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Hypercholesterolemia, characterized by elevated low‐density lipoprotein (LDL) cholesterol levels, is a significant risk factor for cardiovascular disease. Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a crucial role in cholesterol metabolism by regulating LDL receptor degradation, making it a therapeutic target for mitigating hypercholesterolemia‐associated risks. In this context, we aimed to engineer human H ferritin as a scaffold to present 24 copies of a PCSK9‐targeting domain. The rationale behind this protein nanoparticle design was to disrupt the PCSK9–LDL receptor interaction, thereby attenuating the PCSK9‐mediated impairment of LDL cholesterol clearance. The N‐terminal sequence of human H ferritin was engineered to incorporate a 13‐amino acid linear peptide (Pep2‐8), which was previously identified as the smallest PCSK9 inhibitor. Exploiting the quaternary structure of ferritin, engineered nanoparticles were designed to display 24 copies of the targeting peptide on their surface, enabling a multivalent binding effect. Extensive biochemical characterization confirmed precise control over nanoparticle size and morphology, alongside robust PCSK9‐binding affinity (KD in the high picomolar range). Subsequent efficacy assessments employing the HepG2 liver cell line demonstrated the ability of engineered ferritin's ability to disrupt PCSK9–LDL receptor interaction, thereby promoting LDL receptor recycling on cell surfaces and consequently enhancing LDL uptake. Our findings highlight the potential of ferritin‐based platforms as versatile tools for targeting PCSK9 in the management of hypercholesterolemia. This study not only contributes to the advancement of ferritin‐based therapeutics but also offers valuable insights into novel strategies for treating cardiovascular diseases. [ABSTRACT FROM AUTHOR]

Ferritin, a naturally occurring iron storage protein, has gained significant attention as a drug delivery platform due to its inherent biocompatibility and capacity to encapsulate therapeutic agents. In this study, we successfully genetically engineered human H ferritin by incorporating 4 or 6 tryptophan residues per subunit, strategically oriented towards the inner cavity of the nanoparticle. This modification aimed to enhance the encapsulation of hydrophobic drugs into the ferritin cage. Comprehensive characterization of the mutants revealed that only the variant carrying four tryptophan substitutions per subunit retained the ability to disassemble and reassemble properly. As a proof of concept, we evaluated the loading capacity of this mutant with ellipticine, a natural hydrophobic indole alkaloid with multimodal anticancer activity. Our data demonstrated that this specific mutant exhibited significantly higher efficiency in loading ellipticine compared to human H ferritin. Furthermore, to evaluate the versatility of this hydrophobicity‐enhanced ferritin nanoparticle as a drug carrier, we conducted a comparative study by also encapsulating doxorubicin, a commonly used anticancer drug. Subsequently, we tested both ellipticine and doxorubicin‐loaded nanoparticles on a promyelocytic leukemia cell line, demonstrating efficient uptake by these cells and resulting in the expected cytotoxic effect. [ABSTRACT FROM AUTHOR]

Ferritin nanoparticles play many important roles in theranostic and bioengineering applications and have been successfully used as nanovectors for the targeted delivery of drugs due to their ability to specifically bind the transferrin receptor (TfR1, or CD71). They can be either genetically or chemically modified for encapsulating therapeutics or probes in their inner cavity. Here, we analyzed a new engineered ferritin nanoparticle, made of the H chain mouse ferritin (HFt) fused with a specific lanthanide binding tag (LBT). The HFt‐LBT has one high affinity lanthanide binding site per each of the 24 subunits and a tryptophane residue within the tag that acts as an antenna able to transfer the energy to the lanthanide ions via a LRET process. In this study, among lanthanides, we selected europium for its red emission that allows to reduce overlap with tissue auto‐fluorescence. Steady state emission measurements and time‐resolved emission spectroscopy have been employed to investigate the interaction between the HFt‐LBT and the Eu3+ ions. This allowed us to identify the Eu3+ energy states involved in the process and to pave the way for the future use of HFt‐LBT Eu3+ complex in theranostics. [ABSTRACT FROM AUTHOR]

The interaction of the recombinant hemoglobin from Vitreoscilla sp. (VHb) with the bacterial membrane of Escherichia coli cells was investigated by measuring the propensity of VHb to interact with monolayers formed by natural bacterial phosholipids. The data suggest a possible lipid-induced regulation of oxygen affinity as the basis of VHb functioning.

An acidic surface variant (ASV) of the 'truncated' hemoglobin from Thermobifida fusca was designed in order to create a versatile globin scaffold endowed with thermostability and a high level of recombinant expression in its soluble form while keeping the active site unmodified. Molecular dynamics simulations showed that the mutated residues remain solvent-exposed and does not affect the overall protein structure and hence ASV could be used in combinatorial mutagenesis of the distal heme pocket residues.

A complete thermodynamic and kinetic study was performed to assess the relevant sulfide binding parameters in the recombinant proteins extracted from Escherichia coli cells after overexpression. The high affinity for hydrogen sulfide might have a possible physiological significance as [H.sub.2]S is produced in bacteria at metabolic steps involved in cysteine biosynthesis and hence in thiol redox homeostasis.

Research shows that Escherichia coli flavohemoglobin binds to liposomes prepared from lipid extracts of E. coli membranes. Results reveal that protein bound lipids not only affect the binding properties of imidazole to the ferric protein but also modify the reactivity of the ferrous protein with oxygen and carbon monoxide. Data indicate that these effects are due to the involvement of heme iron and a non-heme site in the lipid/ligand recognition.