Production of Recombinant Human Apoferritin Heteromers

Grace, James E.; Eden, Marc E. Van; Aust, Steven D.

doi:10.1006/abbi.2000.2068

Cited by 13 publications

(10 citation statements)

References 25 publications

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“…The band in L-transfected cells had lower mobility than that of the control. This concurs with what has been known about the migration pattern of L-rich ferritin, which is slower than that of H-rich ferritin due to difference in the charge of the chains (19). The difference in the ferritin mobility between control and L-transfected cells indicates that subunit composition of newly synthesized ferritin from L-transfectants differs from that of LEC-specific ferritin.…”

Section: Resultssupporting

confidence: 89%

Identification of a Mechanism by Which Lens Epithelial Cells Limit Accumulation of Overexpressed Ferritin H-chain

Goralska

Holley

McGahan

2003

Journal of Biological Chemistry

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The primary cultures of canine lens epithelial cells were transiently transfected with cDNAs for dog ferritin H-or L-chains in order to study differential expression of these chains. By using chain-specific antibodies, we determined that at 48 h after transfection overexpression of L-chain was much higher (9-fold over control) than that of H-chain (1.7-fold). We discovered that differentially transfected cells secrete overexpressed chains as homopolymeric ferritin into the media. Fortyeight hours after transfection accumulation of H-ferritin in the media was much higher (3-fold) than that of L-ferritin. This resulted in lowering of the concentration of H-chain in the cytosol. Co-transfection of cells with both H-and L-chain cDNAs increased the intracellular levels of H-chain and eliminated secretion of Hferritin to the media. We concluded that lens epithelial cells differentially regulate concentration of both ferritin chains in the cytosol. The overexpressed L-chain accumulated in the cytosol as predominantly homopolymeric L-ferritin. This is in contrast to H-chain, which is removed to the media unless there is an L-chain available to form heteropolymeric ferritin. These data indicate that the inability of cells to more strictly control cytosolic levels of L-chain may augment its accumulation in lenses of humans with hereditary hyperferritinemia cataract syndrome, which is caused by overexpression of L-chain due to mutation in the regulatory element in the untranslated region of the mRNA of the chain.Ferritins are the highly conserved iron storage proteins found in species from bacteria to mammals. Mammalian ferritins, located predominantly in cytoplasm, are heteropolymers of 24 subunits of two types, heavy (H 1 ; 21 kDa) and light (L; 19 kDa). Despite high (ϳ50%) sequence identity and similar three-dimensional structures, the subunits are genetically different and each has a distinct and complementary role in storing iron. The H subunit has a ferroxidase center responsible for uptake and oxidation of Fe 2ϩ into ferric ions. Translocation of these ions into the core of the ferritin shell and their mineralization for long term storage is facilitated by the L subunit. The intracellular level of ferritin correlates with the size of the pool of intracellular iron, which plays the main role in the regulation of ferritin expression. The iron-controlled expression of both chains is primarily translational and involves binding of iron-regulatory proteins IRP1 and IRP2 (iron sensors) to iron-response element (IRE), a stem-loop structure present in the 5Ј-untranslated region of the mRNA (1). The expression of H-chain gene is also regulated transcriptionally by cytokines (2), hormones, oncogenes (3), and inducers of cell differentiation (4).The H and L subunit ratio of mammalian ferritins varies and is highly tissue-specific; however, mechanisms by which cells maintain the chain-specific ratio are not fully understood. Although both chains complement each other in the process of sequestering and storing iron, H-chain has ...

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Section: Resultssupporting

confidence: 89%

Identification of a Mechanism by Which Lens Epithelial Cells Limit Accumulation of Overexpressed Ferritin H-chain

Goralska

Holley

McGahan

2003

Journal of Biological Chemistry

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“…Since only H chain showed affinity for cancer cells through the binding with transferrin receptor, by means of an elegant combination of chemical engineering and fermentation technology, different types of mammalian ferritin variants were formulated by selfassembling by only H subunits (H-APO) [49]. Thus, H-APO was loaded with DOXO following both the previously reported acidic [50,51] or basic dissociation protocols [52].…”

Section: Apo In Drug Delivery the Tumor Casementioning

confidence: 99%

Protein cage nanostructure as drug delivery system: magnifying glass on apoferritin

Belletti

Pederzoli

Forni

et al. 2016

Expert Opinion on Drug Delivery

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New frontiers in nanomedicine are moving towards the research of new biomaterials. Apoferritin (APO), is a uniform regular self-assemblies nano-sized protein with excellent biocompatibility and a unique structure that affords it the ability to stabilize small active molecules in its inner core. Areas covered: APO can be loaded by applying a passive process (mainly used for ions and metals) or by a unique formulative approach based on disassemby/reassembly process. In this article, we aim to organize the experimental evidence provided by a number of studies on the loading, release and targeting. Attention is initially focused on the most investigated antineoplastic drug and contrast agents up to the most recent application in gene therapy. Expert opinion: Various preclinical studies have demonstrated that APO improved the potency and selectivity of some chemotherapeutics. However, in order to translate the use of APO into therapy, some issues must be solved, especially regarding the reproducibility of the loading protocol used, the optimization of nanocarrier characterization, detailed understanding of the final structure of loaded APO, and the real mechanism and timing of drug release.

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“…The purification was followed as described by Grace et al in reference [409]. Cell pellets were resuspended in 50 mM Tris pH 7.0 with DNase I (20 µg mL −1 ), RNase I (µg mL −1 ) and lysozyme (1 mg mL −1 ).…”

Section: Purification Of Large-scale Expression Culture Of H Human Apmentioning

confidence: 99%

Aqueous Near‐Infrared Fluorescent Composites Based on Apoferritin‐Encapsulated PbS Quantum Dots

et al. 2008

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Production of Recombinant Human Apoferritin Heteromers

Cited by 13 publications

References 25 publications

Identification of a Mechanism by Which Lens Epithelial Cells Limit Accumulation of Overexpressed Ferritin H-chain

Identification of a Mechanism by Which Lens Epithelial Cells Limit Accumulation of Overexpressed Ferritin H-chain

Protein cage nanostructure as drug delivery system: magnifying glass on apoferritin

Aqueous Near‐Infrared Fluorescent Composites Based on Apoferritin‐Encapsulated PbS Quantum Dots

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