BBA - Molecular Basis of Disease (v.1772, #5)

Mitochondrial protein import and human health and disease by James A. MacKenzie; R. Mark Payne (509-523).
The targeting and assembly of nuclear-encoded mitochondrial proteins are essential processes because the energy supply of humans is dependent upon the proper functioning of mitochondria. Defective import of mitochondrial proteins can arise from mutations in the targeting signals within precursor proteins, from mutations that disrupt the proper functioning of the import machinery, or from deficiencies in the chaperones involved in the proper folding and assembly of proteins once they are imported. Defects in these steps of import have been shown to lead to oxidative stress, neurodegenerative diseases, and metabolic disorders. In addition, protein import into mitochondria has been found to be a dynamically regulated process that varies in response to conditions such as oxidative stress, aging, drug treatment, and exercise. This review focuses on how mitochondrial protein import affects human health and disease.
Keywords: Mitochondria; Protein targeting; Protein translocation; Human health and disease;

Hyperglycemia: Cell death in a cave by Eric J. Smart; Xiang-An Li (524-526).
Mitochondria play a central role in mediating high glucose-induced apoptosis. A recent study has shown that increases in glucose levels induce significant alterations in caveolae components, suggesting that high glucose may affect apoptotic signaling initiated in caveolae.
Keywords: Glucose; Apoptosis; Mitochondria; SR-BI; Caveolae;

Expression of ferroportin1, hephaestin and ceruloplasmin in rat heart by Zhong Ming Qian; Yan Zhong Chang; Gina Leung; Jun Rong Du; Li Zhu; Qin Wang; Lijian Niu; You Jia Xu; Lei Yang; Kwok Ping Ho; Ya Ke (527-532).
Iron-mediated injury plays an important role in a number of heart disorders. Studies on heart iron are therefore crucial for understanding the causes of excessive heart iron. Heart cells have the ability to accumulate transferrin-bound-iron via the transferrin receptor and non-transferrin-bound-iron probably via the L-type Ca2+ channel and the divalent metal transporter1. However, little is known about the mechanisms of iron export in the heart cells. Here, we investigated expression of iron exporters including ferroportin 1 (Fpn1), ceruloplasmin (CP) and hephaestin (Heph) and provided evidence for their existence in the heart. We demonstrated that iron has a significant effect on expression of Fpn1 and CP, but not Heph. Treatment of a high-iron diet induced a significant increase in Fpn1, a decrease in CP but no change in Heph mRNA and protein. The control of Fpn1 and CP protein expression by iron was parallel to that of their mRNA expression, suggesting a transcriptional regulation of Fpn1 and CP by iron. The existence of these proteins in the heart implies that they might have a role in heart iron homeostasis.
Keywords: Ferroportin1; Ceruloplasmin; Hephaestin; Transferrin receptor; Heart; Iron metabolism;

G11778A in the subunit ND4 gene of NADH dehydrogenase complex is the most common primary mutation found in Leber's hereditary optic neuropathy (LHON) patients. The NDI1 gene, which encodes the internal NADH–quinone oxidoreductase in Saccharomyces cerevisiae, was introduced into the nuclear genome of a mitochondrial defective human cell line, Le1.3.1, carrying the G11778A mutation. In transformant cell lines, LeNDI1-1 and -2, total and complex I-dependent respiration were fully restored and largely resistant to complex I inhibitor, rotenone, indicating a dominant role of NDI1 in the transfer of electrons in the host cells. Whereas the original mutant Le1.3.1 cell grows poorly in medium containing galactose, the transformants have a fully restored growth capacity in galactose medium, although the ATP production was not totally recovered. Furthermore, the increased oxidative stress in the cells carrying the G11778A mutation was alleviated in transformants, demonstrated by a decreased reactive oxygen species (ROS) level. Finally, transformants were also shown to be desensitized to induction to apoptosis and also exhibit greater resistance to paraquat-induced cell death. It is concluded that the yeast NDI1 enzyme can improve the oxidative phosphorylation capacity in cells carrying the G11778A mutation and protect the cells from oxidative stress and cell death.
Keywords: Mitochondrial; Complex I; LHON; NDI1; Oxidative stress; Apoptosis;

Biological roles of anti-GM1 antibodies in patients with Guillain–Barré syndrome for nerve growth factor signaling by Toshifumi Tanaka; Daisuke Furutama; Reiko Sakai; Atsushi Fujita; Fumiharu Kimura; Muneyoshi Tagami; Nakaaki Ohsawa; Toshiaki Hanafusa (543-548).
To reveal the biological and pathological roles of anti-GM1 antibody in Guillain–Barré syndrome (GBS), we examined its effects on nerve growth factor (NGF) induced TrkA autophosphorylation (NGF-TrkA signaling) in PC12 cells, a sympathetic nerve cell line. The NGF-TrkA signaling is enhanced by exogenous GM1 ganglioside and this phenomenon is regarded as one of the functional aspects of GM1. The IgGs purified from patients' sera inhibited the NGF-TrkA signaling in GM1 pre-incubated PC12 cells. The degrees of inhibition by IgGs from patients paralleled their immunological reactivity to GM1. In addition, the IgGs also inhibited the neurite outgrowth of NGF-treated PC12 cells. Immunoglobulins in the rabbit sera, which were immunized by GM1, also caused a similar suppressive phenomenon. These results suggested that the anti-GM1 antibody could play roles in pathophysiology in anti-GM1 antibody positive GBS through interfering with the neurotrophic action of NGF and GM1 mediated signal modulation including NGF-TrkA signaling. It is suggested that the modulation of GM1 function is one important action of antibodies and could be one of the important mechanisms in GBS.
Keywords: Guillain–Barré syndrome; Anti-GM1 antibody; Trk A; Nerve growth factor;

Activation of T-lymphocytes is an important component of inflammatory and infectious processes, including HIV infection. It is regulated via the actions of various cell-surface receptors, including CD4 and CXCR4. We examined the roles of CD4 and CXCR4 in the adhesive interaction of CD4 + T-cells with the vascular endothelium. CD4 + Jurkat cells were incubated in the presence or absence of anti-CD4 to stimulate CD4, or with SDF-1 alpha, a cognate ligand of CXCR4. Stimulation of CD4 or CXCR4 each significantly enhanced cell adhesion. We next stimulated the two receptors together, using gp120, a component of HIV. This enhanced cell adhesion was greater than stimulation of CD4 or CXCR4 individually. Western blotting revealed that stimulation of CXCR4 by SDF-1 alpha significantly increased the phosphorylation of ERK1/2 in Jurkat cells. Treatment with anti-CD4 also activated ERK1/2, although to a lesser extent. When the expression of CD4 was reduced by siRNA transfection, both CD4-dependent adhesion and MAPK activation were diminished. Furthermore, pre-treatment with fluvastatin, significantly attenuated observed Jurkat cell adhesion. These findings indicate novel mechanisms of CD4+ T-cells recruitment to activated endothelium via CD4 and CXCR4, which are modulated by statin.
Keywords: Endothelial cells; T-cells; Adhesion Molecules; Inflammation; Statin;

Lipid abnormalities in succinate semialdehyde dehydrogenase (Aldh5a1 /) deficient mouse brain provide additional evidence for myelin alterations by G. Barcelo-Coblijn; E.J. Murphy; K. Mills; B. Winchester; C. Jakobs; O.C. Snead; K.M. Gibson (556-562).
Earlier work from our laboratory provided evidence for myelin abnormalities (decreased quantities of proteins associated with myelin compaction, decreased sheath thickness) in cortex and hippocampus of Aldh5a1 / mice, which have a complete ablation of the succinate semialdehyde dehydrogenase protein [E.A. Donarum, D.A. Stephan, K. Larkin, E.J. Murphy, M. Gupta, H. Senephansiri, R.C. Switzer, P.L. Pearl, O.C. Snead, C. Jakobs, K.M. Gibson, Expression profiling reveals multiple myelin alterations in murine succinate semialdehyde dehydrogenase deficiency, J. Inher. Metab. Dis. 29 (2006) 143–156]. In the current report, we have extended these findings via comprehensive analysis of brain phospholipid fractions, including quantitation of fatty acids in individual phospholipid subclasses and estimation of hexose-ceramide in Aldh5a1 / brain. In comparison to wild-type littermates (Aldh5a1 +/+ ), we detected a 20% reduction in the ethanolamine glycerophospholipid content of Aldh5a1 /mice, while other brain phospholipids (choline glycerophospholipid, phosphatidylserine and phosphatidylinositol) were within normal limits. Analysis of individual fatty acids in each of these fractions revealed consistent alterations in n-3 fatty acids, primarily increased 22:6n-3 levels (docosahexaenoic acid; DHA). In the phosphatidyl serine fraction there were marked increases in the proportions of polyunsaturated fatty acids with corresponding decreases of monounsaturated fatty acids. Interestingly, the levels of hexose-ceramide (glucosyl- and galactosylceramide, principal myelin cerebrosides) were decreased in Aldh5a1 / brain tissue (one-tailed t test, p  = 0.0449). The current results suggest that lipid and myelin abnormalities in this animal may contribute to the pathophysiology.
Keywords: Succinate semialdehyde dehydrogenase (SSADH); Aldehyde dehydrogenase 5a1 (Aldh5a1); γ-hydroxybutyric acid; γ-aminobutyric acid (GABA); Myelin; Phospholipids; Ethanolamine glycerophospholipid; Ethanolamine plasmalogen; Galactosylceramide; Docosohexaenoic acid (DHA);

Guanidinoacetate methyltransferase (GAMT) deficiency is an inherited neurometabolic disorder biochemically characterized by tissue accumulation of guanidinoacetate (GAA) and depletion of creatine. Affected patients present epilepsy and mental retardation whose etiopathogeny is unclear. In a previous study we showed that instrastriatal administration of GAA caused a reduction of Na+,K+-ATPase and creatine kinase (CK) activities, as well as an increase in TBARS (an index of lipid peroxidation). In the present study we investigated the in vitro and in vivo effects of GAA on glucose uptake from [U-14C] acetate (citric acid cycle activity) and on the activities of complexes II, II–III, III and IV of the respiratory chain in striatum of rats. Results showed that 50 and 100 μM GAA (in vitro studies) and GAA administration (in vivo studies) significantly inhibited complexes II and II–III, respectively, but did not alter complexes III and IV, as well as CO2 production. We also studied the influence of taurine or vitamins E and C on the inhibitory effects caused by intrastriatal administration of GAA on complexes II and II–III, Na+,K+-ATPase and CK activities, and on TBARS in rat striatum. Pre-treatment with taurine and vitamins E and C revealed that taurine prevents the effects of intrastriatal administration of GAA on the inhibition of complex II, complex II–III, and Na+,K+-ATPase activities. Vitamins E and C prevent the effects of intrastriatal administration of GAA on the inhibition of CK and Na+,K+-ATPase activities, and on the increase of TBARS. The data suggest that GAA in vivo and in vitro treatment disturbs important parameters of striatum energy metabolism and that oxidative damage may be mediating these effects. It is presumed that defects in striatum bioenergetics might be involved in the pathophysiology of striatum damage characteristic of patients with GAMT-deficiency.
Keywords: GAMT-deficiency; Metabolic disease; Guanidinoacetate; Respiratory chain; Taurine; Vitamins E and C;

The 16 kDa subunit of vacuolar H+-ATPase is a novel sarcoglycan-interacting protein by Jiwei Chen; Mhairi A. Skinner; Weixing Shi; Qian-Chun Yu; Alan G. Wildeman; Yiu-mo Michael Chan (570-579).
The sarcoglycan complex in muscle consists of α-, β-, γ- and δ-sarcoglycan and is part of the larger dystrophin–glycoprotein complex (DGC), which is essential for maintaining muscle membrane integrity. Mutations in any of the four sarcoglycans cause limb-girdle muscular dystrophies (LGMD). In this report, we have identified a novel interaction between δ-sarcoglycan and the 16 kDa subunit c (16K) of vacuolar H+-ATPase. Co-expression studies in heterologous cell system revealed that 16K interacts specifically with δ-sarcoglycan and the highly related γ-sarcoglycan through the transmembrane domains. In cultured C2C12 myotubes, 16K forms a complex with sarcoglycans at the plasma membrane. Loss of sarcoglycans in the sarcoglycan-deficient BIO14.6 hamster destabilizes the DGC and alters the localization of 16K at the sarcolemma. In addition, the steady state level of β1-integrin is increased. Recent studies have shown that 16K also interacts directly with β1-integrin and our data demonstrated that sarcoglycans, 16K and β1-integrin were immunoprecipitated together in C2C12 myotubes. Since sarcoglycans have been proposed to participate in bi-directional signaling with integrins, our findings suggest that 16K might mediate the communication between sarcoglycans and integrins and play an important role in the pathogenesis of muscular dystrophy.
Keywords: Sarcoglycan; Vacuolar ATPase; 16K; Muscular dystrophy; Integrin; BIO14.6 hamster;

Augmented erythrocyte band-3 phosphorylation in septic mice by Michael R. Condon; Eleonora Feketova; George W. Machiedo; Edwin A. Deitch; Zoltan Spolarics (580-586).
Infection-induced RBC dysfunction has been shown to play a role in the modulation of host response to injury and infection. The underlying biochemical mechanisms are not known. This study investigated alterations in RBC band-3 phosphorylation status and its relationship to anion exchange activity in vitro as well as under in vivo septic conditions induced by cecal ligation and puncture (CLP) in mice. Pervanadate treatment in vitro increased band-3 tyrosine phosphorylation that was accompanied by decreased RBC deformability and anion exchange activity. Following sepsis, band-3 tyrosine phosphorylation in whole RBC ghosts as well as in cytoskeleton-bound or soluble RBC protein fractions were elevated as compared to controls. Although anion exchange activity was similar in RBCs from septic and control animals, band-3 interaction with eosin-5-maleimide (EMA), which binds to band-3 lysine moieties, was increased in cells from septic animals as compared to controls, indicating that sepsis altered band 3 organization within the RBC membrane. Since glucose-6-phosphate dehydrogenase is a major antioxidant enzyme in RBC, in order to assess the potential role of oxidative stress in band-3 tyrosine phosphorylation, sepsis-induced RBC responses were also compared between WT and (G6PD) mutant animals (20% of normal G6PD activity). Band-3 membrane content and EMA staining were elevated in G6PD mutant mice compared to WT under control non-septic conditions. Following sepsis, G6PD mutant animals showed lessened responses in band-3 tyrosine phosphorylation and EMA staining compared to WT. RBC anion exchange activity was similar between mutant and WT animals under all tested conditions. In summary, these studies indicate that sepsis results in elevated band-3 tyrosine phosphorylation and alters band-3 membrane organization without grossly affecting RBC anion exchange activity. The observations also suggest that factors other than oxidative stress are responsible for the sepsis-induced increase in RBC band-3 tyrosine phosphorylation.
Keywords: Sepsis; RBC deformability; Protein phosphorylation; Anion exchange; Infection; G6PD deficiency; Oxidative stress;

Enzyme enhancement activity of N-octyl-β-valienamine on β-glucosidase mutants associated with Gaucher disease by Ke Lei; Haruaki Ninomiya; Michitaka Suzuki; Takehiko Inoue; Miwa Sawa; Masami Iida; Hiroyuki Ida; Yoshikatsu Eto; Seiichiro Ogawa; Kousaku Ohno; Yoshiyuki Suzuki (587-596).
Gaucher disease (GD), caused by a defect of β-glucosidase (β-Glu), is the most common form of sphingolipidosis. We have previously shown that a carbohydrate mimic N-octyl-β-valienamine (NOV), an inhibitor of β-Glu, could increase the protein level and enzyme activity of F213I mutant β-Glu in cultured GD fibroblasts, suggesting that NOV acted as a pharmacological chaperone to accelerate transport and maturation of this mutant enzyme. In the current study, NOV effects were evaluated in GD fibroblasts with various β-Glu mutations and in COS cells transiently expressing recombinant mutant proteins. In addition to F213I, NOV was effective on N188S, G202R and N370S mutant forms of β-Glu, whereas it was ineffective on G193W, D409H and L444P mutants. When expressed in COS cells, the mutant proteins as well as the wild-type protein were localized predominantly in the endoplasmic reticulum and were sensitive to Endo-H treatment. NOV did not alter this localization or Endo-H sensitivity, suggesting that it acted in the endoplasmic reticulum. Profiling of N-alkyl-β-valienamines with various lengths of the acyl chain showed that N-dodecyl-β-valienamine was as effective as NOV. These results suggest a potential therapeutic value of NOV and related compounds for GD with a broad range of β-Glu mutations.
Keywords: Gaucher disease; β-glucosidase; Valienamine; Chaperone;