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

Kenoth, Roopa; Brown, Rhoderick E.; Kamlekar, Ravi Kanth

doi:10.1007/978-1-4939-9136-5_17

Cited by 3 publications

(8 citation statements)

References 95 publications

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“…To assess whether certain PIPs can activate the SL transfer activities of various GLTP superfamily members (human CPTP, plant CPTP-ACD11, human GLTP), we used an established fluorescence resonance energy transfer (FRET) approach that monitors the real-time kinetics of the complete SL transfer reaction, i.e. , SL uptake by protein from “SL-source” membrane vesicles and SL delivery by protein to “destination” membrane vesicles ( 44 ). A more complete description of the FRET assay is provided as Supporting information and illustrated in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Real-time intermembrane transfer rates of fluorescent sphingolipids by CPTP, ACD11, and GLTP were obtained by Förster resonance energy transfer (FRET) using a SPEX FluoroLog3 spectrofluorimeter (Horiba Scientific), with excitation and emission band passes of 2 nm and a stirred (∼100 rpm), temperature-controlled (25 °C ± 0.1 deg. C) sample cuvette holder ( 44 , 48 , 61 ). All fluorescent lipids were localized initially to the sphingolipid-source (donor) POPC vesicles formed by rapid ethanol injection.…”

Section: Methodsmentioning

confidence: 99%

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

Gao

Zhai

Болдырев

et al. 2021

Journal of Biological Chemistry

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Ceramide-1-phosphate transfer proteins (CPTPs) are members of the glycolipid transfer protein (GLTP) superfamily that shuttle ceramide-1-phosphate (C1P) between membranes. CPTPs regulate cellular sphingolipid homeostasis in ways that impact programmed cell death and inflammation. CPTP downregulation specifically alters C1P levels in the plasma and trans -Golgi membranes, stimulating proinflammatory eicosanoid production and autophagy-dependent inflammasome-mediated cytokine release. However, the mechanisms used by CPTP to target the trans -Golgi and plasma membrane are not well understood. Here, we monitored C1P intervesicular transfer using fluorescence energy transfer (FRET) and showed that certain phosphoinositides (phosphatidylinositol 4,5 bisphosphate (PI-(4,5)P 2 ) and phosphatidylinositol 4-phosphate (PI-4P)) increased CPTP transfer activity, whereas others (phosphatidylinositol 3-phosphate (PI-3P) and PI) did not. PIPs that stimulated CPTP did not stimulate GLTP, another superfamily member. Short-chain PI-(4,5)P 2, which is soluble and does not remain membrane-embedded, failed to activate CPTP. CPTP stimulation by physiologically relevant PI-(4,5)P 2 levels surpassed that of phosphatidylserine (PS), the only known non-PIP stimulator of CPTP, despite PI-(4,5)P 2 increasing membrane equilibrium binding affinity less effectively than PS. Functional mapping of mutations that led to altered FRET lipid transfer and assessment of CPTP membrane interaction by surface plasmon resonance indicated that di-arginine motifs located in the α-6 helix and the α3-α4 helix regulatory loop of the membrane-interaction region serve as PI-(4,5)P 2 headgroup-specific interaction sites. Haddock modeling revealed specific interactions involving the PI-(4,5)P 2 headgroup that left the acyl chains oriented favorably for membrane embedding. We propose that PI-(4,5)P 2 interaction sites enhance CPTP activity by serving as preferred membrane targeting/docking sites that favorably orient the protein for function.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

Gao

Zhai

Болдырев

et al. 2021

Journal of Biological Chemistry

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“…Compared to many other fluorophores, BODIPY exhibits photophysical properties that enhance lipid transfer assay performance, i.e., high environmental stability, high photostability, low environmental polarity sensitivity, and high emission intensity. ,− Thus, we relied on SL carrying a pentadecanoyl acyl chain labeled with tetramethyl-BODIPY (Me 4 -BODIPY) as an energy donor and C 18 -DiI as an energy acceptor for real-time kinetic tracking of the complete lipid transfer reaction using fluorescence resonance energy transfer (FRET) technology, which provides an order of magnitude sensitivity increase compared to the anthrylvinyl-SL/3-perylenoyl-PC energy donor/acceptor pair previously used to track lipid transfer. , The lipid transfer reaction involves uptake of SL by protein from “SL-source” model membranes and delivery of SL by protein to “receiver” model membranes. Figure illustrates the lipid fluorophore structures and changing FRET response that reflects SL transfer.…”

Section: Resultsmentioning

confidence: 99%

“…When bound, the lipid cargo is sufficiently shielded from the cellular aqueous milieu so that LTPs effectively become molecular solubilizers of lipid. Interest in LTPs has enjoyed a renaissance stemming from emerging information indicating that certain LTPs can function in vivo as molecular sensors and/or presentation devices involved in lipid metabolic regulation and signaling processes. − …”

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confidence: 99%

Measuring Lipid Transfer Protein Activity Using Bicelle-Dilution Model Membranes

Gao

Zhai

et al. 2020

Anal. Chem.

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In vitro assessment of lipid intermembrane transfer activity by cellular proteins typically involves measurement of either radiolabeled or fluorescently labeled lipid trafficking between vesicle model membranes. Use of bilayer vesicles in lipid transfer assays usually comes with inherent challenges because of complexities associated with the preparation of vesicles and their rather short “shelf life”. Such issues necessitate the laborious task of fresh vesicle preparation to achieve lipid transfer assays of high quality, precision, and reproducibility. To overcome these limitations, we have assessed model membrane generation by bicelle dilution for monitoring the transfer rates and specificity of various BODIPY-labeled sphingolipids by different glycolipid transfer protein (GLTP) superfamily members using a sensitive fluorescence resonance energy transfer approach. Robust, protein-selective sphingolipid transfer is observed using donor and acceptor model membranes generated by dilution of 0.5 q-value mixtures. The sphingolipid transfer rates are comparable to those observed between small bilayer vesicles produced by sonication or ethanol injection. Among the notable advantages of using bicelle-generated model membranes are (i) easy and straightforward preparation by means that avoid lipid fluorophore degradation and (ii) long “shelf life” after production (≥6 days) and resilience to freeze–thaw storage. The bicelle-dilution-based assay is sufficiently robust, sensitive, and stable for application, not only to purified LTPs but also for LTP activity detection in crude cytosolic fractions of cell homogenates.

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“…It is not known whether the principal factors of sphingolipid sorting are determined by the characteristics of lipids or proteins, nor is it known how sphingolipids complete selective trafficking or what molecular mechanisms underlie the two different transport pathways (protein-mediated and vesicular transport). Fluorophore-labeled sphingolipids have been used to track sphingolipid intermembrane transport in microorganisms and mammalian cells ( Hackstadt et al., 1996 ; van Helvoort et al., 1997 ; Kenoth et al., 2019 ). Transgenic plants (usually Arabidopsis ) with different tagged-marker proteins involved in lipid trafficking pathways can be used to study sphingolipid transport.…”

Section: Perspectivesmentioning

confidence: 99%

Sphingolipid metabolism, transport, and functions in plants: Recent progress and future perspectives

Liu

Hou

Bao

et al. 2021

Plant Communications

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Sphingolipids, which comprise membrane systems together with other lipids, are ubiquitous in cellular organisms. They show a high degree of diversity across plant species and vary in their structures, properties, and functions. Benefiting from the development of lipidomic techniques, over 300 plant sphingolipids have been identified. Generally divided into free long-chain bases (LCBs), ceramides, glycosylceramides (GlcCers) and glycosyl inositol phosphoceramides (GIPCs), plant sphingolipids exhibit organized aggregation within lipid membranes to form raft domains with sterols. Accumulating evidence has revealed that sphingolipids obey certain trafficking and distribution rules and confer unique properties to membranes. Functional studies using sphingolipid biosynthetic mutants demonstrate that sphingolipids participate in plant developmental regulation, stimulus sensing, and stress responses. Here, we present an updated metabolism/degradation map and summarize the structures of plant sphingolipids, review recent progress in understanding the functions of sphingolipids in plant development and stress responses, and review sphingolipid distribution and trafficking in plant cells. We also highlight some important challenges and issues that we may face during the process of studying sphingolipids.

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

Cited by 3 publications

References 95 publications

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

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

Measuring Lipid Transfer Protein Activity Using Bicelle-Dilution Model Membranes

Sphingolipid metabolism, transport, and functions in plants: Recent progress and future perspectives

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