A High-Throughput Method for Identifying Novel Genes That Influence Metabolic Pathways Reveals New Iron and Heme Regulation in Pseudomonas aeruginosa

The ability to simultaneously and more directly correlate genes with metabolite levels on a global level would provide novel information for many biological platforms yet has thus far been challenging. Here, we describe a method to help address this problem, which we dub “Met-Seq” (metabolite-coupled Tn sequencing).

Heme, A Metabolic Sensor, Directly Regulates the Activity of the KDM4 Histone Demethylase Family and Their Interactions with Partner Proteins

The KDM4 histone demethylase subfamily is constituted of yeast JmjC domain-containing proteins, such as Gis1, and human Gis1 orthologues, such as KDM4A/B/C. KDM4 proteins have important functions in regulating chromatin structure and gene expression in response to metabolic and nutritional stimuli. Heme acts as a versatile signaling molecule to regulate important cellular functions in diverse organisms ranging from bacteria to humans. Here, using purified KDM4 proteins containing the JmjN/C domain, we showed that heme stimulates the histone demethylase activity of the JmjN/C domains of KDM4A and Cas well as full-length Gis1. Furthermore, we found that the C-terminal regions of KDM4 proteins, like that of Gis1, can confer heme regulation when fused to an unrelated transcriptional activator. Interestingly, biochemical pull-down of Gis1-interacting proteins followed by mass spectrometry identified 147 unique proteins associated with Gis1 under heme-sufficient and/or heme-deficient conditions. These 147 proteins included a significant number of heterocyclic compound-binding proteins, Ubl-conjugated proteins, metabolic enzymes/proteins, and acetylated proteins. These results suggested that KDM4s interact with diverse cellular proteins to form a complex network to sense metabolic and nutritional conditions like heme levels and respond by altering their interactions with other proteins and functional activities, such as histone demethylation.

TheprrF-Encoded Small Regulatory RNAs Are Required for Iron Homeostasis and Virulence of Pseudomonas aeruginosa

Pseudomonas aeruginosais an opportunistic pathogen that requires iron to cause infection, but it also must regulate the uptake of iron to avoid iron toxicity. The iron-responsive PrrF1 and PrrF2 small regulatory RNAs (sRNAs) are part ofP. aeruginosa'siron regulatory network and affect the expression of at least 50 genes encoding iron-containing proteins. The genes encoding the PrrF1 and PrrF2 sRNAs are encoded in tandem inP. aeruginosa, allowing for the expression of a distinct, heme-responsive sRNA named PrrH that appears to regulate genes involved in heme metabolism. Using a combination of growth, mass spectrometry, and gene expression analysis, we showed that the ΔprrF1,2mutant, which lacks expression of the PrrF and PrrH sRNAs, is defective for both iron and heme homeostasis. We also identifiedphuS, encoding a heme binding protein involved in heme acquisition, andvreR, encoding a previously identified regulator ofP. aeruginosavirulence genes, as novel targets ofprrF-mediated heme regulation. Finally, we showed that theprrFlocus encoding the PrrF and PrrH sRNAs is required forP. aeruginosavirulence in a murine model of acute lung infection. Moreover, we showed that inoculation with a ΔprrF1,2deletion mutant protects against future challenge with wild-typeP. aeruginosa. Combined, these data demonstrate that theprrF-encoded sRNAs are critical regulators ofP. aeruginosavirulence.

Protective Action of Antioxidants on Hepatic Damage Induced by Griseofulvin

Erythropoietic protoporphyria (EPP) is a disease associated with ferrochelatase deficiency and characterized by the accumulation of protoporphyrin IX (PROTO IX) in erythrocytes, liver, and skin. In some cases, a severe hepatic failure and cholestasis were observed. Griseofulvin (Gris) develops an experimental EPP with hepatic manifestations in mice such as PROTO IX accumulation followed by cellular damage as wells as necrotic and inflammatory processes. The antioxidant defense system was also altered. The aim was to evaluate the possible protective effect of different antioxidant compounds: trolox (Tx), ascorbic acid (Asc), the combination Tx and Asc, melatonin (Mel), and the polyphenols: ellagic acid, quercetin, chlorogenic acid, caffeic acid, gallic acid, and ferulic acid on liver damage and oxidative stress markers in a mouse model of EPP. Coadministration of Gris with Tx, Asc, and its combination, or Mel mainly affected heme biosynthetic pathway, resulting in a decrease in ALA-S activity which was increased by Gris, while the tested polyphenols exerted a protective effect on oxidative stress, decreasing lipid peroxidation and the activity of some antioxidant enzymes. In conclusion, antioxidant compounds can only protect partially against the liver damage induced by Gris, reducing oxidative stress or acting on heme regulation.

Heme Regulation in Traumatic Brain Injury: Relevance to the Adult and Developing Brain

Intracranial bleeding is one of the most prominent aspects in the clinical diagnosis and prognosis of traumatic brain injury (TBI). Substantial amounts of blood products, such as heme, are released because of traumatic subarachnoid hemorrhages, intraparenchymal contusions, and hematomas. Despite this, surprisingly few studies have directly addressed the role of blood products, in particular heme, in the setting of TBI. Heme is degraded by heme oxygenase (HO) into three highly bioactive products: iron, bilirubin, and carbon monoxide. The HO isozymes, in particular HO-1 and HO-2, exhibit significantly different expression patterns and appear to have specific roles after injury. Developmentally, differences between the adult and immature brain have implications for endogenous protection from oxidative stress. The aim of this paper is to review recent advances in the understanding of heme regulation and metabolism after brain injury and its specific relevance to the developing brain. These findings suggest novel clinical therapeutic options for further translational study.