Erythropoietin signaling regulates heme biosynthesis

Heme is required for survival of all cells, and in most eukaryotes, is produced through a series of eight enzymatic reactions. Although heme production is critical for many cellular processes, how it is coupled to cellular differentiation is unknown. Here, using zebrafish, murine, and human models, we show that erythropoietin (EPO) signaling, together with the GATA1 transcriptional target, AKAP10, regulates heme biosynthesis during erythropoiesis at the outer mitochondrial membrane. This integrated pathway culminates with the direct phosphorylation of the crucial heme biosynthetic enzyme, ferrochelatase (FECH) by protein kinase A (PKA). Biochemical, pharmacological, and genetic inhibition of this signaling pathway result in a block in hemoglobin production and concomitant intracellular accumulation of protoporphyrin intermediates. Broadly, our results implicate aberrant PKA signaling in the pathogenesis of hematologic diseases. We propose a unifying model in which the erythroid transcriptional program works in concert with post-translational mechanisms to regulate heme metabolism during normal development

Safe Nanoparticles: Are We There Yet?

The field of nanotechnology has grown over the last two decades and made the transition from the benchtop to applied technologies. Nanoscale-sized particles, or nanoparticles, have emerged as promising tools with broad applications in drug delivery, diagnostics, cosmetics and several other biological and non-biological areas. These advances lead to questions about nanoparticle safety. Despite considerable efforts to understand the toxicity and safety of these nanoparticles, many of these questions are not yet fully answered. Nevertheless, these efforts have identified several approaches to minimize and prevent nanoparticle toxicity to promote safer nanotechnology. This review summarizes our current knowledge on nanoparticles, their toxic effects, their interactions with mammalian cells and finally current approaches to minimizing their toxicity

Liposomes Targeting P21 Activated Kinase-1 (PAK-1) and Selective for Secretory Phospholipase A2 (sPLA2) Decrease Cell Viability and Induce Apoptosis in Metastatic Triple-Negative Breast Cancer Cells

P21 activated kinases (or group I PAKs) are serine/threonine kinases whose expression is altered in prostate and breast cancers. PAK-1 activity is inhibited by the small molecule “Inhibitor targeting PAK-1 activation-3” (IPA-3), which has selectivity for PAK-1 but is metabolically unstable. Secretory Group IIA phospholipase A2 (sPLA2) expression correlates to increased metastasis and decreased survival in many cancers. We previously designed novel liposomal formulations targeting both PAK-1 and sPLA2, called Secretory Phospholipase Responsive liposomes or SPRL-IPA-3, and demonstrated their ability to alter prostate cancer growth. The efficacy of SPRL against other types of cancers is not well understood. We addressed this limitation by determining the ability of SPRL to induce cell death in a diverse panel of cells representing different stages of breast cancer, including the invasive but non-metastatic MCF-7 cells, and metastatic triple-negative breast cancer (TNBC) cells such as MDA-MB-231, MDA-MB-468, and MDA-MB-435. We investigated the role of sPLA2 in the disposition of these liposomes by comparing the efficacy of SPRL-IPA-3 to IPA-3 encapsulated in sterically stabilized liposomes (SSL-IPA-3), a formulation shown to be less sensitive to sPLA2. Both SSL-IPA-3 and SPRL-IPA-3 induced time- and dose-dependent decreases in MTT staining in all cell lines tested, but SPRL-IPA-3-induced effects in metastatic TNBC cell lines were superior over SSL-IPA-3. The reduction in MTT staining induced by SPRL-IPA-3 correlated to the expression of Group IIA sPLA2. sPLA2 expression also correlated to increased induction of apoptosis in TNBC cell lines by SPRL-IPA-3. These data suggest that SPRL-IPA-3 is selective for metastatic TNBC cells and that the efficacy of SPRL-IPA-3 is mediated, in part, by the expression of Group IIA sPLA2

Lyophilization of Nanoparticles, Does It Really Work? Overview of the Current Status and Challenges

Nanoparticles are being increasingly used as drug delivery systems to enhance the delivery to and uptake by target cells and to reduce off-target toxicity of free drugs. However, although the advantages of nanoparticles as drug carriers are clear, there are still some limitations, especially in maintaining their long-term stability. Lyophilization, also known as freeze-drying, has been heavily investigated as a solution to this problem. This strategy has been shown to be effective in increasing both the long-term stability of nanoparticles and the shelf life of the drug product. However, the process is still in need of improvement in several aspects, such as the process parameters, formulation factors, and characterization techniques. This review summarizes the advantages and limitations of nanoparticles for the treatment of disease, advantages and limitations, and the status of the lyophilization of nanoparticles for therapeutic use and provides insight into both the advantages and the limitations

Production and characterization of erythropoietic protoporphyric heterodimeric ferrochelatases

Mutations resulting in diminished activity of the dimeric enzyme ferrochelatase are a prerequisite for the inherited disorder erythropoietic protoporphyria (EPP). Patients with clinical EPP have only 10% to 30% of normal levels of ferrochelatase activity, and although many patients with EPP have one mutant allele and one “low-expression” normal allele, the possibility remains that, for some, low ferrochelatase activity may result from an EPP mutation that has an impact on both subunits of the wild-type/mutant heterodimer. Here we present data for 12 ferrochelatase wild-type/EPP mutant heterodimers showing that some mutations result in heterodimers with the residual activity anticipated from individual constituents, whereas others result in heterodimers with significantly lower activity than would be predicted. Although the data do not allow an a priori prediction of heterodimeric residual activity based solely on the in vitro activity of EPP homodimers or the position of the mutated residue within ferrochelatase, mutations that affect the dimer interface or [2Fe-2S] cluster have a significantly greater impact on residual activity than would be predicted. These data suggest that some EPP mutations may result in clinically overt EPP in the absence of a low-expression, wild-type allele; this is of potential significance for genetic counseling of patients with EPP

Liposomes in Cancer Therapy: How Did We Start and Where Are We Now

Since their first discovery in the 1960s by Alec Bangham, liposomes have been shown to be effective drug delivery systems for treating various cancers. Several liposome-based formulations received approval by the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA), with many others in clinical trials. Liposomes have several advantages, including improved pharmacokinetic properties of the encapsulated drug, reduced systemic toxicity, extended circulation time, and targeted disposition in tumor sites due to the enhanced permeability and retention (EPR) mechanism. However, it is worth noting that despite their efficacy in treating various cancers, liposomes still have some potential toxicity and lack specific targeting and disposition. This explains, in part, why their translation into the clinic has progressed only incrementally, which poses the need for more research to focus on addressing such translational limitations. This review summarizes the main properties of liposomes, their current status in cancer therapy, and their limitations and challenges to achieving maximal therapeutic efficacy

Identification and Characterization of Solvent-Filled Channels in Human Ferrochelatase

Ferrochelatase catalyzes the formation of protoheme from two potentially cytotoxic products, iron and protoporphyrin IX. While much is known from structural and kinetic studies on human ferrochelatase of the dynamic nature of the enzyme during catalysis and the binding of protoporphyrin IX and heme, little is known about how metal is delivered to the active site and how chelation occurs. Analysis of all ferrochelatase structures available to date reveals the existence of several solvent-filled channels that originate at the protein surface and continue to the active site. These channels have been proposed to provide a route for substrate entry, water entry, and proton exit during the catalytic cycle. To begin to understand the functions of these channels, we investigated in vitro and in vivo a number of variants that line these solvent-filled channels. Data presented herein support the role of one of these channels, which originates at the surface residue H240, in the delivery of iron to the active site. Structural studies of the arginyl variant of the conserved residue F337, which resides at the back of the active site pocket, suggest that it not only regulates the opening and closing of active site channels but also plays a role in regulating the enzyme mechanism. These data provide insight into the movement of the substrate and water into and out of the active site and how this movement is coordinated with the reaction mechanism

Discovery and Characterization of HemQ: AN ESSENTIAL HEME BIOSYNTHETIC PATHWAY COMPONENT*

Here we identify a previously undescribed protein, HemQ, that is required for heme synthesis in Gram-positive bacteria. We have characterized HemQ from Bacillus subtilis and a number of Actinobacteria. HemQ is a multimeric heme-binding protein. Spectroscopic studies indicate that this heme is high spin ferric iron and is ligated by a conserved histidine with the sixth coordination site available for binding a small molecule. The presence of HemQ along with the terminal two pathway enzymes, protoporphyrinogen oxidase (HemY) and ferrochelatase, is required to synthesize heme in vivo and in vitro. Although the exact role played by HemQ remains to be characterized, to be fully functional in vitro it requires the presence of a bound heme. HemQ possesses minimal peroxidase activity, but as a catalase it has a turnover of over 104 min−1. We propose that this activity may be required to eliminate hydrogen peroxide that is generated by each turnover of HemY. Given the essential nature of heme synthesis and the restricted distribution of HemQ, this protein is a potential antimicrobial target for pathogens such as Mycobacterium tuberculosis