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1.
J Biol Chem ; 299(4): 104607, 2023 04.
Artículo en Inglés | MEDLINE | ID: mdl-36924944

RESUMEN

The glycolipid transfer protein (GLTP) has been linked to many cellular processes aside from its best-known in vitro function as a lipid transport protein. It has been proposed to act as a sensor and regulator of glycosphingolipid homeostasis in cells. Furthermore, through its previously determined interaction with the endoplasmic reticulum membrane protein VAP-A (vesicle-associated membrane protein-associated protein A), GLTP may also be involved in facilitating vesicular transport in cells. In this study, we characterized the phenotype of CRISPR/Cas9-mediated GLTP KO HeLa cells. We showed that motility, three-dimensional growth, and cellular metabolism were all altered by GLTP knockout. Expression of a GLTP mutant incapable of binding VAP disrupted cell spheroid formation, indicating that the GLTP-VAP interaction is linked to cellular adhesion, cohesion, and three-dimensional growth. Most notably, we found evidence that GLTP, through its interaction with VAP-A, affects vesicular trafficking, marking the first cellular process discovered to be directly impacted by a change in GLTP expression.


Asunto(s)
Transporte Biológico , Proteínas Portadoras , Membrana Celular , Humanos , Transporte Biológico/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Células HeLa , Técnicas de Inactivación de Genes , Unión Proteica/genética , Regulación de la Expresión Génica/genética , Citosol/metabolismo , Movimiento Celular/genética
2.
J Cell Sci ; 132(4)2018 08 20.
Artículo en Inglés | MEDLINE | ID: mdl-30072441

RESUMEN

Integrin transmembrane receptors control a wide range of biological interactions by triggering the assembly of large multiprotein complexes at their cytoplasmic interface. Diverse methods have been used to investigate interactions between integrins and intracellular proteins, and predominantly include peptide-based pulldowns and biochemical immuno-isolations from detergent-solubilised cell lysates. However, quantitative methods to probe integrin-protein interactions in a more biologically relevant context where the integrin is embedded within a lipid bilayer have been lacking. Here, we describe 'protein-liposome interactions by flow cytometry' (denoted ProLIF), a technique to reconstitute recombinant integrin transmembrane domains (TMDs) and cytoplasmic tail (CT) fragments in liposomes as individual subunits or as αß heterodimers and, via flow cytometry, allow rapid and quantitative measurement of protein interactions with these membrane-embedded integrins. Importantly, the assay can analyse binding of fluorescent proteins directly from cell lysates without further purification steps. Moreover, the effect of membrane composition, such as PI(4,5)P2 incorporation, on protein recruitment to the integrin CTs can be analysed. ProLIF requires no specific instrumentation and can be applied to measure a broad range of membrane-dependent protein-protein interactions with the potential for high-throughput/multiplex analyses.This article has associated First Person interviews with the first authors of the paper (see doi: 10.1242/jcs.223644 and doi: 10.1242/jcs.223719).


Asunto(s)
Membrana Celular/metabolismo , Integrinas/metabolismo , Liposomas/metabolismo , Proteolípidos/metabolismo , Adhesión Celular/fisiología , Citoplasma/metabolismo , Dimerización , Citometría de Flujo/métodos , Humanos , Unión Proteica/fisiología
3.
Nature ; 501(7465): 116-20, 2013 Sep 05.
Artículo en Inglés | MEDLINE | ID: mdl-23913272

RESUMEN

Newly synthesized proteins and lipids are transported across the Golgi complex via different mechanisms whose respective roles are not completely clear. We previously identified a non-vesicular intra-Golgi transport pathway for glucosylceramide (GlcCer)--the common precursor of the different series of glycosphingolipids-that is operated by the cytosolic GlcCer-transfer protein FAPP2 (also known as PLEKHA8) (ref. 1). However, the molecular determinants of the FAPP2-mediated transfer of GlcCer from the cis-Golgi to the trans-Golgi network, as well as the physiological relevance of maintaining two parallel transport pathways of GlcCer--vesicular and non-vesicular--through the Golgi, remain poorly defined. Here, using mouse and cell models, we clarify the molecular mechanisms underlying the intra-Golgi vectorial transfer of GlcCer by FAPP2 and show that GlcCer is channelled by vesicular and non-vesicular transport to two topologically distinct glycosylation tracks in the Golgi cisternae and the trans-Golgi network, respectively. Our results indicate that the transport modality across the Golgi complex is a key determinant for the glycosylation pattern of a cargo and establish a new paradigm for the branching of the glycosphingolipid synthetic pathway.


Asunto(s)
Glucosilceramidas/metabolismo , Glicosilación , Aparato de Golgi/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Transporte Biológico , Línea Celular , Globósidos/biosíntesis , Globósidos/química , Globósidos/metabolismo , Glucosilceramidas/química , Glicoesfingolípidos/biosíntesis , Glicoesfingolípidos/química , Glicoesfingolípidos/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Fosfatos de Fosfatidilinositol/metabolismo , Red trans-Golgi/metabolismo
4.
Cell Mol Life Sci ; 75(18): 3393-3410, 2018 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-29549423

RESUMEN

The UDP-glucose ceramide glucosyltransferase (UGCG) is a key enzyme in the synthesis of glycosylated sphingolipids, since this enzyme generates the precursor for all complex glycosphingolipids (GSL), the GlcCer. The UGCG has been associated with several cancer-related processes such as maintaining cancer stem cell properties or multidrug resistance induction. The precise mechanisms underlying these processes are unknown. Here, we investigated the molecular mechanisms occurring after UGCG overexpression in breast cancer cells. We observed alterations of several cellular properties such as morphological changes, which enhanced proliferation and doxorubicin resistance in UGCG overexpressing MCF-7 cells. These cellular effects seem to be mediated by an altered composition of glycosphingolipid-enriched microdomains (GEMs), especially an accumulation of globotriaosylceramide (Gb3) and glucosylceramide (GlcCer), which leads to an activation of Akt and ERK1/2. The induction of the Akt and ERK1/2 signaling pathway results in an increased gene expression of multidrug resistance protein 1 (MDR1) and anti-apoptotic genes and a decrease of pro-apoptotic gene expression. Inhibition of the protein kinase C (PKC) and phosphoinositide 3 kinase (PI3K) reduced MDR1 gene expression. This study discloses how changes in UGCG expression impact several cellular signaling pathways in breast cancer cells resulting in enhanced proliferation and multidrug resistance.


Asunto(s)
Proliferación Celular , Resistencia a Antineoplásicos , Glucosiltransferasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Apoptosis/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Colesterol/análisis , Doxorrubicina/farmacología , Resistencia a Antineoplásicos/genética , Femenino , Glucosiltransferasas/genética , Humanos , Células MCF-7 , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Transducción de Señal/genética , Esfingolípidos/análisis , Esfingolípidos/metabolismo , Esfingomielina Fosfodiesterasa/genética , Esfingomielina Fosfodiesterasa/metabolismo , Esfingosina N-Aciltransferasa/genética , Esfingosina N-Aciltransferasa/metabolismo
5.
Toxicol Appl Pharmacol ; 360: 160-184, 2018 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-30268580

RESUMEN

The topobiological behaviour of Nrf1 dictates its post-translational modification and its ability to transactivate target genes. Here, we have elucidated that topovectorial mechanisms control the juxtamembrane processing of Nrf1 on the cyto/nucleoplasmic side of endoplasmic reticulum (ER), whereupon it is cleaved and degraded to remove various lengths of its N-terminal domain (NTD, also refolded into a UBL module) and acidic domain-1 (AD1) to yield multiple isoforms. Notably, an N-terminal ~12.5-kDa polypeptide of Nrf1 arises from selective cleavage at an NHB2-adjoining region within NTD, whilst other longer UBL-containing isoforms may arise from proteolytic processing of the protein within AD1 around PEST1 and Neh2L degrons. The susceptibility of Nrf1 to proteolysis is determined by dynamic repositioning of potential UBL-adjacent degrons and cleavage sites from the ER lumen through p97-driven retrotranslocation and -independent pathways into the cyto/nucleoplasm. These repositioned degrons and cleavage sites within NTD and AD1 of Nrf1 are coming into their bona fide functionality, thereby enabling it to be selectively processed by cytosolic DDI-1/2 proteases and also partiality degraded via 26S proteasomes. The resultant proteolytic processing of Nrf1 gives rise to a mature ~85-kDa CNC-bZIP transcription factor, which regulates transcriptional expression of cognate target genes. Furthermore, putative ubiquitination of Nrf1 is not a prerequisite necessary for involvement of p97 in the client processing. Overall, the regulated juxtamembrane proteolysis (RJP) of Nrf1, though occurring in close proximity to the ER, is distinctive from the mechanism that regulates the intramembrane proteolytic (RIP) processing of ATF6 and SREBP1.


Asunto(s)
Factor 1 Relacionado con NF-E2/metabolismo , Péptidos/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Línea Celular , Núcleo Celular/metabolismo , Chlorocebus aethiops , Citosol/metabolismo , Retículo Endoplásmico/metabolismo , Regulación de la Expresión Génica/fisiología , Células HEK293 , Humanos , Ratones , Complejo de la Endopetidasa Proteasomal/metabolismo , Isoformas de Proteínas/metabolismo , Proteolisis , Alineación de Secuencia , Activación Transcripcional/fisiología
6.
BMC Cancer ; 18(1): 153, 2018 02 06.
Artículo en Inglés | MEDLINE | ID: mdl-29409484

RESUMEN

The UDP-glucose ceramide glycosyltransferase (UGCG) is a key enzyme in the sphingolipid metabolism by generating glucosylceramide (GlcCer), the precursor for all glycosphingolipids (GSL), which are essential for proper cell function. Interestingly, the UGCG is also overexpressed in several cancer types and correlates with multidrug resistance protein 1 (MDR1) gene expression. This membrane protein is responsible for efflux of toxic substances and protects cancer cells from cell damage through chemotherapeutic agents. Studies showed a connection between UGCG and MDR1 overexpression and multidrug resistance development, but the precise underlying mechanisms are unknown. Here, we give an overview about the UGCG and its connection to MDR1 in multidrug resistant cells. Furthermore, we focus on UGCG transcriptional regulation, the impact of UGCG on cellular signaling pathways and the effect of UGCG and MDR1 on the lipid composition of membranes and how this could influence multidrug resistance development. To our knowledge, this is the first review presenting an overview about UGCG with focus on the relationship to MDR1 in the process of multidrug resistance development.


Asunto(s)
Resistencia a Múltiples Medicamentos/genética , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica , Glucosiltransferasas/genética , Neoplasias/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Animales , Glucosiltransferasas/metabolismo , Humanos , Modelos Genéticos , Neoplasias/metabolismo , Neoplasias/patología
7.
Int J Mol Sci ; 19(10)2018 Sep 26.
Artículo en Inglés | MEDLINE | ID: mdl-30261635

RESUMEN

Normal growth and development, as well as adaptive responses to various intracellular and environmental stresses, are tightly controlled by transcriptional networks. The evolutionarily conserved genomic sequences across species highlights the architecture of such certain regulatory elements. Among them, one of the most conserved transcription factors is the basic-region leucine zipper (bZIP) family. Herein, we have performed phylogenetic analysis of these bZIP proteins and found, to our surprise, that there exist a few homologous proteins of the family members Jun, Fos, ATF2, BATF, C/EBP and CNC (cap'n'collar) in either viruses or bacteria, albeit expansion and diversification of this bZIP superfamily have occurred in vertebrates from metazoan. Interestingly, a specific group of bZIP proteins is identified, designated Nach (Nrf and CNC homology), because of their strong conservation with all the known CNC and NF-E2 p45 subunit-related factors Nrf1 and Nrf2. Further experimental evidence has also been provided, revealing that Nach1 and Nach2 from the marine bacteria exert distinctive functions, when compared with human Nrf1 and Nrf2, in the transcriptional regulation of antioxidant response element (ARE)-battery genes. Collectively, further insights into these Nach/CNC-bZIP subfamily transcription factors provide a novel better understanding of distinct biological functions of these factors expressed in distinct species from the marine bacteria to humans.


Asunto(s)
Organismos Acuáticos/genética , Bacterias/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Evolución Molecular , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/clasificación , Regulación de la Expresión Génica , Variación Genética , Humanos , Filogenia , Especificidad de la Especie
8.
Biochim Biophys Acta Mol Cell Res ; 1871(2): 119644, 2024 02.
Artículo en Inglés | MEDLINE | ID: mdl-37996059

RESUMEN

Since Nrf1 and Nrf2 are essential for regulating the lipid metabolism pathways, their dysregulation has thus been shown to be critically involved in the non-controllable inflammatory transformation into cancer. Herein, we have explored the molecular mechanisms underlying their distinct regulation of lipid metabolism, by comparatively analyzing the changes in those lipid metabolism-related genes in Nrf1α-/- and/or Nrf2-/- cell lines relative to wild-type controls. The results revealed that loss of Nrf1α leads to lipid metabolism disorders. That is, its lipid synthesis pathway was up-regulated by the JNK-Nrf2-AP1 signaling, while its lipid decomposition pathway was down-regulated by the nuclear receptor PPAR-PGC1 signaling, thereby resulting in severe accumulation of lipids as deposited in lipid droplets. By contrast, knockout of Nrf2 gave rise to decreases in lipid synthesis and uptake capacity. These demonstrate that Nrf1 and Nrf2 contribute to significant differences in the cellular lipid metabolism profiles and relevant pathological responses. Further experimental evidence unraveled that lipid deposition in Nrf1α-/- cells resulted from CD36 up-regulation by activating the PI3K-AKT-mTOR pathway, leading to abnormal activation of the inflammatory response. This was also accompanied by a series of adverse consequences, e.g., accumulation of reactive oxygen species (ROS) in Nrf1α-/- cells. Interestingly, treatment of Nrf1α-/- cells with 2-bromopalmitate (2BP) enabled the yield of lipid droplets to be strikingly alleviated, as accompanied by substantial abolishment of CD36 and critical inflammatory cytokines. Such Nrf1α-/- -led inflammatory accumulation of lipids, as well as ROS, was significantly ameliorated by 2BP. Overall, this study provides a potential strategy for cancer prevention and treatment by precision targeting of Nrf1, Nrf2 alone or both.


Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Factor 1 Relacionado con NF-E2 , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Factor 2 Relacionado con NF-E2/genética , Palmitatos , Fosfatidilinositol 3-Quinasas , Especies Reactivas de Oxígeno , Factor 1 Relacionado con NF-E2/genética
9.
Nature ; 449(7158): 62-7, 2007 Sep 06.
Artículo en Inglés | MEDLINE | ID: mdl-17687330

RESUMEN

The molecular machinery responsible for the generation of transport carriers moving from the Golgi complex to the plasma membrane relies on a tight interplay between proteins and lipids. Among the lipid-binding proteins of this machinery, we previously identified the four-phosphate adaptor protein FAPP2, the pleckstrin homology domain of which binds phosphatidylinositol 4-phosphate and the small GTPase ARF1. FAPP2 also possesses a glycolipid-transfer-protein homology domain. Here we show that human FAPP2 is a glucosylceramide-transfer protein that has a pivotal role in the synthesis of complex glycosphingolipids, key structural and signalling components of the plasma membrane. The requirement for FAPP2 makes the whole glycosphingolipid synthetic pathway sensitive to regulation by phosphatidylinositol 4-phosphate and ARF1. Thus, by coupling the synthesis of glycosphingolipids with their export to the cell surface, FAPP2 emerges as crucial in determining the lipid identity and composition of the plasma membrane.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Glucosilceramidas/metabolismo , Glicoesfingolípidos/biosíntesis , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Transporte Biológico , Línea Celular , Membrana Celular/química , Membrana Celular/metabolismo , Ceramidas/metabolismo , Humanos , Modelos Biológicos , Fosfatos de Fosfatidilinositol/metabolismo , Esfingosina/metabolismo , Red trans-Golgi/metabolismo
10.
Biochim Biophys Acta ; 1808(12): 2886-93, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-21910967

RESUMEN

The glycolipid transfer protein (GLTP) is capable of transporting glycolipids from a donor membrane, through the aqueous environment, to an acceptor membrane. The GLTP mediated glycolipid transfer from sphingomyelin membranes is very slow. In contrast, the transfer is fast from membranes composed of phosphatidylcholine. The lateral glycolipid membrane organization is known to be driven by their tendency to mix non-randomly with different membrane lipids. Consequently, the properties of the membrane lipids surrounding the glycolipids play an important role in the ability of GLTP to bind and transfer its substrates. Since GLTP transfer of glycolipids is almost nonexistent from sphingomyelin membranes, we have used this exceptionality to investigate if membrane intercalators can alter the membrane packing and induce glycolipid transfer. We found that the bile salts cholate, deoxycholate, taurocholate and taurodeoxycholate, cause glucosylceramide to become transferrable by GLTP. Other compounds, such as single chain lipids, ceramide and nonionic surfactants, that have membrane-perturbing effects, did not affect the transfer capability of GLTP. We speculate that the strong hydrogen bonding network formed in the interfacial region of glycosphingolipid-sphingomyelin membranes is disrupted by the membrane partition of the bile salts causing the glycosphingolipid to become transferrable.


Asunto(s)
Ácidos y Sales Biliares/farmacología , Glucosilceramidas/química , Membranas Artificiales , Polarización de Fluorescencia , Temperatura
11.
Biochim Biophys Acta ; 1808(1): 47-54, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-20804726

RESUMEN

The glycolipid transfer protein (GLTP) is a protein capable of binding and transferring glycolipids. GLTP is cytosolic and it can interact through its FFAT-like (two phenylalanines in an acidic tract) motif with proteins localized on the surface of the endoplasmic reticulum. Previous in vitro work with GLTP has focused mainly on the complete transfer reaction of the protein, that is, binding and subsequent removal of the glycolipid from the donor membrane, transfer through the aqueous environment, and the final release of the glycolipid to an acceptor membrane. Using bilayer vesicles and surface plasmon resonance spectroscopy, we have now, for the first time, analyzed the binding and lipid removal capacity of GLTP with a completely label-free technique. This technique is focused on the initial steps in GLTP-mediated transfer and the parameters affecting these steps can be more precisely determined. We used the new approach for detailed structure-function studies of GLTP by examining the glycolipid transfer capacity of specific GLTP tryptophan mutants. Tryptophan 96 is crucial for the transfer activity of the protein and tryptophan 142 is an important part of the proteins membrane interacting domain. Further, we varied the composition of the used lipid vesicles and gained information on the effect of membrane properties on GLTP activity. GLTP prefers to interact with more tightly packed membranes, although GLTP-mediated transfer is faster from more fluid membranes. This technique is very useful for the study of membrane-protein interactions and lipid-transfer rates and it can easily be adapted to other membrane-interacting proteins.


Asunto(s)
Proteínas Portadoras/química , Glucolípidos/química , Resonancia por Plasmón de Superficie/métodos , Secuencias de Aminoácidos , Biofisica , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Escherichia coli/metabolismo , Lípidos/química , Mutación , Fenilalanina/química , Estructura Terciaria de Proteína , Factores de Tiempo , Triptófano/química
12.
Biochim Biophys Acta ; 1808(1): 229-35, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-20875392

RESUMEN

The in vitro activity of the ceramide transporter, CERT has been studied using a fluorescence assay. CERT is responsible for the in vivo non-vesicular trafficking of ceramide between the endoplasmic reticulum and Golgi. In this study we have examined how the membrane environment surrounding the ceramide substrate, the membrane packing density and the membrane charge, are affecting the ceramide transfer activity. To examine this we have used an anthrylvinyl-labeled ceramide analogue. We found that if ceramide is in a tightly packed environment such as in sphingomyelin or dipalmitoylphosphatidylcholine containing membranes, the CERT transfer activity is markedly reduced. Ceramide in fluid membranes on the other hand are available for CERT mediated transfer. CERT also favors membranes that contain phosphatidylinositol 4-monophospate, due to its binding capacity of the pleckstrin homology domain towards phosphatidylinositol 4-monophospate. From this study we conclude that the membrane matrix surrounding ceramide, that is ceramide miscibility, is largely affecting the transfer activity of CERT.


Asunto(s)
Ceramidas/química , Lípidos/química , Proteínas Serina-Treonina Quinasas/química , 1,2-Dipalmitoilfosfatidilcolina/química , Catálisis , Colesterol/química , Escherichia coli/metabolismo , Polarización de Fluorescencia , Humanos , Membrana Dobles de Lípidos/química , Mutación , Fosfatos/química , Fosfatidilcolinas/química , Fosfatidilinositoles/química , Fosfolípidos/química , Esfingomielinas/química
13.
J Cell Biol ; 179(1): 101-15, 2007 Oct 08.
Artículo en Inglés | MEDLINE | ID: mdl-17923531

RESUMEN

Glycosphingolipids are controlled by the spatial organization of their metabolism and by transport specificity. Using immunoelectron microscopy, we localize to the Golgi stack the glycosyltransferases that produce glucosylceramide (GlcCer), lactosylceramide (LacCer), and GM3. GlcCer is synthesized on the cytosolic side and must translocate across to the Golgi lumen for LacCer synthesis. However, only very little natural GlcCer translocates across the Golgi in vitro. As GlcCer reaches the cell surface when Golgi vesicular trafficking is inhibited, it must translocate across a post-Golgi membrane. Concanamycin, a vacuolar proton pump inhibitor, blocks translocation independently of multidrug transporters that are known to translocate short-chain GlcCer. Concanamycin did not reduce LacCer and GM3 synthesis. Thus, GlcCer destined for glycolipid synthesis follows a different pathway and transports back into the endoplasmic reticulum (ER) via the late Golgi protein FAPP2. FAPP2 knockdown strongly reduces GM3 synthesis. Overall, we show that newly synthesized GlcCer enters two pathways: one toward the noncytosolic surface of a post-Golgi membrane and one via the ER toward the Golgi lumen LacCer synthase.


Asunto(s)
Glucosilceramidas/metabolismo , Glicoesfingolípidos/biosíntesis , Aparato de Golgi/metabolismo , Animales , Antígenos CD/metabolismo , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Brefeldino A/farmacología , Proteínas Portadoras/metabolismo , Proteínas Portadoras/fisiología , Bovinos , Línea Celular , Cricetinae , Cricetulus , Retículo Endoplásmico/metabolismo , Glicosiltransferasas/metabolismo , Aparato de Golgi/enzimología , Humanos , Membranas Intracelulares/metabolismo , Lactosilceramidos/metabolismo , Macrólidos/farmacología , Ratones , Microscopía Fluorescente , Modelos Biológicos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Inhibidores de la Bomba de Protones/farmacología , Ratas
14.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1866(11): 159021, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34339859

RESUMEN

Lipid bilayers function as boundaries that enclose their content from the surrounding media, and the composition of different membrane types is accurately and dynamically tailored so that they can perform their function. To achieve this balance, lipid biosynthetic machinery and lipid trafficking events are intertwined into an elegant network. In this review, we focus on the intracellular movement of sphingolipids mediated by sphingolipid transfer proteins. Additionally, we will focus on the best characterized and understood mammalian sphingolipid transfer proteins and provide an overview of how they are hypothesized to function. Some are already well understood, while others remain enigmatic. A few are actual lipid transfer proteins, moving lipids from membrane to membrane, while others may have more of a sensor role, possibly reacting to changes in the concentrations of their ligands. Considering the substrates available for cytosolic sphingolipid transfer proteins, one open question that is discussed is whether galactosylceramide is a target. Another question is the exact mechanics by which sphingolipid transfer proteins are targeted to different organelles, such as how four phosphate adapter protein-2, FAPP2 is targeted to the endoplasmic reticulum. The aim of this review is to discuss what is known within the field today and to provide a basic understanding of how these proteins may work.


Asunto(s)
Proteínas Portadoras/metabolismo , Esfingolípidos/metabolismo , Animales , Transporte Biológico , Humanos
15.
Artículo en Inglés | MEDLINE | ID: mdl-33181324

RESUMEN

Lysosome Associated Protein Transmembrane 4B (LAPTM4B) is a four-membrane spanning ceramide interacting protein that regulates mTORC1 signaling. Here, we show that LAPTM4B is sorted into intraluminal vesicles (ILVs) of multivesicular endosomes (MVEs) and released in small extracellular vesicles (sEVs) into conditioned cell culture medium and human urine. Efficient sorting of LAPTM4B into ILV membranes depends on its third transmembrane domain containing a sphingolipid interaction motif (SLim). Unbiased lipidomic analysis reveals a strong enrichment of glycosphingolipids in sEVs secreted from LAPTM4B knockout cells and from cells expressing a SLim-deficient LAPTM4B mutant. The altered sphingolipid profile is accompanied by a distinct SLim-dependent co-modulation of ether lipid species. The changes in the lipid composition of sEVs derived from LAPTM4B knockout cells is reflected by an increased stability of membrane nanodomains of sEVs. These results identify LAPTM4B as a determinant of the glycosphingolipid profile and membrane properties of sEVs.


Asunto(s)
Exosomas/metabolismo , Glicoesfingolípidos/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Oncogénicas/metabolismo , Línea Celular Tumoral , Endosomas/metabolismo , Técnicas de Inactivación de Genes , Humanos , Metabolismo de los Lípidos , Lipidómica , Proteínas de la Membrana/genética , Proteínas Oncogénicas/genética
16.
Biochim Biophys Acta ; 1788(1): 267-72, 2009 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-19007748

RESUMEN

The glycolipid transfer protein is found from animals and fungi to plants and red micro-alga. Some eukaryotes that do not encode the glucosylceramide synthase like the yeast Schizosaccharomyces pombe and Saccharomyces cerevisiae do neither produce glycolipid transfer like proteins. On the other hand yeast like Eremothecium gossypii that do synthesize glucosylceramide also express glycolipid transfer protein. Based on this novel genetic relationship it is not far fetched to assume that there must be a strong correlation between the synthesis of the glycolipid precursor and the glycolipid transfer protein. Because the glycolipid transfer protein is localized in the cytosol it is unlikely that it would participate in events associated with lipid rafts or caveolar structures, since they are found on the outer leaflet of the plasma membrane. Rather, GLTP is likely to be involved in events at the cytosolic side of the plasma membrane or the endoplasmic reticulum, maybe function as a reporter or sensor of glycolipid levels. A similar function has been proposed for other proteins with affinity for lipids like the oxysterol binding proteins and phosphatidylinositol transfer proteins that are thought to be able act as lipid sensors. Recent discoveries in the glycolipid transfer protein field are discussed.


Asunto(s)
Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , Membrana Celular/química , Humanos , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Homología de Secuencia de Aminoácido
17.
Int J Biochem Cell Biol ; 127: 105834, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32827762

RESUMEN

Identifying co-expression of lipid species is challenging, but indispensable to identify novel therapeutic targets for breast cancer treatment. Lipid metabolism is often dysregulated in cancer cells, and changes in lipid metabolism affect cellular processes such as proliferation, autophagy, and tumor development. In addition to mRNA analysis of sphingolipid metabolizing enzymes, we performed liquid chromatography time-of-flight mass spectrometry analysis in three breast cancer cell lines. These breast cancer cell lines differ in estrogen receptor and G-protein coupled estrogen receptor 1 status. Our data show that sphingolipids and non-sphingolipids are strongly increased in SKBr3 cells. SKBr3 cells are estrogen receptor negative and G-protein coupled estrogen receptor 1 positive. Treatment with G15, a G-protein coupled estrogen receptor 1 antagonist, abolishes the effect of increased sphingolipid and non-sphingolipid levels in SKBr3 cells. In particular, ether lipids are expressed at much higher levels in cancer compared to normal cells and are strongly increased in SKBr3 cells. Our analysis reveals that this is accompanied by increased sphingolipid levels such as ceramide, sphingadiene-ceramide and sphingomyelin. This shows the importance of focusing on more than one lipid class when investigating molecular mechanisms in breast cancer cells. Our analysis allows unbiased screening for different lipid classes leading to identification of co-expression patterns of lipids in the context of breast cancer. Co-expression of different lipid classes could influence tumorigenic potential of breast cancer cells. Identification of co-regulated lipid species is important to achieve improved breast cancer treatment outcome.


Asunto(s)
Neoplasias de la Mama/metabolismo , Receptor alfa de Estrógeno/metabolismo , Lipidómica/métodos , Lípidos/biosíntesis , Éteres Fosfolípidos/metabolismo , Esfingolípidos/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Ceramidas/metabolismo , Receptor alfa de Estrógeno/genética , Femenino , Humanos , Receptores de Estrógenos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo
18.
Biochem Biophys Res Commun ; 388(2): 395-9, 2009 Oct 16.
Artículo en Inglés | MEDLINE | ID: mdl-19665998

RESUMEN

The glycolipid transfer protein (GLTP) is a cytoplasmic protein with an ability to bind glycolipids and catalyze their in vitro transfer. In this study, we have found a FFAT-like motif in GLTP. The FFAT (two phenylalanines in an acidic tract) motif in lipid-binding proteins has previously been shown to interact with the VAPs (vesicle-associated membrane protein-associated proteins) in the endoplasmic reticulum. Here we used glutathione S-transferase pull-down experiments to confirm that GLTP and VAP-A interact. By displacing different amino acids in the motif we clearly show that the interaction is dependent on the FFAT-like motif in GLTP. The potential role of GLTP in the endoplasmic reticulum association is discussed.


Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Proteínas Portadoras/genética , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Humanos , Datos de Secuencia Molecular , Mutación , Proteínas de Transporte Vesicular/genética
19.
Methods Mol Biol ; 1949: 105-114, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30790252

RESUMEN

Here we summarize how glycosphingolipid production can be followed using metabolic labeling with radiolabeled lipid precursors. No assays are available yet that directly would address the lipid transfer protein activity in vivo. Therefore, these approaches can serve as tools to indirectly study the lipid transfer protein activity in cells, by monitoring their impact on the glycosphingolipid homeostasis.


Asunto(s)
Proteínas Portadoras/metabolismo , Glicoesfingolípidos/metabolismo , Ceramidas/metabolismo , Cromatografía en Capa Delgada , Glucolípidos/metabolismo , Metabolismo de los Lípidos , Redes y Vías Metabólicas , Esfingosina/análogos & derivados , Esfingosina/metabolismo
20.
Biochim Biophys Acta ; 1771(11): 1353-63, 2007 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-17980653

RESUMEN

Glycolipid transfer proteins (GLTPs) are small proteins that specifically transfer glycolipids from one bilayer membrane to another in vitro. However, the precise biological function is still unknown. In this study the intracellular distribution of GLTP was determined. We have used several independent methods, including differential and discontinuous density gradient centrifugation, plasma membrane permeabilization and confocal microscopy imaging, and we demonstrate that GLTP has a cytosolic location. The GLTP is not located in the Golgi apparatus, endoplasmic reticulum, nucleus, lysosomes, mitochondria or peroxisomes in HeLa cells. We have also used a fluorescence resonance energy transfer assay to detect transfer of fluorescently labeled BODIPY-glucosylceramide in the cytosolic fraction from both wild-type and GLTP-overexpressing HeLa cells. Furthermore, we have studied de novo sphingolipid changes in cells overexpressing GLTP using sphinganine metabolic labeling. The results show a significant increase in the synthesis of glucosylceramide (GlcCer) and a decrease in the sphingomyelin (SM) synthesis. However, no changes were detected in the de novo sphingolipid synthesis in GLTP-knockdown cells compared to control cells. We propose that GLTP is not likely involved in the de novo synthesis of glycosphingolipids, but could rather have a role as a glycolipid sensor for the cellular levels of glucosylceramide.


Asunto(s)
Proteínas Portadoras/metabolismo , Esfingolípidos/biosíntesis , Especificidad de Anticuerpos , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Citosol/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Expresión Génica , Glucosilceramidas/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Microscopía Confocal , Interferencia de ARN , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Fracciones Subcelulares/metabolismo , Transfección
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