BBA - Molecular Basis of Disease (v.1822, #7)

β2 long-acting and anticholinergic drugs control TGF-β1-mediated neutrophilic inflammation in COPD by Mirella Profita; Anna Bonanno; Angela Marina Montalbano; Giusy Daniela Albano; Loredana Riccobono; Liboria Siena; Maria Ferraro; Paola Casarosa; Michael Paul Pieper; Mark Gjomarkaj (1079-1089).
We quantified TGF-β1 and acetylcholine (ACh) concentrations in induced sputum supernatants (ISSs) from 18 healthy controls (HC), 22 healthy smokers (HS) and 21 COPDs. ISSs from HC, HS and COPD as well as rhTGF-β1 were also tested in neutrophil adhesion and in mAChR2, mAChR3 and ChAT expression experiments in human bronchial epithelial cells (16-HBE). Finally, we evaluated the effects of Olodaterol (a novel inhaled β2-adrenoceptor agonist) and Tiotropium Spiriva®, alone or in combination, on neutrophil adhesion and mAChRs and ChAT expression in stimulated 16-HBE. The results showed that 1) TGF-β1 and ACh concentrations are increased in ISSs from COPD in comparison to HC and HS, and TGF-β1 in HS is higher than in HC; 2) ISSs from COPD and HS caused increased neutrophil adhesion to 16-HBE when compared to ISSs from HC. The effect of ISSs from COPD was significantly reduced by TGF-β1 depletion or by the pretreatment with Olodaterol or Tiotropium alone or in combination, while the effect of ISSs from HS was significantly reduced by the pretreatment with Olodaterol alone; 3) mAChR2, mAChR3 and ChAT expression was increased in 16-HBE stimulated with ISSs from COPD and TGF-β1 depletion significantly reduced this effect on mAChR3 and ChAT expression; 4) rhTGF-β1 increased mAChR2, mAChR3 and ChAT expression in 16-HBE; 5) Olodaterol did not affect the expression of mAChRs and ChAT in 16-HBE. Our findings support the use of β2 long-acting and anticholinergic drugs to control the bronchoconstriction and TGF-β1-mediated neutrophilic inflammation in COPD.► Both TGF-β and acetylcholine increase in induced sputum during airway inflammation of COPD. ► TGF-β increases expression of muscarinic receptors and choline acetyltransferase in epithelial cells. ► ACh promotes neutrophil adhesion to epithelial cells targeting muscarinic receptors. ► TGF-β and ACh interact to promote neutrophilic inflammation in the airways. ► β2 long-acting and anticholinergic drugs might control TGF-β and ACh neutrophilic airway inflammation in COPD.
Keywords: TGF-β1; COPD; Neutrophilic inflammation; Beta2 long acting; Anticholinergic drug;

Adipocytes are highly specialized cells that play a central role in lipid homeostasis and the maintenance of energy balance. Obesity, an excessive accumulation of adipose tissue, is a major risk factor for the development of Type 2 diabetes mellitus (T2DM), cardiovascular disease, and hypertension. A variety of studies suggest that obesity and T2DM can be linked to a breakdown in the regulatory mechanisms that control the expression and transcriptional activity of PPARγ. PPARγ is a nuclear hormone receptor that functions as a master switch in controlling adipocyte differentiation and development. Also important in controlling glucose homeostasis and insulin sensitivity, PPARγ is a ligand-dependent transcription factor that is the functional receptor for the anti-diabetic thiazolidinediones (TZDs). In the last fifteen years, a variety of covalent modifications of PPARγ activity have been identified and studied. These covalent modifications include phosphorylation, ubiquitylation, O-GlcNAcylation and SUMOylation. Covalent modifications of PPARγ represent key regulatory mechanisms that control both PPARγ protein stability and transcriptional activity. A variety of PPARγ transgenic models, including mice heterozygous for PPARγ, have demonstrated the importance of PPARγ expression in glucose homeostasis and insulin resistance. In the following review, we have highlighted the regulation of PPARγ by covalent modifications, the interplay between these interactions and how these post-translational modifications impact metabolic disease states.► PPARγ is important in fat cell development. ► PPARγ plays a role in glucose homeostasis and is an anti-diabetic drug target. ► The activity of PPARγ is largely effected by covalent modifications. ► The activity of PPARγ is highly regulated by phosphorylation, SUMOylation and ubiquitylation.
Keywords: Adipocyte; PPARγ; Adipogenesis; Ubiquitylation; SUMOylation;

ß-Ureidopropionase deficiency: Phenotype, genotype and protein structural consequences in 16 patients by André B.P. van Kuilenburg; Doreen Dobritzsch; Judith Meijer; Michael Krumpel; Laila A. Selim; Mohamed S. Rashed; Birgit Assmann; Rutger Meinsma; Bernhard Lohkamp; Tetsuya Ito; Nico G.G.M. Abeling; Kayoko Saito; Kaoru Eto; Martin Smitka; Martin Engvall; Chunhua Zhang; Wang Xu; Lida Zoetekouw; Raoul C.M. Hennekam (1096-1108).
ß-Ureidopropionase is the third enzyme of the pyrimidine degradation pathway and catalyzes the conversion of N-carbamyl-ß-alanine and N-carbamyl-ß-aminoisobutyric acid to ß-alanine and ß-aminoisobutyric acid, ammonia and CO2. To date, only five genetically confirmed patients with a complete ß-ureidopropionase deficiency have been reported. Here, we report on the clinical, biochemical and molecular findings of 11 newly identified ß-ureidopropionase deficient patients as well as the analysis of the mutations in a three-dimensional framework. Patients presented mainly with neurological abnormalities (intellectual disabilities, seizures, abnormal tonus regulation, microcephaly, and malformations on neuro-imaging) and markedly elevated levels of N-carbamyl-ß-alanine and N-carbamyl-ß-aminoisobutyric acid in urine and plasma. Analysis of UPB1, encoding ß-ureidopropionase, showed 6 novel missense mutations and one novel splice-site mutation. Heterologous expression of the 6 mutant enzymes in Escherichia coli showed that all mutations yielded mutant ß-ureidopropionase proteins with significantly decreased activity. Analysis of a homology model of human ß-ureidopropionase generated using the crystal structure of the enzyme from Drosophila melanogaster indicated that the point mutations p.G235R, p.R236W and p.S264R lead to amino acid exchanges in the active site and therefore affect substrate binding and catalysis. The mutations L13S, R326Q and T359M resulted most likely in folding defects and oligomer assembly impairment. Two mutations were identified in several unrelated ß-ureidopropionase patients, indicating that ß-ureidopropionase deficiency may be more common than anticipated.► Largest cohort of ß-ureidopropionase deficient patients studied so far. ► Patients presented mainly with neurological abnormalities. ► Analysis of UPB1 showed 7 novel mutations. ► Detailed analysis of purified wild-type and mutant ß-ureidopropionase. ► Mutations affected catalysis, global folding and structural integrity.
Keywords: ß-Ureidopropionase; UPB1; Neurological abnormalities; Homology modeling; Functional and structural protein analysis;

Activation of a cryptic splice site in a potentially lethal coagulation defect accounts for a functional protein variant by Nicola Cavallari; Dario Balestra; Alessio Branchini; Iva Maestri; Ampaiwan Chuamsunrit; Werasak Sasanakul; Guglielmo Mariani; Franco Pagani; Francesco Bernardi; Mirko Pinotti (1109-1113).
Changes at the invariable donor splice site + 1 guanine, relatively frequent in human genetic disease, are predicted to abrogate correct splicing, and thus are classified as null mutations. However, their ability to direct residual expression, which might have pathophysiological implications in several diseases, has been poorly investigated. As a model to address this issue, we studied the IVS6 + 1G > T mutation found in patients with severe deficiency of the protease triggering coagulation, factor VII (FVII), whose absence is considered lethal. In expression studies, the IVS6 + 1G > T induced exon 6 skipping and frame-shift, and prevented synthesis of correct FVII transcripts detectable by radioactive/fluorescent labelling or real-time RT-PCR. Intriguingly, the mutation induced the activation of a cryptic donor splice site in exon 6 and production of an in-frame 30 bp deleted transcript (8 ± 2%). Expression of this cDNA variant, lacking 10 residues in the activation domain, resulted in secretion of trace amounts (0.2 ± 0.04%) of protein with appreciable specific activity (48 ± 16% of wt-FVII). Altogether these data indicate that the IVS6 + 1G > T mutation is compatible with the synthesis of functional FVII molecules (~ 0.01% of normal, 1 pM), which could trigger coagulation. The low but detectable thrombin generation (352 ± 55 nM) measured in plasma from an IVS6 + 1G > T homozygote was consistent with a minimal initiation of the enzymatic cascade. In conclusion, we provide experimental clues for traces of FVII expression, which might have reverted an otherwise perinatally lethal genetic condition.► + 1G donor splice site mutations virtually suppress correct gene expression. ► Complete deficiency of coagulation factor VII (FVII) is lethal. ► The F7 IVS6 + 1G/T induces exon skipping and use of an in-frame cryptic 5'ss. ► The latter transcript encoded a protein lacking 10 residues in the activation domain. ► The residual activity of this variant might have reverted a lethal condition.
Keywords: Aberrant splicing; Residual gene expression; Human genetic disease; Coagulation factor deficiency;

Adaptation of respiratory chain biogenesis to cytochrome c oxidase deficiency caused by SURF1 gene mutations by Nikola Kovářová; Alena Čížková Vrbacká; Petr Pecina; Viktor Stránecký; Ewa Pronicka; Stanislav Kmoch; Josef Houštěk (1114-1124).
The loss of Surf1 protein leads to a severe COX deficiency manifested as a fatal neurodegenerative disorder, the Leigh syndrome (LSCOX). Surf1 appears to be involved in the early step of COX assembly but its function remains unknown. The aim of the study was to find out how SURF1 gene mutations influence expression of OXPHOS and other pro-mitochondrial genes and to further characterize the altered COX assembly. Analysis of fibroblast cell lines from 9 patients with SURF1 mutations revealed a 70% decrease of the COX complex content to be associated with 32–54% upregulation of respiratory chain complexes I, III and V and accumulation of Cox5a subunit. Whole genome expression profiling showed a general decrease of transcriptional activity in LSCOX cells and indicated that the adaptive changes in OXPHOS complexes are due to a posttranscriptional compensatory mechanism. Electrophoretic and WB analysis showed that in mitochondria of LSCOX cells compared to controls, the assembled COX is present entirely in a supercomplex form, as I–III2–IV supercomplex but not as larger supercomplexes. The lack of COX also caused an accumulation of I–III2 supercomplex. The accumulated Cox5a was mainly present as a free subunit. We have found out that the major COX assembly subcomplexes accumulated due to SURF1 mutations range in size between approximately 85–140 kDa. In addition to the originally proposed S2 intermediate they might also represent Cox1-containing complexes lacking other COX subunits. Unlike the assembled COX, subcomplexes are unable to associate with complexes I and III.► SURF1 mutations are frequent cause of severe COX deficiency. ► Altered energy provision leads to increase of respiratory chain complexes I, III, and V. ► Adaptive changes in mitochondrial biogenesis are due to posttranscriptional events. ► All residual COX is incorporated into I–III2–IV1 supercomplex.
Keywords: Mitochondrial disorder; SURF1 gene; Leigh syndrome; Gene expression; Oxidative phosphorylation; Cytochrome c oxidase;

Overexpression of VMAT-2 and DT-diaphorase protects substantia nigra-derived cells against aminochrome neurotoxicity by Patricia Muñoz; Irmgard Paris; Laurie H. Sanders; J. Timothy Greenamyre; Juan Segura-Aguilar (1125-1136).
We tested the hypothesis that both VMAT-2 and DT-diaphorase are an important cellular defense against aminochrome-dependent neurotoxicity during dopamine oxidation. A cell line with VMAT-2 and DT-diaphorase over-expressed was created. The transfection of RCSN-3 cells with a bicistronic plasmid coding for VMAT-2 fused with GFP-IRES-DT-diaphorase cDNA induced a significant increase in protein expression of VMAT-2 (7-fold; P < 0.001) and DT-diaphorase (9-fold; P < 0.001), accompanied by a 4- and 5.5-fold significant increase in transport and enzyme activity, respectively. Studies with synaptic vesicles from rat substantia nigra revealed that VMAT-2 uptake of 3H-aminochrome 6.3 ± 0.4 nmol/min/mg was similar to dopamine uptake 6.2 ± 0.3 nmol/min/mg that which were dependent on ATP. Interestingly, aminochrome uptake was inhibited by 2 μM lobeline but not reserpine (1 and 10 μM). Incubation of cells overexpressing VMAT-2 and DT-diaphorase with 20 μM aminochrome resulted in (i) a significant decrease in cell death (6-fold, P < 0.001); (ii) normal ultra structure determined by transmission electron microscopy contrasting with a significant increase of autophagosome and a dramatic remodeling of the mitochondrial inner membrane in wild type cells; (iii) normal level of ATP (256 ± 11 μM) contrasting with a significant decrease in wild type cells (121 ± 11 μM, P < 0.001); and (iv) a significant decrease in DNA laddering (21 ± 8 pixels, P < 0.001) cells in comparison with wild type cells treated with 20 μM aminochrome (269 ± 9). These results support our hypothesis that VMAT-2 and DT-diaphorase are an important defense system against aminochrome formed during dopamine oxidation.► VMAT‐2 take up aminochrome. ► Aminochrome uptake mediated by VMAT‐2 is inhibited by lobeline but not reserpine. ► VMAT‐2 and DT‐diaphorase overexpression prevents aminochrome neurotoxicity. ► VMAT‐2 and DT‐diaphorase overexpression prevents increase of autophagosome. ► VMAT‐2 and DT‐diaphorase overexpression prevents mitochondria remodeling.
Keywords: Dopamine; Neuromelanin; VMAT-2; DT-diaphorase; Aminochrome; Parkinson's disease;

Lysosomal di-N-acetylchitobiase-deficient mouse tissues accumulate Man2GlcNAc2 and Man3GlcNAc2 by Emanuele Persichetti; Katharina Klein; Silvia Paciotti; Karine Lecointe; Chiara Balducci; Sebastian Franken; Sandrine Duvet; Ulrich Matzner; Rita Roberti; Dieter Hartmann; Volkmar Gieselmann; Tommaso Beccari (1137-1146).
Most lysosomal storage diseases are caused by defects in genes encoding for acidic hydrolases. Deficiency of an enzyme involved in the catabolic pathway of N-linked glycans leads to the accumulation of the respective substrate and consequently to the onset of a specific storage disorder. Di-N-acetylchitobiase and core specific α1–6mannosidase represent the only exception. In fact, to date no lysosomal disease has been correlated to the deficiency of these enzymes. We generated di-N-acetylchitobiase-deficient mice by gene targeting of the Ctbs gene in murine embryonic stem cells. Accumulation of Man2GlcNAc2 and Man3GlcNAc2 was evaluated in all analyzed tissues and the tetrasaccharide was detected in urines. Multilamellar inclusion bodies reminiscent of polar lipids were present in epithelia of a scattered subset of proximal tubules in the kidney. Less constantly, enlarged Kupffer cells were observed in liver, filled with phagocytic material resembling partly digested red blood cells. These findings confirm an important role for lysosomal di-N-acetylchitobiase in glycans degradation and suggest that its deficiency could be the cause of a not yet described lysosomal storage disease.► A lysosomal di-N-acetylchitobiase deficient mouse model has been generated. ► KO mice accumulate oligosaccharides in all tissues and lipids in kidney. ► KO mice present pathological features common to other storage disorder mouse models.
Keywords: Lysosomal di-N-acetylchitobiase; Lysosomal storage disease; Knockout mouse; N-linked glycan;

Quantitative magnetic analysis reveals ferritin-like iron as the most predominant iron-containing species in the murine Hfe-haemochromatosis by Lucía Gutiérrez; Maja Vujić Spasić; Martina U. Muckenthaler; Francisco J. Lázaro (1147-1153).
Quantitative analysis of the temperature dependent AC magnetic susceptibility of freeze-dried mouse tissues from an Hfe hereditary haemochromatosis disease model indicates that iron predominantly appears biomineralised, like in the ferritin cores, in the liver, the spleen and duodenum. The distribution of the amount of ferritin-like iron between genders and genotypes coincides with that of elemental iron and nonheme iron. Importantly, the so-called paramagnetic iron, a quantity also determined from the magnetic data and indicative of nonmineralised iron forms, appears only marginally increased when iron overload takes place.Display Omitted► Iron speciation has been analysed in a murine model of Hfe-haemochromatosis. ► Magnetic measurements allow quantitative analysis of iron-containing species. ► Ferritin-like iron is the most predominant species in the liver, spleen and duodenum. ► The proportion of paramagnetic iron is enhanced in the spleen.
Keywords: Ferritin; Iron; AC susceptibility; Paramagnetic; Hfe; Haemochromatosis;

A relation between TGF-β and mast cell tryptase in experimental emphysema models by Esmaeil Mortaz; Masoumeh E. Givi; Carla A. Da Silva; Gert Folkerts; Frank A. Redegeld (1154-1160).
Chronic obstructive pulmonary disease (COPD) is a multicomponent disease characterized by emphysema and/or chronic bronchitis. The aim of this study was to investigate the effect of cigarette smoke exposure on mast cells and mast cell function in vitro and in vivo in order to get further insight in the role of mast cells in the pathogenesis of emphysema. Cigarette smoke conditioned medium (CSM) induced the expression of mast cell tryptase (MMCP-6) in primary cultured mast cells. This tryptase expression was caused by the CSM-stimulated production of TGF-β in culture and neutralization of TGF-β suppressed the CSM-induced expression of tryptase in mast cells. An increase in mast cell tryptase expression was also found in an experimental model for emphysema. Exposure of mice to cigarette smoke increased the number of mast cells in the airways and the expression of mast cell tryptase. In accordance with the in vitro findings, TGF-β in bronchoalveolar lavage fluid of smoke-exposed animals was significantly increased. Our study indicates that mast cells may be a source of TGF-β production after cigarette smoke exposure and that in turn TGF-β may change the tryptase expression in mast cells.► This study shows that cigarette smoke causes an increase in the mast cell population and a change in phenotype of mast cells. ► Cigarette smoke induced the release of TGF-β from mast cells which was responsible for the induction of tryptase expression. ► To what respect these effects contribute to pathological airway remodeling in lung emphysema remains to be elucidated in future research.
Keywords: Emphysema; FcεRI; COPD; Mast cell; Tryptase;

Transcriptional changes in OXPHOS complex I deficiency are related to anti-oxidant pathways and could explain the disturbed calcium homeostasis by A.M. Voets; M. Huigsloot; P.J. Lindsey; A.M. Leenders; W.J.H. Koopman; P.H.G.M. Willems; R.J. Rodenburg; J.A.M. Smeitink; H.J.M. Smeets (1161-1168).
Defective complex I (CI) is the most common type of oxidative phosphorylation disease, with an incidence of 1 in 5000 live births. Here, whole genome expression profiling of fibroblasts from CI deficient patients was performed to gain insight into the cell pathological mechanism. Our results suggest that patient fibroblasts responded to oxidative stress by Nrf2-mediated induction of the glutathione antioxidant system and Gadd45-mediated activation of the DNA damage response pathway. Furthermore, the observed reduced expression of selenoproteins, might explain the disturbed calcium homeostasis previously described for the patient fibroblasts and might be linked to endoplasmic reticulum stress. These results suggest that both glutathione and selenium metabolism are potentially therapeutic targets in CI deficiency.► Defective complex I (CI) is the most common type of oxidative phosphorylation disease. ► Transcriptomics identified novel molecular processes underlying CI deficiency in fibroblasts of patients. ► The Nrf2 pathway is induced in fibroblasts of complex I deficient patients. ► Disturbed calcium homeostasis in complex I deficiency can be related to selenoproteins. ► Glutathione and selenium metabolism are potentially therapeutic targets in CI deficiency.
Keywords: Gene expression; Complex I deficiency; Mitochondrion; Oxidative stress; Selenoprotein; Nrf2;

Increased cone sensitivity to ABCA4 deficiency provides insight into macular vision loss in Stargardt's dystrophy by Shannon M. Conley; Xue Cai; Rasha Makkia; Yalin Wu; Janet R. Sparrow; Muna I. Naash (1169-1179).
Autosomal recessive Stargardt macular dystrophy is caused by mutations in the photoreceptor disc rim protein ABCA4/ABCR. Key clinical features of Stargardt disease include relatively mild rod defects such as delayed dark adaptation, coupled with severe cone defects reflected in macular atrophy and central vision loss. In spite of this clinical divergence, there has been no biochemical study of the effects of ABCA4 deficiency on cones vs. rods. Here we utilize the cone-dominant Abca4 −/− /Nrl −/− double knockout mouse to study this issue. We show that as early as post-natal day (P) 30, Abca4 −/− /Nrl −/− retinas have significantly fewer rosettes than Abca4 +/+ /Nrl −/− retinas, a phenotype often associated with accelerated degeneration. Abca4-deficient mice in both the wild-type and cone-dominant background accumulate more of the toxic bisretinoid A2E than their ABCA4-competent counterparts, but Abca4 −/− /Nrl −/− eyes generate significantly more A2E per mole of 11-cis-retinal (11-cisRAL) than Abca4 −/− eyes. At P120, Abca4 −/− /Nrl −/− produced 340 ± 121 pmoles A2E/nmol 11-cisRAL while Abca4 −/− produced 50.4 ± 8.05 pmoles A2E/nmol 11-cisRAL. Nevertheless, the retinal pigment epithelium (RPE) of Abca4 −/− /Nrl −/− eyes exhibits fewer lipofuscin granules than the RPE of Abca4 −/− eyes; at P120: Abca4 −/− /Nrl −/− exhibit 0.045 ± 0.013 lipofuscin granules/μm2 of RPE vs. Abca4 −/− 0.17 ± 0.030 lipofuscin granules/μm2 of RPE. These data indicate that ABCA4-deficient cones simultaneously generate more A2E than rods and are less able to effectively clear it, and suggest that primary cone toxicity may contribute to Stargardt's-associated macular vision loss in addition to cone death secondary to RPE atrophy.► Stargardt's disease is associated with mutations in Abca4 and causes macular degeneration. ► We use the Abca4 −/−/Nrl −/− double knockout mouse to model Stargardt's disease. ► We show that cones accumulate more toxic A2E per mole of retinoid than rods. ► We also show that cones lacking Abca4 are less effective at clearing A2E than rods. ► These data suggest that cones are more sensitive to the loss of Abca4 than rods.
Keywords: ABCA4; Cone; Retinal degeneration; Stargardt's; A2E;