Characterization of the promotor of the gene for the lysosomal integral membrane protein type-2 (LIMP-2/SCARB2)

Dem Lysosomalen Integralen Membranprotein Typ-2 (LIMP-2) wird als Transporter für die beta-Glukocerebrosidase (beta-GC) zunehmend eine wichtige Rolle in der Pathogenese der Erkrankungen Morbus Gaucher und Parkinson-Syndrom zugesprochen. Dabei scheint LIMP-2 vor allem einen modulierenden Einfluss auf das Krankheitsgeschehen zu haben. Wie gezeigt werden konnte, führt die heterologe Expression von LIMP-2 in einem Zell-Modell für Morbus Gaucher zu einem gesteigerten Transport des fehlgefalteten Enzyms beta-GC in post-ER-Kompartimente. In der vorliegenden Arbeit wurde zunächst der Promotor von SCARB2 genauer charakterisiert. Hierbei ließ sich eine Sequenz von 34 Basenpaaren als ein wichtiges regulatorisches Element identifizieren. Mittels einer in silico Analyse konnte der Kreis der Transkriptionsfaktoren, die an dieser Sequenz binden, auf drei Transkriptionsfaktoren eingekreist werden. Ein wichtiges Ergebnis dieser Arbeit ist, dass es durch Inkubation mit hypoosmolarer Saccharose zu einem signifikanten Anstieg der SCARB2-Promotoraktivität kommt. Gibt man diesen Zucker in das Nährmedium der Zellkultur, führt dies zur Aufnahme der Saccharose in die Zelle und zur Ausbildung großer Vakuolen, sogenannter „Sucrosomen“. Nach Stimulation mit Saccharose konnte sowohl im Luziferase-Assay eine Aktivierung des SCARB2-Promotors gemessen, als auch im Western blot eine gesteigerte Expression von LIMP-2 und beta-GC festgestellt werden. Außerdem kam es in LIMP-2-defizienten Zellen zu einem gesteigerten Transport von beta-GC in post-ER-Kompartimente sowie zu einem Anstieg der beta-GC-Aktivität. In einem abschließenden Experiment konnte nachgewiesen werden, dass das Einwirken von Saccharose auch zu einer Reduktion der alpha-Synuclein-Menge in den Zellen führt. Solch eine Reduktion von alpha-Synuclein ist ein Ansatzpunkt in der Entwicklung neuer Therapiemöglichkeiten des Parkinson-Syndroms.The lysosomal integral membrane protein type-2 (LIMP-2) as the transporter of beta-glucocerebrosidase (beta-GC) is involved in the pathogenesis of the diseases Parkinson’s disease and Gaucher Disease. Thereby LIMP-2 seems to have a modulating influence on the development and severity of these diseases. Recently it has been shown that the heterologous expression of LIMP-2 leads to an increased transport of misfolded beta-GC in a model of Gaucher Disease. In the present work the promotor of SCARB2 has been characterized more specifically. In this issue a sequence of 34 base pairs has been identified which seems to have a major impact on the regulation of the promotor activity. An in silico analysis of this regulatory element revealed three transcription factors binding to this sequence. Further it could be shown that incubation with hypoosmolar sucrose leads to a significantly elevated SCARB2 promotor activity. Cultivating cells in sucrose enriched medium induces the uptake of the sugar into the cells and the vacuolation, building so called “sucrosomes”. After the stimulation with sucrose there was a mensurable increase of the activity of the SCARB2 promotor in the luciferase assay as well as an increased protein expression of LIMP-2 and beta-GC in Western blotting. Furthermore there was an elevation in the transport of beta-GC into post-ER compartments in LIMP-2 deficient cells and an increase of beta-GC activity. In a concluding experiment it could be shown that the incubation of cells with sucrose leads to a reduction of alpha-synuclein inside the cells. Such a reduction of alpha-synuclein is a promising approach for the development of new therapeutic strategies concerning Parkinson’s disease

Characterization of the complex formed by beta-glucocerebrosidase and the lysosomal integral membrane protein type-2

Zunke F, Andresen L, Wesseler S, et al. Characterization of the complex formed by beta-glucocerebrosidase and the lysosomal integral membrane protein type-2. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA. 2016;113(14):3791-3796.The lysosomal integral membrane protein type-2 (LIMP-2) plays a pivotal role in the delivery of beta-glucocerebrosidase (GC) to lysosomes. Mutations in GC result in Gaucher's disease (GD) and are the major genetic risk factor for the development of Parkinson's disease (PD). Variants in the LIMP-2 gene cause action myoclonus-renal failure syndrome and also have been linked to PD. Given the importance of GC and LIMP-2 in disease pathogenesis, we studied their interaction sites in more detail. Our previous data demonstrated that the crystal structure of LIMP-2 displays a hydrophobic three-helix bundle composed of helices 4, 5, and 7, of which helix 5 and 7 are important for ligand binding. Here, we identified a similar helical motif in GC through surface potential analysis. Coimmunoprecipitation and immunofluorescence studies revealed a triple-helical interface region within GC as critical for LIMP-2 binding and lysosomal transport. Based on these findings, we generated a LIMP-2 helix 5-derived peptide that precipitated and activated recombinant wild-type and GD-associated N370S mutant GC in vitro. The helix 5 peptide fused to a cell-penetrating peptide also activated endogenous lysosomal GC and reduced alpha-synuclein levels, suggesting that LIMP-2-derived peptides can be used to activate endogenous as well as recombinant wild-type or mutant GC efficiently. Our data also provide a structural model of the LIMP-2/GC complex that will facilitate the development of GC chaperones and activators as potential therapeutics for GD, PD, and related synucleinopathies