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BBA - Molecular Basis of Disease (v.1802, #5)

Editorial Board (pp. i).

Mitochondrial bioenergetics and dynamics interplay in complex I-deficient fibroblasts by Moran M. Morán; H. Rivera; Sanchez-Arago M. Sánchez-Aragó; Blazquez A. Blázquez; B. Merinero; C. Ugalde; J. Arenas; J.M. Cuezva; Martin M.A. Martín (pp. 443-453).

Background: Complex I (CI) deficiency is the most frequent cause of OXPHOS disorders. Recent studies have shown increases in reactive oxygen species (ROS) production and mitochondrial network disturbances in patients' fibroblasts harbouring mutations in CI subunits. Objectives: The present work evaluates the impact of mutations in the NDUFA1 and NDUFV1 genes of CI on mitochondrial bioenergetics and dynamics, in fibroblasts from patients suffering isolated CI deficiency. Results: Decreased oxygen consumption rate and slow growth rate were found in patients with severe CI deficiency. Mitochondrial diameter was slightly increased in patients' cells cultured in galactose or treated with 2′-deoxyglucose without evidence of mitochondrial fragmentation. Expression levels of the main proteins involved in mitochondrial dynamics, OPA1, MFN2, and DRP1, were slightly augmented in all patients' cells lines. The study of mitochondrial dynamics showed delayed recovery of the mitochondrial network after treatment with the uncoupler carbonyl cyanide m-chlorophenyl hydrazone (cccp) in patients with severe CI deficiency. Intracellular ROS levels were not increased neither in glucose nor galactose medium in patients' fibroblasts. Conclusion: Our main finding was that severe CI deficiency in patients harbouring mutations in the NDUFA1 and NDUFV1 genes is linked to a delayed mitochondrial network recovery after cccp treatment. However, the CI deficiency is neither associated with massive mitochondrial fragmentation nor with increased ROS levels. The different genetic backgrounds of patients with OXPHOS disorders would explain, at least partially, differences in the pathophysiological manifestations of CI deficiency.

Keywords: Mitochondrial dynamics; Mitochondrial disease; NDUFA1; NDUFV1; Bioenergetics

Oral cancer, cigarette smoke and mitochondrial 18kDa translocator protein (TSPO) — In vitro, in vivo, salivary analysis by Rafael Nagler; Ofer Ben-Izhak; Dana Savulescu; Ella Krayzler; Sharon Akrish; Svetlana Leschiner; Irina Otradnov; Sivan Zeno; Leo Veenman; Moshe Gavish (pp. 454-461).

Oral cancer features high rates of mortality and morbidity, and is in dire need for new approaches. In the present study we analyzed 18kDa translocator protein (TSPO) expression in oral (tongue) cancer tumors by immunohistochemistry. We also assayed TSPO binding in human tongue cancer cell lines and in the cellular fraction of saliva from tongue cancer patients, heavy cigarette smokers, and non-smoking healthy people as controls. Concurrently, TSPO protein levels, cell viability, mitochondrial membrane potential (Δ ψ_m), and general protein levels were analyzed. TSPO expression could be significantly enhanced in oral cancer tumors, compared to unaffected adjacent tissue. We also found that five-year survival probability dropped from 65% in patients with TSPO negative tumors to 7% in patients with highly expressed TSPO ( p<0.001). TSPO binding capacity was also pronounced in the human oral cancer cell lines SCC-25 and SCC-15 (3133±643fmol/mg protein and 6956±549fmol/mg protein, respectively). Binding decreased by 56% and 72%, in the SCC-25 and SCC-15 cell lines, respectively ( p<0.05) following CS exposure in cell culture. In the cellular fraction of saliva of heavy smokers TSPO binding was lower than in non-smokers (by 53%, p<0.05). Also the cellular fraction of saliva exposed to CS in vitro showed decreased TSPO binding compared to unexposed saliva (by 30%, p<0.001). Interestingly, oral cancer patients also displayed significantly lower TSPO binding in the cellular fraction of saliva compared to healthy controls (by 40%, p<0.01). Our results suggest that low TSPO binding found in the cellular fraction of saliva may depend on genetic background as well as result from exposure to CS. We suggest that this may be related to a predisposition for occurrence of oral cancer.

Keywords: TSPO; Cigarette smoke; Oral cancer; Prognosis; Saliva

Increased SOD1 association with chromatin, DNA damage, p53 activation, and apoptosis in a cellular model of SOD1-linked ALS by Livea F. Barbosa; Fernanda M. Cerqueira; Antero F.A. Macedo; Camila C.M. Garcia; José Pedro F. Angeli; Robert I. Schumacher; Mari Cleide Sogayar; Ohara Augusto; Carri Maria Teresa Carrì; Paolo Di Mascio; Marisa H.G. Medeiros (pp. 462-471).

Mutations in the gene encoding cytosolic Cu,Zn-superoxide dismutase (SOD1) have been linked to familial amyotrophic lateral sclerosis (FALS). However the molecular mechanisms of motor neuron death are multi-factorial and remain unclear. Here we examined DNA damage, p53 activity and apoptosis in SH-SY5Y human neuroblastoma cells transfected to achieve low-level expression of either wild-type or mutant Gly⁹³→Ala (G93A) SOD1, typical of FALS. DNA damage was investigated by evaluating the levels of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) and DNA strand breaks. Significantly higher levels of DNA damage, increased p53 activity, and a greater percentage of apoptotic cells were observed in SH-SY5Y cells transfected with G93A SOD1 when compared to cells overexpressing wild-type SOD1 and untransfected cells. Western blot, FACS, and confocal microscopy analysis demonstrated that G93A SOD1 is present in the nucleus in association with DNA. Nuclear G93A SOD1 has identical superoxide dismutase activity but displays increased peroxidase activity when compared to wild-type SOD1. These results indicate that the G93A mutant SOD1 association with DNA might induce DNA damage and trigger the apoptotic response by activating p53. This toxic activity of mutant SOD1 in the nucleus may play an important role in the complex mechanisms associated with motor neuron death observed in ALS pathogenesis.

Keywords: Abbreviations; SOD1; Cu,Zn-superoxide dismutase; ALS; amyotrophic lateral sclerosis; FALS; familial amyotrophic lateral sclerosis; dGuo; 2′-deoxyguanosine; 8-oxodGuo; 8-oxo-7,8-dihydro-2′-deoxyguanosine; MDA; malondialdehyde; TBA; 2-thiobarbituric acid; HPLC; high-performance liquid chromatography; EC; electrochemical detection; ER; endoplasmic reticulum; NBT; nitro blue tetrazolium; DHR; dihydrorhodamineALS; SOD1; p53; Nucleus; DNA damage

Arylsulfatase B regulates interaction of chondroitin-4-sulfate and kininogen in renal epithelial cells by Sumit Bhattacharyya; Kumar Kotlo; Robert Danziger; Joanne K. Tobacman (pp. 472-477).

The enzyme arylsulfatase B ( N-acetylgalactosamine 4-sulfatase; ASB; ARSB), which removes 4-sulfate groups from the nonreducing end of chondroitin-4-sulfate (C4S;CSA) and dermatan sulfate, has cellular effects, beyond those associated with the lysosomal storage disease mucopolysaccharidosis VI. Previously, reduced ASB activity was reported in cystic fibrosis patients and in malignant human mammary epithelial cell lines in tissue culture compared to normal cells. ASB silencing and overexpression were associated with alterations in syndecan-1 and decorin expression in MCF-7 cells and in IL-8 secretion in human bronchial epithelial cells. In this report, we present the role of ASB in the regulation of the kininogen–bradykinin axis owing to its effect on chondroitin-4-sulfation and the interaction of C4S with kininogen. Silencing or overexpression of ASB in normal rat kidney epithelial cells in tissue culture modified the content of total sulfated glycosaminoglycans (sGAGs), C4S, kininogen, and bradykinin in spent media and cell lysates. Treatment of the cultured cells with chondroitinase ABC also increased the secretion of bradykinin into the spent media and reduced the C4S-associated kininogen. When ASB was overexpressed, the cellular kininogen that associated with C4S declined, suggesting a vital role for chondroitin-4-sulfation in regulating the kininogen–C4S interaction. These findings suggest that ASB, owing to its effect on chondroitin-4-sulfation, may impact on the kininogen–bradykinin axis and, thereby, may influence blood pressure.Because ASB activity is influenced by several ions, including chloride and phosphate, ASB activity may provide a link between salt responsiveness and the bradykinin-associated mechanism of blood pressure regulation.

Keywords: Arylsulfatase B; Chondroitin-4-sulfate; Kininogen; Bradykinin; Hypertension; Sulfate; Glycosaminoglycan

Compared effects of missense mutations in Very-Long-Chain Acyl-CoA Dehydrogenase deficiency: Combined analysis by structural, functional and pharmacological approaches by Stéphanie Gobin-Limballe; Ryan P. McAndrew; Fatima Djouadi; Jung-Ja Kim; Jean Bastin (pp. 478-484).

Very-Long-Chain Acyl-CoA Dehydrogenase deficiency (VLCADD) is an autosomal recessive disorder considered as one of the more common ß-oxidation defects, possibly associated with neonatal cardiomyopathy, infantile hepatic coma, or adult-onset myopathy. Numerous gene missense mutations have been described in these VLCADD phenotypes, but only few of them have been structurally and functionally analyzed, and the molecular basis of disease variability is still poorly understood. To address this question, we first analyzed fourteen disease-causing amino acid changes using the recently described crystal structure of VLCAD. The predicted effects varied from the replacement of amino acid residues lining the substrate binding cavity, involved in holoenzyme–FAD interactions or in enzyme dimerisation, predicted to have severe functional consequences, up to amino acid substitutions outside key enzyme domains or lying on near enzyme surface, with predicted milder consequences. These data were combined with functional analysis of residual fatty acid oxidation (FAO) and VLCAD protein levels in patient cells harboring these mutations, before and after pharmacological stimulation by bezafibrate. Mutations identified as detrimental to the protein structure in the 3-D model were generally associated to profound FAO and VLCAD protein deficiencies in the patient cells, however, some mutations affecting FAD binding or monomer–monomer interactions allowed a partial response to bezafibrate. On the other hand, bezafibrate restored near-normal FAO rates in some mutations predicted to have milder consequences on enzyme structure. Overall, combination of structural, biochemical, and pharmacological analysis allowed assessment of the relative severity of individual mutations, with possible applications for disease management and therapeutic approach.

Keywords: Fatty acid ß-oxidation; Very-Long-Chain-Acyl-CoA Dehydrogenase; 3-D modeling; Structure–function analysis; VLCAD deficiency; Pharmacological therapy

ISG15 and immune diseases by Young Joo Jeon; Hee Min Yoo; Chin Ha Chung (pp. 485-496).

ISG15, the product of interferon (IFN)-stimulated gene 15, is the first identified ubiquitin-like protein, consisting of two ubiquitin-like domains. ISG15 is synthesized as a precursor in certain mammals and, therefore, needs to be processed to expose the C-terminal glycine residue before conjugation to target proteins. A set of three-step cascade enzymes, an E1 enzyme (UBE1L), an E2 enzyme (UbcH8), and one of several E3 ligases (e.g., EFP and HERC5), catalyzes ISG15 conjugation (ISGylation) of a specific protein. These enzymes are unique among the cascade enzymes for ubiquitin and other ubiquitin-like proteins in that all of them are induced by type I IFNs or other stimuli, such as exposure to viruses and lipopolysaccharide. Mass spectrometric analysis has led to the identification of several hundreds of candidate proteins that can be conjugated by ISG15. Some of them are type I IFN-induced proteins, such as PKR and RIG-I, and some are the key regulators that are involved in IFN signaling, such as JAK1 and STAT1, implicating the role of ISG15 and its conjugates in type I IFN-mediated innate immune responses. However, relatively little is known about the functional significance of ISG15 induction due to the lack of information on the consequences of its conjugation to target proteins. Here, we describe the recent progress made in exploring the biological function of ISG15 and its reversible modification of target proteins and thus in their implication in immune diseases.

Keywords: ISG15; UBP43; UBE1L; UbcH8; Type I interferons; Viral infection; Immune diseases

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BBA - Molecular Basis of Disease (v.1802, #5)