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Membrane folate-binding proteins are responsible for folate-protein conjugate endocytosis into cultured cells.

ABSTRACT: Folate-protein conjugates have been shown to bind to and enter HeLa and KB cells by receptor-mediated endocytosis [Leamon and Low (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 5572-5576]. Although these cells contain a membrane folate-binding protein (FBP) involved in the uptake of free folate, no studies have been conducted to evaluate whether the folate-protein conjugates enter cells via the same protein. To address this issue, HeLa cell monolayers were treated with folate-labelled 125I-RNAase under various conditions characteristic of FBP-mediated folate uptake. Folate-labelled 125I-RNAase was found to bind to cells with high affinity (Kd = 24 nM), and like the free vitamin, its binding could be competitively blocked by excess free folate. Furthermore, binding could be reversed by either washing the cells with acid/saline, pH 3.0, or by treating the cells with phosphatidyl-inositol-specific phospholipase C, an enzyme known to release FBP from cell surfaces. Because cells pretreated with anti-FBP serum were unable to bind folate conjugates, and since the same antiserum identified a single 65 kDa band reminiscent of FBPs found in many other tissues, we conclude that a classical FBP is responsible for the uptake of folate-protein conjugates by receptor-bearing cells.

SUBMITTER: Leamon CP

PROVIDER: S-EPMC1132447 | biostudies-other | 1993 May

REPOSITORIES: biostudies-other

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Project description:BackgroundMany integral membrane proteins, like their non-membrane counterparts, form either transient or permanent multi-subunit complexes in order to carry out their biochemical function. Computational methods that provide structural details of these interactions are needed since, despite their importance, relatively few structures of membrane protein complexes are available.ResultsWe present a method for predicting which residues are in protein-protein binding sites within the transmembrane regions of membrane proteins. The method uses a Random Forest classifier trained on residue type distributions and evolutionary conservation for individual surface residues, followed by spatial averaging of the residue scores. The prediction accuracy achieved for membrane proteins is comparable to that for non-membrane proteins. Also, like previous results for non-membrane proteins, the accuracy is significantly higher for residues distant from the binding site boundary. Furthermore, a predictor trained on non-membrane proteins was found to yield poor accuracy on membrane proteins, as expected from the different distribution of surface residue types between the two classes of proteins. Thus, although the same procedure can be used to predict binding sites in membrane and non-membrane proteins, separate predictors trained on each class of proteins are required. Finally, the contribution of each residue property to the overall prediction accuracy is analyzed and prediction examples are discussed.ConclusionGiven a membrane protein structure and a multiple alignment of related sequences, the presented method gives a prioritized list of which surface residues participate in intramembrane protein-protein interactions. The method has potential applications in guiding the experimental verification of membrane protein interactions, structure-based drug discovery, and also in constraining the search space for computational methods, such as protein docking or threading, that predict membrane protein complex structures.

Project description:The regular accumulation of nanoparticles in the liver makes them hepatotoxic and decreases the circulation time, thus reducing their therapeutic effect. Resolving this problem will be significant in improving bioavailability and reducing side effects. In this study, we reduced the phagocytosis of epirubicin (EPI)-loaded folic acid-conjugated pullulan acetate (FPA/EPI) nanoparticles by Kupffer cells (KCs) through internalization and nuclear factor kappa B (NF-kB) signal pathway inhibitors, thus allowing development of FPA/EPI nanoparticles as a nanodrug delivery system (NDDS) based on our previous study. FPA/EPI nanoparticles were prepared by the dialysis method. Rat KCs were preincubated with the following individual or compound inhibitors: chlorpromazine (CPZ), nystatin (NY), colchicine (Col), amiloride (AMR), and pyrrolidine dithiocarbamate (PDTC). Dose- and time-dependent cellular uptake effects of inhibitors on FPA/EPI nanoparticles were determined through fluorometry. The cytokine levels of tumor necrosis factor alpha (TNF-?), interleukin-1 beta (IL-1?), and IL-6 were tested in culture supernatants by bead-based multiplex flow cytometry. The uptake study demonstrated that inhibitors had an obvious inhibitory effect (P<0.05 or P<0.01), with NY, AMR and Col all showing time-dependent inhibitory effects. PDTC + NY had the strongest inhibitory effect, with an uptake rate of 14.62%. The levels of the three proinflammatory cytokines were changed significantly by the compound inhibitors. TNF-? was significantly inhibited (P<0.05 or P<0.01), but IL-1? and IL-6 showed smaller decreases. These results suggested that clathrin- and caveolae-mediated endocytosis were the main routes via which nanoparticles entered KCs and that the NF-kB signal pathway was very important too. In summary, multiple mechanisms, including clathrin- and caveolae-mediated endocytosis, contribute to cytokine production in macrophages following exposure to folic acid-conjugated pullulan acetate nanoparticles. Thus, the endocytosis inhibition strategy has great potential for improving therapy and reducing toxicity of an NDDS in the treatment of cancer.

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Membrane folate-binding proteins are responsible for folate-protein conjugate endocytosis into cultured cells.

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