Tamás Janáky

Biogenesis of cytosolic ribosomes requires the essential iron-sulphur protein Rli1p and mitochondria.

Mitochondria perform a central function in the biogenesis of cellular iron–sulphur (Fe/S) proteins. It is unknown to date why this biosynthetic pathway is indispensable for life, the more so as no essential mitochondrial Fe/S proteins are known. Here, we show that the soluble ATP‐binding cassette (ABC) protein Rli1p carries N‐terminal Fe/S clusters that require the mitochondrial and cytosolic Fe/S protein biogenesis machineries for assembly. Mutations in critical cysteine residues of Rli1p abolish association with Fe/S clusters and lead to loss of cell viability. Hence, the essential character of Fe/S clusters in Rli1p explains the indispensable character of mitochondria in eukaryotes. We further report that Rli1p is associated with ribosomes and with Hcr1p, a protein involved in rRNA processing and translation initiation. Depletion of Rli1p causes a nuclear export defect of the small and large ribosomal subunits and subsequently a translational arrest. Thus, ribosome biogenesis and function are intimately linked to the crucial role of mitochondria in the maturation of the essential Fe/S protein Rli1p.

Cholesterol Potentiates ABCG2 Activity in a Heterologous Expression System: Improved in Vitro Model to Study Function of Human ABCG2

ABCG2, a transporter of the ATP-binding cassette family, is known to play a prominent role in the absorption, distribution, metabolism, and excretion of xenobiotics. Drug-transporter interactions are commonly screened by high-throughput systems using transfected insect and/or human cell lines. The determination of ABCG2-ATPase activity is one method to identify ABCG2 substrate and inhibitors. We demonstrate that the ATPase activities of the human ABCG2 transfected Sf9 cell membranes (MXR-Sf9) and ABCG2-overexpressing human cell membranes (MXR-M) differ. Variation due to disparity in the glycosylation level of the protein had no effect on the transporter. The influence of cholesterol on ABCG2-ATPase activity was investigated because the lipid compositions of insect and human cells are largely different from each other. Differences in cholesterol content, shown by cholesterol loading and depletion experiments, conferred the difference in stimulation of basal ABCG2-ATPase of the two cell membranes. Basal ABCG2-ATPase activity could be stimulated by sulfasalazine, prazosin, and topotecan, known substrates of ABCG2 in cholesterol-loaded MXR-Sf9 and MXR-M cell membranes. In contrast, ABCG2-ATPase could not be stimulated in MXR-Sf9 or in cholesterol-depleted MXR-M membranes. Moreover, cholesterol loading significantly improved the drug transport into inside-out membrane vesicles prepared from MXR-Sf9 cells. MXR-M and cholesterol-loaded MXR-Sf9 cell membranes displayed similar ABCG2-ATPase activity and vesicular transport. Our study indicates an essential role of membrane cholesterol for the function of ABCG2.

Decreased serum and red blood cell kynurenic acid levels in Alzheimer's disease

Kynurenine aminotransferases (KAT I and KAT II) are responsible for the transamination of kynurenine (KYN) to form kynurenic acid (KYNA), an excitatory amino acid receptor antagonist. Since these members of the kynurenine pathway (KP) are proposed to be involved in the pathogenesis of Alzheimers dementia (AD), the activities of these enzymes and the levels of these metabolites were measured in the plasma and red blood cells (RBCs) of AD and control subjects together with the inheritance of the apolipoprotein (APOE) epsilon4 allele. KYNA levels were significantly decreased both in the plasma and in the RBCs in AD, but the levels of KYN and the activities of KAT I and KAT II remained unchanged. No association has been found with the possession of the epsilon4 allele. These findings indicate an altered peripheral KP in AD regardless of the APOE status of the probands.

Kynurenine metabolism in multiple sclerosis

Objective –  Excitatory amino acid receptors are involved in the normal physiology of the brain, and may play a role in the pathogenesis of neurological disorders such as Huntingtons disease, Parkinsons disease, amyotrophic lateral sclerosis, etc. It has been demonstrated that the blockade of one of these receptors ameliorates the symptoms of experimental allergic encephalomyelitis, an animal model of multiple sclerosis (MS). In a recent study, a decreased level of kynurenic acid was found in the cerebrospinal fluid of patients with MS. The only known endogenous excitotoxin receptor antagonist is the tryptophan metabolite kynurenic acid. Another metabolite is quinolinic acid, which exerts different action: it is an excitotoxin receptor agonist. The ratio of these two metabolites is determined by the activities of kynurenine aminotransferase I and II (KAT I and KAT II). In this study, we measured the activities of these enzymes and the concentration of kynurenic acid in the red blood cells (RBC) and in the plasma of patients with MS. KAT activities were detected both in the RBC and in the plasma. As compared with the control subjects, the KAT I and KAT II activities were significantly higher in the RBC of the patients. The concentration of kynurenic acid is elevated in the plasma of MS patients, and there is a tendency to an elevation in the RBC. These changes may indicate a compensatory protective mechanism against excitatory neurotoxic effects. Our data demonstrate the involvement of the kynurenine system in the pathogenesis of MS, which may predict a novel therapeutic intervention.

Lovastatin interferes with the infarct size-limiting effect of ischemic preconditioning and postconditioning in rat hearts

Statins have been shown to be cardioprotective; however, their interaction with endogenous cardioprotection by ischemic preconditioning and postconditioning is not known. In the present study, we examined if acute and chronic administration of the 3-hydroxy-3-methylglutaryl CoA reductase inhibitor lovastatin affected the infarct size-limiting effect of ischemic preconditioning and postconditioning in rat hearts. Wistar rats were randomly assigned to the following three groups: 1) vehicle (1% methylcellulose per os for 12 days), 2) chronic lovastatin (15 mg.kg(-1).day(-1) per os for 12 days), and 3) acute lovastatin (1% methylcellulose per os for 12 days and 50 micromol/l lovastatin in the perfusate). Hearts isolated from the three groups were either subjected to a nonconditioning (aerobic perfusion followed by 30-min coronary occlusion and 120-min reperfusion, i.e., test ischemia-reperfusion), preconditioning (three intermittent periods of 5-min ischemia-reperfusion cycles before test ischemia-reperfusion), or postconditioning (six cycles of 10-s ischemia-reperfusion after test ischemia) perfusion protocol. Preconditioning and postconditioning significantly decreased infarct size in vehicle-treated hearts. However, preconditioning failed to decrease infarct size in acute lovastatin-treated hearts, but the effect of postconditioning remained unchanged. Chronic lovastatin treatment abolished postconditioning but not preconditioning; however, it decreased infarct size in the nonconditioned group. Myocardial levels of coenzyme Q9 were decreased in both acute and chronic lovastatin-treated rats. Western blot analysis revealed that both acute and chronic lovastatin treatment attenuated the phoshorylation of Akt; however, acute but not chronic lovastatin treatment increased the phosphorylation of p42 MAPK/ERK. We conclude that, although lovastatin may lead to cardioprotection, it interferes with the mechanisms of cardiac adaptation to ischemic stress.

A mouse model of anxiety molecularly characterized by altered protein networks in the brain proteome

Recently, several attempts have been made to describe changes related to certain anxiety states in the proteome of experimental animal models. However, these studies are restricted by limitations regarding the number and correct identification of separated proteins. Moreover, the application of a systems biology approach to discover the molecular mechanisms of anxiety requires genetically homogenous inbred animal models. Therefore, we developed a novel mouse model of anxiety using a combination of crossbreeding (inbred for 35 generations) and behavioral selection. We found significant changes in 82 proteins in the total brain proteome compared to the control proteome. Thirty-four of these proteins had been previously identified in other anxiety, depression or repeated psychosocial stress studies. The identified proteins are associated with different cellular functions, including synaptic transmission, metabolism, proteolysis, protein biosynthesis and folding, cytoskeletal proteins, brain development and neurogenesis, oxidative stress, signal transduction. Our proteomics data suggest that alterations in serotonin receptor-associated proteins, in the carbohydrate metabolism, in the cellular redox system and in synaptic docking are all involved in anxiety.

P-glycoprotein inhibition by membrane cholesterol modulation

P-glycoprotein (Pgp) is a transmembrane protein that actively exports lipophilic chemotherapeutics from the cells causing multidrug resistance. Pgp molecules are partially localized in TX-100-resistant rafts, and the activity of the transporter is highly sensitive to the presence of cholesterol. To better understand these relationships, the influence of membrane cholesterol content on Pgp function, as measured via calcein accumulation, was studied in correlation with changes elicited in membrane structure. Membrane cholesterol was modulated by heptakis(2,6-di-O-methyl)-beta-cyclodextrin (DIMEB) and the cholesterol inclusion complex of DIMEB (Chol-DIMEB). Changes in membrane cholesterol level were reflected by alterations in the overall lipid packing as measured by Merocyanine 540 (MC540) staining and were also accompanied by changes in the raft association of the pump. DIMEB and Chol-DIMEB treatments have also lead to increased permeability of the cell membrane in both directions, raising the possibility that the effects on pumping efficiency reflect leakage of ATP also from the non-permeabilized cells. However, the treatments did not influence the intracellular ATP levels of the non-permeabilized cells. Our data suggest that Pgp inhibition by cyclodextrin treatments arises through modulation of its membrane microenvironment, rather than as a result of concomitant cytotoxicity.

ABCG2 (breast cancer resistance protein/mitoxantrone resistance-associated protein) ATPase assay: a useful tool to detect drug-transporter interactions.

The ATPase assay using membrane preparations from recombinant baculovirus-infected Spodoptera frugiperda ovarian (Sf9) cells is widely used to detect the interaction of compounds with different ATP-binding cassette transporters. However, Sf9 membrane preparations containing the wild-type ABCG2 transporter show an elevated baseline vanadate-sensitive ATPase activity, which cannot be further stimulated by substrates of ABCG2. Therefore, this assay system cannot be used for the detection of ABCG2 substrates. To overcome this difficulty we 1) purified membranes from a selected human cell line expressing wild-type ABCG2, and 2) inhibited the baseline ATPase activity with different inhibitors. In our modified assay, ABCG2 substrates were able to stimulate the baseline ATPase activity of ABCG2 expressed in membranes of human cells. Furthermore, using the specific ABCG2 inhibitors Ko143 or Ko134 allowed us to suppress the baseline vanadate-sensitive ATPase activity. Substrates of ABCG2 could stimulate this suppressed baseline ATPase, resulting in a better signal-to-background ratio and a robust assay to detect substrates of the ABCG2 transporter. The ATPase assay and the direct vesicular transport measurements for estrone-3-sulfate were in good accordance.

Kynurenine metabolism in plasma and in red blood cells in Parkinson's disease

Substantial evidence indicates that neuroactive kynurenine metabolites play a role in the normal physiology of the human brain, and are involved in the pathology of neurodegenerative disorders such as Parkinsons disease (PD). A sidearm product of the pathway, kynurenic acid (KYNA), which is synthesized by the irreversible transamination of kynurenine (KYN) by kynurenine aminotransferases (KAT I and KAT II), is an excitatory amino acid receptor antagonist. In the present study we measured the level of KYNA and the activities of the biosynthetic enzyme isoforms KAT I and KAT II in the plasma and in the erythrocytes (RBC) of 19 PD patients and 17 age-matched controls. The KAT I and KAT II activities were significantly lower in the plasma of PD patients, followed by a tendency to a decrease in plasma KYNA. An elevated KYNA level correlated with a significant increase in KAT II activity in the RBC of PD patients. These data support the contribution of an altered KYNA metabolism in the RBC to the pathogenesis of PD. The increased activity of KAT II in correlation with the elevated KYNA level in the RBC may mediate a consecutive protective response against excitatory neurotoxic effects.

Kynurenine administered together with probenecid markedly inhibits pentylenetetrazol-induced seizures. An electrophysiological and behavioural study

The kynurenine pathway converts tryptophan into various compounds, including l-kynurenine, which in turn can be converted to the excitatory amino acid receptor antagonist kynurenic acid, which may therefore serve as a protective agent in such neurological disorders as epileptic seizures. Kynurenic acid, however, has a very limited ability to cross the blood-brain barrier, whereas kynurenine passes the barrier easily. In this study, we tested the hypothesis that kynurenine administered systemically together with probenecid, which inhibits kynurenic acid excretion from the cerebrospinal fluid, results in an increased level of kynurenic acid in the brain that is sufficiently high to provide protection against the development of pentylentetrazol-induced epileptic seizures. CA3 stimulation-evoked population spike activity was recorded from the pyramidal layer of area CA1 of the rat hippocampus, and in another series of behavioural experiments, water maze and open-field studies were carried out to test the presumed protective effect of kynurenine + probenecid pre-treatment against pentylenetetrazol-induced seizures. This study has furnished the first electrophysiological proof that systemic kynurenine (300 mg/kg, i.p.) and probenecid (200 mg/kg, i.p.) administration protects against pentylenetetrazol-induced (60 mg/kg, i.p.) epileptic seizures.