BBA - Molecular Basis of Disease (v.1832, #3)
Editorial Board (i).
Is transketolase-like protein, TKTL1, transketolase? by Ludmilla E. Meshalkina; Valeriy L. Drutsa; Olga N. Koroleva; Olga N. Solovjeva; German A. Kochetov (387-390).
Until recently it was assumed that the transketolase-like protein (TKTL1) detected in the tumor tissue, is catalytically active mutant form of human transketolase (hTKT). Human TKT shares 61% sequence identity with TKTL1. And the two proteins are 77% homologous at the amino acid level. The major difference is the absence of 38 amino acid residues in the N-terminal region of TKTL1. Site-specific mutagenesis was used for modifying hTKT gene; the resulting construct had a 114-bp deletion corresponding to a deletion of 38 amino acid residues in hTKT protein. Wild type hTKT and mutant variant (DhTKT) were expressed in Escherichia coli and isolated using Ni-agarose affinity chromatography. We have demonstrated here that DhTKT is devoid of transketolase activity and lacks bound thiamine diphosphate (ThDP). In view of these results, it is unlikely that TKTL1 may be a ThDP-dependent protein capable of catalyzing the transketolase reaction, as hypothesized previously.► Transketolase-like protein 1 (TKTL1) is a marker of certain tumor tissues. ► Site-specific mutagenesis was used for modifying human transketolase (hTKT) gene. ► The mutant (DhTKT) had a deletion of 38 amino acid residues in hTKT protein. ► This deletion is the major difference between hTKT and TKTL1. ► We demonstrate that DhTKT is devoid of transketolase activity and lacks bound ThDP.
Keywords: Human transketolase; Thiamine diphosphate; Transketolase-like protein 1; TKTL1; Oncology; Site-specific mutagenesis;
Insulin-like growth factor-1 regulates glutathione peroxidase expression and activity in vascular endothelial cells: Implications for atheroprotective actions of insulin-like growth factor-1 by Yusuke Higashi; Arvind Pandey; Brett Goodwin; Patrice Delafontaine (391-399).
Oxidative stress promotes endothelial cell senescence and endothelial dysfunction, important early steps in atherogenesis. To investigate potential antioxidant effects of IGF-1 we treated human aortic endothelial cells (hAECs) with 0–100 ng/mL IGF-1 prior to exposure to native or oxidized low-density lipoprotein (oxLDL). IGF-1 dose- and time- dependently reduced basal- and oxLDL-induced ROS generation. IGF-1 did not alter superoxide dismutase or catalase activity but markedly increased activity of glutathione peroxidase (GPX), a crucial antioxidant enzyme, via a phosphoinositide-3 kinase dependent pathway. IGF-1 did not increase GPX1 mRNA levels but increased GPX1 protein levels by 2.6-fold at 24 h, and altered selenocysteine-incorporation complex formation on GPX1 mRNA. Furthermore, IGF-1 blocked hydrogen peroxide induced premature cell senescence in hAECs. In conclusion, IGF-1 upregulates GPX1 expression in hAECs via a translational mechanism, which may play an important role in the ability of IGF-1 to reduce endothelial cell oxidative stress and premature senescence. Our findings have major implications for understanding vasculoprotective effects of IGF-1.► Potential antioxidant effects of IGF-1 were tested in vascular endothelial cells. ► IGF-1 upregulated GPX activity via a phosphoinositide-3 kinase dependent pathway. ► IGF-1 modulated selenocysteine-incorporation complex formation on GPX1 mRNA. ► IGF-1 blocked oxidant-induced premature senescence in vascular endothelial cells.
Keywords: Oxidative stress; Senescence; Endothelial dysfunction; Atherosclerosis;
Characterization of human DAAO variants potentially related to an increased risk of schizophrenia by Laura Caldinelli; Silvia Sacchi; Gianluca Molla; Marco Nardini; Loredano Pollegioni (400-410).
Considering the key role of d-serine in N-methyl-d-aspartate receptor-mediated neurotransmission, it is highly relevant to define the role that enzymes play in d-serine synthesis and degradation. In particular, the details of regulation of the d-serine catabolic human enzyme d-amino acid oxidase (hDAAO) are unknown although different lines of evidence have shown it to be involved in schizophrenia susceptibility. Here we investigated the effect of three single nucleotide polymorphisms and known mutations in hDAAO, i.e., D31H, R279A, and G331V. A very low amount of soluble G331V hDAAO is produced in E. coli cells: the recombinant variant enzyme is fully active. Human U87 glioblastoma cells transiently transfected for G331V hDAAO show a low viability, a significant amount of protein aggregates, and augmented apoptosis. The recombinant D31H and R279A hDAAO variants do not show alterations in tertiary and quaternary structures, thermal stability, binding affinity for inhibitors, and the modulator pLG72, whereas the kinetic efficiency and the affinity for d-serine and for FAD were higher than for the wild-type enzyme. While these effects for the substitution at position 31 cannot be structurally explained, the R279A mutation might affect the hDAAO FAD-binding affinity by altering the “structurally ambivalent” peptide V47–L51. In agreement with the observed increased activity, expression of D31H and R279A hDAAO variants in U87 cells produces a higher decrease in cellular d/(d + l) serine ratio than the wild-type counterpart. In vivo, these substitutions could affect cellular d-serine concentration and its release at synapsis and thus might be relevant for schizophrenia susceptibility.► DAAO controls the cellular concentration of the neuromodulator d-serine. ► Here, three point variants of human DAAO have been investigated. ► G331V DAAO yields protein aggregation, augments apoptosis and low cell viability. ► D31H and R279A increase DAAO activity and FAD binding and decrease cellular d-serine level. ► The latter condition correlates to an increased risk to schizophrenia.
Keywords: Schizophrenia; d-Serine; Neurotransmission; d-Amino acid oxidase; Enzyme variants;
Oct-1 recruitment to the nuclear envelope in adult-onset autosomal dominant leukodystrophy by Marta Columbaro; Elisabetta Mattioli; Nadir M. Maraldi; Michela Ortolani; Laura Gasparini; Maria Rosaria D'Apice; Diana Postorivo; Anna Maria Nardone; Sofia Avnet; Pietro Cortelli; Rocco Liguori; Giovanna Lattanzi (411-420).
Adult-onset autosomal dominant leukodystrophy (ADLD) is a slowly progressive neurological disorder characterised by pyramidal, cerebellar, and autonomic disturbances. Duplication of the LMNB1 gene is the genetic cause of ADLD, yet the pathogenetic mechanism is not defined. In this study, we analysed cells and muscle tissue from three patients affected by ADLD, carrying an extra copy of the LMNB1 gene. Lamin B1 levels were dramatically increased in ADLD nuclei, both in skin fibroblasts and skeletal muscle fibres. Since lamin B1 is known to bind Oct-1, a transcription factor involved in the oxidative stress pathway, we investigated Oct-1 fate in ADLD. Oct-1 recruitment to the nuclear periphery was increased in ADLD cells, while nucleoplasmic localisation of the transcription factor under oxidative stress conditions was reduced. Importantly, lamin B1 degradation occurring in some, but not all ADLD cell lines, slowed down lamin B1 and Oct-1 accumulation. In skeletal muscle, focal disorganisation of sarcomeres was observed, while IIB-myosin heavy chain, an Oct-1 target gene, was under-expressed and rod-containing fibres were formed. These data show that a high degree of regulation of lamin B1 expression is implicated in the different clinical phenotypes observed in ADLD and show that altered Oct-1 nuclear localisation contributes to the disease phenotype.► Lamin B1 is accumulated in the nuclear lamina of skin and muscle cells in ADLD. ► Lamin B1 accumulation in ADLD is associated with Oct-1 recruitment. ► Lamin B1 and Oct-1 amount in nuclei are affected by protein degradation rate. ► Skeletal muscle tissue abnormalities are involved in ADLD. ► The severity of ADLD phenotype is related to the amount of accumulated lamin B1.
Keywords: Oct-1; Lamin B1; Autosomal dominant leukodystrophy (ADLD); Protein degradation; IIB-myosin heavy chain;
Quercetin supplementation is effective in improving mitochondrial dysfunctions induced by 3-nitropropionic acid: Implications in Huntington's disease by Rajat Sandhir; Arpit Mehrotra (421-430).
The study was designed to investigate the beneficial effect of quercetin supplementation in 3-nitropropionic acid (3-NP) induced model of Huntington's disease (HD). HD was induced in rats by administering sub-chronic dose of 3-NP, intraperitoneally, twice daily for 17 days. Quercetin was supplemented at a dose of 25 mg/kg body weight by oral gavage for 21 days. At the end of treatment, mitochondrial bioenergetics, mitochondrial swelling, oxidative stress, neurobehavioral deficits and histopathological changes were analyzed. Quercetin supplementation was able to reverse 3-NP induced inhibition of respiratory chain complexes, restore ATP levels, attenuate mitochondrial oxidative stress in terms of lipid peroxidation and prevent mitochondrial swelling. Quercetin administration also restored the activities of superoxide dismutase and catalase along with thiol content in 3-NP treated animals. Beneficial effect of quercetin administration was observed on 3-NP induced motor deficits analyzed by narrow beam walk and footprint analysis. Histopathological analysis of 3-NP treated rats revealed pyknotic nuclei and astrogliosis in striatum, which were reduced or absent in quercetin supplemented animals. Altogether, our results show that quercetin supplementation to 3-NP induced HD animals ameliorated mitochondrial dysfunctions, oxidative stress and neurobehavioral deficits in rats showing potential of this flavonoid in maintaining mitochondrial functions, suggesting a putative role of quercetin in HD management.► Quercetin improved mitochondrial dysfunctions in 3-NP induced rodent model of HD. ► Mitochondrial antioxidant status was improved on quercetin supplementation. ► Quercetin ameliorated behavioral deficits along with histopathological changes. ► A putative role of quercetin supplementation in HD management is suggested.
Keywords: Flavonoid; Huntington's disease; Mitochondrion; Neurodegeneration; 3-Nitropropionic acid; Quercetin;
Balanced translocation t(3;18)(p13;q22.3) and points mutation in the ZNF407 gene detected in patients with both moderate non-syndromic intellectual disability and autism by Cong-mian Ren; Yan Liang; Fengxiang Wei; Ya-nan Zhang; Shou-qiang Zhong; Heng Gu; Xing-Sheng Dong; Yang-Yu Huang; Hua Ke; Xin-ming Son; Damu Tang; Zheng Chen (431-438).
Intellectual disability (ID) is a common disease. While the etiology remains incompletely understood, genetic defects are a major contributor, which include mutations in genes encoding zinc finger proteins. These proteins modulate gene expression via binding to DNA. Consistent with this knowledge, we report here the identification of mutations in the ZNF407 gene in ID/autistic patients. In our study of an ID patient with autism, a reciprocal translocation 46,XY,t(3;18)(p13;q22.3) was detected. By using FISH and long-range PCR approaches, we have precisely mapped the breakpoints associated with this translocation in a gene-free region in chromosome 3 and in the third intron of the ZNF407 gene in chromosome18. The latter reduces ZNF407 expression. Consistent with this observation, in our subsequent investigation of 105 ID/autism patients with similar clinical presentations, two missense mutations Y460C and P1195A were identified. These mutations cause non-conservative amino acid substitutions in the linker regions between individual finger structures. In line with the linker regions being critical for the integrity of zinc finger motifs, both mutations may result in loss of ZNF407 function. Taken together, we demonstrate that mutations in the ZNF407 gene contribute to the pathogenesis of a group of ID patients with autism.► We examined gene mutations contributing to intellectual disability (ID) and autism. ► We mapped the breakpoints to ZNF407 in an ID/autistic patient with a reciprocal translocation between chromosomes 3 and 18. ► This break inhibits ZNF407 expression. ► Two ZNF407 mutants were found among 105 ID/autistic patients. ► Mutations in ZNF407 contribute to ID/autism.
Keywords: Non-syndromic intellectual disability; ZNF407 gene; Reciprocal translocation; Fluorescence in situ hybridization;
Leptin regulates amyloid β production via the γ-secretase complex by Dana M. Niedowicz; Christa M. Studzinski; Adam M. Weidner; Thomas L. Platt; Kristen N. Kingry; Tina L. Beckett; Annadora J. Bruce-Keller; Jeffrey N. Keller; M. Paul Murphy (439-444).
Alzheimer's disease (AD) is the most common age-related neurodegenerative disease, affecting an estimated 5.3 million people in the United States. While many factors likely contribute to AD progression, it is widely accepted that AD is driven by the accumulation of β-amyloid (Aβ), a small, fibrillogenic peptide generated by the sequential proteolysis of the amyloid precursor protein by the β- and γ-secretases. Though the underlying causes of Aβ accumulation in sporadic AD are myriad, it is clear that lifestyle and overall health play a significant role. The adipocyte-derived hormone leptin has varied systemic affects, including neuropeptide release and neuroprotection. A recent study by Lieb et al. (2009) showed that individuals with low plasma leptin levels are at greater risk of developing AD, through unknown mechanisms. In this report, we show that plasma leptin is a strong negative predictor of Aβ levels in the mouse brain, supporting a protective role for the hormone in AD onset. We also show that the inhibition of Aβ accumulation is due to the downregulation of transcription of the γ-secretase components. On the other hand, β-secretase expression is either unchanged (BACE1) or increased (BACE2). Finally, we show that only presenilin 1 (PS1) is negatively correlated with plasma leptin at the protein level (p < 0.0001). These data are intriguing and may highlight a role for leptin in regulating the onset of amyloid pathology and AD.► Plasma leptin is negatively correlated with brain β-amyloid. ► Leptin treatment reduces β-amyloid in neuronal cells. ► Leptin treatment inhibits γ-secretase expression in neuronal cells. ► Plasma leptin in negatively correlated with presenilin 1 expression in the brain.
Keywords: Leptin; Presenilin; γ-secretase; β-amyloid; Alzheimer's disease;
Cybrid studies establish the causal link between the mtDNA m.3890G>A/MT-ND1 mutation and optic atrophy with bilateral brainstem lesions by Leonardo Caporali; Anna Maria Ghelli; Luisa Iommarini; Alessandra Maresca; Maria Lucia Valentino; Chiara La Morgia; Rocco Liguori; Claudia Zanna; Piero Barboni; Vera De Nardo; Andrea Martinuzzi; Giovanni Rizzo; Caterina Tonon; Raffaele Lodi; Maria Antonietta Calvaruso; Martina Cappelletti; Anna Maria Porcelli; Alessandro Achilli; Maria Pala; Antonio Torroni; Valerio Carelli (445-452).
Complex I (CI) deficiency is a frequent cause of mitochondrial disorders and, in most cases, is due to mutations in CI subunit genes encoded by mitochondrial DNA (mtDNA). In this study, we establish the pathogenic role of the heteroplasmic mtDNA m.3890G>A/MT-ND1 (p.R195Q) mutation, which affects an extremely conserved amino acid position in ND1 subunit of CI. This mutation was found in a young-adult male with optic atrophy resembling Leber's hereditary optic neuropathy (LHON) and bilateral brainstem lesions. The only previously reported case with this mutation was a girl with fatal infantile Leigh syndrome with bilateral brainstem lesions. Transfer of the mutant mtDNA in the cybrid cell system resulted in a marked reduction of CI activity and CI-dependent ATP synthesis in the presence of a normally assembled enzyme.These findings establish the pathogenicity of the m.3890G>A/MT-ND1 mutation and remark the link between CI mutations affecting the mtDNA-encoded ND subunits and LHON-like optic atrophy, which may be complicated by bilateral and symmetric lesions affecting the central nervous system. Peculiar to this mutation is the distribution of the brainstem lesions, with sparing of the striatum in both patients.► Heteroplasmic m.3890G>A/MT-ND1 mutation causes optic atrophy and bilateral brainstem lesions ► This mutation affects a conserved amino acid in a functional domain of ND1 subunit ► This mutation significantly affects CI redox activity and function
Keywords: Mitochondrial disorder; Vision loss; MT-ND1; Complex I;
PSAP induces a unique Apaf-1 and Smac-dependent mitochondrial apoptotic pathway independent of Bcl-2 family proteins by Ting Li; Linlin Zeng; Wei Gao; Mei-Zhen Cui; Xueqi Fu; Xuemin Xu (453-474).
Presenilin-associated protein (PSAP) has been identified as a mitochondrial proapoptotic protein. However, the mechanism by which PSAP induces apoptosis remains unknown. To this end, we have established an inducible expression system. Using this system, we have examined the roles of B-cell lymphoma 2 (Bcl-2) family proteins, cytochrome c, Smac (Smac/Diablo, second mitochondria-derived activator of caspases/direct IAP binding protein with low PI), and Apaf-1 (apoptotic protease-activating factor) in PSAP-induced apoptosis. Our results demonstrate that knockdown of Apaf-1 abolished PSAP-induced caspase activation and poly(ADP ribose) polymerase (PARP) cleavage, indicating that the apoptosome formation triggered by cytochrome c is crucial for PSAP-induced apoptosis. Our data also demonstrate that knockdown of Smac abolished PSAP-induced caspase activation and PARP cleavage, indicating that, in addition to Apaf-1 or apoptosome formation, Smac is also essential for PSAP-induced apoptosis. However, interestingly, our data demonstrate that overexpression of Bcl-2 and Bcl-xL did not protect cells from PSAP-induced apoptosis, and that knockdown of Bid, Bax, and Bak had no effect on PSAP-induced cytochrome c and Smac release, indicating that PSAP-induced apoptosis is not regulated by Bcl-2 family proteins. These results strongly suggest that PSAP evokes mitochondrial apoptotic cascades via a novel mechanism that is not regulated by Bcl-2 family proteins, but that both the formation of cytochrome c-Apaf-1 apoptosome and the presence of Smac are absolutely required for PSAP-induced apoptosis.In the extrinsic pathway, apoptosis is induced by ligation of a death receptor such as Fas by ligand FasL. In the intrinsic pathway, intracellular death signals are transmitted to the mitochondria through pro-apoptotic Bcl-2 family proteins, resulting in the release of cytochrome c and/or Smac/DIABLO leading to apoptosis. However, the mitochondrial pro-apoptotic protein PSAP induces release of cytochrome c and Smac/DIABLO in a Bcl-2 protein-independent manner, suggesting a new mitochondrial apoptotic pathway.Display Omitted► PSAP was identified as an apoptotic protein and associated with Alzheimer presenilin. ► First, we found that induces apoptosis dependent on Smac and Apaf-1; ► Second, BAx, tBid, and Bak are not required for PSAP-induced cytochrome c release; ► Third, PSAP-induced apoptosis was not protected by Bcl-2 protein. ► PSAP opens a new avenue to study the mechanism of mitochondria-mediated apoptosis.
Keywords: Alzheimer's disease; Apoptosis; Mitochondria; Bcl-2; Oxidative stress;
Involvement of kinase PKC-zeta in the p62/p62P392L-driven activation of NF-κB in human osteoclasts by Estelle Chamoux; Stephen McManus; Gino Laberge; Martine Bisson; Sophie Roux (475-484).
Mutations of the gene encoding sequestosome1 (SQSTM1/p62), clustering in or near the UBA domain, have been described in Paget's disease of bone (PDB); among these the P392L substitution is the most prevalent. Protein p62 mediates several cell functions, including the control of NF-κB signaling, and autophagy. This scaffolding protein interacts with atypical PKCζ in the RANKL-induced signaling complex. We have previously shown that osteoclasts (OCs) overexpressing the p62P392L variant were in a constitutively activated state, presenting activated kinase p-PKCζ/λ and activated NF-κB prior to RANKL stimulation. In the present study, we investigated the relationships between PKCζ and NF-κB activation in human OCs transfected with p62 variants. We showed that PKCζ and p-PKCζ/λ co-localize with p62, and that PKCζ is involved in the RANKL-induced NF-κB activation and in the RANKL-independent activation of NF-κB observed in p62P392L-transfected cells. We also observed a basal and RANKL-induced increase in IκBα levels in the presence of the p62P392L mutation that contrasted with the NF-κB activation. In this study we propose that PKCζ plays a role in the activation of NF-κB by acting as a p65 (RelA) kinase at Ser536, independently of IκBα; this alternative pathway could be used preferentially in the presence of the p62P392L mutation, which may hinder the ubiquitin–proteasome pathway. Overall, our results highlight the importance of p62-associated PKCζ in the overactive state of pagetic OCs and in the activation of NF-κB, particularly in the presence of the p62P392L mutation.► PKCζ is involved in the RANKL-induced NF-κB activation in human osteoclasts (OCs). ► PKCζ is involved in the basal activation of NF-κB observed in p62P392L-OCs. ► Increased IκBα levels in p62P392L-OCs contrasted with simultaneous NF-κB activation. ► PKCζ contributes to p65 (RelA) phosphorylation at Ser536 in RANKL-induced NF-κB activation. ► PKCζ-induced phosphorylation of Ser536p65 in p62P392L containing OCs contributes to IκBα-independent NF-κB activation.
Keywords: Paget's disease of bone; Human osteoclast; SQSTM1/p62; PKCζ; p65;
Trypanosoma cruzi, the causative agent of Chagas disease, modulates interleukin-6-induced STAT3 phosphorylation via gp130 cleavage in different host cells by Nicolás Eric Ponce; Eugenio Antonio Carrera-Silva; Andrea Vanina Pellegrini; Silvia Inés Cazorla; Emilio Luis Malchiodi; Ana Paula Lima; Susana Gea; Maria Pilar Aoki (485-494).
Interleukin-6 mediates host defense and cell survival mainly through the activation of the transcription factor STAT3 via the glycoprotein gp130, a shared signal-transducing receptor for several IL-6-type cytokines. We have reported that the cardiotrophic parasite Trypanosoma cruzi protects murine cardiomyocytes from apoptosis. In agreement, an intense induction of the anti-apoptotic factor Bcl-2 is found in cardiac fibers during the acute phase of infection, establishing a higher threshold against apoptosis. We report here that inactive cruzipain, the main cysteine protease secreted by the parasite, specifically triggered TLR2 and the subsequent release of IL-6, which acted as an essential anti-apoptotic factor for cardiomyocyte cultures. Although comparable IL-6 levels were found under active cruzipain stimulation, starved cardiac cell monolayers could not be rescued from apoptosis. Moreover, cardiomyocytes treated with active cruzipain completely abrogated the STAT3 phosphorylation and nuclear translocation induced by recombinant IL-6. This inhibition was also observed on splenocytes, but it was reverted when the enzyme was complexed with chagasin, a parasite cysteine protease inhibitor. Furthermore, the inhibition of IL-6-induced p-STAT3 was evidenced in spleen cells stimulated with pre-activated supernatants derived from trypomastigotes. To account for these observations, we found that cruzipain enzymatically cleaved recombinant gp130 ectodomain, and induced the release of membrane-distal N-terminal domain of this receptor on human peripheral blood mononuclear cells. These results demonstrate, for the first time, that the parasite may modify the IL-6-induced response through the modulation of its cysteine protease activity, suggesting that specific inhibitors may help to improve the immune cell activation and cardioprotective effects.► Inactive cruzipain triggers TLR2 and IL-6 secretion, leading to cardioprotection. ► Cruzipain enzymatically cleaves the IL-6 receptor gp130 in different host cells. ► This is reverted when the enzyme is complexed with chagasin, a parasite inhibitor. ► Cruzipain induces the release of Ig-like domain of gp130 on human leukocytes. ► Trypomastigote supernatants abolish STAT3 phosphorylation in splenocytes.
Keywords: Trypanosoma cruzi; TLR2; IL-6 signaling; gp130 Ig-like domain; Human leukocytes; Cardiomyocytes;