Heme-catalyzed degradation of linoleate 9-hydroperoxide (9-HPODE) forms two allylic epoxy-ketones via a proposed pseudo-symmetrical diepoxy radical intermediate.

Heme-initiated decomposition of unsaturated fatty acid hydroperoxides creates alkoxyl radicals that propagate a complex series of reactions to hydroxy, keto, epoxy and aldehydic products. Herein, among the products from the hematin-catalyzed degradation of 9-hydroperoxy-linoleic acid (9-HPODE), we observed a double peak on normal-phase HPLC that resolved on RP-HPLC into equal proportions of two epoxy-allylic ketones with identical UV spectra. Their proton NMR spectra were also indistinguishable and consistent with 9,10-trans-epoxy-11E-13-keto- and 9-keto-10E-12,13-trans-epoxy-octadecenoic acids. Acid hydrolysis to the corresponding dihydroxy-ketones and GC-MS analysis identified the earlier eluting product on RP-HPLC as the 9,10-epoxy regio-isomer. Starting from the C9-hydroperoxide, recovery of the two epoxy-ketones in equal proportions suggests their formation from a common intermediate. Earlier work has proposed formation of a pseudo-symmetrical diepoxy radical (9,10-epoxy-11(•)-12,13-epoxy, derived from an epoxy allylic hydroperoxide precursor) in the carbon chain fragmentation leading to aldehydic products. This intermediate in pathways of alkoxyl radical reactions forms equal pairs of aldehydes, and now also a pair of epoxy-ketones, and based on mechanism the same products arise from either 9-HPODE or 13-HPODE. Our results point to the intermediacy of this diepoxy-carbinyl radical in the origin of at least two classes of linoleate peroxidation products, and it should be considered as a viable intermediate for homo-conjugated diene peroxidation in general. The reactions could contribute to the aldehydes and epoxy-ketones in tissues undergoing oxidative transformations of polyunsaturated fatty acids.

Silymarin ameliorates diazinon-induced subacute nephrotoxicity in rats via the Keap1-Nrf2/heme oxygenase-1 signaling pathway.

PURPOSE: The goal of the current study was to clarify the potential molecular mechanism underlying the protective effects of silymarin (SIL) administration against diazinon-induced subacute nephrotoxicity, with a special emphasis on the role of the Kelch-like-associated protein-1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2)-heme oxygenase-1 (HO-1) signaling pathway in minimizing the oxidative stress induced by diazinon (DZN). METHODS: Five equal groups of thirty adult male Wistar rats were created at random. Group 1 (G1) was maintained under typical control conditions and administered saline intragastrically (I/G) once daily for 4 weeks; G2 was administered olive oil I/G for 4 weeks; G3 was I/G administered silymarin daily for 4 weeks; G4 was I/G administered diazinon daily for 4 weeks. G5 was I/G administered silymarin daily 1 h before the I/G administration of the diazinon for 4 weeks. Blood samples were collected at the end of the experiment for the determination of complete blood cell count, and kidney function tests. Kidney specimens were collected for the evaluation of the oxidative markers, mRNA gene expression, protein markers, and histopathological examination. RESULTS: SIL reduced the renal dysfunction caused by DZN by restoring urea and creatinine levels, as well as oxidative indicators. Although the expression of Keap-1 was also elevated, overexpression of Nrf2 also enhanced the expression of HO-1, a crucial target enzyme of Nrf2. CONCLUSIONS: SIL is hypothesized to potentially aid in the prevention and management of nephrotoxicity caused by DZN.

Generating high purity and yield of capsanthin and capsorubin carrot germplasm through synthetic metabolic engineering.

Capsanthin and capsorubin are red κ-xanthophylls exclusively found in a handful of other plant species. Currently, capsanthin and capsorubin are only extracted from red pepper. Here, high purity production of capsanthin and capsorubin has been achieved in carrot taproot by synthetic metabolic engineering strategy. Expression of a capsanthin-capsorubin synthase gene (CaCCS) from pepper resulted in dominant production of capsanthin whereas expression of a LiCCS gene from tiger lily resulted in production of both capsanthin and capsorubin in carrot taproot. The highest content of capsanthin and capsorubin was obtained in LiC-1 carrot taproot hosting the LiCCS gene, 150.09 µg/g DW (dry weight). Co-expression of DcBCH1 with CCS could improve the purity of capsanthin and capsorubin by eliminating the non-target carotenoids (eg. α-carotene and ß-carotene). The highest purity of capsanthin and capsorubin was obtained in BLiC-1 carrot taproot hosting DcBCH1+LiCCS genes, 91.10% of total carotenoids. The non-native pigments were esterified partially and stored in the globular chromoplast of carrot taproot. Our results demonstrated the possibility of employing carrot taproot as green factories for high purity production of capsanthin and capsorubin. The capsanthin/capsorubin carrot germplasms were also valuable materials for breeding colorful carrots cultivars.

Heme (dys)homeostasis and liver disease.

Heme is essential for a variety of proteins involved in vital physiological functions in the body, such as oxygen transport, drug metabolism, biosynthesis of steroids, signal transduction, antioxidant defense and mitochondrial respiration. However, free heme is potentially cytotoxic due to the capacity of heme iron to promote the oxidation of cellular molecules. The liver plays a central role in heme metabolism by significantly contributing to heme synthesis, heme detoxification, and recycling of heme iron. Conversely, enzymatic defects in the heme biosynthetic pathway originate multisystemic diseases (porphyrias) that are highly associated with liver damage. In addition, there is growing evidence that heme contributes to the outcomes of inflammatory, metabolic and malignant liver diseases. In this review, we summarize the contribution of the liver to heme metabolism and the association of heme dyshomeostasis with liver disease.

A new strategy for Astragaloside IV in the treatment of diabetic kidney disease: Analyzing the regulation of ferroptosis and mitochondrial function of renal tubular epithelial cells.

In China, the Astragalus membranaceus root is used to treat chronic kidney disease. Astragaloside IV (AS-IV), the primary bioactive compound, exhibits anti-inflammatory and antioxidative properties; however, its renoprotective mechanism in diabetic kidney disease (DKD) remains unclear. The study aimed to investigate the protective effects of AS-IV on DKD revealing the underlying mechanisms. We established an early diabetic rat model by feeding a high-fat diet and administering low-dose streptozotocin. Twelve weeks post-treatment, renal function was evaluated using functional assays, histological analyses, immunohistochemistry, western blotting, and transmission electron microscopy. HK-2 cells exposed to high glucose conditions were used to examine the effect of AS-IV on oxidative stress, iron levels, reactive oxygen species (ROS), and lipid peroxidation. Network pharmacology, proteomics, molecular docking, and molecular dynamics simulation techniques were employed to elucidate the role of AS-IV in DKD. The results revealed that AS-IV effectively enhanced renal function and mitigated disease pathology, oxidative stress, and ferroptosis markers in DKD rats. In HK-2 cells, AS-IV lowered the levels of lipid peroxides, Fe2+, and glutathione, indicating the repair of ferroptosis-related mitochondrial damage. AS-IV reduced mitochondrial ROS while enhancing mitochondrial membrane potential and ATP production, indicating its role in combating mitochondrial dysfunction. Overall, in silico analyses revealed that AS-IV interacts with HMOX1, FTH1, and TFR1 proteins, supporting its efficacy in alleviating renal injury by targeting mitochondrial dysfunction and ferroptosis. AS-IV may play a renoprotective role by regulating mitochondrial dysfunction and inhibiting. HMOX1/FTH1/TFR1-induced ferroptosis. Accordingly, AS-IV could be developed for the clinical treatment of DKD-related renal injury.

Decreased vascular contraction and changes in oxidative state in middle-aged Wistar rats after exposure to increased levels of dietary zinc.

Both copper and zinc are known to be important for maintaining health, but most research has focused on deficiencies of these elements. Recent studies have shown that high levels of Cu can be toxic, especially to the cardiovascular (CV) system. However, little research has been done on the effects of higher levels of Zn on the CV system. In this study, male Wistar rats aged 12 months were given a diet with twice the recommended daily allowance of zinc (31.8 mg/kg of diet) and compared to a control group (15.9 mg/kg of diet) after 8 weeks. Blood plasma and internal organs of both groups were examined for levels of copper, zinc, selenium and iron, as well as several key enzymes. Aortic rings from both groups were also examined to determine vascular functioning. There were very few changes in the vascular system functioning after chronic exposure to zinc, and only one enzyme, heme oxygenase-1 (HO-1) was elevated, whereas vascular contraction to noradrenaline decreased with no changes in vasodilation to acetylcholine. Of the micronutrients, zinc and selenium were elevated in the blood plasma, while copper decreased. Meanwhile, the total antioxidant status increased. These were not observed in the liver. Therefore, it is proposed that there is a mechanism in place within the vascular system to protect against the overproduction of heme, caused by chronic zinc exposure.

Hemin-Induced Transient Senescence Via DNA Damage Response: A Neuroprotective Mechanism Against Ferroptosis in Intracerebral Hemorrhage.

Intracerebral hemorrhage (ICH) poses acute fatality and long-term neurological risks due to hemin and iron accumulation from hemoglobin breakdown. Our observation that hemin induces DNA double-strand breaks (DSBs), prompting a senescence-like phenotype in neurons, necessitating deeper exploration of cellular responses. Using experimental ICH models and human ICH patient tissue, we elucidate hemin-mediated DNA damage response (DDR) inducing transient senescence and delayed expression of heme oxygenase (HO-1). HO-1 co-localizes with senescence-associated ß-Galactosidase (SA-ß-Gal) in ICH patient tissues, emphasizing clinical relevance of inducible HO-1 expression in senescent cells. We reveal a reversible senescence state protective against acute cell death by hemin, while repeat exposure leads to long-lasting senescence. Inhibiting early senescence expression increases cell death, supporting the protective role of senescence against hemin toxicity. Hemin-induced senescence is attenuated by a pleiotropic carbon nanoparticle that is a catalytic mimic of superoxide dismutase, but this treatment increased lipid peroxidation, consistent with ferroptosis from hemin breakdown released iron. When coupled with iron chelator deferoxamine (DEF), the nanoparticle reduces hemin-induced senescence and upregulates factors protecting against ferroptosis. Our study suggests transient senescence induced by DDR as an early potential neuroprotective mechanism in ICH, but the risk or iron-related toxicity supports a multi-pronged therapeutic approach.

Ca2+-controlled Mn(II) removal process in Aurantimonas sp. HBX-1: Microbially-induced carbonate precipitation (MICP) versus Mn(II) oxidation.

The application of manganese-oxidizing bacteria (MnOB) to produce manganese oxides (MnOx) has been widely studied, but often overlooking the concurrent formation of MnCO3. In this study, we found Ca2+ plays a crucial role in controlling Mn(II) removal in the bacterium Aurantimonas sp. HBX-1. Under conditions with 6.8 mM Ca2+ and without adding Ca2+, 100 µM Mn(II) was removed by 96.96 % and 38.28 % within 8 days, respectively. X-ray photoelectron spectroscopy (XPS) showed that adding Ca2+ increased the average oxidation state (AOS) of the solid products from 2.05 to 2.37. X-ray absorption fine structure (XAFS) analysis revealed the product proportions as follows: under Ca2+-supplemented condition, the ratio of MnOx (1 < x ≤ 2) to MnCO3 was 52 % to 28.1 %, while under Ca2+-free condition, the ratio shifted to 4.6 % for MnOx (1 < x ≤ 2) and 55.2 % for MnCO3. Urease activity assay and proteomic analysis confirmed the expression of urease and carbonic anhydrase, leading to the formation of MnCO3. Additionally, animal heme peroxidase (AHP) in strain HBX-1 was found to be responsible for Mn(II) oxidation through superoxide production, with Ca2+ addition promoting its expression level. Given the widespread presence of Ca2+ in wastewater, its potential impact on the biogeochemical Mn(II) cycle driven by bacteria should be reconsidered.

The Increase in the Peroxidase Activity of the Cytochrome C with Substitutions in the Universal Binding Site Is Associated with Changes in the Ability to Interact with External Ligands.

Cytochrome c (CytC), a one-electron carrier, transfers electrons from complex bc1 to cytochrome c oxidase (CcO) in the electron-transport chain. Electrostatic interaction with the partners, complex bc1 and CcO, is ensured by a lysine cluster near the heme forming the Universal Binding Site (UBS). We constructed three mutant variants of mitochondrial CytC with one (2Mut), four (5Mut), and five (8Mut) Lys->Glu substitutions in the UBS and some compensating Glu->Lys substitutions at the periphery of the UBS for charge compensation. All mutants showed a 4-6 times increased peroxidase activity and accelerated binding of cyanide to the ferric heme of CytC. In contrast, decomposition of the cyanide complex with ferrous CytC, as monitored by magnetic circular dichroism spectroscopy, was slower in mutants compared to WT. Molecular dynamic simulations revealed the increase in the fluctuations of Cα atoms of individual residues of mutant CytC compared to WT, especially in the Ω-loop (70-85), which can cause destabilization of the Fe…S(Met80) coordination link, facilitation of the binding of exogenous ligands cyanide and peroxide, and an increase in peroxidase activity. It was found that only one substitution K72E is enough to induce all these changes, indicating the significance of K72 and the Ω-loop (70-85) for the structure and physiology of mitochondrial CytC. In this work, we also propose using a ferro-ferricyanide buffer as a substrate to monitor the peroxidase activity of CytC. This new approach allows us to determine the rate of peroxidase activity at moderate (200 µM) concentrations of H2O2 and avoid complications of radical formation during the reaction.

Entrance channels to coproheme in coproporphyrin ferrochelatase probed by exogenous imidazole binding.

Iron insertion into porphyrins is an essential step in heme biosynthesis. In the coproporphyrin-dependent pathway, specific to monoderm bacteria, this reaction is catalyzed by the monomeric enzyme coproporphyrin ferrochelatase. In addition to the mechanistic details of the metalation of the porphyrin, the identification of the substrate access channel for ferrous iron to the active site is important to fully understand this enzymatic system. In fact, whether the iron reaches the active site from the distal or the proximal porphyrin side is still under debate. In this study we have thoroughly addressed this question in Listeria monocytogenes coproporphyrin ferrochelatase by X-ray crystallography, steady-state and pre-steady-state imidazole ligand binding studies, together with a detailed spectroscopic characterization using resonance Raman and UV-vis absorption spectroscopies in solution. Analysis of the X-ray structures of coproporphyrin ferrochelatase-coproporphyrin III crystals soaked with ferrous iron shows that iron is present on both sides of the porphyrin. The kinetic and spectroscopic study of imidazole binding to coproporphyrin ferrochelatase­iron coproporphyrin III clearly indicates the presence of two possible binding sites in this monomeric enzyme that influence each other, which is confirmed by the observed cooperativity at steady-state and a biphasic behavior in the pre-steady-state experiments. The current results are discussed in the context of the entire heme biosynthetic pathway and pave the way for future studies focusing on protein-protein interactions.

Expression of a stress-inducible heme oxygenase-1 in NK cells is maintained in the process of human aging.

Introduction: Heme oxygenase-1 (HO-1) is a stress-inducible heat shock protein (HSP32) that exerts cytoprotective effects against oxidative stress and inflammation, and is involved in the maintenance of cellular homeostasis. This study aimed to evaluate the expression of HO-1 in natural killer (NK) cells from individuals of different age groups after stimulation with various factors, and to analyze the relationships between the concentration of this cytoprotective protein and parameters corresponding to oxidative stress and inflammation, that is, NOD-like receptor protein 3 (NLRP3), glutathione (GSH), GSH disulfide (GSSG), and interleukin 6 (IL-6). Methods: The study population comprised three age groups: young adults (age range, 19-23 years), older adults aged under 85 years (age range, 73-84 years), and older adults aged over 85 years (age range, 85-92 years). NLRP3, GSH, and GSSG concentrations were measured in serum, whereas the HO-1 concentration and IL-6 expression were studied in NK cells cultivated for 48 h and stimulated with IL-2, lipopolysaccharide (LPS), or phorbol 12-myristate 13-acetate (PMA) with ionomycin. Results: The analysis of serum NLRP3, GSH, and GSSG concentrations revealed no statistically significant differences among the studied age groups. However, some typical trends of aging were observed, such as a decrease in GSH concentration and an increase in both GSSG level, and GSSG/GSH ratio. The highest basal expression of IL-6 and lowest basal content of HO-1 were found in NK cells of adults over 85 years of age. The NK cells in this age group also showed the highest sensitivity to stimulation with the applied factors. Moreover, statistically significant negative correlations were observed between HO-1 and IL-6 expression levels in the studied NK cells. Conclusions: These results showed that NK cells can express HO-1 at a basal level, which was significantly increased in activated cells, even in the oldest group of adults. The reciprocal relationship between HO-1 and IL-6 expression suggests a negative feedback loop between these parameters.

Analyzing the FMN-heme interdomain docking interactions in neuronal and inducible NOS isoforms by pulsed EPR experiments and conformational distribution modeling.

Nitric oxide synthases (NOSs), a family of flavo-hemoproteins with relatively rigid domains linked by flexible regions, require optimal FMN domain docking to the heme domain for efficient interdomain electron transfer (IET). To probe the FMN-heme interdomain docking, the magnetic dipole interactions between the FMN semiquinone radical (FMNH•) and the low-spin ferric heme centers in oxygenase/FMN (oxyFMN) constructs of neuronal and inducible NOS (nNOS and iNOS, respectively) were measured using the relaxation-induced dipolar modulation enhancement (RIDME) technique. The FMNH• RIDME data were analyzed using the mesoscale Monte Carlo calculations of conformational distributions of NOS, which were improved to account for the native degrees of freedom of the amino acid residues constituting the flexible interdomain tethers. This combined computational and experimental analysis allowed for the estimation of the stabilization energies and populations of the docking complexes of calmodulin (CaM) and the FMN domain with the heme domain. Moreover, combining the five-pulse and scaled four-pulse RIDME data into a single trace has significantly reduced the uncertainty in the estimated docking probabilities. The obtained FMN-heme domain docking energies for nNOS and iNOS were similar (-3.8 kcal/mol), in agreement with the high degree of conservation of the FMN-heme domain docking interface between the NOS isoforms. In spite of the similar energetics, the FMN-heme domain docking probabilities in nNOS and iNOS oxyFMN were noticeably different (~ 0.19 and 0.23, respectively), likely due to differences in the lengths of the FMN-heme interdomain tethers and the docking interface topographies. The analysis based on the IET theory and RIDME experiments indicates that the variations in conformational dynamics may account for half of the difference in the FMN-heme IET rates between the two NOS isoforms.

Heme pocket hydrogen bonding residue interactions within the Pectobacterium Diguanylate cyclase-containing globin coupled sensor: A resonance Raman study.

Heme-based sensor proteins are used by organisms to control signaling and physiological effects in response to their gaseous environment. Globin-coupled sensors (GCS) are oxygen-sensing proteins that are widely distributed in bacteria. These proteins consist of a heme globin domain linked by a middle domain to various output domains, including diguanylate cyclase domains, which are responsible for synthesizing c-di-GMP, a bacterial second messenger crucial for regulating biofilm formation. To understand the roles of heme pocket residues in controlling activity of the diguanylate cyclase domain, variants of the Pectobacterium carotovorum GCS (PccGCS) were characterized by enzyme kinetics and resonance Raman (rR) spectroscopy. Results of these studies have identified roles for hydrogen bonding and heme edge residues in modulating heme pocket conformation and flexibility. Better understanding of the ligand-dependent GCS signaling mechanism and the residues involved may allow for future development of methods to control O2-dependent c-di-GMP production.

Targeting heme in sickle cell disease: new perspectives on priapism treatment.

Men with sickle cell disease (SCD) frequently experience priapism, defined as prolonged, painful erections occurring without sexual arousal or desire. This urological emergency can lead to penile fibrosis and permanent erectile dysfunction if not treated adequately. Due to its complex pathophysiology, there is currently no effective preventative treatment for this condition. Recent studies have highlighted the dysfunction of the nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) pathway in erectile tissues as a critical mechanism in developing priapism in SCD. Additionally, further research indicates that intravascular hemolysis promotes increased smooth muscle relaxation in the corpus cavernosum and that excess heme may significantly contribute to priapism in SCD. Pharmacological treatments should ideally target the pathophysiological basis of the disease. Agents that reduce excess free heme in the plasma have emerged as potential therapeutic candidates. This review explores the molecular mechanisms underlying the excess of heme in SCD and its contribution to developing priapism. We discuss pharmacological approaches targeting the excess free heme in the plasma, highlighting it as a potential therapeutic target for future interventions in managing priapism.

A Cytochrome P450 TxtE Model System with Mechanistic and Theoretical Evidence for a Heme Peroxynitrite Active Species.

The cytochrome P450 homolog, TxtE, efficiently catalyzes the direct and regioselective aromatic nitration of the indolyl moiety of L-tryptophan to 4-nitro-L-tryptophan, using nitric oxide and dioxygen as co-substrates. Pathways for such direct and selective nitration of heteroaromatic motifs present platforms for engineering new nitration biocatalysts for pharmacologically beneficial targets, among a medley of other pivotal industrial applications. Precise mechanistic details concerning this pathway are only weakly understood, albeit a heme iron(III)-peroxynitrite active species has been postulated. To shed light on this unique reaction landscape, we investigated the indole nitration pathway of a series of biomimetic ferric heme superoxide mimics, [(Por)FeIII(O2-•)], in the presence of NO. Therein, our model systems gave rise to three distinct nitroindole products, including 4-nitroindole, the product analogous to that obtained with TxtE. Moreover, 15N and 18O isotope labeling studies, along with meticulously designed control experiments lend credence to a heme peroxynitrite active nitrating agent, drawing close similarities to the tryptophan nitration mechanism of TxtE. All organic and inorganic reaction components have been fully characterized using spectroscopic methods. Theoretical investigation into several mechanistic possibilities deem a unique indolyl radical based reaction pathway as the most energetically favorable, products of which, are in excellent agreement with experimental findings.

Repurposing myoglobin into a carbene transferase for a [2,3]-sigmatropic Sommelet-Hauser rearrangement.

New-to-Nature biocatalysis has emerged as a promising tool in organic synthesis thanks to progress in protein engineering. Notably, hemeproteins have been evolved into robust catalysts for carbene and nitrene transfers and related sigmatropic rearrangements. In this work, we report the first example of a [2,3]-sigmatropic Sommelet-Hauser rearrangement initiated by a carbene transfer of the sperm whale myoglobin mutant L29S,H64V,V68F that was previously reported to catalyze the mechanistically similar [2,3]-sigmatropic Doyle-Kirmse rearrangement. This repurposed heme enzyme catalyzes the Sommelet-Hauser rearrangement between ethyl diazoacetate and benzyl thioethers bearing strong electron-withdrawing substituents with good yields and enantiomeric excess. Optimized catalytic conditions in the absence of any reductant led to an increased asymmetric induction with up to 59% enantiomeric excess. This myoglobin mutant is therefore one of the few catalysts for the asymmetric Sommelet-Hauser rearrangement. This work broadens the scope of abiological reactions catalyzed by iron-carbene transferases with a new example of asymmetric sigmatropic rearrangement.

Minocycline alleviates microglia ferroptosis by inhibiting HO-1 during cerebral ischemia-reperfusion injury.

OBJECTIVE: Ischemic stroke is a leading cause of death and disability in individuals worldwide. Cerebral ischemia-reperfusion injury (CIRI) typically results in severe secondary injury and complications following reperfusion therapy. Microglia play critical roles in the inflammatory reaction of CIRI. However, less attention has been given to microglial death in this process. Our study aims to explore microglial death in CIRI and the effects and mechanism of minocycline treatment on microglia. METHODS: A middle cerebral artery occlusion (MCAO) model was applied to induce CIRI in rats. At 0 h, 24 h and 48 h post-operation, rats were intraperitoneally injected with 45 mg/kg minocycline. Neurological deficit scoring, 2,3,5-triphenyltetrazolium chloride (TTC) staining, assessment of activated microglia and examination of mitochondrial structure were conducted and checked at 72 h after reperfusion. Additionally, an in vitro model of oxygen-glucose deprivation/reperfusion (OGD/R) model was established. BV-2 cells were treated with various pharmacological inhibitors of cell death or minocycline. Cell viability, lipid peroxidation, mitochondrial structure and function, and labile Fe2+ and ferroptosis-associated gene/protein levels were measured. Hemin was used for further validation after transcriptome analysis. RESULTS: In the MCAO and OGD/R models, ferroptosis was identified as a major form of microglial death. Minocycline inhibited microglia ferroptosis by reducing HO-1 expression. In addition, minocycline improved mitochondrial membrane potential, mitochondrial structures and microglial survival in vivo. Minocycline also decreased labile Fe2+ levels, lipid peroxidation, and expression of ferritin heavy chain (FTH) and it improved mitochondrial structure and function in vitro. Upregulation of HO-1 counteracted the protective effect of minocycline. CONCLUSION: Ferroptosis is a major form of microglial death in CIRI. The protective mechanism of minocycline in CIRI partially hinges on its ability to effectively ameliorate microglia ferroptosis by downregulating HO-1 expression. Consequently, targeting microglia ferroptosis is a promising treatment for CIRI.

Novel UROD mutation for porphyria cutanea tarda, type 2: a case report.

Background: Porphyria cutanea tarda (PCT) is usually caused by acquired defects in uroporphyrinogen decarboxylase (UROD) activity in the liver. This more common form of PCT is called type 1 PCT. Major known risk factors for PCT include iron overload, such as occurs due to mutations in HFE, associated with classical hereditary hemochromatosis, chronic hepatitis C infection, heavy alcohol use, tobacco use, and estrogen therapy. In addition, in about 25% of patients with PCT, namely, those with PCT type 2, an inherited partial defect in UROD activity is found. In such persons, this partial defect, which is found in all cells, including hepatocytes, red blood cells, and others, contributes to the development of biochemically and clinically active disease. Case Description: Herein we describe salient features of a man in his eighth decade of life with onset of clinical PCT. Among risk factors were heavy alcohol and tobacco use. Genetic testing revealed a novel mutation in one of his alleles of the UROD gene, namely, c.224 G>C; p. Arg 75 Pro, and enzymatic testing revealed that red blood cell UROD activity was decreased by 50%. This mutation in the UROD gene is predicted to have a major effect on protein structure and function, confirmed by the 50% decrease in activity of the enzyme. Conclusions: The previously undescribed mutation in UROD, found in this man, namely, c.224 G>C; p. Arg 75 Pro is pathogenic.

Targeting heme degradation pathway augments prostate cancer cell sensitivity to docetaxel-induced apoptosis and attenuates migration.

Introduction: Chemotherapy, notably docetaxel (Doc), stands as the primary treatment for castration-resistant prostate cancer (CRPC). However, its efficacy is hindered by side effects and chemoresistance. Hypoxia in prostate cancer (PC) correlates with chemoresistance to Doc-induced apoptosis via Heme Oxygenase-1 (HO-1) modulation, a key enzyme in heme metabolism. This study investigated targeting heme degradation pathway via HO-1 inhibition to potentiate the therapeutic efficacy of Doc in PC. Methods: Utilizing diverse PC cell lines, we evaluated HO-1 inhibition alone and with Doc on viability, apoptosis, migration, and epithelial- to- mesenchymal transition (EMT) markers and elucidated the underlying mechanisms. Results: HO-1 inhibition significantly reduced PC cell viability under hypoxic and normoxic conditions, enhancing Doc-induced apoptosis through interconnected mechanisms, including elevated reactive oxygen species (ROS) levels, glutathione cycle disruption, and modulation of Signal Transducer and Activator of Transcription 1 (STAT1) pathway. The interplay between STAT1 and HO-1 suggests its reliance on HO-1 activation. Additionally, a decrease in cell migration and downregulation of EMT markers (vimentin and snail) were observed, indicating attenuation of mesenchymal phenotype. Discussion: In conclusion, the combination of HO-1 inhibition with Doc holds promise for improving therapeutic outcomes and advancing clinical management in PC.

Resonance Raman spectral analysis of the heme site structure of cytochrome c oxidase with its positive regulator CHCHD2.

Cytochrome c oxidase (CcO) reduces O2, pumps protons in the mitochondrial respiratory chain, and is essential for oxygen consumption in the cell. The coiled-coil-helix-coiled-coil-helix domain-containing 2 (CHCHD2; also known as mitochondrial nuclear retrograde regulator 1 [MNRR1], Parkinson's disease 22 [PARK22] and aging-associated gene 10 protein [AAG10]) is a protein that binds to CcO from the intermembrane space and positively regulates the activity of CcO. Despite the importance of CHCHD2 in mitochondrial function, the mechanism of action of CHCHD2 and structural information regarding its binding to CcO remain unknown. Here, we utilized visible resonance Raman spectroscopy to investigate the structural changes around the hemes in CcO in the reduced and CO-bound states upon CHCHD2 binding. We found that CHCHD2 has a significant impact on the structure of CcO in the reduced state. Mapping of the heme peripheries that result in Raman spectral changes in the structure of CcO highlighted helices IX and X near the hemes as sites where CHCHD2 takes action. Part of helix IX is exposed in the intermembrane space, whereas helix X, located between both hemes, may play a key role in proton uptake to a proton-loading site in the reduced state for proton pumping. Taken together, our results suggested that CHCHD2 binds near helix IX and induces a structural change in helix X, accelerating proton uptake.