Browsing by Subject "LIPID RAFTS"

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  • Siljamaki, Elina; Rintanen, Nina; Kirsi, Maija; Upla, Paula; Wang, Wei; Karjalainen, Mikko; Ikonen, Elina; Marjomaki, Varpu (2013)
  • Chmielarz, Piotr; Er, Safak; Konovalova, Julia; Bandres, Laura; Hlushchuk, Irena; Albert, Katrina; Panhelainen, Anne; Luk, Kelvin; Airavaara, Mikko; Domanskyi, Andrii (2020)
    Background Parkinson's disease (PD) is associated with proteostasis disturbances and accumulation of misfolded alpha-synuclein (alpha-syn), a cytosolic protein present in high concentrations at pre-synaptic neuronal terminals. It is a primary constituent of intracellular protein aggregates known as Lewy neurites or Lewy bodies. Progression of Lewy pathology caused by the prion-like self-templating properties of misfolded alpha-syn is a characteristic feature in the brains of PD patients. Glial cell line-derived neurotrophic factor (GDNF) promotes survival of mature dopamine (DA) neurons in vitro and in vivo. However, the data on its effect on Lewy pathology is controversial. Objectives We studied the effects of GDNF on misfolded alpha-syn accumulation in DA neurons. Methods Lewy pathology progression was modeled by the application of alpha-syn preformed fibrils in cultured DA neurons and in the adult mice. Results We discovered that GDNF prevented accumulation of misfolded alpha-syn in DA neurons in culture and in vivo. These effects were abolished by deletion of receptor tyrosine kinase rearranged during transfection (RET) or by inhibitors of corresponding signaling pathway. Expression of constitutively active RET protected DA neurons from fibril-induced alpha-syn accumulation. Conclusions For the first time, we have shown the neurotrophic factor-mediated protection against the misfolded alpha-syn propagation in DA neurons, uncovered underlying receptors, and investigated the involved signaling pathways. These results demonstrate that activation of GDNF/RET signaling can be an effective therapeutic approach to prevent Lewy pathology spread at early stages of PD. (c) 2020 International Parkinson and Movement Disorder Society
  • Mobarak, Edouard; Håversen, Liliana; Manna, Moutusi; Rutberg, Mikael; Levin, Malin; Perkins, Rosie; Rog, Tomasz; Vattulainen, Ilpo; Boren, Jan (2018)
    Toll-like receptor 4 (TLR4) is activated by bacterial lipopolysaccharide (LPS), which drives the production of proinflammatory cytokines. Earlier studies have indicated that cholesterol-and glycosphingolipid-rich subregions of the plasma membrane (lipid domains) are important for TLR4-mediated signaling. We report that inhibition of glucosylceramide (GluCer) synthase, which resulted in decreased concentrations of the glycosphingolipid GluCer in lipid domains, reduced the LPS-induced inflammatory response in both mouse and human macrophages. Atomistic molecular dynamics simulations of the TLR4 dimer complex (with and without LPS in its MD-2 binding pockets) in membranes (in the presence and absence of GluCer) showed that: (1) LPS induced a tilted orientation of TLR4 and increased dimer integrity; (2) GluCer did not affect the integrity of the LPS/TLR4 dimer but reduced the LPS-induced tilt; and (3) GluCer increased electrostatic interactions between the membrane and the TLR4 extracellular domain, which could potentially modulate the tilt. We also showed that GCS inhibition reduced the interaction between TLR4 and the intracellular adaptor protein Mal. We conclude that the GluCer-induced effects on LPS/TLR4 orientation may influence the signaling capabilities of the LPS/TLR4 complex by affecting its interaction with downstream signaling proteins.
  • Merezhko, Maria; Pakarinen, Emmi; Uronen, Riikka-Liisa; Huttunen, Henri J. (2020)
    The plasma membrane consists of a variety of discrete domains differing from the surrounding membrane in composition and properties. Selective partitioning of protein to these microdomains is essential for membrane functioning and integrity. Studying the nanoscale size and dynamic nature of the membrane microdomains requires advanced imaging approaches with a high spatiotemporal resolution and, consequently, expensive and specialized equipment, unavailable for most researchers and unsuited for large-scale studies. Thus, understanding of protein partitioning to the membrane microdomains in health and disease is still hampered by the lack of inexpensive live-cell approaches with an appropriate spatial resolution. Here, we have developed a novel approach based on Gaussia princeps luciferase protein-fragment complementation assay to quantitively investigate protein partitioning to cholesterol and sphingomyelin-rich domains, sometimes called 'lipid rafts', in intact living cells with a high-spatial resolution. In the assay, the reporter construct, carrying one half of the luciferase protein, is targeted to lipid microdomains through the fused acetylation motif from Src-family kinase Fyn. A protein of interest carries the second half of the luciferase protein. Together, this serves as a reversible real-time sensor of raft recruitment for the studied protein. We demonstrated that the assay can efficiently detect the dynamic alterations in raft localization of two disease-associated proteins: Akt and APP. Importantly, this method can be used in high-throughput screenings and other large-scale studies in living cells. This inexpensive, and easy to implement raft localization assay will benefit all researchers interested in protein partitioning in rafts.
  • Tigistu-Sahle, Feven; Lampinen, Milla; Kilpinen, Lotta; Holopainen, Minna; Lehenkari, Petri; Laitinen, Saara; Käkelä, Reijo (2017)
    High arachidonic acid (20:4n-6) and low n-3 PUFA levels impair the capacity of cultured human bone marrow mesenchymal stromal cells (hBMSCs) to modulate immune functions. The capacity of the hBMSCs to modify PUFA structures was found to be limited. Therefore, different PUFA supplements given to the cells resulted in very different glycerophospholipid (GPL) species profiles and substrate availability for phospholipases, which have preferences for polar head group and acyl chains when liberating PUFA precursors for production of lipid mediators. When supplemented with 20:4n-6, the cells increased prostaglandin E2 secretion. However, they elongated 20:4n-6 to the less active precursor, 22:4n-6, and also incorporated it into triacylglycerols, which may have limited the proinflammatory signaling. The n-3 PUFA precursor, 18:3n-3, had little potency to reduce the GPL 20:4n-6 content, while the eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acid supplements efficiently displaced the 20:4n-6 acyls, and created diverse GPL species substrate pools allowing attenuation of inflammatory signaling.(Jlr) The results emphasize the importance of choosing appropriate PUFA supplements for in vitro hBMSC expansion and suggests that for optimal function they require an exogenous fatty acid source providing 20:5n-3 and 22:6n-3 sufficiently, but 20:4n-6 moderately, which calls for specifically designed optimal PUFA supplements for the cultures.
  • Koponen, Annika; Pan, Guoping; Kivelä, Annukka M.; Ralko, Arthur; Taskinen, Juuso H.; Arora, Amita; Kosonen, Riikka; Kari, Otto K.; Ndika, Joseph; Ikonen, Elina; Cho, Wonhwa; Yan, Daoguang; Olkkonen, Vesa M. (2020)
    Oxysterol-binding protein-related protein 2 (ORP2), a cholesterol-PI(4,5)P(2)countercurrent transporter, was recently identified as a novel regulator of plasma membrane (PM) cholesterol and PI(4,5)P(2)content in HeLa cells. Here, we investigate the role of ORP2 in endothelial cell (EC) cholesterol and PI(4,5)P(2)distribution, angiogenic signaling, and angiogenesis. We show that ORP2 knock-down modifies the distribution of cholesterol accessible to a D4H probe, between late endosomes and the PM. Depletion of ORP2 from ECs inhibits their angiogenic tube formation capacity, alters the gene expression of angiogenic signaling pathways such as VEGFR2, Akt, mTOR, eNOS, and Notch, and reduces EC migration, proliferation, and cell viability. We show that ORP2 regulates the integrity of VEGFR2 at the PM in a cholesterol-dependent manner, the depletion of ORP2 resulting in proteolytic cleavage by matrix metalloproteinases, and reduced activity of VEGFR2 and its downstream signaling. We demonstrate that ORP2 depletion increases the PM PI(4,5)P(2)coincident with altered F-actin morphology, and reduces both VEGFR2 and cholesterol in buoyant raft membranes. Moreover, ORP2 knock-down suppresses the expression of the lipid raft-associated proteins VE-cadherin and caveolin-1. Analysis of the retinal microvasculature in ORP2 knock-out mice generated during this study demonstrates the subtle alterations of morphology characterized by reduced vessel length and increased density of tip cells and perpendicular sprouts. Gene expression changes in the retina suggest disturbance of sterol homeostasis, downregulation of VE-cadherin, and a putative disturbance of Notch signaling. Our data identifies ORP2 as a novel regulator of EC cholesterol and PI(4,5)P(2)homeostasis and cholesterol-dependent angiogenic signaling.
  • Mahato, Arun Kumar; Sidorova, Yulia A. (2020)
    Rearranged during transfection (RET) is the tyrosine kinase receptor that under normal circumstances interacts with ligand at the cell surface and mediates various essential roles in a variety of cellular processes such as proliferation, differentiation, survival, migration, and metabolism. RET plays a pivotal role in the development of both peripheral and central nervous systems. RET is expressed from early stages of embryogenesis and remains expressed throughout all life stages. Mutations either activating or inhibiting RET result in several aggressive diseases, namely cancer and Hirschsprung disease. However, the physiological ligand-dependent activation of RET receptor is important for the survival and maintenance of several neuronal populations, appetite, and weight gain control, thus providing an opportunity for the development of disease-modifying therapeutics against neurodegeneration and obesity. In this review, we describe the structure of RET, its signaling, and its role in both normal conditions as well as in several disorders. We highlight the differences in the signaling and outcomes of constitutive and ligand-induced RET activation. Finally, we review the data on recently developed small molecular weight RET agonists and their potential for the treatment of various diseases.