speculate that the average area per POPC lipid that corresponds to the cutoff length of lipid–ion interactions generates an equal but opposite impact on ion bridges and separate lipid–ion pairs. As a result of the dynamic equilibrium, the overall structural properties of bilayers are not affected. As the molecular mechanism proposed is affected by the structural properties of a particular lipid, it might help us to understand the fundamentals of processes occurring in complex multicomponent membrane systems.
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Affiliation(s)
- Sergei Kurakin
- Frank Laboratory of Neutron Physics, Joint Institute for Nuclear Research, Dubna, Russia
- Institute of Physics, Kazan Federal University, Kazan, Russia
| | - Oleksandr Ivankov
- Frank Laboratory of Neutron Physics, Joint Institute for Nuclear Research, Dubna, Russia
| | - Vadim Skoi
- Frank Laboratory of Neutron Physics, Joint Institute for Nuclear Research, Dubna, Russia
- Moscow Institute of Physics and Technology, Dolgoprudnyi, Russia
| | - Alexander Kuklin
- Frank Laboratory of Neutron Physics, Joint Institute for Nuclear Research, Dubna, Russia
- Moscow Institute of Physics and Technology, Dolgoprudnyi, Russia
| | - Daniela Uhríková
- Department of Physical Chemistry of Drugs, Faculty of Pharmacy, Comenius University Bratislava, Bratislava, Slovakia
| | - Norbert Kučerka
- Frank Laboratory of Neutron Physics, Joint Institute for Nuclear Research, Dubna, Russia
- Department of Physical Chemistry of Drugs, Faculty of Pharmacy, Comenius University Bratislava, Bratislava, Slovakia
- *Correspondence: Norbert Kučerka,
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32
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Low-Frequency Magnetic Field Exposure System for Cells Electromagnetic Biocompatibility Studies. APPLIED SCIENCES-BASEL 2022. [DOI: 10.3390/app12146846] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
The advancement in science and technology has resulted in the invention and widespread usage of many electrical devices in the daily lives of humans. The exponential use of modern electronic facilities has increased electromagnetic field exposure in the current population. Therefore, the presented article deals with designing, constructing, and testing a new applicator system developed for cells electromagnetic biocompatibility studies. The applicator system is intended for studying the non-thermal impacts of low-frequency magnetic field on cell cultures growth. Main attention is focused on increasing the capacity of the applicator and effectivity of the experiments. The key idea is to reach high level of the magnetic field homogeneity in an area of interest and the temperature stability during the biocompatibility studies. The applicator system is designed based on numerical simulations and its construction, measurements, and properties evaluation are also reported for proving the applicator’s functionality. The new applicator allows performing five parallel experiments at the same time under the same conditions. The simulation together with the experimental results confirm that the magnetic field homogeneity reaches 99% in the area of interest and the maximum temperature instability is lower than 2% during the experiments. The effectiveness of new applicator is tested and proved during preliminary experiments with Saccharomyces Cerevisiae cells. The observed effects of MF exposure represent maximal stimulation of 74% and maximal inhibition of 49%. The reason why MF with the same parameters induces inhibition in one sample and stimulation in the other will be the subject of further research.
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33
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Haro-Reyes T, Díaz-Peralta L, Galván-Hernández A, Rodríguez-López A, Rodríguez-Fragoso L, Ortega-Blake I. Polyene Antibiotics Physical Chemistry and Their Effect on Lipid Membranes; Impacting Biological Processes and Medical Applications. MEMBRANES 2022; 12:681. [PMID: 35877884 PMCID: PMC9316096 DOI: 10.3390/membranes12070681] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/25/2022] [Revised: 06/21/2022] [Accepted: 06/23/2022] [Indexed: 01/27/2023]
Abstract
This review examined a collection of studies regarding the molecular properties of some polyene antibiotic molecules as well as their properties in solution and in particular environmental conditions. We also looked into the proposed mechanism of action of polyenes, where membrane properties play a crucial role. Given the interest in polyene antibiotics as therapeutic agents, we looked into alternative ways of reducing their collateral toxicity, including semi-synthesis of derivatives and new formulations. We follow with studies on the role of membrane structure and, finally, recent developments regarding the most important clinical applications of these compounds.
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Affiliation(s)
- Tammy Haro-Reyes
- Instituto de Ciencias Físicas, Universidad Nacional Autónoma de México, Av. Universidad s/n, Col. Chamilpa, Cuernavaca 62210, Morelos, Mexico; (T.H.-R.); (L.D.-P.); (A.G.-H.)
| | - Lucero Díaz-Peralta
- Instituto de Ciencias Físicas, Universidad Nacional Autónoma de México, Av. Universidad s/n, Col. Chamilpa, Cuernavaca 62210, Morelos, Mexico; (T.H.-R.); (L.D.-P.); (A.G.-H.)
| | - Arturo Galván-Hernández
- Instituto de Ciencias Físicas, Universidad Nacional Autónoma de México, Av. Universidad s/n, Col. Chamilpa, Cuernavaca 62210, Morelos, Mexico; (T.H.-R.); (L.D.-P.); (A.G.-H.)
| | - Anahi Rodríguez-López
- Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, Cuernavaca 62210, Morelos, Mexico; (A.R.-L.); (L.R.-F.)
| | - Lourdes Rodríguez-Fragoso
- Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, Cuernavaca 62210, Morelos, Mexico; (A.R.-L.); (L.R.-F.)
| | - Iván Ortega-Blake
- Instituto de Ciencias Físicas, Universidad Nacional Autónoma de México, Av. Universidad s/n, Col. Chamilpa, Cuernavaca 62210, Morelos, Mexico; (T.H.-R.); (L.D.-P.); (A.G.-H.)
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Thomas FB, Omnus DJ, Bader JM, Chung GH, Kono N, Stefan CJ. Tricalbin proteins regulate plasma membrane phospholipid homeostasis. Life Sci Alliance 2022; 5:5/8/e202201430. [PMID: 35440494 PMCID: PMC9018018 DOI: 10.26508/lsa.202201430] [Citation(s) in RCA: 18] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2022] [Revised: 04/03/2022] [Accepted: 04/04/2022] [Indexed: 12/26/2022] Open
Abstract
The evolutionarily conserved extended synaptotagmin (E-Syt) proteins are calcium-activated lipid transfer proteins that function at contacts between the ER and plasma membrane (ER-PM contacts). However, roles of the E-Syt family members in PM lipid organisation remain incomplete. Among the E-Syt family, the yeast tricalbin (Tcb) proteins are essential for PM integrity upon heat stress, but it is not known how they contribute to PM maintenance. Using quantitative lipidomics and microscopy, we find that the Tcb proteins regulate phosphatidylserine homeostasis at the PM. Moreover, upon heat-induced membrane stress, Tcb3 co-localises with the PM protein Sfk1 that is implicated in PM phospholipid asymmetry and integrity. The Tcb proteins also control the PM targeting of the known phosphatidylserine effector Pkc1 upon heat-induced stress. Phosphatidylserine has evolutionarily conserved roles in PM organisation, integrity, and repair. We propose that phospholipid regulation is an ancient essential function of E-Syt family members required for PM integrity.
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Affiliation(s)
- Ffion B Thomas
- Medical Research Council Laboratory for Molecular Cell Biology, University College London, London, UK
| | - Deike J Omnus
- Medical Research Council Laboratory for Molecular Cell Biology, University College London, London, UK
| | - Jakob M Bader
- Medical Research Council Laboratory for Molecular Cell Biology, University College London, London, UK
| | - Gary Hc Chung
- Medical Research Council Laboratory for Molecular Cell Biology, University College London, London, UK
| | - Nozomu Kono
- Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan
| | - Christopher J Stefan
- Medical Research Council Laboratory for Molecular Cell Biology, University College London, London, UK
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Structural Characterization of Mono- and Dimethylphosphatidylethanolamines from Various Organisms Using a Complex Analytical Strategy Including Chiral Chromatography. Symmetry (Basel) 2022. [DOI: 10.3390/sym14030616] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022] Open
Abstract
Two minor phospholipids, i.e., mono- and/or dimethylphosphatidylethanolamines, are widespread in many organisms, from bacteria to higher plants and animals. A molecular mixture of methyl-PE and dimethyl-PE was obtained from total lipids by liquid chromatography and further identified by mass spectrometry. Total methyl-PE and dimethyl-PE were cleaved by phospholipase C, and the resulting diacylglycerols, in the form of acetyl derivatives, were separated into alkyl-acyl, alkenyl-acyl, and diacylglycerols. Reversed-phase LC/MS allowed dozens of molecular species to be identified and further analyzed. This was performed on a chiral column, and identification by tandem positive ESI revealed that diacyl derivatives from all four bacteria were mixtures of both R and S enantiomers. The same applied to alkenyl-acyl derivatives of anaerobic bacteria. Analysis thus confirmed that some bacteria biosynthesize phospholipids having both sn-glycerol-3-phosphate and sn-glycerol-1-phosphate as precursors. These findings were further supported by data already published in GenBank. The use of chiral chromatography made it possible to prove that both enantiomers of glycerol phosphate of some molecular species of mono- and dimethylphosphatidylethanolamines are present. The result of the analysis can be interpreted that the cultured bacteria do not have homochiral membranes but, on the contrary, have an asymmetric, i.e., heterochiral membranes.
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Need for more focus on lipid species in studies of biological and model membranes. Prog Lipid Res 2022; 86:101160. [DOI: 10.1016/j.plipres.2022.101160] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2021] [Accepted: 03/06/2022] [Indexed: 11/23/2022]
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Han X, Gross RW. The foundations and development of lipidomics. J Lipid Res 2022; 63:100164. [PMID: 34953866 PMCID: PMC8953652 DOI: 10.1016/j.jlr.2021.100164] [Citation(s) in RCA: 120] [Impact Index Per Article: 40.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2021] [Revised: 12/13/2021] [Accepted: 12/14/2021] [Indexed: 12/15/2022] Open
Abstract
For over a century, the importance of lipid metabolism in biology was recognized but difficult to mechanistically understand due to the lack of sensitive and robust technologies for identification and quantification of lipid molecular species. The enabling technological breakthroughs emerged in the 1980s with the development of soft ionization methods (Electrospray Ionization and Matrix Assisted Laser Desorption/Ionization) that could identify and quantify intact individual lipid molecular species. These soft ionization technologies laid the foundations for what was to be later named the field of lipidomics. Further innovative advances in multistage fragmentation, dramatic improvements in resolution and mass accuracy, and multiplexed sample analysis fueled the early growth of lipidomics through the early 1990s. The field exponentially grew through the use of a variety of strategic approaches, which included direct infusion, chromatographic separation, and charge-switch derivatization, which facilitated access to the low abundance species of the lipidome. In this Thematic Review, we provide a broad perspective of the foundations, enabling advances, and predicted future directions of growth of the lipidomics field.
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Affiliation(s)
- Xianlin Han
- Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA; Departments of Medicine - Diabetes, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA.
| | - Richard W Gross
- Division of Bioorganic Chemistry and Molecular Pharmacology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO, USA; Department of Chemistry, Washington University, St. Louis, MO, USA
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Yeast cells actively tune their membranes to phase separate at temperatures that scale with growth temperatures. Proc Natl Acad Sci U S A 2022; 119:2116007119. [PMID: 35046036 PMCID: PMC8795566 DOI: 10.1073/pnas.2116007119] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 12/06/2021] [Indexed: 02/06/2023] Open
Abstract
Phase separation in membranes creates domains enriched in specific components. To date, the best example of micrometer-scale phase separation in the membrane of an unperturbed, living cell occurs in a yeast (Saccharomyces cerevisiae) organelle called the vacuole. Recent studies indicate that the phases are functionally important, enabling yeast survival during periods of stress. We discovered that yeast regulate this phase transition; the temperature at which membrane components mix into a single phase is ∼15 °C above the growth temperature. To maintain this offset, yeast may tune the level of ergosterol (a molecule that is structurally similar to cholesterol) in their membranes. Surprisingly, depleting sterols in vacuole membranes causes them to phase separate, in contrast to previous assumptions. Membranes of vacuoles, the lysosomal organelles of Saccharomyces cerevisiae (budding yeast), undergo extraordinary changes during the cell’s normal growth cycle. The cycle begins with a stage of rapid cell growth. Then, as glucose becomes scarce, growth slows, and vacuole membranes phase separate into micrometer-scale domains of two liquid phases. Recent studies suggest that these domains promote yeast survival by organizing membrane proteins that play key roles in a central signaling pathway conserved among eukaryotes (TORC1). An outstanding question in the field has been whether cells regulate phase transitions in response to new physical conditions and how this occurs. Here, we measure transition temperatures and find that after an increase of roughly 15 °C, vacuole membranes appear uniform, independent of growth temperature. Moreover, populations of cells grown at a single temperature regulate this transition to occur over a surprisingly narrow temperature range. Remarkably, the transition temperature scales linearly with the growth temperature, demonstrating that the cells physiologically adapt to maintain proximity to the transition. Next, we ask how yeast adjust their membranes to achieve phase separation. We isolate vacuoles from yeast during the rapid stage of growth, when their membranes do not natively exhibit domains. Ergosterol is the major sterol in yeast. We find that domains appear when ergosterol is depleted, contradicting the prevalent assumption that increases in sterol concentration generally cause membrane phase separation in vivo, but in agreement with previous studies using artificial and cell-derived membranes.
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Hsieh MK, Yu Y, Klauda JB. All-Atom Modeling of Complex Cellular Membranes. LANGMUIR : THE ACS JOURNAL OF SURFACES AND COLLOIDS 2022; 38:3-17. [PMID: 34962814 DOI: 10.1021/acs.langmuir.1c02084] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/14/2023]
Abstract
Cell membranes are composed of a variety of lipids and proteins where they interact with each other to fulfill their roles. The first step in modeling these interactions in molecular simulations is to have reliable mimetics of the membrane's lipid environment. This Feature Article presents our recent efforts to model complex cellular membranes using all-atom force fields. A short review of the CHARMM36 (C36) lipid force field and its recent update to incorporate the long-range dispersion is presented. Key examples of model membranes mimicking various species and organelles are given. These include single-celled organisms such as bacteria (E. coli., chlamydia, and P. aeruginosa) and yeast (plasma membrane, endoplasmic reticulum, and trans-Golgi network) and more advanced ones such as plants (soybean and Arabidopsis thaliana) and mammals (ocular lens, stratum corneum, and peripheral nerve myelin). Leaflet asymmetry in composition has also been applied to some of these models. With the increased lipid diversity in the C36 lipid FF, these complex models can better reflect the structural, mechanical, and dynamic properties of realistic membranes and open an opportunity to study biological processes involving other molecules.
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Abstract
Emerging studies have shown that lipid metabolism plays an important role in aging. High resolution in situ imaging of lipid metabolic dynamics inside cells and tissues affords a novel and potent approach for understanding many biological processes such as aging. Here we established a new optical imaging platform that combines D2O-probed stimulated Raman scattering (DO-SRS) imaging microscopy and a Drosophila model to directly visualize metabolic activities in situ during aging. The sub-cellular spatial distribution of de novo lipogenesis in the fat body was quantitatively imaged and examined. We discovered a dramatic decrease in lipid turnover in 35-day-old flies. Decreases in protein turnover occurred earlier than lipids (25-day vs. 35-day), and there are many proteins localized on the cell and lipid droplet membrane. This suggests that protein metabolism may act as a prerequisite for lipid metabolism during aging. This alteration of maintenance of protein turnover indicates disrupted lipid metabolism. We further found a significantly higher lipid turnover rate in large LDs, indicating more active metabolism in large LDs, suggesting that large and small LDs play different roles in metabolism to maintain cellular homeostasis. This is the first study that directly visualizes spatiotemporal alterations of lipid (and protein) metabolism in Drosophila during the aging process. Our study not only demonstrates a new imaging platform for studying lipid metabolism, but also unravels the important interconnections between lipid metabolism and aging.
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Affiliation(s)
- Yajuan Li
- Department of Bioengineering, University of California San Diego, USA.
| | - Wenxu Zhang
- Department of Bioengineering, University of California San Diego, USA.
| | - Anthony A Fung
- Department of Bioengineering, University of California San Diego, USA.
| | - Lingyan Shi
- Department of Bioengineering, University of California San Diego, USA.
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Ikhlef S, Lipp NF, Delfosse V, Fuggetta N, Bourguet W, Magdeleine M, Drin G. Functional analyses of phosphatidylserine/PI(4)P exchangers with diverse lipid species and membrane contexts reveal unanticipated rules on lipid transfer. BMC Biol 2021; 19:248. [PMID: 34801011 PMCID: PMC8606082 DOI: 10.1186/s12915-021-01183-1] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2021] [Accepted: 11/04/2021] [Indexed: 11/14/2022] Open
Abstract
Background Lipid species are accurately distributed in the eukaryotic cell so that organelle and plasma membranes have an adequate lipid composition to support numerous cellular functions. In the plasma membrane, a precise regulation of the level of lipids such as phosphatidylserine, PI(4)P, and PI(4,5)P2, is critical for maintaining the signaling competence of the cell. Several lipid transfer proteins of the ORP/Osh family contribute to this fine-tuning by delivering PS, synthesized in the endoplasmic reticulum, to the plasma membrane in exchange for PI(4)P. To get insights into the role of these PS/PI(4)P exchangers in regulating plasma membrane features, we question how they selectively recognize and transfer lipid ligands with different acyl chains, whether these proteins exchange PS exclusively for PI(4)P or additionally for PI(4,5)P2, and how sterol abundance in the plasma membrane impacts their activity. Results We measured in vitro how the yeast Osh6p and human ORP8 transported PS and PI(4)P subspecies of diverse length and unsaturation degree between membranes by fluorescence-based assays. We established that the exchange activity of Osh6p and ORP8 strongly depends on whether these ligands are saturated or not, and is high with representative cellular PS and PI(4)P subspecies. Unexpectedly, we found that the speed at which these proteins individually transfer lipid ligands between membranes is inversely related to their affinity for them and that high-affinity ligands must be exchanged to be transferred more rapidly. Next we determined that Osh6p and ORP8 cannot use PI(4,5)P2 for exchange processes, because it is a low-affinity ligand, and do not transfer more PS into sterol-rich membranes. Conclusions Our study provides new insights into PS/PI(4)P exchangers by indicating the degree to which they can regulate the acyl chain composition of the PM, and how they control PM phosphoinositide levels. Moreover, we establish general rules on how the activity of lipid transfer proteins relates to their affinity for ligands. Supplementary Information The online version contains supplementary material available at 10.1186/s12915-021-01183-1.
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Affiliation(s)
- Souade Ikhlef
- Université Côte d'Azur, Centre National de la Recherche Scientifique, Institut de Pharmacologie Moléculaire et Cellulaire, 660 route des lucioles, 06560, Valbonne, France
| | - Nicolas-Frédéric Lipp
- Université Côte d'Azur, Centre National de la Recherche Scientifique, Institut de Pharmacologie Moléculaire et Cellulaire, 660 route des lucioles, 06560, Valbonne, France.,Current position: Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA, USA
| | - Vanessa Delfosse
- Centre de Biologie Structurale, INSERM, CNRS, Université de Montpellier, Montpellier, France
| | - Nicolas Fuggetta
- Université Côte d'Azur, Centre National de la Recherche Scientifique, Institut de Pharmacologie Moléculaire et Cellulaire, 660 route des lucioles, 06560, Valbonne, France
| | - William Bourguet
- Centre de Biologie Structurale, INSERM, CNRS, Université de Montpellier, Montpellier, France
| | - Maud Magdeleine
- Université Côte d'Azur, Centre National de la Recherche Scientifique, Institut de Pharmacologie Moléculaire et Cellulaire, 660 route des lucioles, 06560, Valbonne, France
| | - Guillaume Drin
- Université Côte d'Azur, Centre National de la Recherche Scientifique, Institut de Pharmacologie Moléculaire et Cellulaire, 660 route des lucioles, 06560, Valbonne, France.
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Torng T, Wickner W. Phosphatidylinositol and phosphatidylinositol-3-phosphate activate HOPS to catalyze SNARE assembly, allowing small headgroup lipids to support the terminal steps of membrane fusion. Mol Biol Cell 2021; 32:ar19. [PMID: 34495682 PMCID: PMC8693972 DOI: 10.1091/mbc.e21-07-0373] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Intracellular membrane fusion requires Rab GTPases, tethers, SNAREs of the R, Qa, Qb, and Qc families, and SNARE chaperones of the Sec17 (SNAP), Sec18 (NSF), and SM (Sec1/Munc18) families. The vacuolar HOPS complex combines the functions of membrane tethering and SM catalysis of SNARE assembly. HOPS is activated for this catalysis by binding to the vacuolar lipids and Rab. Of the eight major vacuolar lipids, we now report that phosphatidylinositol and phosphatidylinositol-3-phosphate are required to activate HOPS for SNARE complex assembly. These lipids plus ergosterol also allow full trans-SNARE complex assembly, yet do not support fusion, which is reliant on either phosphatidylethanolamine (PE) or on phosphatidic acid (PA), phosphatidylserine (PS), and diacylglycerol (DAG). Fusion with a synthetic tether and without HOPS, or even without SNAREs, still relies on either PE or on PS, PA, and DAG. These lipids are thus required for the terminal bilayer rearrangement step of fusion, distinct from the lipid requirements for the earlier step of activating HOPS for trans-SNARE assembly.
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Affiliation(s)
- Thomas Torng
- Department of Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, NH 03755-3844
| | - William Wickner
- Department of Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, NH 03755-3844
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Li J, Wang K, Ji M, Zhang T, Yang C, Liu H, Chen S, Li H, Li H. Cys-SH based quantitative redox proteomics of salt induced response in sugar beet monosomic addition line M14. BOTANICAL STUDIES 2021; 62:16. [PMID: 34661775 PMCID: PMC8523603 DOI: 10.1186/s40529-021-00320-x] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/26/2021] [Accepted: 09/04/2021] [Indexed: 06/01/2023]
Abstract
BACKGROUND Salt stress is a major abiotic stress that limits plant growth, development and productivity. Studying the molecular mechanisms of salt stress tolerance may help to enhance crop productivity. Sugar beet monosomic addition line M14 exhibits tolerance to salt stress. RESULTS In this work, the changes in the BvM14 proteome and redox proteome induced by salt stress were analyzed using a multiplex iodoTMTRAQ double labeling quantitative proteomics approach. A total of 80 proteins were differentially expressed under salt stress. Interestingly, A total of 48 redoxed peptides were identified for 42 potential redox-regulated proteins showed differential redox change under salt stress. A large proportion of the redox proteins were involved in photosynthesis, ROS homeostasis and other pathways. For example, ribulose bisphosphate carboxylase/oxygenase activase changed in its redox state after salt treatments. In addition, three redox proteins involved in regulation of ROS homeostasis were also changed in redox states. Transcription levels of eighteen differential proteins and redox proteins were profiled. (The proteomics data generated in this study have been submitted to the ProteomeXchange and can be accessed via username: reviewer_pxd027550@ebi.ac.uk, password: q9YNM1Pe and proteomeXchange# PXD027550.) CONCLUSIONS: The results showed involvement of protein redox modifications in BvM14 salt stress response and revealed the short-term salt responsive mechanisms. The knowledge may inform marker-based breeding effort of sugar beet and other crops for stress resilience and high yield.
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Affiliation(s)
- Jinna Li
- Ministry of Education, School of Chemistry and Materials Science, Heilongjiang University, Harbin, 150080, China
- Key Laboratory of Molecular Biology of Heilongjiang Province, College of Life Sciences, Heilongjiang University, Harbin, 150080, China
| | - Kun Wang
- Engineering Research Center of Agricultural Microbiology Technology, Ministry of Education, Heilongjiang University, Harbin, 150080, China
| | - Meichao Ji
- Key Laboratory of Molecular Biology of Heilongjiang Province, College of Life Sciences, Heilongjiang University, Harbin, 150080, China
| | - Tingyue Zhang
- Key Laboratory of Molecular Biology of Heilongjiang Province, College of Life Sciences, Heilongjiang University, Harbin, 150080, China
| | - Chao Yang
- Key Laboratory of Molecular Biology of Heilongjiang Province, College of Life Sciences, Heilongjiang University, Harbin, 150080, China
| | - He Liu
- Key Laboratory of Molecular Biology of Heilongjiang Province, College of Life Sciences, Heilongjiang University, Harbin, 150080, China
| | - Sixue Chen
- Proteomics and Mass Spectrometry, Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL, 32610, USA
- Department of Biology, Genetics Institute, Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL, 32610, USA
| | - Hongli Li
- Key Laboratory of Molecular Biology of Heilongjiang Province, College of Life Sciences, Heilongjiang University, Harbin, 150080, China.
- Engineering Research Center of Agricultural Microbiology Technology, Ministry of Education, Heilongjiang University, Harbin, 150080, China.
| | - Haiying Li
- Ministry of Education, School of Chemistry and Materials Science, Heilongjiang University, Harbin, 150080, China.
- Key Laboratory of Molecular Biology of Heilongjiang Province, College of Life Sciences, Heilongjiang University, Harbin, 150080, China.
- Engineering Research Center of Agricultural Microbiology Technology, Ministry of Education, Heilongjiang University, Harbin, 150080, China.
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Lenoir G, D'Ambrosio JM, Dieudonné T, Čopič A. Transport Pathways That Contribute to the Cellular Distribution of Phosphatidylserine. Front Cell Dev Biol 2021; 9:737907. [PMID: 34540851 PMCID: PMC8440936 DOI: 10.3389/fcell.2021.737907] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2021] [Accepted: 08/10/2021] [Indexed: 12/05/2022] Open
Abstract
Phosphatidylserine (PS) is a negatively charged phospholipid that displays a highly uneven distribution within cellular membranes, essential for establishment of cell polarity and other processes. In this review, we discuss how combined action of PS biosynthesis enzymes in the endoplasmic reticulum (ER), lipid transfer proteins (LTPs) acting within membrane contact sites (MCS) between the ER and other compartments, and lipid flippases and scramblases that mediate PS flip-flop between membrane leaflets controls the cellular distribution of PS. Enrichment of PS in specific compartments, in particular in the cytosolic leaflet of the plasma membrane (PM), requires input of energy, which can be supplied in the form of ATP or by phosphoinositides. Conversely, coupling between PS synthesis or degradation, PS flip-flop and PS transfer may enable PS transfer by passive flow. Such scenario is best documented by recent work on the formation of autophagosomes. The existence of lateral PS nanodomains, which is well-documented in the case of the PM and postulated for other compartments, can change the steepness or direction of PS gradients between compartments. Improvements in cellular imaging of lipids and membranes, lipidomic analysis of complex cellular samples, reconstitution of cellular lipid transport reactions and high-resolution structural data have greatly increased our understanding of cellular PS homeostasis. Our review also highlights how budding yeast has been instrumental for our understanding of the organization and transport of PS in cells.
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Affiliation(s)
- Guillaume Lenoir
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell, Gif-sur-Yvette, France
| | - Juan Martín D'Ambrosio
- Centre de Recherche en Biologie Cellulaire de Montpellier (CRBM), Université de Montpellier, CNRS, Montpellier, France
| | - Thibaud Dieudonné
- Danish Research Institute of Translational Neuroscience - DANDRITE, Nordic EMBL Partnership for Molecular Medicine, Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark
| | - Alenka Čopič
- Centre de Recherche en Biologie Cellulaire de Montpellier (CRBM), Université de Montpellier, CNRS, Montpellier, France
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Encinar Del Dedo J, Fernández-Golbano IM, Pastor L, Meler P, Ferrer-Orta C, Rebollo E, Geli MI. Coupled sterol synthesis and transport machineries at ER-endocytic contact sites. J Cell Biol 2021; 220:212484. [PMID: 34283201 PMCID: PMC8294947 DOI: 10.1083/jcb.202010016] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2020] [Revised: 05/27/2021] [Accepted: 06/29/2021] [Indexed: 12/17/2022] Open
Abstract
Sterols are unevenly distributed within cellular membranes. How their biosynthetic and transport machineries are organized to generate heterogeneity is largely unknown. We previously showed that the yeast sterol transporter Osh2 is recruited to endoplasmic reticulum (ER)–endocytic contacts to facilitate actin polymerization. We now find that a subset of sterol biosynthetic enzymes also localizes at these contacts and interacts with Osh2 and the endocytic machinery. Following the sterol dynamics, we show that Osh2 extracts sterols from these subdomains, which we name ERSESs (ER sterol exit sites). Further, we demonstrate that coupling of the sterol synthesis and transport machineries is required for endocytosis in mother cells, but not in daughters, where plasma membrane loading with accessible sterols and endocytosis are linked to secretion.
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Affiliation(s)
| | | | - Laura Pastor
- Institute for Molecular Biology of Barcelona, Spanish Research Council, Barcelona, Spain
| | - Paula Meler
- Institute for Molecular Biology of Barcelona, Spanish Research Council, Barcelona, Spain
| | - Cristina Ferrer-Orta
- Institute for Molecular Biology of Barcelona, Spanish Research Council, Barcelona, Spain
| | - Elena Rebollo
- Institute for Molecular Biology of Barcelona, Spanish Research Council, Barcelona, Spain
| | - Maria Isabel Geli
- Institute for Molecular Biology of Barcelona, Spanish Research Council, Barcelona, Spain
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Wei CX, Burrow MF, Botelho MG, Leung WK. Analysing Complex Oral Protein Samples: Complete Workflow and Case Analysis of Salivary Pellicles. J Clin Med 2021; 10:2801. [PMID: 34202147 PMCID: PMC8267628 DOI: 10.3390/jcm10132801] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2021] [Revised: 06/21/2021] [Accepted: 06/22/2021] [Indexed: 11/17/2022] Open
Abstract
Studies on small quantity, highly complex protein samples, such as salivary pellicle, have been enabled by recent major technological and analytical breakthroughs. Advances in mass spectrometry-based computational proteomics such as Multidimensional Protein Identification Technology have allowed precise identification and quantification of complex protein samples on a proteome-wide scale, which has enabled the determination of corresponding genes and cellular functions at the protein level. The latter was achieved via protein-protein interaction mapping with Gene Ontology annotation. In recent years, the application of these technologies has broken various barriers in small-quantity-complex-protein research such as salivary pellicle. This review provides a concise summary of contemporary proteomic techniques contributing to (1) increased complex protein (up to hundreds) identification using minute sample sizes (µg level), (2) precise protein quantification by advanced stable isotope labelling or label-free approaches and (3) the emerging concepts and techniques regarding computational integration, such as the Gene Ontology Consortium and protein-protein interaction mapping. The latter integrates the structural, genomic, and biological context of proteins and genes to predict protein interactions and functional connections in a given biological context. The same technological breakthroughs and computational integration concepts can also be applied to other low-volume oral protein complexes such as gingival crevicular or peri-implant sulcular fluids.
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Affiliation(s)
- Chen-Xuan Wei
- Faculty of Dentistry, The University of Hong Kong, Hong Kong, China; (C.-X.W.); (M.F.B.); (M.G.B.)
- School of Dentistry, University of Michigan, Ann Arbor, MI 48104, USA
| | - Michael Francis Burrow
- Faculty of Dentistry, The University of Hong Kong, Hong Kong, China; (C.-X.W.); (M.F.B.); (M.G.B.)
| | - Michael George Botelho
- Faculty of Dentistry, The University of Hong Kong, Hong Kong, China; (C.-X.W.); (M.F.B.); (M.G.B.)
| | - W. Keung Leung
- Faculty of Dentistry, The University of Hong Kong, Hong Kong, China; (C.-X.W.); (M.F.B.); (M.G.B.)
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Binotti B, Jahn R, Pérez-Lara Á. An overview of the synaptic vesicle lipid composition. Arch Biochem Biophys 2021; 709:108966. [PMID: 34139199 DOI: 10.1016/j.abb.2021.108966] [Citation(s) in RCA: 45] [Impact Index Per Article: 11.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2021] [Revised: 06/10/2021] [Accepted: 06/10/2021] [Indexed: 11/29/2022]
Abstract
Chemical neurotransmission is the major mechanism of neuronal communication. Neurotransmitters are released from secretory organelles, the synaptic vesicles (SVs) via exocytosis into the synaptic cleft. Fusion of SVs with the presynaptic plasma membrane is balanced by endocytosis, thus maintaining the presynaptic membrane at steady-state levels. The protein machineries responsible for exo- and endocytosis have been extensively investigated. In contrast, less is known about the role of lipids in synaptic transmission and how the lipid composition of SVs is affected by dynamic exo-endocytotic cycling. Here we summarize the current knowledge about the composition, organization, and function of SV membrane lipids. We also cover lipid biogenesis and maintenance during the synaptic vesicle cycle.
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Affiliation(s)
- Beyenech Binotti
- Department of Biochemistry, University of Würzburg, Am Hubland, 97074, Würzburg, Germany
| | - Reinhard Jahn
- Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, Am Faßberg 11, 37077, Göttingen, Germany.
| | - Ángel Pérez-Lara
- Department of Physical Chemistry, University of Granada, Campus Universitario de Cartuja, 18071, Granada, Spain.
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Robustelli J, Baumgart T. Membrane partitioning and lipid selectivity of the N-terminal amphipathic H0 helices of endophilin isoforms. BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 2021; 1863:183660. [PMID: 34090873 DOI: 10.1016/j.bbamem.2021.183660] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Received: 04/01/2021] [Revised: 05/23/2021] [Accepted: 05/26/2021] [Indexed: 11/26/2022]
Abstract
Endophilin is an N-BAR protein, which is characterized by a crescent-shaped BAR domain and an amphipathic helix that contributes to the membrane binding of these proteins. The exact function of that H0 helix has been a topic of debate. In mammals, there are five different endophilin isoforms, grouped into A (three members) and B (two members) subclasses, which have been described to differ in their subcellular localization and function. We asked to what extent molecular properties of the H0 helices of these members affect their membrane targeting behavior. We found that all H0 helices of the endophilin isoforms display a two-state equilibrium between disordered and α-helical states in which the helical secondary structure can be stabilized through trifluoroethanol. The helicities in high TFE were strikingly different among the H0 peptides. We investigated H0-membrane partitioning by the monitoring of secondary structure changes via CD spectroscopy. We found that the presence of anionic phospholipids is critical for all H0 helices partitioning into membranes. Membrane partitioning is found to be sensitive to variations in membrane complexity. Overall, the H0 B subfamily displays stronger membrane partitioning than the H0 A subfamily. The H0 A peptide-membrane binding occurs predominantly through electrostatic interactions. Variation among the H0 A subfamily may be attributed to slight alterations in the amino acid sequence. Meanwhile, the H0 B subfamily displays greater specificity for certain membrane compositions, and this may link H0 B peptide binding to endophilin B's cellular function.
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Affiliation(s)
- Jaclyn Robustelli
- Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104, United States
| | - Tobias Baumgart
- Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104, United States.
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Mioka T, Guo T, Wang S, Tsuji T, Kishimoto T, Fujimoto T, Tanaka K. Characterization of micron-scale protein-depleted plasma membrane domains in phosphatidylserine-deficient yeast cells. J Cell Sci 2021; 135:261783. [PMID: 34000034 DOI: 10.1242/jcs.256529] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2020] [Accepted: 03/16/2021] [Indexed: 12/30/2022] Open
Abstract
Membrane phase separation to form micron-scale domains of lipids and proteins occurs in artificial membranes; however, a similar large-scale phase separation has not been reported in the plasma membrane of the living cells. We show here that a stable micron-scale protein-depleted region is generated in the plasma membrane of yeast mutants lacking phosphatidylserine at high temperatures. We named this region the 'void zone'. Transmembrane proteins and certain peripheral membrane proteins and phospholipids are excluded from the void zone. The void zone is rich in ergosterol, and requires ergosterol and sphingolipids for its formation. Such properties are also found in the cholesterol-enriched domains of phase-separated artificial membranes, but the void zone is a novel membrane domain that requires energy and various cellular functions for its formation. The formation of the void zone indicates that the plasma membrane in living cells has the potential to undergo phase separation with certain lipid compositions. We also found that void zones were frequently in contact with vacuoles, in which a membrane domain was also formed at the contact site.
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Affiliation(s)
- Tetsuo Mioka
- Division of Molecular Interaction, Institute for Genetic Medicine, Hokkaido University Graduate School of Life Science, Sapporo, Hokkaido 060-0815, Japan
| | - Tian Guo
- Division of Molecular Interaction, Institute for Genetic Medicine, Hokkaido University Graduate School of Life Science, Sapporo, Hokkaido 060-0815, Japan
| | - Shiyao Wang
- Division of Molecular Interaction, Institute for Genetic Medicine, Hokkaido University Graduate School of Life Science, Sapporo, Hokkaido 060-0815, Japan
| | - Takuma Tsuji
- Laboratory of Molecular Cell Biology, Research Institute for Diseases of Old Age, Juntendo University Graduate School of Medicine, Tokyo 113-8421, Japan
| | - Takuma Kishimoto
- Division of Molecular Interaction, Institute for Genetic Medicine, Hokkaido University Graduate School of Life Science, Sapporo, Hokkaido 060-0815, Japan
| | - Toyoshi Fujimoto
- Laboratory of Molecular Cell Biology, Research Institute for Diseases of Old Age, Juntendo University Graduate School of Medicine, Tokyo 113-8421, Japan
| | - Kazuma Tanaka
- Division of Molecular Interaction, Institute for Genetic Medicine, Hokkaido University Graduate School of Life Science, Sapporo, Hokkaido 060-0815, Japan
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Ghorbani M, Wang E, Krämer A, Klauda JB. Molecular dynamics simulations of ethanol permeation through single and double-lipid bilayers. J Chem Phys 2021; 153:125101. [PMID: 33003717 DOI: 10.1063/5.0013430] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
Abstract
Permeation of small molecules through membranes is a fundamental biological process, and molecular dynamics simulations have proven to be a promising tool for studying the permeability of membranes by providing a precise characterization of the free energy and diffusivity. In this study, permeation of ethanol through three different membranes of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS), PO-phosphatidylethanolamine (POPE), and PO-phosphatidylcholine (POPC) is studied. Permeabilities are calculated and compared with two different approaches based on Fick's first law and the inhomogeneous solubility-diffusion model. Microsecond simulation of double bilayers of these membranes provided a direct measurement of permeability by a flux-based counting method. These simulations show that a membrane of POPC has the highest permeability, followed by POPE and POPS. Due to the membrane-modulating properties of ethanol, the permeability increases as functions of concentration and saturation of the inner leaflet in a double bilayer setting, as opposed to the customary definition as a proportionality constant. This concentration dependence is confirmed by single bilayer simulations at different ethanol concentrations ranging from 1% to 18%, where permeability estimates are available from transition-based counting and the inhomogeneous solubility-diffusion model. We show that the free energy and diffusion profiles for ethanol lack accuracy at higher permeant concentrations due to non-Markovian kinetics caused by collective behavior. In contrast, the counting method provides unbiased estimates. Finally, the permeabilities obtained from single bilayer simulations are combined to represent natural gradients felt by a cellular membrane, which accurately models the non-equilibrium effects on ethanol permeability from single bilayer simulations in equilibrium.
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Affiliation(s)
- Mahdi Ghorbani
- Department of Chemical and Biomolecular Engineering, University of Maryland, College Park, Maryland 20742, USA
| | - Eric Wang
- Department of Chemical and Biomolecular Engineering, University of Maryland, College Park, Maryland 20742, USA
| | - Andreas Krämer
- Laboratory of Computational Biology, National, Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20824, USA
| | - Jeffery B Klauda
- Department of Chemical and Biomolecular Engineering, University of Maryland, College Park, Maryland 20742, USA
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