Stem Cells and Experimental Leukemia Can Be Distinguished by Lipid Raft Protein Composition

Mesenchymal stem cells (MSCs) are promising tools for cancer therapy, but there is a risk of malignant transformation in their clinical application. Our previous work revealed that the paracrine protein S100B in the glioma microenvironment induces malignant transformation of MSCs and upregulates intracellular S100B, which could affect cell homeostasis by interfering with p53. The purpose of this study was to investigate whether extracellular S100B can be internalized by MSCs and the specific endocytic pathway involved in S100B internalization. By using real-time confocal microscopy and structured illumination microscopy (SIM), we visualized the uptake of fluorescently labeled S100B protein (S100B-Alexa488) and monitored the intracellular trafficking of internalized vesicles. The results showed that S100B-Alexa488 was efficiently internalized into MSCs in a time-dependent manner and transported through endolysosomal pathways. After that, we used chemical inhibitors and RNA interference approaches to investigate possible mechanisms involved in S100B-Alexa488 uptake. The internalization of S100B-Alexa488 was inhibited by pitstop-2 or dyngo-4a treatment or RNA-mediated silencing of clathrin or dynamin, and the lipid raft-mediated endocytosis inhibitors nystatin and MβCD. In conclusion, our findings show that clathrin and lipid rafts contribute to the internalization of S100B-Alexa488, which provides promising interventions for the safe application of MSCs in glioma therapy.

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RaftProt V2: understanding membrane microdomain function through lipid raft proteomes

10.1101/286039 ◽

2018 ◽

Author(s):

Ahmed Mohamed ◽

Anup Shah ◽

David Chen ◽

Michelle M. Hill

Keyword(s):

Experimental Evidence ◽

Lipid Rafts ◽

Lipid Raft ◽

Protein Composition ◽

Biological Data ◽

Tissue Type ◽

Membrane Microdomains ◽

Membrane Rafts ◽

Link Type ◽

Membrane Microdomain

ABSTRACTCellular membranes feature dynamic submicrometer-scale lateral membrane domainsvariously referred to as lipid rafts, membrane rafts or glycosphingolipid-enriched microdomains (GEM). In order to understand the molecular functions of lipid rafts, numerous studies have utilized various biochemical methods to isolate and examine the protein composition of membrane rafts. However, interpretation of individual raft proteomics studies are confounded by the limitations of isolation methods and the dynamic nature of rafts. Knowledge-based approaches can facilitate biological data interpretation by integrating experimental evidence from existing studies. To this end, we previously developed RaftProt (http://lipid-raft-database.di.uq.edu.au/), a searchable database of mammalian lipid raft-associated proteins. Despite being a valuable and highly used resource, improvements in search capabilities and visualisation were still needed. Here, we present RaftProt V2 (http://raftprot.org), an improved update of RaftProt, enabling interrogation and integration of datasets at the cell/tissue type and UniRef/Gene level. Besides the addition of new datasets and re-mapping of all entries to both UniProt and UniRef IDs, we have annotated the level of experimental evidence for each protein entry. The search engine now allows for multiple protein or experiment searches where correlations, interactions or overlaps can be investigated. The web-interface has been completely re-designed and offers new interactive tools for data and subset selection, correlation analysis and network visualization. Overall, RaftProt aims to advance our understanding of lipid raft function by revealing the proteomes and pathways that are associated with membrane microdomains in diverse tissue and conditions.Database URL: http://raftprot.org

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Prion Protein in Stem Cells: A Lipid Raft Component Involved in the Cellular Differentiation Process

International Journal of Molecular Sciences ◽

10.3390/ijms21114168 ◽

2020 ◽

Vol 21 (11) ◽

pp. 4168 ◽

Cited By ~ 1

Author(s):

Stefano Martellucci ◽

Costantino Santacroce ◽

Francesca Santilli ◽

Valeria Manganelli ◽

Maurizio Sorice ◽

...

Keyword(s):

Stem Cells ◽

Prion Protein ◽

Lipid Raft ◽

Cellular Differentiation ◽

Cell Types ◽

Pleiotropic Effect ◽

Cellular Prion Protein ◽

Differentiation Process ◽

Differentiation Degree

The prion protein (PrP) is an enigmatic molecule with a pleiotropic effect on different cell types; it is localized stably in lipid raft microdomains and it is able to recruit downstream signal transduction pathways by its interaction with various biochemical partners. Since its discovery, this lipid raft component has been involved in several functions, although most of the publications focused on the pathological role of the protein. Recent studies report a key role of cellular prion protein (PrPC) in physiological processes, including cellular differentiation. Indeed, the PrPC, whose expression is modulated according to the cell differentiation degree, appears to be part of the multimolecular signaling pathways of the neuronal differentiation process. In this review, we aim to summarize the main findings that report the link between PrPC and stem cells.

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Isolation of Lipid Raft Proteins from CD133+ Cancer Stem Cells

Methods in Molecular Biology - Lipidomics ◽

10.1007/978-1-4939-6996-8_3 ◽

2017 ◽

pp. 25-31 ◽

Cited By ~ 2

Author(s):

Vineet K. Gupta ◽

Sulagna Banerjee

Keyword(s):

Stem Cells ◽

Cancer Stem Cells ◽

Lipid Raft

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Proteomic Analysis of Lipid Rafts from RBL-2H3 Mast Cells

International Journal of Molecular Sciences ◽

10.3390/ijms20163904 ◽

2019 ◽

Vol 20 (16) ◽

pp. 3904 ◽

Cited By ~ 2

Author(s):

Edismauro Garcia Freitas Filho ◽

Luiz Augusto Marin Jaca ◽

Lilian Cristiane Baeza ◽

Célia Maria de Almeida Soares ◽

Clayton Luiz Borges ◽

...

Keyword(s):

Mast Cells ◽

Lipid Rafts ◽

Lipid Raft ◽

Protein Composition ◽

Enrichment Analysis ◽

Functional Enrichment Analysis ◽

Cell Types ◽

Cellular Membrane ◽

Functional Enrichment ◽

Membrane Microdomains

Lipid rafts are highly ordered membrane microdomains enriched in cholesterol, glycosphingolipids, and certain proteins. They are involved in the regulation of cellular processes in diverse cell types, including mast cells (MCs). The MC lipid raft protein composition was assessed using qualitative mass spectrometric characterization of the proteome from detergent-resistant membrane fractions from RBL-2H3 MCs. Using two different post-isolation treatment methods, a total of 949 lipid raft associated proteins were identified. The majority of these MC lipid raft proteins had already been described in the RaftProtV2 database and are among highest cited/experimentally validated lipid raft proteins. Additionally, more than half of the identified proteins had lipid modifications and/or transmembrane domains. Classification of identified proteins into functional categories showed that the proteins were associated with cellular membrane compartments, and with some biological and molecular functions, such as regulation, localization, binding, catalytic activity, and response to stimulus. Furthermore, functional enrichment analysis demonstrated an intimate involvement of identified proteins with various aspects of MC biological processes, especially those related to regulated secretion, organization/stabilization of macromolecules complexes, and signal transduction. This study represents the first comprehensive proteomic profile of MC lipid rafts and provides additional information to elucidate immunoregulatory functions coordinated by raft proteins in MCs.

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Fibronectin stimulates migration through lipid raft dependent NHE-1 activation in mouse embryonic stem cells: Involvement of RhoA, Ca2+/CaM, and ERK

Biochimica et Biophysica Acta (BBA) - General Subjects ◽

10.1016/j.bbagen.2012.05.013 ◽

2012 ◽

Vol 1820 (10) ◽

pp. 1618-1627 ◽

Cited By ~ 12

Author(s):

Jae Hong Park ◽

Jung Min Ryu ◽

Seung Pil Yun ◽

Mi Ok Kim ◽

Ho Jae Han

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Lipid Raft ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cells

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Mechanism of PKA-Dependent and Lipid-Raft Independent Stimulation of Connexin43 Expression by Oxytoxin in Mouse Embryonic Stem Cells

Molecular Endocrinology ◽

10.1210/me.2011-1343 ◽

2012 ◽

Vol 26 (7) ◽

pp. 1144-1157 ◽

Cited By ~ 11

Author(s):

Seung Pil Yun ◽

Su Shin Park ◽

Jung Min Ryu ◽

Jae Hong Park ◽

Mi Ok Kim ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Lipid Raft ◽

Inner Cell Mass ◽

Cell Mass ◽

Embryonic Stem ◽

Early Embryo ◽

Creb Binding Protein ◽

Gap Junctional Intercellular Communication ◽

Cx43 Expression

Abstract Previous studies shows that connexins appear very early during murine embryo development, the gap junctional intercellular communication found in the inner cell mass of early embryo is also maintained in embryonic stem cells (ESC), and expression of oxytocin receptor (OTR) is developmentally regulated at early embryonic development. However, effect of oxytocin (OT) on the regulation of the connexin43 (Cx43) and maintenance of undifferentiation is not fully understood in stem cells. Therefore, we investigated the effect of OT on Cx43 expression and related signaling cascades in mouse ESC. OT increased Cx43 expression that was inhibited by the OTR inhibitor atosiban. In experiments to examine whether the effect of OT depends on lipid rafts, caveolin-1 (cav-1), cav-2, and flotillin-2, but not OTR, were detected in lipid raft fractions. Also, colocalization of OTR, cav-1, and cav-2 was not detected. Moreover, the lipid raft disruptor methyl-β-cyclodextrin did not attenuate OT-induced Cx43 expression. In experiments to examine related signaling pathways, OT activated cAMP/protein kinase A (PKA) which was inhibited by adenylyl cyclase inhibitor SQ 22536 and PKA inhibitor PKI. OT increased nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) phosphorylation which was inhibited by PKI. OT also increased cAMP response element-binding (CREB)/CREB-binding protein (CBP) expression in the nucleus and induced the formation of CREB1/NF-κB/CBP complexes, which was blocked by the NF-κB-specific small interfering RNA, NF-κB inhibitors, SN50, and bay11–7082. Complex disruption by NF-κB inhibitors decreased OT-induced Cx43 expression. In conclusion, OT stimulates Cx43 expression through the NF-κB/CREB/CBP complex via the lipid raft-independent OTR-mediated cAMP/PKA in mouse ESC.

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Colony Forming Potential and Protein Composition of Commercial Umbilical Cord Allograft Products in Comparison With Autologous Orthobiologics

The American Journal of Sports Medicine ◽

10.1177/03635465211031275 ◽

2021 ◽

pp. 036354652110312

Author(s):

Dustin R. Berger ◽

Christopher J. Centeno ◽

John D. Kisiday ◽

C. Wayne McIlwraith ◽

Neven J. Steinmetz

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Growth Factors ◽

Growth Factor ◽

Umbilical Cord ◽

Protein Composition ◽

Living Cells ◽

Cell Phenotype ◽

Viable Cells ◽

Bone Marrow Plasma

Background: Umbilical cord (UC) connective tissues contain plastic-adherent, colony forming unit–fibroblasts (CFU-Fs) amenable to culture expansion for potential therapeutic use. Recently, UC-derived allograft products have been made available to practitioners in orthopaedics and other specialties, by companies purporting “stem cell”–based healing. However, such marketing claims conflict with existing regulations for these human tissues, generating questions over the cellular and protein composition of current commercially available UC allograft products. Purpose: To evaluate commercial UC allograft products for viable cells, CFU-Fs, and protein makeup. Study Design: Descriptive laboratory study. Methods: Five commercial UC allograft products claiming to contain viable, undescribed “stem cells,” 2 obtained from UC blood (UCB) and 3 from UC tissue (UCT), were analyzed. Image-based methods were used to measure cell concentration and viability, a traditional CFU-F assay was used to evaluate in vitro behavior indicative of a connective tissue progenitor cell phenotype often referred to as mesenchymal stem/stromal cells, and quantitative immunoassay arrays were used to measure a combination of cytokines and growth factors. Bone marrow concentrate (BMC) and plasma derived from the blood and bone marrow of middle-aged individuals served as comparative controls for cell culture and protein analyses, respectively. Results: Viable cells were identified within all 5 UC allograft products, with those derived from UCB having greater percentages of living cells (40%-59%) than those from UCT (1%-22%). Compared with autologous BMC (>95% viability and >300 million living cells), no CFU-Fs were observed within any UC allograft product (<15 million living cells). Moreover, a substantial number of proteins, particularly those within UCB allograft products, were undetectable or present at lower concentrations compared with blood and bone marrow plasma controls. Interestingly, several important growth factors and cytokines, including basic fibroblast growth factor, hepatocyte growth factor, interleukin-1 receptor antagonist, and osteoprotegerin, were most prevalent in 1 or more UCT allograft products as compared with blood and bone marrow plasma. Conclusion: CFU-Fs, often referred to as stem cells, were not found within any of the commercial UC allograft products analyzed, and clinicians should remain wary of marketing claims stating otherwise. Clinical Relevance: Any therapeutic benefit of current UC allograft products in orthopaedic medicine is more likely to be attributed to their protein composition (UCT > UCB) or inclusion of cells without colony forming potential (UCB > UCT).

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