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Biochemical and Molecular Actions of Nutrients

Subcellular Localization of Soybean 7S Globulin in HepG2 Cells and LDL Receptor Up-Regulation by Its ' Constituent Subunit

Cristina Manzoni^*, Marcello Duranti, Ivano Eberini^*, Hubert Scharnag^**, Winfried März^**, Silvia Castiglioni^* and Maria R. Lovati^*,2

^* Department of Pharmacological Sciences, and Department of Agrofood Molecular Sciences, University of Milan, 20133 Milan, Italy and ^** Department of Medicine, Division of Clinical Chemistry, Albert Ludwigs-University, 79106 Freiburg, Germany

²To whom correspondence should be addressed. E-mail: mariarosa.lovati{at}unimi.it.

The aims of this work were to monitor the subcellular localization of soybean 7S globulin in HepG2 cells and determine its interaction with cell protein components, by using laser-induced fluorescence capillary electrophoresis (LIF-CE). Furthermore, we evaluated in the same cell line the involvement of the ' constituent subunit from 7S globulin in the modulation of LDL catabolism. The results indicated a main fluorescein isothiocyanate-tagged 7S globulin (FITC-7S) component in the cytosolic fraction, that was not present in the nuclear compartment. The electrophoretic mobility of this tagged component suggested either a dissociation of the 7S oligomer or its partial intracellular degradation. Interactions of soybean 7S globulin with FITC-thioredoxin 1 and FITC-cyclophilin B, HepG2 cell membrane proteins, were demonstrated in in vitro assays. In a separate experiment with HepG2 cells, the ability of the ' subunit purified from soybean 7S globulin to modulate the activity of the LDL receptors was evaluated by tracking the uptake and degradation of labeled LDL. The up-regulation of LDL receptors by the ' subunit, as further confirmed by a LDL receptor promoter assay, was significantly greater than that found in the control cells. In conclusion, this study, while confirming our previous indirect evidence of the key role of ' subunit on the cell cholesterol homeostasis, reveals a potentially interesting association of soybean 7S globulin with proteins, such as thioredoxin 1 and cyclophilin B, that are involved in cell protection against oxidative stress.

KEY WORDS: • soybean 7S globulin • capillary electrophoresis • subcellular localization • LDL receptors • cholesterol homeostasis

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