BBA - Molecular Basis of Disease (v.1782, #5)
Editorial Board (ii).
Differential metabolic consequences of fumarate hydratase and respiratory chain defects by Nuno Raimundo; Jouni Ahtinen; Ksenija Fumić; Ivo Barić; Anne M. Remes; Risto Renkonen; Risto Lapatto; Anu Suomalainen (287-294).
Defects of the oxidative ATP production pathway lead to an amazing variety of disease phenotypes, ranging from childhood encephalomyopathies to hereditary tumor formation. A key enzyme of tricarboxylic cycle, fumarate hydratase (FH), is involved in encephalopathies, but also in leiomyoma formation, and occasionally also in various types of cancer. MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) and NARP (neuropathy ataxia retinitis pigmentosa) are progressive neurological disorders, caused by mitochondrial DNA mutations and respiratory chain (RC) deficiency. These diseases lead to disability and premature death, but not to tumorigenesis. We studied the cellular consequences of FH and RC deficiencies, aiming to identify general responses to energy metabolism defect and those specific for FH-deficiency, suggestively connected to tumorigenesis. Unlike in RC deficiency, the FH-deficient diploid human fibroblasts showed no signs of oxidative stress, but had a reduced redox state with high glutathione levels. The cytoplasmic FH isoform, previously described, but with an unknown function, was completely lacking in all FH-deficient lines. Fumarate was increased in two of our FH-lines, but accumulation of HIF-1α was not detected. Glycolysis was induced in both MELAS and in FH-deficiency. Accumulation of fumarate in primary fibroblasts did not activate a hypoxia response, suggesting that hypoxia activation due to fumarate accumulation may be a tissue-specific response. The lack of cytoplasmic form of FH and the reduced redox environment were typical for all FH-mutant lines, and their role in FH-related tumorigenesis requires further attention.
Keywords: Mitochondria; Fumarate hydratase; Redox state; Respiratory chain defect;
High glucose induction of DNA-binding activity of the transcription factor NFκB in patients with diabetic nephropathy by Bingmei Yang; Andrea Hodgkinson; Peter J. Oates; Beverley A. Millward; Andrew G. Demaine (295-302).
The aim of this study was to investigate whether high glucose induces aldose reductase (AKR1B1) expression through NFκB, which may contribute to the pathogenesis of diabetic nephropathy. 34 Caucasoid patients with type 1 diabetes were recruited; 20 nephropaths and 14 long-term uncomplicated subjects. Peripheral blood mononuclear cells (PBMCs) were cultured under normal or high glucose (25 mmol/l of d-glucose) with or without an aldose reductase inhibitor (ARI). High glucose increased NFκB binding activities in the PBMCs from nephropaths compared to the uncomplicated subjects (1.77 ± 0.22 vs. 1.16 ± 0.04, p = 0.02). ARI induced a substantially greater decrease of NFκB binding activities in the nephropaths compared to the uncomplicated subjects (0.58 ± 0.06 vs. 0.79 ± 0.06, p = 0.032). AKR1B1 protein levels in the nephropaths were increased under high glucose conditions and decreased in the presence of an ARI, whilst the silencing of the NFκB p65 gene in vitro reduced the transcriptional activities of AKR1B1 in luciferase assays. These results show that NFκB induces AKR1B1expression under high glucose conditions, and the pattern of expression differs between nephropaths and the uncomplicated subjects.
Keywords: Nuclear factor kappa B; Diabetic nephropathy; Aldose reductase; Aldose reductase inhibitor; siRNA;
Cataract mutation P20S of αB-crystallin impairs chaperone activity of αA-crystallin and induces apoptosis of human lens epithelial cells by Hui Li; Chang Li; Qiulun Lu; Ting Su; Tie Ke; David Wan-Cheng Li; Mingxiong Yuan; Jingyu Liu; Xiang Ren; Zhihong Zhang; Shaoqiong Zeng; Qing K. Wang; Mugen Liu (303-309).
Cataract is a common cause of childhood blindness worldwide. α-crystallin, which is comprised of two homologous subunits, αA- and αB-crystallin, plays a key role in the maintenance of lens transparency. Recently, we have identified a missense mutation in αB-crystallin that changes the proline residue at codon 20 to a serine residue (P20S) in a large Chinese family with autosomal dominant posterior polar congenital cataract. To explore the molecular mechanism by which the P20S mutation causes cataract, we examined the quaternary structure, subunit exchange and chaperone activity of the reconstituted heteroaggregates of α-crystallins containing wild type (WT) αA in combination with either WT-αB- or mutant αB-crystallin, respectively. Compared with heteroaggregates of WT-αA and WT-αB, heteroaggregates containing WT-αA and mutant αB showed nearly the same molecular mass, but the subunit-exchange rate and chaperone activity were decreased markedly. In human lens epithelial cells, unlike WT-αB-crystallin, the P20S mutant protein showed abnormal nuclear localization, and unusual ability to trigger apoptosis. These results suggest that the changes in the structure and function of the α-crystallin complex and cytotoxicity are vital factors in the pathogenesis of congenital cataract linked to the P20S mutation in the αB-crystallin.
Keywords: Cataract; Crystallin αA and αB; Mutation; Apoptosis; Chaperone activity; Lens transparency;
Influence of PDZK1 on lipoprotein metabolism and atherosclerosis by Olivier Kocher; Ayce Yesilaltay; Ching-Hung Shen; Songwen Zhang; Kathleen Daniels; Rinku Pal; Jianzhu Chen; Monty Krieger (310-316).
PDZK1 is a scaffold protein containing four PDZ protein interaction domains, which bind to the carboxy termini of a number of membrane transporter proteins, including ion channels (e.g., CFTR) and cell surface receptors. One of these, the HDL receptor, scavenger receptor class B type I (SR-BI), exhibits a striking, tissue-specific dependence on PDZK1 for its expression and activity. In PDZK1 knockout (KO) mice there is a marked reduction of SR-BI protein expression (~ 95%) in the liver, but not in steroidogenic tissues or, as we show in this report, in bone marrow- or spleen-derived macrophages, or lung-derived endothelial cells. Because of hepatic SR-BI deficiency, PDZK1 KO mice exhibit dyslipidemia characterized by elevated plasma cholesterol carried in abnormally large HDL particles. Here, we show that inactivation of the PDZK1 gene promotes the development of aortic root atherosclerosis in apolipoprotein E (apoE) KO mice fed with a high fat/high cholesterol diet. However, unlike complete SR-BI-deficiency in SR-BI/apoE double KO mice, PDZK1 deficiency in PDZK1/apoE double knockout mice did not result in development of occlusive coronary artery disease or myocardial infarction, presumably because of their residual expression of SR-BI. These findings demonstrate that deficiency of an adaptor protein essential for normal expression of a lipoprotein receptor promotes atherosclerosis in a murine model. They also define PDZK1 as a member of the family of proteins that is instrumental in preventing cardiovascular disease by maintaining normal lipoprotein metabolism.
Keywords: Atherosclerosis; PDZ domains; HDL; Cholesterol; PDZK1; SR-BI;
The impact of mitochondrial tRNA mutations on the amount of ATP synthase differs in the brain compared to other tissues by Daniela Fornuskova; Olga Brantova; Marketa Tesarova; Lukas Stiburek; Tomas Honzik; Laszlo Wenchich; Evzenie Tietzeova; Hana Hansikova; Jiri Zeman (317-325).
The impact of point mutations in mitochondrial tRNA genes on the amount and stability of respiratory chain complexes and ATP synthase (OXPHOS) has been broadly characterized in cultured skin fibroblasts, skeletal muscle samples, and mitochondrial cybrids. However, less is known about how these mutations affect other tissues, especially the brain. We have compared OXPHOS protein deficiency patterns in skeletal muscle mitochondria of patients with Leigh (8363G>A), MERRF (8344A>G), and MELAS (3243A>G) syndromes. Both mutations that affect mt-tRNALys (8363G>A, 8344A>G) resulted in severe combined deficiency of complexes I and IV, compared to an isolated severe defect of complex I in the 3243A>G sample (mt-tRNALeu(UUR)). Furthermore, we compared obtained patterns with those found in the heart, frontal cortex, and liver of 8363G>A and 3243A>G patients. In the frontal cortex mitochondria of both patients, the patterns of OXPHOS deficiencies differed substantially from those observed in other tissues, and this difference was particularly striking for ATP synthase. Surprisingly, in the frontal cortex of the 3243A>G patient, whose ATP synthase level was below the detection limit, the assembly of complex IV, as inferred from 2D-PAGE immunoblotting, appeared to be hindered by some factor other than the availability of mtDNA-encoded subunits.
Keywords: Brain; COX — cytochrome c oxidase; Leigh syndrome; MELAS syndrome; MERRF syndrome; Tissue specificity;
Alzheimer's disease protein Aβ1–42 does not disrupt isolated synaptic vesicles by Peter B. Allen; Daniel T. Chiu (326-334).
Synaptic vesicles are central to neurotransmission and cognition. Studies of the Alzheimer's disease (AD) associated peptide, amyloid beta (Aβ), suggest that it has the potential to non-specifically solubilize or permeabilize membranes and that it has detergent and pore-forming properties. Damage to the membrane or integrity of synaptic vesicles could compromise its function. We test the hypothesis that the intact synaptic vesicle is a direct site of attack by Aβ1–42 in AD pathology by examining the properties of individual isolated vesicles exposed to Aβ1–42. In particular, we compared the rate of leakage of dye molecules from synaptic vesicles, the rate of proton permeation across the membrane of the vesicle, and the rate of active proton transport into the vesicle interior in the presence and absence of Aβ1–42. From these experiments, we conclude that isolated synaptic vesicles are not disrupted by Aβ1–42.
Keywords: Amyloid beta; Synaptic vesicle; Alzheimer's disease; Total internal reflection fluorescence; Integrity; Leakage;
Subcellular localization of prion proteins and the 37 kDa/67 kDa laminin receptor fused to fluorescent proteins by Daphne Nikles; Karen Vana; Sabine Gauczynski; Heike Knetsch; Heike Ludewigs; Stefan Weiss (335-340).
The 37 kDa/67 kDa laminin receptor LRP/LR acts as a receptor for both PrPc and PrPSc at the cell surface. Here, we further analyzed the subcellular localization of fluorescent labeled prion protein (PrP) and laminin receptor (LRP/LR) molecules. We show that EGFP-PrP is localized at the cell surface and in a perinuclear compartment, respectively. In contrast, a DsRed-ΔSP-PrP mutant lacking the signal peptide is almost exclusively found in the nucleus but does not colocalize with heterochromatin. Interestingly, LRP-DsRed efficiently colocalizes with EGFP-PrP in the perinuclear compartment and LRP-ECFP partly colocalizes with DsRed-ΔSP-PrP in the nucleus, respectively. We conclude that the interactions of PrP and LRP/LR are not restricted to the cell surface but occur also in intracellular compartments suggesting a putative role of LRP/LR in the trafficking of PrP molecules.
Keywords: Prion protein; PrP; Laminin receptor; LRP/LR; Subcellular localization; Cancer; Heterochromatin;
Dietary fructose induces a wide range of genes with distinct shift in carbohydrate and lipid metabolism in fed and fasted rat liver by Hyun-Young Koo; Matthew A. Wallig; Byung Hong Chung; Takayuki Y. Nara; B.H. Simon Cho; Manabu T. Nakamura (341-348).
Dietary fructose has been suspected to contribute to development of metabolic syndrome. However, underlying mechanisms of fructose effects are not well characterized. We investigated metabolic outcomes and hepatic expression of key regulatory genes upon fructose feeding under well defined conditions. Rats were fed a 63% (w/w) glucose or fructose diet for 4 h/day for 2 weeks, and were killed after feeding or 24-hour fasting. Liver glycogen was higher in the fructose-fed rats, indicating robust conversion of fructose to glycogen through gluconeogenesis despite simultaneous induction of genes for de novo lipogenesis and increased liver triglycerides. Fructose feeding increased mRNA of previously unidentified genes involved in macronutrient metabolism including fructokinase, aldolase B, phosphofructokinase-1, fructose-1,6-bisphosphatase and carbohydrate response element binding protein (ChREBP). Activity of glucose-6-phosphate dehydrogenase, a key enzyme for ChREBP activation, remained elevated in both fed and fasted fructose groups. In the fasted liver, the fructose group showed lower non-esterified fatty acids, triglycerides and microsomal triglyceride transfer protein mRNA, suggesting low VLDL synthesis even though plasma VLDL triglycerides were higher. In conclusion, fructose feeding induced a broader range of genes than previously identified with simultaneous increase in glycogen and triglycerides in liver. The induction may be in part mediated by ChREBP.
Keywords: Fatty acid synthase; Glycerol-3-phosphate acyltransferase; Glucose-6-phosphatase; Max-like factor X Pyruvate kinase; Sterol regulatory element binding protein 1;
Fatty acids influence binding of cobalt to serum albumin in patients with fatty liver by G. Jayakumar Amirtharaj; Sathish Kumar Natarajan; Ashis Mukhopadhya; Uday George Zachariah; Sudheer K. Hegde; George Kurian; K.A. Balasubramanian; Anup Ramachandran (349-354).
Human serum albumin binds ligands such as fatty acids and metals in circulation. Oxidative stress can modify albumin and affect ligand binding. This study examines the role of oxidative stress and fatty acids in modulating cobalt binding to albumin in patients with fatty liver. Elevated levels of malondialdehyde and protein carbonyls, indicative of oxidative stress were evident in serum of patients with fatty liver. A significant decrease in albumin–cobalt binding was also observed. Albumin isolated from patient serum also showed an increase in bound fatty acids. In vitro experiments indicated that while oxidant exposure or removal of fatty acids independently decreased cobalt binding to albumin, removal of fatty acids from the protein prior to oxidant exposure did not influence the oxidant effect on albumin–cobalt binding. These results suggest that oxidative stress and fatty acids on albumin can influence albumin–cobalt binding in patients with fatty liver by independent mechanisms.
Keywords: Fatty liver; Oxidative stress; Albumin; Cobalt; Fatty acid;
Increase in P-glycoprotein accompanied by activation of protein kinase Cα and NF-κB p65 in the livers of rats with streptozotocin-induced diabetes by Natsumi Kameyama; Sakiko Arisawa; Jun Ueyama; Satomi Kagota; Kazumasa Shinozuka; Ai Hattori; Yasuaki Tatsumi; Hisao Hayashi; Kenji Takagi; Shinya Wakusawa (355-360).
It is known that protein kinase C (PKC) signal transduction is enhanced in a diabetic state, and that PKC activator phorbol esters increase the gene expression of MDR1 in human tumor cells. To clarify the expression of the liver transporters under diabetic conditions and the roles of PKCα and the transcription factor NF-κB, we investigated the expression levels of Mdr1a, Mdr1b, Mdr2, Mrp2, Bcrp, Bsep, Oct1, Oat2, and Oat3 transporters, PKCα, IκB, and NF-κB in the liver of rats with STZ-induced hyperglycemia. A selective increase in the gene expression of Mdr1b was detected by RT-PCR. Western blotting with C219 antibody revealed an increase in P-glycoprotein. Although the mRNA level of PKCα was not affected, translocation of PKCα to the microsomal fraction was detected. NF-κB p65, IκBα and IκBβ mRNA levels were increased as was the level of nuclear NF-κB p65. From these findings, it was suggested that STZ-induced hyperglycemia caused the upregulation of Mdr1b P-gp expression through the activation of PKCα and NF-κB.
Keywords: Mdr1b; PKCα; NF-κB p65; IκB; Diabetes; Rat; Liver; Streptozotocin;
Corrigendum to “Normal levels of DM RNA and myotonin protein kinase in skeletal muscle from adult myotonic dystrophy (DM) patients” [Biochim. Biophys. Acta 1317 (1996) 155–157] by Satyakam Bhagavati; Ashwini Ghatpande; Betty Leung (361).
Corrigendum to “(CTG)n repeats markedly inhibit differentiation of the C2C12 myoblast cell line: Implications for congenital myotonic dystrophy” [Biochim. Biophys. Acta 1453 (1999) 221–229] by Satyakam Bhagavati; S.A. Shafiq; Weimin Xu (362).