Resveratrol Partially Prevents Rotenone-Induced Neurotoxicity in Dopaminergic SH-SY5Y Cells through Induction of Heme Oxygenase-1 Dependent Autophagy

Parkinson disease (PD) is a complex neurodegenerative disorder characterized by a progressive loss of dopaminergic neurons. Mitochondrial dysfunction, oxidative stress or protein misfolding and aggregation may underlie this process. Autophagy is an intracellular catabolic mechanism responsible for protein degradation and recycling of damaged proteins and cytoplasmic organelles. Autophagic dysfunction may hasten the progression of neuronal degeneration. In this study, resveratrol promoted autophagic flux and protected dopaminergic neurons against rotenone-induced apoptosis. In an in vivo PD model, rotenone induced loss of dopaminergic neurons, increased oxidation of mitochondrial proteins and promoted autophagic vesicle development in brain tissue. The natural phytoalexin resveratrol prevented rotenone-induced neuronal apoptosis in vitro, and this pro-survival effect was abolished by an autophagic inhibitor. Although both rotenone and resveratrol promoted LC3-II accumulation, autophagic flux was inhibited by rotenone and augmented by resveratrol. Further, rotenone reduced heme oxygenase-1 (HO-1) expression, whereas resveratrol increased HO-1 expression. Pharmacological inhibition of HO-1 abolished resveratrol-mediated autophagy and neuroprotection. Notably, the effects of a pharmacological inducer of HO-1 were similar to those of resveratrol, and protected against rotenone-induced cell death in an autophagy-dependent manner, validating the hypothesis of HO-1 dependent autophagy in preventing neuronal death in the in vitro PD model. Collectively, our findings suggest that resveratrol induces HO-1 expression and prevents dopaminergic cell death by regulating autophagic flux; thus protecting against rotenone-induced neuronal apoptosis.     

Critical Role of Hemopexin Mediated Cytoprotection in the Pathophysiology of Sickle Cell Disease

Circulating hemopexin is the primary protein responsible for the clearance of heme; therefore, it is a systemic combatant against deleterious inflammation and oxidative stress induced by the presence of free heme. This role of hemopexin is critical in hemolytic pathophysiology. In this review, we outline the current research regarding how the dynamic activity of hemopexin is implicated in sickle cell disease, which is characterized by a pathological aggregation of red blood cells and excessive hemolysis. This pathophysiology leads to symptoms such as acute kidney injury, vaso-occlusion, ischemic stroke, pain crises, and pulmonary hypertension exacerbated by the presence of free heme and hemoglobin. This review includes in vivo studies in mouse, rat, and guinea pig models of sickle cell disease, as well as studies in human samples. In summary, the current research indicates that hemopexin is likely protective against these symptoms and that rectifying depleted hemopexin in patients with sickle cell disease could improve or prevent the symptoms. The data compiled in this review suggest that further preclinical and clinical research should be conducted to uncover pathways of hemopexin in pathological states to evaluate its potential clinical function as both a biomarker and therapy for sickle cell disease and related hemoglobinopathies.     

Bioactive dietary polyphenols inhibit heme iron absorption in a dose-dependent manner in human intestinal Caco-2 cells

Abstract: Although heme iron is an important form of dietary iron, its intestinal absorption mechanism remains elusive. Our previous study revealed that (‐)‐epigallocatechin‐3‐gallate (EGCG) and grape seed extract (GSE) markedly inhibited intestinal heme iron absorption by reducing the basolateral iron export in Caco‐2 cells. The aim of this study was to examine whether small amounts of EGCG, GSE, and green tea extract (GT) could inhibit heme iron absorption, and to test whether the inhibitory action of polyphenols could be offset by ascorbic acid. A heme‐⁵⁵Fe absorption study was conducted by adding various concentrations of EGCG, GSE, and GT to Caco‐2 cells in the absence and presence of ascorbic acid. Polyphenolic compounds significantly inhibited heme‐⁵⁵Fe absorption in a dose‐dependent manner. The addition of ascorbic acid did not modulate the inhibitory effect of dietary polyphenols on heme iron absorption when the cells were treated with polyphenols at a concentration of 46 mg/L. However, ascorbic acid was able to offset or reverse the inhibitory effects of polyphenolic compounds when lower concentrations of polyphenols were added (≤ 4.6 mg/L). Ascorbic acid modulated the heme iron absorption without changing the apical heme uptake, the expression of the proteins involved in heme metabolism and basolateral iron transport, and heme oxygenase activity, indicating that ascorbic acid may enhance heme iron absorption by modulating the intracellular distribution of ⁵⁵Fe. These results imply that the regular consumption of dietary ascorbic acid can easily counteract the inhibitory effects of low concentrations of dietary polyphenols on heme iron absorption but cannot counteract the inhibitory actions of high concentrations of polyphenols. Practical Application: Bioactive dietary polyphenols inhibit heme iron absorption in a dose‐dependent manner. The small amounts of polyphenolic compounds present in foods are capable of reducing heme iron transport across the intestinal enterocyte. However, the inhibitory effects of dietary polyphenolic compounds on heme iron absorption can be offset by ascorbic acid and can possibly be avoided by decreasing the consumption of polyphenols while simultaneously taking ascorbic acid.     

Isopenicillin N synthase binds δ-(L-α-aminoadipoyl)-L-cysteinyl-D-thia-allo-isoleucine through both sulfur atoms

Isopenicillin N synthase (IPNS) catalyses the synthesis of isopenicillin N (IPN), the biosynthetic precursor to penicillin and cephalosporin antibiotics. IPNS is a non‐heme iron(II) oxidase that mediates the oxidative cyclisation of the tripeptide δ‐L ‐α‐aminoadipoyl‐L ‐cysteinyl‐D ‐valine (ACV) to IPN with a concomitant reduction of molecular oxygen to water. Solution‐phase incubation experiments have shown that, although IPNS can turn over analogues with a diverse range of hydrocarbon side chains in the third (valinyl) position of its substrate, the enzyme is much less tolerant of polar residues in this position. Thus, although IPNS converts δ‐L ‐α‐aminoadipoyl‐L ‐cysteinyl‐D ‐isoleucine (ACI) and AC‐D ‐allo‐isoleucine (ACaI) to penam products, the isosteric sulfur‐containing peptides AC‐D ‐thiaisoleucine (ACtI) and AC‐D ‐thia‐allo‐isoleucine (ACtaI) are not turned over. To determine why these peptides are not substrates, we crystallized ACtaI with IPNS. We report the synthesis of ACtaI and the crystal structure of the IPNS:Fe^II:ACtaI complex to 1.79 Å resolution. This structure reveals direct ligation of the thioether side chain to iron: the sulfide sulfur sits 2.66 Å from the metal, squarely in the oxygen binding site. This result articulates a structural basis for the failure of IPNS to turn over these substrates.     

Porphyromonas gingivalis HmuY and Bacteroides vulgatus Bvu—A Novel Competitive Heme Acquisition Strategy

Human oral and gut microbiomes are crucial for maintenance of homeostasis in the human body. Porphyromonas gingivalis, the key etiologic agent of chronic periodontitis, can cause dysbiosis in the mouth and gut, which results in local and systemic infectious inflammatory diseases. Our previous work resulted in extensive biochemical and functional characterization of one of the major P. gingivalis heme acquisition systems (Hmu), with the leading role played by the HmuY hemophore-like protein. We continued our studies on the homologous heme acquisition protein (Bvu) expressed by Bacteroides vulgatus, the dominant species of the gut microbiome. Results from spectrophotometric experiments showed that Bvu binds heme preferentially under reducing conditions using Met145 and Met172 as heme iron-coordinating ligands. Bvu captures heme bound to human serum albumin and only under reducing conditions. Importantly, HmuY is able to sequester heme complexed to Bvu. This is the first study demonstrating that B. vulgatus expresses a heme-binding hemophore-like protein, thus increasing the number of members of a novel HmuY-like family. Data gained in this study confirm the importance of HmuY in the context of P. gingivalis survival in regard to its ability to cause dysbiosis also in the gut microbiome.     

Heme Degradation in Pathophysiology of and Countermeasures to Inflammation-Associated Disease

The class of tetrapyrrol “coordination complexes” called hemes are prosthetic group components of metalloproteins including hemoglobin, which provide functionality to these physiologically essential macromolecules by reversibly binding diatomic gasses, notably O₂, which complexes to ferrous (reduced/Fe(II)) iron within the heme porphyrin ring of hemoglobin in a pH- and PCO₂-dependent manner—thus allowing their transport and delivery to anatomic sites of their function. Here, pathologies associated with aberrant heme degradation are explored in the context of their underlying mechanisms and emerging medical countermeasures developed using heme oxygenase (HO), its major degradative enzyme and bioactive metabolites produced by HO activity. Tissue deposits of heme accumulate as a result of the removal of senescent or damaged erythrocytes from circulation by splenic macrophages, which destroy the cells and internal proteins, including hemoglobin, leaving free heme to accumulate, posing a significant toxicogenic challenge. In humans, HO uses NADPH as a reducing agent, along with molecular oxygen, to degrade heme into carbon monoxide (CO), free ferrous iron (FeII), which is sequestered by ferritin protein, and biliverdin, subsequently metabolized to bilirubin, a potent inhibitor of oxidative stress-mediated tissue damage. CO acts as a cellular messenger and augments vasodilation. Nevertheless, disease- or trauma-associated oxidative stressors sufficiently intense to overwhelm HO may trigger or exacerbate a wide range of diseases, including cardiovascular and neurologic syndromes. Here, strategies are described for counteracting the effects of aberrant heme degradation, with a particular focus on “bioflavonoids” as HO inducers, shown to cause amelioration of severe inflammatory diseases.     

Coculturing of Mosquito-Microbiome Bacteria Promotes Heme Degradation in Elizabethkingia anophelis

Anopheles mosquito microbiomes are intriguing ecological niches. Within the gut, microbes adapt to oxidative stress due to heme and iron after blood meals. Although metagenomic sequencing has illuminated spatial and temporal fluxes of microbiome populations, limited data exist on microbial growth dynamics. Here, we analyze growth interactions between a dominant microbiome species, Elizabethkingia anophelis, and other Anopheles-associated bacteria. We find E. anophelis inhibits a Pseudomonas sp. via an antimicrobial-independent mechanism and observe biliverdins, heme degradation products, upregulated in cocultures. Purification and characterization of E. anophelis HemS demonstrates heme degradation, and we observe hemS expression is upregulated when cocultured with Pseudomonas sp. This study reveals a competitive microbial interaction between mosquito-associated bacteria and characterizes the stimulation of heme degradation in E. anophelis when grown with Pseudomonas sp.     

Redox Potentials of High-Valent Iron-, Cobalt-, and Nickel-Oxido Complexes: Evidence for Exchange Enhanced Reactivity

The ferrocene titration method has been employed to determine the one-electron reduction potentials of a series of oxido-iron(IV), oxido-cobalt(IV) and oxido/hydroxido-nickel(III) complexes based on the same tetradentate TMG₃tren ligand (TMG₃tren=tris[2-(N-tetramethylguanidyl)ethyl]amine). The S=2 ground state of the [(TMG₃tren)Fe^IV=O]²⁺ complex allows an exchange enhanced reactivity, which enables it to perform efficient oxygen atom transfer (OAT) and hydrogen atom abstraction (HAA) reactions with a low one-electron reduction potential of 270 mV vs. SCE. In the absence of exchange enhanced reactivity, the OAT and HAA abilities of the S=3/2 [(TMG₃tren)Co^IV−O(Sc(OTf)₃)]²⁺, S=1/2 [(TMG₃tren)Ni^III−O(H)]²⁺ and the previously reported S=1 [(TMC)(CH₃CN)Fe^IV=O]²⁺ and [(N4Py)Fe^IV=O]²⁺ complexes can be directly correlated to their reduction potentials. Notably, [(N4Py)Fe^IV=O]²⁺ and [(TMG₃tren)Fe^IV=O]²⁺ exhibit similar OAT and HAA reactivities although the reduction potential of [(N4Py)Fe^IV=O]²⁺ is 0.24 V more positive than that of [(TMG₃tren)Fe^IV=O]²⁺. The present study therefore provides experimental evidence for exchange enhanced reactivity and rationalizes nature's choice for employing S=2 oxido-iron(IV) cores to achieve difficult oxidation reactions at biologically viable potentials.