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BBA - Molecular Basis of Disease (v.1802, #6)

Editorial Board (pp. i).

Intracellular delivery of full length recombinant human mitochondrial L-Sco2 protein into the mitochondria of permanent cell lines and SCO2 deficient patient's primary cells by Parthena F. Foltopoulou; Asterios S. Tsiftsoglou; Ioannis D. Bonovolias; Alexandra T. Ingendoh; Lefkothea C. Papadopoulou (pp. 497-508).

Mutations in human SCO2 gene, encoding the mitochondrial inner membrane Sco2 protein, have been found to be responsible for fatal infantile cardioencephalomyopathy and cytochrome c oxidase (COX) deficiency. One potentially fruitful therapeutic approach for this mitochondrial disorder should be considered the production of human recombinant full length L-Sco2 protein and its deliberate transduction into the mitochondria. Recombinant L-Sco2 protein, fused with TAT, a Protein Transduction Domain (PTD), was produced in bacteria and purified from inclusion bodies (IBs). Following solubilisation withl-arginine, this fusion L-Sco2 protein was transduced in cultured mammalian cells of different origin (U-87 MG, T24, K-562, and patient's primary fibroblasts) and assessed for stability, transduction into mitochondria, processing and impact on recovery of COX activity. Our results indicate that: a)l-Arg solution was effective in solubilising recombinant fusion L-Sco2 protein, derived from IBs; b) fusion L-Sco2 protein was delivered successfully via a time- and concentration-dependent process into the mitochondria of human U-87 MG and T24 cells; c) fusion L-Sco2 protein was also transduced in human K-562 cells, transiently depleted of SCO2 transcripts and thus COX deficient; transduction of this fusion protein led to partial recovery of COX activity in such cells; d) [³⁵S]Methionine-labelled fusion L-Sco2 protein, produced in a cell free transcription/translation system and incubated with intact isolated mitochondria derived from K-562 cells, was efficiently processed to yield the corresponding mature Sco2 protein, thus justifying the potential of the transduced fusion L-Sco2 protein to successfully activate COX holoenzyme; and finally, e) recombinant fusion L-Sco2 protein was successfully transduced into the mitochondria of primary fibroblasts derived from SCO2/COX deficient patient and facilitated recovery of COX activity. These findings provide the rationale of delivering recombinant proteins via PTD technology as a model for therapeutic approach of mitochondrial disorders.

Keywords: Abbreviations; COX; Cytochrome; c; oxidase; fusion L-Sco2 protein; recombinant fusion His; ₁₀; -Xa; _SITE; -TAT-L-Sco2-HA protein; PTD; Protein Transduction Domain; TAT; HIV-1 TAT-derived PTD; L; Leader peptide; MTS; Mitochondrial Targeting Signal peptide; MPP; the matrix-located mitochondrial processing peptidase; HA; Hemagglutinin; CDS; Coding sequence; IBs; Inclusion bodies; l; -Arg; l; -Arginine; IM; Imatinib; PK; Proteinase K; CS; Citrate synthase SCO2; /Sco2; Protein transduction; Mitochondrion; Cytochrome; c; oxidase

Glucose deprivation causes oxidative stress and stimulates aggresome formation and autophagy in cultured cardiac myocytes by Paola Marambio; Barbra Toro; Carlos Sanhueza; Rodrigo Troncoso; Valentina Parra; Hugo Verdejo; Garcia Lorena García; Clara Quiroga; Daniela Munafo; Diaz-Elizondo Jessica Díaz-Elizondo; Roberto Bravo; Gonzalez María-Julieta González; Guilermo Diaz-Araya; Zully Pedrozo; Mario Chiong; María Isabel Colombo; Sergio Lavandero (pp. 509-518).

Aggresomes are dynamic structures formed when the ubiquitin–proteasome system is overwhelmed with aggregation-prone proteins. In this process, small protein aggregates are actively transported towards the microtubule-organizing center. A functional role for autophagy in the clearance of aggresomes has also been proposed. In the present work we investigated the molecular mechanisms involved on aggresome formation in cultured rat cardiac myocytes exposed to glucose deprivation. Confocal microscopy showed that small aggregates of polyubiquitinated proteins were formed in cells exposed to glucose deprivation for 6h. However, at longer times (18h), aggregates formed large perinuclear inclusions (aggresomes) which colocalized with γ-tubulin (a microtubule-organizing center marker) and Hsp70. The microtubule disrupting agent vinblastine prevented the formation of these inclusions. Both small aggregates and aggresomes colocalized with autophagy markers such as GFP-LC3 and Rab24. Glucose deprivation stimulates reactive oxygen species (ROS) production and decreases intracellular glutathione levels. ROS inhibition by N-acetylcysteine or by the adenoviral overexpression of catalase or superoxide dismutase disrupted aggresome formation and autophagy induced by glucose deprivation. In conclusion, glucose deprivation induces oxidative stress which is associated with aggresome formation and activation of autophagy in cultured cardiac myocytes.

Keywords: Aggresome; Autophagy; Cardiac myocytes; Reactive oxygen species

Amyloid fibrillation and cytotoxicity of insulin are inhibited by the amphiphilic surfactants by Steven S.-S. Wang; Kuan-Nan Liu; Tzu-Chiang Han (pp. 519-530).

Amyloid fibrils have been associated with at least 25 different degenerative diseases. The 51-residue polypeptide hormone insulin, which is associated with type II diabetes, has been shown to self-assemble to form amyloid fibrils in vitro. With bovine insulin as a model, the research presented here explores the effects of two amphiphilic surfactants (1,2-dihexanoyl-sn-glycero-3-phosphocholine (di-C7-PC) and 1,2-diheptanoyl-sn-glycero-3-phosphocholine (di-C7-PC)) on the in vitro fibrillation process of bovine insulin at pH 2.0 and 55°C. We demonstrated that insulin fibrillation may be inhibited by both surfactants in a dose-dependent fashion. The best inhibition of fibril formation is observed when insulin is incubated with 4mM di-C7-PC. Moreover, the addition of either surfactant at the concentrations studied attenuated insulin fibril-induced cytotoxicity in both PC12 and SH-SY5Y cell lines. The results from this work may contribute to the understanding of the molecular factors affecting amyloid fibrillation and the molecular mechanism(s) of the interactions between the membrane and amyloid proteins.

Keywords: Insulin; Surfactant; Phospholipid; Amyloid fibril; Cytotoxicity; Inhibition

Evidence that yeast frataxin is not an iron storage protein in vivo by Alexandra Seguin; Robert Sutak; Anne-Laure Bulteau; Richard Garcia-Serres; Jean-Louis Oddou; Sophie Lefevre; Renata Santos; Andrew Dancis; Jean-Michel Camadro; Jean-Marc Latour; Emmanuel Lesuisse (pp. 531-538).

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 (Δ yfh1, Δ ggc1 and Δ ssq1) displayed a nearly identical distribution. This was also the case for Δ ggc1 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 4500molecules per cell. Our results do not privilege the hypothesis of Yfh1p being an iron storage protein in vivo.

Keywords: Yeast frataxin; Iron; Oxidative stress; yfh1; ggc1; Mössbauer; Mitochondrion

Differences in RNA processing underlie the tissue specific phenotype of ISCU myopathy by Petter S. Sanaker; Marina Toompuu; Vanessa E. Hogan; Langping He; Charalampos Tzoulis; Zofia M.A. Chrzanowska-Lightowlers; Robert W. Taylor; Laurence A. Bindoff (pp. 539-544).

Hereditary myopathy with lactic acidosis, or myopathy with exercise intolerance, Swedish type (OMIM #255125) is caused by mutations in the iron–sulfur cluster scaffold ( ISCU) gene. The g.7044G>C ISCU mutation induces a splicing error in the pre-mRNA that strengthens a weak intronic splice site leading to inclusion of a new exon and subsequent loss of mRNA and protein. While ISCU is widely expressed, homozygosity for this particular intronic mutation gives rise to a pure myopathy. In order to investigate tissue specificity and disease mechanism, we studied muscle, myoblasts, fibroblasts and blood cells from the first non-Swedish case of this disease. Consistent with the recognised role of ISCU, we found abnormal activities of respiratory chain complexes containing iron–sulfur clusters in patient muscle. We confirmed that, in the presence of the g.7044G>C mutation, splicing produces both abnormally and normally spliced mRNA in all tissues. The ratio of these products varies dramatically between tissues, being most abnormal in mature skeletal muscle that also has the lowest relative starting levels of ISCU mRNA compared with other tissues. Myoblasts and fibroblasts have more of the normally spliced variant as well as higher starting levels of ISCU mRNA. Up-regulation of mtDNA copy number was found in skeletal muscle and myoblasts, but not fibroblasts, and is thought to represent a compensatory response. Tissue specificity in this disorder appears therefore to be dependent on the mRNA starting level, the amount of remaining normally spliced RNA, and the degree to which compensatory mechanisms can respond.

Keywords: Metabolic myopathy; Exercise intolerance; ISCU; Mitochondrial respiratory chain; Splice affecting mutation; RNA processing

Functional analysis of H. sapiens DNA polymerase γ spacer mutation W748S with and without common variant E1143G by Eino J.H. Palin; Annamari Lesonen; Carol L. Farr; Liliya Euro; Anu Suomalainen; Laurie S. Kaguni (pp. 545-551).

Mitochondrial DNA polymerase, POLG, is the sole DNA polymerase found in animal mitochondria. In humans, POLGα W748S in cis with an E1143G mutation has been linked to a new type of recessive ataxia, MIRAS, which is the most common inherited ataxia in Finland. We investigated the biochemical phenotypes of the W748S amino acid change, using recombinant human POLG. We measured processive and non-processive DNA polymerase activity, DNA binding affinity, enzyme processivity, and subunit interaction with recombinant POLGβ. In addition, we studied the effects of the W748S and E1143G mutations in primary human cell cultures using retroviral transduction. Here, we examined cell viability, mitochondrial DNA copy number, and products of mitochondrial translation. Our results indicate that the W748S mutant POLGα does not exhibit a clear biochemical phenotype, making it indistinguishable from wild type POLGα and as such, fail to replicate previously published results. Furthermore, results from the cell models were concurrent with the findings from patients, and support our biochemical findings.

Keywords: Abbreviations; MIRAS; Mitochondrial Recessive Ataxia Syndrome; pol; DNA polymerase; apu; Average Processive Unit; POLG; DNA polymerase gamma holoenzyme; POLGα; catalytic subunit of DNA polymerase gamma; POLGβ; accessory subunit of DNA polymerase gamma; dsDNA; double stranded DNA; EMSA; Electrophoretic Mobility-Shift Assay; Twinkle; mitochondrial DNA helicase; mtSSB; mitochondrial single-stranded DNA-binding protein; mtDNA; mitochondrial DNA; IOSCA; Infantile Onset Spinocerebellar Ataxia; PCR; polymerase chain reaction; RT-PCR; real time polymerase chain reaction; nt; nucleotide; CNS; central nervous system; CT-DNA; calf thymus DNA; tRNA; transfer RNADNA polymerase γ; POLG; POLG1; Ataxia; MIRAS; Alpers

Glutaric aciduria type I and methylmalonic aciduria: Simulation of cerebral import and export of accumulating neurotoxic dicarboxylic acids in in vitro models of the blood–brain barrier and the choroid plexus by Sven W. Sauer; Silvana Opp; Anne Mahringer; Marcin M. Kamiński; Christian Thiel; Jürgen G. Okun; Gert Fricker; Marina A. Morath; Kolker Stefan Kölker (pp. 552-560).

Intracerebral accumulation of neurotoxic dicarboxylic acids (DCAs) plays an important pathophysiological role in glutaric aciduria type I and methylmalonic aciduria. Therefore, we investigated the transport characteristics of accumulating DCAs – glutaric (GA), 3-hydroxyglutaric (3-OH-GA) and methylmalonic acid (MMA) – across porcine brain capillary endothelial cells (pBCEC) and human choroid plexus epithelial cells (hCPEC) representing in vitro models of the blood–brain barrier (BBB) and the choroid plexus respectively. We identified expression of organic acid transporters 1 (OAT1) and 3 (OAT3) in pBCEC on mRNA and protein level. For DCAs tested, transport from the basolateral to the apical site (i.e. efflux) was higher than influx. Efflux transport of GA, 3-OH-GA, and MMA across pBCEC was Na⁺-dependent, ATP-independent, and was inhibited by the OAT substrates para-aminohippuric acid (PAH), estrone sulfate, and taurocholate, and the OAT inhibitor probenecid. Members of the ATP-binding cassette transporter family or the organic anion transporting polypeptide family, namely MRP2, P-gp, BCRP, and OATP1B3, did not mediate transport of GA, 3-OH-GA or MMA confirming the specificity of efflux transport via OATs. In hCPEC, cellular import of GA was dependent on Na⁺-gradient, inhibited by NaCN, and unaffected by probenecid suggesting a Na⁺-dependent DCA transporter. Specific transport of GA across hCPEC, however, was not found. In conclusion, our results indicate a low but specific efflux transport for GA, 3-OH-GA, and MMA across pBCEC, an in vitro model of the BBB, via OAT1 and OAT3 but not across hCPEC, an in vitro model of the choroid plexus.

Keywords: Abbreviations; BBB; blood–brain barrier; BCB; blood–cerebrospinal fluid barrier; BCRP; breast cancer resistance protein; bp; base pairs; C; ₀; initial concentration; ¹⁴; C-GA; ¹⁴; C-glutaric acid; CNS; central nervous system; CoA; coenzyme A; CPM; counts per minute; CSF; cerebrospinal fluid; d; ₄; -GA; d; ₄; -glutaric acid; d; ₅; -3-OH-GA; d; ₅; -3-hydroxyglutaric acid; d; ₃; -MMA; d; ₃; -methylmalonic acid; DCAs; dicarboxylic acids; GA; glutaric acid; GA-I; glutaric aciduria type I; GCDH; glutaryl-CoA dehydrogenase; hCPEC; human choroid plexus epithelial cells; 3-OH-GA; 3-hydroxyglutaric acid; KRB; Krebs–Ringer buffer; MCM; methylmalonyl-CoA mutase; MMA; methylmalonic acid; MMAuria; methylmalonic aciduria; MCT1; monocarboxylic acid/proton co-transporter; MRP2; multidrug resistance protein 2; MTT; tetrazolium salt 3-[4,5-dimethylthiazolyl-2]-2,5-diphenyltetrazolium bromide; NaDC; Na; ⁺; -dependent dicarboxylic acid transporter; NMG; n; -methylglucamine; OAT; organic acid transporter; OATP; organic anion transporting polypeptide; P; _app; apparent permeability coefficient; PAH; para-; aminohippuric acid; pBCEC; porcine brain capillary endothelial cells; P-gp; P-glycoprotein; TBST; tris-buffered saline Tween-20; V; _ac; volume of the acceptor compartmentGlutaric aciduria type I; Methylmalonic aciduria; Dicarboxylic acids; Blood–brain barrier; Organic acid transporter; Plexus choroideus

Deleterious effects of reactive aldehydes and glycated proteins on macrophage proteasomal function: Possible links between diabetes and atherosclerosis by Fatemeh Moheimani; Philip E. Morgan; David M. van Reyk; Michael J. Davies (pp. 561-571).

People with diabetes experience chronic hyperglycemia and are at a high risk of developing atherosclerosis and microvascular disease. Reactions of glucose, or aldehydes derived from glucose (e.g. methylglyoxal, glyoxal, or glycolaldehyde), with proteins result in glycation that ultimately yield advanced glycation end products (AGE). AGE are present at elevated levels in plasma and atherosclerotic lesions from people with diabetes, and previous in vitro studies have postulated that the presence of these materials is deleterious to cell function. This accumulation of AGE and glycated proteins within cells may arise from either increased formation and/or ineffective removal by cellular proteolytic systems, such as the proteasomes, the major multi-enzyme complex that removes proteins within cells. In this study it is shown that whilst high glucose concentrations fail to modify proteasome enzyme activities in J774A.1 macrophage-like cell extracts, reactive aldehydes enhanced proteasomal enzyme activities. In contrast BSA, pre-treated with high glucose for 8weeks, inhibited both the chymotrypsin-like and caspase-like activities. BSA glycated using methylglyoxal or glycolaldehyde, also inhibited proteasomal activity though to differing extents. This suppression of proteasome activity by glycated proteins may result in further intracellular accumulation of glycated proteins with subsequent deleterious effects on cellular function.

Keywords: Abbreviations; AMC; 7-amino-4-methylcoumarin; BSA; bovine serum albumin; DTT; dithiothreitol; GA; glycolaldehyde; GO; glyoxal; MGO; methylglyoxal; PBS; phosphate-buffered salineDiabetes-associated atherosclerosis; Proteasome; Glycated proteins; Advanced glycation end products; Reactive aldehydes; Protein turnover

A novel germline mutation in Peroxisome Proliferator-Activated Receptor γ gene associated with large intestine polyp formation and dyslipidemia by D. Capaccio; A. Ciccodicola; L. Sabatino; A. Casamassimi; M. Pancione; A. Fucci; A. Febbraro; A. Merlino; G. Graziano; V. Colantuoni (pp. 572-581).

We report a novel PPARG germline mutation in a patient affected by colorectal cancer that replaces serine 289 with cysteine in the mature protein (S289C). The mutant has impaired transactivation potential and acts as dominant negative to the wild type receptor. In addition, it no longer restrains cell proliferation both in vitro and in vivo. Interestingly, the S289C mutant poorly activates target genes and interferes with the inflammatory pathway in tumor tissues and proximal normal mucosa. Consistently, only mutation carriers exhibit colonic lesions that can evolve to dysplastic polyps. The proband presented also dyslipidemia, hypertension and overweight, not associated to type 2 diabetes; of note, family members tested positive for the mutation and display only a dyslipidemic profile at variable penetrance with other biochemical parameters in the normal range. Finally, superimposing the mutation to the crystal structure of the ligand binding domain, the new Cys289 becomes so closely positioned to Cys285 to form an S–S bridge. This would reduce the depth of the ligand binding pocket and impede agonist positioning, explaining the biological effects and subcellular distribution of the mutant protein. This is the first PPARG germline mutation associated with dyslipidemia and colonic polyp formation that can progress to full-blown adenocarcinoma.

Keywords: Abbreviations; PPAR; Peroxisome Proliferator-Activated Receptors; CRC; colorectal cancer; LOH; loss of heterozygosity; APC; adenomatous poliposis coliPeroxisome Proliferator-Activated Receptor; Loss-of-function mutation; Colorectal cancer; Dyslipidemia

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BBA - Molecular Basis of Disease (v.1802, #6)