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BBA - Molecular Basis of Disease (v.1812, #5)

Editorial Board (pp. i).

Insulin receptor substrate-1 and -2 mediate resistance to glucose-induced caspase-3 activation in human neuroblastoma cells by Stohr Oliver Stöhr; Johann Hahn; Lorna Moll; Uschi Leeser; Susanna Freude; Corinna Bernard; Katharina Schilbach; Andreas Markl; Michael Udelhoven; Wilhelm Krone; Markus Schubert (pp. 573-580).

Hyperglycemia in patients with type 2 diabetes causes multiple neuronal complications, e.g., diabetic polyneuropathy, cognitive decline, and embryonic neural crest defects due to increased apoptosis. Possible mechanisms of neuronal response to increased glucose burden are still a matter of debate. Insulin and insulin-like growth factor-1 (IGF-1) receptor signaling inhibits glucose-induced caspase-3 activation and apoptotic cell death. The insulin receptor substrates (IRS) are intracellular adapter proteins mediating insulin's and IGF-1's intracellular effects. Even though all IRS proteins have similar function and structure, recent data suggest different actions of IRS-1 and IRS-2 in mediating their anti-apoptotic effects in glucose neurotoxicity. We therefore investigated the role of IRS-1/-2 in glucose-induced caspase-3 activation using human neuroblastoma cells. Overexpression of IRS-1 or IRS-2 caused complete resistance to glucose-induced caspase-3 cleavage. Inhibition of PI3-kinase reversed this protective effect of IRS-1 or IRS-2. However, MAP-kinases inhibition had only minor impact. IRS overexpression increased MnSOD abundance as well as BAD phosphorylation while Bim and BAX levels remained unchanged. Since Akt promotes cell survival at least partially via phosphorylation and inhibition of downstream forkhead box-O (FoxO) transcription factors, we generated neuroblastoma cells stably overexpressing a dominant negative mutant of FoxO1 mimicking activation of the insulin/IGF-1 pathway on FoxO-mediated transcription. Using these cells we showed that FoxO1 is not involved in neuronal protection mediated by increased IRS-1/-2 expression. Thus, overexpression of both IRS-1 and IRS-2 induces complete resistance to glucose-induced caspase-3 activation via PI3-kinase mediated BAD phosphorylation and MnSOD expression independent of FoxO1.► Insulin receptor substrate-1 and -2 mediate resistance to glucose-induced caspase-3 activation in human neuroblastoma cells. ► IRS-1/-2 overexpression induces resistance to glucose-induced caspase-3 activation. ► Inhibition of PI3-kinase reverses the protective effect of IRS-1/-2. ► FoxO1 is not involved in neuronal protection mediated by increased IRS-1/-2 expression. ► IRS-1/-2 regulates neuronal MnSOD expression.

Keywords: Glucose; Apoptosis; Insulin receptor substrate; MnSOD; Neuron

Circadian rhythm of adrenal glucocorticoid: Its regulation and clinical implications by Sooyoung Chung; Gi Hoon Son; Kyungjin Kim (pp. 581-591).

Glucocorticoid (GC) is an adrenal steroid hormone that controls a variety of physiological processes such as metabolism, immune response, cardiovascular activity, and brain function. In addition to GC induction in response to stress, even in relatively undisturbed states its circulating level is subjected to a robust daily variation with a peak around the onset of the active period of the day. It has long been believed that the synthesis and secretion of GC are primarily regulated by the hypothalamus–pituitary–adrenal (HPA) neuroendocrine axis. However, recent chronobiological research strongly supports the idea that multiple regulatory mechanisms along with the classical HPA neuroendocrine axis underlie the diurnal rhythm of circulating GC. Most notably, recent studies demonstrate that the molecular circadian clockwork is heavily involved in the daily GC rhythm at multiple levels. The daily GC rhythm is implicated in various human diseases accompanied by abnormal GC levels. Patients with such diseases frequently show a blunted GC rhythmicity and, more importantly, circadian rhythm-related symptoms. In this review, we focus on recent advances in the understanding of the circadian regulation of adrenal GC and its implications in human health and disease.► Current understanding of circadian control of the adrenal gland. ► Multiple regulatory mechanisms underlying the daily rhythm of glucocorticoid (GC). ► Role of the adrenal local clockwork in circadian GC production. ► Importance of GC rhythm in human health and diseases.

Keywords: Glucocorticoid; Adrenal gland; HPA axis; Circadian rhythm; Biological clock

MicroRNAs and the cell cycle by María José Bueno; Marcos Malumbres (pp. 592-601).

The control of cell proliferation by microRNAs (miRNAs) is well established and the alteration of these small, non-coding RNAs may contribute to tumor development by perturbing critical cell cycle regulators. Oncogenic miRNAs may facilitate cell cycle entry and progression by targeting CDK inhibitors or transcriptional repressors of the retinoblastoma family. On the other hand, tumor suppressor miRNAs induce cell cycle arrest by downregulating multiple components of the cell cycle machinery. Recent data also suggest that miRNAs act co-ordinately with transcriptional factors involved in cell cycle regulation such as c-MYC, E2F or p53. These miRNAs not only can potentiate the function of these factors but they may also limit the excessive translation of cell cycle proteins upon mitogenic or oncogenic stimuli to protect cells from replicative stress. The implications of these regulatory networks in cell proliferation and human disease are discussed.► Multiple cell-cycle regulators are controlled by miRNAs. ► miRNA expression is also controlled by cell-cycle-dependent transcription factors. ► miRNAs may help to prevent replicative stress. ► miRNAs and transcription factors regulate each other through multiple networks. ► The control of the cell cycle by miRNAs has relevant consequences in tumor development.

Keywords: E2F; Cancer; Cell cycle; Cyclin; Cyclin-dependent kinase; microRNA; Mitosis; pRB; Transcription

Heterotetrameric forms of human phenylalanine hydroxylase: Co-expression of wild-type and mutant forms in a bicistronic system by João Leandro; Paula Leandro; Torgeir Flatmark (pp. 602-612).

Hybrid forms of human phenylalanine hydroxylase (hPAH) mutants have been found to present catalytic activities lower than predicted from the individual recombinant forms, indicating that interallelic complementation could be a major determinant of the metabolic phenotype of compound heterozygous phenylketonuric (PKU) patients. To provide a molecular explanation for interallelic complementation we have here developed a bicistronic expression system and a purification strategy to obtain isolated hPAH heteromeric forms. On co-expression of WT-hPAH (~50% tetramer; ~10% dimer) and the N- and C-terminally truncated form ΔN102/ΔC24-hPAH (~80% dimer) no heterodimers were recovered. Moreover, by co-expression of WT-hPAH and the N-terminally truncated form ΔN102-hPAH (~95% tetramer), heterotetramers, as a result of an assembly of two different homodimers, were isolated. The recovered (WT)/(ΔN102)-hPAH heterotetramers revealed a catalytic activity deviating significantly from that calculated by averaging the respective recombinant homotetrameric forms. The heterotetramer assembly also results in conformational changes in the WT-hPAH protomer, as detected by trypsin limited proteolysis. The finding that the presence of two homodimers with different kinetic parameters influences the properties of the resulting heterotetrameric protein indicates that the dimers exhibit interactions which are transmitted across the assembled tetramer. The bicistronic expression system developed here allowed the isolation of hybrid forms that exhibit negative interallelic complementation, and may represent a model system for studying the molecular pathogenic mechanisms of PAH gene mutations in compound heterozygous PKU patients, providing the rationale to understand the observed inconsistencies both in genotype/phenotype correlations and in the response to BH₄ supplementation.►A bicistronic expression system allowed the isolation of individual hPAH hybrid forms. ►No heterodimeric forms were isolated on co-expression of WT/truncated mutant dimers. ►Heterotetrameric forms were isolated on co-expression of WT/truncated mutant tetramers. ►(WT)/(ΔN102)-hPAH heterotetramer reveals negative interallelic complementation. ►Conformational changes occur in the WT protomer of (WT)/(ΔN102)-hPAH heterotetramer.

Keywords: Abbreviations; 6His; hexahistidyl tag; BH; ₄; (6; R; )-; l; -; erythro; -5,6,7,8-tetrahydrobiopterin; hPAH; human phenylalanine hydroxylase; IC; interallelic complementation; IPTG; isopropyl-; β; -; d; -thio-galactoside; l; -Phe; l; -phenylalanine; MBP; maltose binding protein; PKU; phenylketonuria; PRA; predicted residual activity; WT; wild-typePhenylketonuria; Human phenylalanine hydroxylase; Interallelic complementation; Bicistronic system; Hybrid protein; Heteroallelic protein

Ethanol exposure modulates hepatic S-adenosylmethionine and S-adenosylhomocysteine levels in the isolated perfused rat liver through changes in the redox state of the NADH/NAD⁺ system by Walter H. Watson; Zhenyuan Song; Irina A. Kirpich; Ion V. Deaciuc; Theresa Chen; Craig J. McClain (pp. 613-618).

Methionine metabolism is disrupted in patients with alcoholic liver disease, resulting in altered hepatic concentrations of S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), and other metabolites. The present study tested the hypothesis that reductive stress mediates the effects of ethanol on liver methionine metabolism. Isolated rat livers were perfused with ethanol or propanol to induce a reductive stress by increasing the NADH/NAD⁺ ratio, and the concentrations of SAM and SAH in the liver tissue were determined by high-performance liquid chromatography. The increase in the NADH/NAD⁺ ratio induced by ethanol or propanol was associated with a marked decrease in SAM and an increase in SAH liver content. 4-Methylpyrazole, an inhibitor the NAD⁺-dependent enzyme alcohol dehydrogenase, blocked the increase in the NADH/NAD⁺ ratio and prevented the alterations in SAM and SAH. Similarly, co-infusion of pyruvate, which is metabolized by the NADH-dependent enzyme lactate dehydrogenase, restored the NADH/NAD⁺ ratio and normalized SAM and SAH levels. The data establish an initial link between the effects of ethanol on the NADH/NAD⁺ redox couple and the effects of ethanol on methionine metabolism in the liver.►Decreased hepatic SAM and increased SAH are hallmarks of alcoholic liver disease. ►A decrease in the SAM/SAH ratio inhibits numerous transmethylation reactions. ►The mechanisms by which ethanol affects SAM and SAH are unclear. ►We have identified the NADH/NAD⁺ system as a major regulator of SAM and SAH levels.

Keywords: Abbreviations; SAM; S-adenosylmethionine; MAT; methionine adenosyltransferase; SAHH; S-adenosylhomocysteine hydrolase; G; +; M; glucose; +; methionine; 4-MP; 4-methylpyrazole; Pyr; pyruvate; EtOH; ethanol; Prop; propanolPerfused rat liver; Alcohol; Redox state; Methionine metabolism; Reductive stress

Clinical and molecular characterization of five patients with succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency by Toshiyuki Fukao; Jörn Oliver Sass; Petri Kursula; Eva Thimm; Udo Wendel; Can Ficicioglu; Kamel Monastiri; Nathalie Guffon; Baric Ivo Barić; Marie-therese Zabot; Naomi Kondo (pp. 619-624).

Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency is an inborn error of ketone body metabolism and causes episodic ketoacidosis. We report clinical and molecular analyses of 5 patients with SCOT deficiency. Patients GS07, GS13, and GS14 are homozygotes of S405P, L327P, and R468C, respectively. GS17 and GS18 are compound heterozygotes for S226N and A215V, and V404F and E273X, respectively. These mutations have not been reported previously. Missense mutations were further characterized by transient expression analysis of mutant cDNAs. Among 6 missense mutations, mutants L327P, R468C, and A215V retained some residual activities and their mutant proteins were detected in immunoblot analysis following expression at 37°C. They were more stable at 30°C than 37°C, indicating their temperature sensitive character. The R468C mutant is a distinct temperature sensitive mutant which retained 12% and 51% of wild-type residual activities at 37 and 30°C, respectively. The S226N mutant protein was detected but retained no residual activity. Effects of missense mutations were predicted from the tertiary structure of the SCOT molecule. Main effects of these mutations were destabilization of SCOT molecules, and some of them also affected catalytic activity. Among 5 patients, GS07 and GS18 had null mutations in both alleles and the other three patients retained some residual SCOT activities. All 5 developed a first severe ketoacidotic crisis with blood gas pH <7.1, and experienced multiple ketoacidotic decompensations (two of them had seven such episodes). In general, the outcome was good even following multiple ketoacidotic events. Permanent ketosis or ketonuria is considered a pathognomonic feature of SCOT deficiency. However, this condition depends not only on residual activity but also on environmental factors.►Five patients with SCOT deficiency were reported. ►All developed multiple severe ketoacidosis but their outcome was good. ►Six missense mutations were analyzed by expression analysis of mutant cDNAs. ►Their effects were viewed from the tertiary structure of SCOT molecule. ►Main effects were destabilization of SCOT molecules; some also affected catalytic activity.

Keywords: Ketoacidosis; OXCT; Mutation; SCOT; Succinyl-CoA:3-ketoacid CoA transferase; Structure–function analysis

The interplay between SUCLA2, SUCLG2, and mitochondrial DNA depletion by Chaya Miller; Liya Wang; Elsebet Ostergaard; Phyllis Dan; Ann Saada (pp. 625-629).

SUCLA2-related mitochondrial DNA (mtDNA) depletion syndrome is a result of mutations in the β subunit of the ADP-dependent isoform of the Krebs cycle succinyl-CoA synthase (SCS). The mechanism of tissue specificity and mtDNA depletion is elusive but complementation by the GDP-dependent isoform encoded by SUCLG2, and the association with mitochondrial nucleoside diphosphate kinase (NDPK), is a plausible link.We have investigated this relationship by studying SUCLA2 deficient fibroblasts derived from patients and detected normal mtDNA content and normal NDPK activity. However, knockdown of SUCLG2 by shRNA in both patient and control fibroblasts resulted in a significant decrease in mtDNA amount, decreased NDPK and cytochrome c oxidase activities, and a marked growth impairment. This suggests that, SUCLG2, to a higher degree than SUCLA2, is crucial for mtDNA maintenance and that mitochondrial NDPK is involved. Although results pertain to a cell culture system, the findings might explain the pathomechanism and tissue specificity in mtDNA depletion caused by defective SUCLA2.►SUCLA2 deficient patient cells have normal mtDNA content and mitochondrial NDPK activity. ►Knockdown of SUCLG2 in both normal and SUCLA2 deficient cells causes decreased mtDNA content, decreased cytochrome c oxidase and mitochondrial NDPK activities, and impaired growth. ►The findings explain the pathomechanism and tissue specificity in SUCLA2-related mtDNA depletion syndrome.

Keywords: mtDNA depletion; Succinyl-CoA synthase; SUCLA2; SUCLG2; NDPK; Mitochondrial respiratory chain

Analysis of the mitochondrial proteome in multiple sclerosis cortex by Laurie Broadwater; Ashish Pandit; Robert Clements; Sausan Azzam; Jonathan Vadnal; Michael Sulak; V. Wee Yong; Ernest J. Freeman; Roger B. Gregory; Jennifer McDonough (pp. 630-641).

Mitochondrial dysfunction has been proposed to play a role in the neuropathology of multiple sclerosis (MS). Previously, we reported significant alterations in the transcription of nuclear-encoded electron transport chain genes in MS and confirmed translational alterations for components of Complexes I and III that resulted in reductions in their activity. To more thoroughly and efficiently elucidate potential alterations in the expression of mitochondrial and related proteins, we have characterized the mitochondrial proteome in postmortem MS and control cortex using Surface-Enhanced Laser Desorption Ionization Time of Flight Mass Spectrometry (SELDI-TOF-MS). Using principal component analysis (PCA) and hierarchical clustering techniques we were able to analyze the differential patterns of SELDI-TOF spectra to reveal clusters of peaks which distinguished MS from control samples. Four proteins in particular were responsible for distinguishing disease from control. Peptide fingerprint mapping unambiguously identified these differentially expressed proteins. Three proteins identified are involved in respiration including cytochrome c oxidase subunit 5b (COX5b), the brain specific isozyme of creatine kinase, and hemoglobin β-chain. The fourth protein identified was myelin basic protein (MBP). We then investigated whether these alterations were consistent in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. We found that MBP was similarly altered in EAE but the respiratory proteins were not. These data indicate that while the EAE mouse model may mimic aspects of MS neuropathology which result from inflammatory demyelinating events, there is another distinct mechanism involved in mitochondrial dysfunction in gray matter in MS which is not modeled in EAE.►Analysis of the mitochondrial proteome in MS reveals altered respiratory proteins. ►Nitrated respiratory proteins in MS are consistent with the EAE mouse model. ►Altered expression of respiratory proteins in MS is not modeled in EAE. ►A distinct mechanism is involved in gray matter mitochondrial pathology in MS.

Keywords: Abbreviations; SELDI-TOF-MS; Surface-Enhanced Laser Desorption Ionization Time of Flight Mass Spectrometry; PCA; principal component analysis; NAGM; normal appearing gray matter; MS; multiple sclerosis; EAE; experimental autoimmune encephalomyelitis; MBP; myelin basic protein; COX5b; cytochrome; c; oxidase subunit 5bMitochondrial proteomics; Multiple sclerosis; Experimental autoimmune encephalomyelitis

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BBA - Molecular Basis of Disease (v.1812, #5)