Structural insight into AMPK regulation: ADP comes into play

The AMP-activated protein kinase (AMPK), a sensor of cellular energy status found in all eukaryotes, responds to changes in intracellular adenosine nucleotide levels resulting from metabolic stresses. Here we describe crystal structures of a heterotrimeric regulatory core fragment from Schizosaccharomyces pombe AMPK in complex with ADP, ADP/AMP, ADP/ATP, and 5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranotide (AICAR phosphate, or ZMP), a well-characterized AMPK activator. Prior crystallographic studies had revealed a single site in the gamma subunit that binds either ATP or AMP within Bateman domain B. Here we show that ZMP binds at this site, mimicking the binding of AMP. An analogous site in Bateman domain A selectively accommodates ADP, which binds in a distinct manner that also involves direct ligation to elements from the beta subunit. These observations suggest a possible role for ADP in regulating AMPK response to changes in cellular energy status.     

The role of frataxin in fission yeast iron metabolism: Implications for Friedreich's ataxia

Background

The neurodegenerative disease Friedreich's ataxia is the result of frataxin deficiency. Frataxin is a mitochondrial protein involved in iron–sulfur cluster (Fe–S) cofactor biogenesis, but its functional role in this pathway is debated. This is due to the interconnectivity of iron metabolic and oxidative stress response pathways that make distinguishing primary effects of frataxin deficiency challenging. Since Fe–S cluster assembly is conserved, frataxin overexpression phenotypes in a simple eukaryotic organism will provide additional insight into frataxin function.

Methods

The Schizosaccharomyces pombe frataxin homologue (fxn1) was overexpressed from a plasmid under a thiamine repressible promoter. The S. pombe transformants were characterized at several expression strengths for cellular growth, mitochondrial organization, iron levels, oxidative stress, and activities of Fe–S cluster containing enzymes.

Results

Observed phenotypes were dependent on the amount of Fxn1 overexpression. High Fxn1 overexpression severely inhibited S. pombe growth, impaired mitochondrial membrane integrity and cellular respiration, and led to Fxn1 aggregation. Cellular iron accumulation was observed at moderate Fxn1 overexpression but was most pronounced at high levels of Fxn1. All levels of Fxn1 overexpression up-regulated oxidative stress defense and mitochondrial Fe–S cluster containing enzyme activities.

Conclusions

Despite the presence of oxidative stress and accumulated iron, activation of Fe–S cluster enzymes was common to all levels of Fxn1 overexpression; therefore, Fxn1 may regulate the efficiency of Fe–S cluster biogenesis in S. pombe.

General Significance

We provide evidence that suggests that dysregulated Fe–S cluster biogenesis is a primary effect of both frataxin overexpression and deficiency as in Friedreich's ataxia.     

Mitochondrial iron metabolism in the yeast Saccharomyces cerevisiae

Iron is fundamental to many biological processes, but is also detrimental as it fosters the synthesis of destructive oxygen radicals. Recent experiments have increased our knowledge of the critical process of regulation of mitochondrial iron metabolism. A number of genes directly involved in iron homeostasis in this organelle have been identified. Intriguingly, a minor Hsp70 molecular chaperone of the mitochondrial matrix has been implicated as a player in this process as well.     

Structure of frataxin iron cores: an X-ray absorption spectroscopic study

X-ray absorption spectroscopy at the iron K-edge indicates that the iron cores of human and yeast frataxin polymers assembled in vitro are identical to each other and are similar but not identical to ferritin cores. Both frataxin polymers contain ferrihydrite, a biomineral composed of ferric oxide/hydroxide octahedra. The ferrihydrite in frataxin is less ordered than iron cores of horse spleen ferritin, having fewer face-sharing Fe-Fe interactions but similar double corner-sharing interactions. The extended X-ray absorption fine structure (EXAFS) analysis agrees with previous electron microscopy data showing that frataxin cores are composed of very small ferrihydrite crystallites.     

Drosophila frataxin: an iron chaperone during cellular Fe-S cluster bioassembly

Frataxin, a mitochondrial protein that is directly involved in regulating cellular iron homeostasis, has been suggested to serve as an iron chaperone during cellular Fe-S cluster biosynthesis. In humans, decreased amounts or impaired function of frataxin causes the autosomal recessive neurodegenerative disorder Friedreich's ataxia. Cellular production of Fe-S clusters is accomplished by the Fe cofactor assembly platform enzymes Isu (eukaryotes) and IscU (prokaryotes). In this report, we have characterized the overall stability and iron binding properties of the Drosophila frataxin homologue (Dfh). Dfh is highly folded with secondary structural elements consistent with the structurally characterized frataxin orthologs. While the melting temperature ( T M approximately 59 degrees C) and chemical stability ([urea] 50% approximately 2.4 M) of Drosophila frataxin, measured using circular dichroism (CD) and fluorescence spectroscopy, closely match values determined for the human ortholog, pure Dfh is more stable against autodegradation than both the human and yeast proteins. The ferrous iron binding affinity ( K d approximately 6.0 microM) and optimal metal to protein stoichiometry (1:1) for Dfh have been measured using isothermal titration calorimetry (ITC). Under anaerobic conditions with salt present, holo-Dfh is a stable iron-loaded protein monomer. Frataxin prevents reactive oxygen species-induced oxidative damage to DNA when presented with both Fe(II) and H 2O 2. Ferrous iron bound to Dfh is high-spin and held in a partially symmetric Fe-(O/N) 6 coordination environment, as determined by X-ray absorption spectroscopy (XAS). Extended X-ray absorption fine structure (EXAFS) simulations indicate the average Fe-O/N bond length in Dfh is 2.13 A, consistent with a ligand geometry constructed by water and carboxylate oxygens most likely supplied in part by surface-exposed conserved acidic residues located on helix 1 and strand 1 in the structurally characterized frataxin orthologs. The iron-dependent binding affinity ( K d approximately 0.21 microM) and optimal holo-Dfh to Isu monomer stoichiometry (1:1) have also been determined using ITC. Finally, frataxin mediates the delivery of Fe(II) to Isu, promoting Fe-S cluster assembly in vitro. The Dfh-assisted assembly of Fe-S clusters occurs with an observed kinetic rate constant ( k obs) of 0.096 min (-1).     

Axis formation: squint comes into focus

Gene products provided by the mother to the embryo determine the body axes in most animals. A recent study in zebrafish proposes that the TGFss signal Squint is one such factor.     

Mitochondrial retrograde regulation in plants

Plant cells must react to a variety of adverse environmental conditions that they may experience on a regular basis. Part of this response centers around (1) ROS as damaging molecules and signaling molecules; (2) redox status, which can be influenced by ROS production; and (3) availability of metabolites. All of these are also likely to interface with changes in hormone levels [Desikan, R., Hancock, J., Neill, S., 2005. Reactive oxygen species as signalling molecules. In: Smirnoff, N. (ed.), Antioxidants and reactive oxygen species in plants. Blackwell Pub. Ltd., Oxford, pp. 169-196; Kwak, J.M., Nguyen, V., Schroeder, J.I., 2006. The role of reactive oxygen species in hormonal responses. Plant Physiol. 141, 323-329]. Each of these areas can be strongly influenced by changes in mitochondrial function. Such changes trigger altered nuclear gene expression by a poorly understood process of mitochondrial retrograde regulation (MRR), which is likely composed of several distinct signaling pathways. Much of what is known about plant MRR centers around the response to a dysfunctional mtETC and subsequent induction of genes encoding proteins involved in recovery of mitochondrial functions, such as AOX and alternative NAD(P)H dehydrogenases, and genes encoding enzymes aimed at regaining ROS level/redox homeostasis, such as glutathione transferases, catalases, ascorbate peroxidases and superoxide dismutases. However, as evidence of new and interesting targets of MRR emerge, this picture is likely to change and the complexity and importance of MRR in plant responses to stresses and the decision for cells to either recover or switch into programmed cell death mode is likely to become more apparent.     

Myotonic dystrophy: RNA pathogenesis comes into focus

Myotonic dystrophy (DM)--the most common form of muscular dystrophy in adults, affecting 1/8000 individuals--is a dominantly inherited disorder with a peculiar and rare pattern of multisystemic clinical features affecting skeletal muscle, the heart, the eye, and the endocrine system. Two genetic loci have been associated with the DM phenotype: DM1, on chromosome 19, and DM2, on chromosome 3. In 1992, the mutation responsible for DM1 was identified as a CTG expansion located in the 3' untranslated region of the dystrophia myotonica-protein kinase gene (DMPK). How this untranslated CTG expansion causes myotonic dystrophy type 1(DM1) has been controversial. The recent discovery that myotonic dystrophy type 2 (DM2) is caused by an untranslated CCTG expansion, along with other discoveries on DM1 pathogenesis, indicate that the clinical features common to both diseases are caused by a gain-of-function RNA mechanism in which the CUG and CCUG repeats alter cellular function, including alternative splicing of various genes. We discuss the pathogenic mechanisms that have been proposed for the myotonic dystrophies, the clinical and molecular features of DM1 and DM2, and the characterization of murine and cell-culture models that have been generated to better understand these diseases.     

Evidence that yeast frataxin is not an iron storage protein in vivo

Yeast cells deficient in the yeast frataxin homolog (Yfh1p) accumulate iron in their mitochondria. Whether this iron is toxic, however, remains unclear. We showed that large excesses of iron in the growth medium did not inhibit growth and did not decrease cell viability. Increasing the ratio of mitochondrial iron-to-Yfh1p by decreasing the steady-state level of Yfh1p to less than 100 molecules per cell had very few deleterious effects on cell physiology, even though the mitochondrial iron concentration greatly exceeded the iron-binding capacity of Yfh1p in these conditions. Mössbauer spectroscopy and FPLC analyses of whole mitochondria or of isolated mitochondrial matrices showed that the chemical and biochemical forms of the accumulated iron in mitochondria of mutant yeast strains (Δyfh1, Δggc1 and Δssq1) displayed a nearly identical distribution. This was also the case for Δggc1 cells, in which Yfh1p was overproduced. In these mitochondria, most of the iron was insoluble, and the ratio of soluble-to-insoluble iron did not change when the amount of Yfh1p was increased up to 4500 molecules per cell. Our results do not privilege the hypothesis of Yfh1p being an iron storage protein in vivo.     

Hepatic subcellular metabolism of heme from heme-hemopexin: incorporation of iron into ferritin

The subcellular distribution and metabolic fate of [⁵⁹Fe]heme-[¹²⁵I]-labeled hemopexin after receptor-mediated interaction with the liver was examined in the rat. After intravenous injection, [⁵⁹Fe]heme from the complex and ⁵⁹Fe from hepatic catabolism of this heme accumulate in the liver and undergo changes in their subcellular distribution over 2 hours. The amounts of [⁵⁹Fe]heme and particularly of ⁵⁹Fe increase in the cytosol while remaining constant or decreasing in membranous fractions. In contrast, [¹²⁵I]-labeled hemopexin associated with the liver during heme transport is always a small fraction of the dose and is not measurably catabolized under these conditions.Gel filtration of the cytosol showed that ⁵⁹Fe increased linearly with time in a high molecular weight fraction which was identified immunologically as ferritin. We conclude that heme transported by hemopexin is metabolized by the liver and the iron conserved.     

Mitochondrial quality control mediated by PINK1 and PRKN: links to iron metabolism and tumor immunity

Mitochondrial quality control is an essential process required to maintain cellular homeostasis and functions. Mutations of PINK1 and PRKN/PARK2 contribute to the risk of Parkinson disease. Our recent findings indicate that depletion of Pink1 and Prkn promotes pancreatic tumorigenesis in KRAS-driven engineered mouse models. Mechanistically, PINK1- and PRKN-mediated autophagic degradation of mitochondrial iron importers (e.g., SLC25A37 and SLC25A28) suppresses pancreatic tumor growth by attenuating mitochondrial iron accumulation, inflammasome activation, HMGB1 release, and subsequent immune checkpoint expression. Consequently, pharmacological or genetic inhibition of mitochondrial iron-dependent signals prolongs animal survival and reverses pancreatic tumor phenotype in vivo. Thus, PINK1- and PRKN-mediated immunometabolism provides new insights into the tumor microenvironment and could be a suitable target for new pancreatic cancer treatments.     

Mitochondrial metabolism in cancer metastasis

We have recently proposed a new two-compartment model for understanding the Warburg effect in tumor metabolism. In this model, glycolytic stromal cells produce mitochondrial fuels (L-lactate and ketone bodies) that are then transferred to oxidative epithelial cancer cells, driving OXPHOS and mitochondrial metabolism. Thus, stromal catabolism fuels anabolic tumor growth via energy transfer. We have termed this new cancer paradigm the “reverse Warburg effect,” because stromal cells undergo aerobic glycolysis, rather than tumor cells. To assess whether this mechanism also applies during cancer cell metastasis, we analyzed the bioenergetic status of breast cancer lymph node metastases, by employing a series of metabolic protein markers. For this purpose, we used MCT4 to identify glycolytic cells. Similarly, we used TO MM20 and COX staining as markers of mitochondrial mass and OXPHOS activity, respectively. Consistent with the “reverse Warburg effect,” our results indicate that metastatic breast cancer cells amplify oxidative mitochondrial metabolism (OXPHOS) and that adjacent stromal cells are glycolytic and lack detectable mitochondria. Glycolytic stromal cells included cancer-associated fibroblasts, adipocytes and inflammatory cells. Double labeling experiments with glycolytic (MCT4) and oxidative (TO MM20 or COX) markers directly shows that at least two different metabolic compartments co-exist, side-by-side, within primary tumors and their metastases. Since cancer-associated immune cells appeared glycolytic, this observation may also explain how inflammation literally “fuels” tumor progression and metastatic dissemination, by “feeding” mitochondrial metabolism in cancer cells. Finally, MCT4(+) and TO MM20(-) “glycolytic” cancer cells were rarely observed, indicating that the conventional “Warburg effect” does not frequently occur in cancer-positive lymph node metastases.     

Amorphous no longer: the centrosome comes into focus

Recent genetic and biochemical studies have provided new insights into the molecular basis of centrosome-mediated microtubule nucleation. In addition, molecules and mechanisms involved in microtubule severing and stabilization at the centrosome, assembly of proteins onto centrosomes and regulation of centrosome duplication and separation are being defined. Characterization of centrosome function, together with studies implicating centrosomes in tumorigenesis and demonstrating that centrosomes are highly organized, are beginning to bring into focus an organelle once viewed as an 'amorphous cloud'.     

Silicon nutrition potentiates the antioxidant metabolism of rice plants under iron toxicity

Iron toxicity reduces growth of rice plants in acidic lowlands. Silicon nutrition may alleviate many stresses including heavy metal toxicity in plants. In the present study, the ameliorating effects of silicon nutrition on rice (Oryza sativa L.) plants under toxic Fe levels were investigated. Plants were cultivated in greenhouse in hydroponics under different Fe treatments including 10, 50, 100, and 250 mg L^?1 as Fe-EDTA and silicon nutrition including 0 and 1.5 mM sodium silicate. Iron toxicity imposed significant reduction in plant fresh weight, tiller, and leaf number. The activity of catalase, cell wall, and soluble peroxidases, and polyphenol oxidase in shoots decreased due to moderate Fe toxicity (50 and 100 mg L^?1), but increased at greater Fe concentration. Ascorbate peroxidase activity increased in both roots and shoots of Fe-stressed plants. Iron toxicity led to increased tissue hydrogen peroxide and lipid peroxidation. Silicon nutrition improved plant growth under all Fe treatments and alleviated Fe toxicity symptoms, probably due to lower Fe concentration of Si-treated plants. Silicon application could improve the activity of antioxidant enzymes such as catalase, ascorbate peroxidase, and soluble peroxidase under moderate Fe toxicity, which resulted in greater hydrogen peroxide detoxification and declined lipid peroxidation. Thus, silicon nutrition could ameliorate harmful effects of Fe toxicity possibly through reduction of plant Fe concentration and improvement of antioxidant enzyme activity.