Molecular characterization of Osh6p, an oxysterol binding protein homolog in the yeast Saccharomyces cerevisiae

Oxysterol binding protein (OSBP) and its homologs have been shown to regulate lipid metabolism and vesicular transport. However, the exact molecular function of individual OSBP homologs remains uncharacterized. Here we demonstrate that the yeast OSBP homolog, Osh6p, bound phosphatidic acid and phosphoinositides via its N-terminal half containing the conserved OSBP-related domain (ORD). Using a green fluorescent protein fusion chimera, Osh6p was found to localize to the cytosol and patch-like or punctate structures in the vicinity of the plasma membrane. Further examination by domain mapping demonstrated that the N-terminal half was associated with FM4-64 positive membrane compartments; however, the C-terminal half containing a putative coiled-coil was localized to the nucleoplasm. Functional analysis showed that the deletion of OSH6 led to a significant increase in total cellular ergosterols, whereas OSH6 overexpression caused both a significant decrease in ergosterol levels and resistance to nystatin. Oleate incorporation into sterol esters was affected in OSH6 overexpressing cells. However, Lucifer yellow internalization, and FM4-64 uptake and transport were unaffected in both OSH6 deletion and overexpressing cells. Furthermore, osh6Δ exhibited no defect in carboxypeptidase Y transport and maturation. Lastly, we demonstrated that both the conserved ORD and the putative coiled-coil motif were indispensable for the in vivo function of Osh6p. These data suggest that Osh6p plays a role primarily in regulating cellular sterol metabolism, possibly stero transport.

Copper in mammals: mechanisms of homeostasis and pathophysiology

The ability of mammals to tightly regulate systemic copper levels is vital for
health as demonstrated by the severity of the genetic copper deficiency and copper toxicity disorders, Menkes disease and Wilson disease, respectively. Analysis of these genetic disorders has led to a substantial increase in the understanding of the role of copper in health and disease. The isolation of the genes involved in these diseases and use of yeast mutants with altered copper and iron homeostasis has revealed a range of molecular mechanisms governing copper homeostasis. These mechanisms include regulation of cellular copper uptake and efflux and involve the use of chaperones for safe intracellular copper distribution. Here we provide an overview of the physiological role of copper and the molecular mechanisms
regulating systemic and cellular copper levels in mammals. Furthermore, we discuss the pathophysiological mechanisms and consequences of copper deficiency/overload in relation to disease.

Exercise training increases lipid metabolism gene expression in human skeletal muscle

The effects of a single bout of exercise and exercise training on the expression of genes necessary for the transport and beta -oxidation of fatty acids (FA), together with the gene expression of transcription factors implicated in the regulation of FA homeostasis were investigated. Seven human subjects (3 male, 4 female, 28.9 ± 3.1 yr of age, range 20-42 yr, body mass index 22.6 kg/m², range 17-26 kg/m²) underwent a 9-day exercise training program of 60 min cycling per day at 63% peak oxygen uptake (V_{O2 peak}; 104 ± 14 W). On days 1 and 9 of the program, muscle biopsies were sampled from the vastus lateralis muscle at rest, at the completion of exercise, and again 3 h postexercise. Gene expression of key components of FA transport [FA translocase (FAT/CD36), plasma membrane-associated FA-binding protein], beta -oxidation [carntine palmitoyltransferase(CPT) I, beta -hydroxyacyl-CoA dehydrogenase] and transcriptional control [peroxisome proliferator-activated receptor (PPAR)alpha , PPARgamma , PPARgamma coactivator 1, sterol regulatory element-binding protein-1c] were unaltered by exercise when measured at the completion and at 3 h postexercise. Training increased total lipid oxidation by 24% (P < 0.05) for the 1-h cycling bout. This increased capacity for lipid oxidation was accompanied by an increased expression of FAT/CD36 and CPT I mRNA. Similarly, FAT/CD36 protein abundance was also upregulated by exercise training. We conclude that enhanced fat oxidation after exercise training is most closely associated with the genes involved in regulating FA uptake across the plasma membrane (FAT/CD36) and across the mitochondrial membrane (CPT I).

The molecular basis of copper homeostasis and copper-related disorders

Copper is an essential trace element that can be extremely toxic in excess due to the pro-oxidant activity of copper ions. Inherited disorders of copper transport, Menkes disease (copper deficiency), and Wilson disease (copper toxicosis) are caused by mutations of two closely related Cu transporting-ATPases, and demonstrate the essentiality and potential toxicity of copper. Other copper toxicosis conditions in humans and animals have been described, but are not well understood at a molecular level. Copper homeostatic mechanisms are being discovered. One such mechanism is copper-induced trafficking of the Cu-ATPases, which allows cells to provide copper to secreted cupro-proteins but also to efflux excess copper. Oxidative damage induced by copper may be involved in the pathogenesis of neurodegenerative conditions such as Alzheimer's disease, familial amyotrophic lateral sclerosis, and prion diseases.

Copper-induced trafficking of the Cu-ATPase: a key mechanism for copper homeostasis

The Menkes protein (MNK) and Wilson protein (WND) are transmembrane, CPX-type Cu-ATPases with six metal binding sites (MBSs) in the N-terminal region containing the motif GMXCXXC. In cells cultured in low copper concentration MNK and WND localize to the transGolgi network but in high copper relocalize either to the plasma membrane (MNK) or a vesicular compartment (WND). In this paper we investigate the role of the MBSs in Cu-transport and trafficking. The copper transport activity of MBS mutants of MNK was determined by their ability to complement a strain of Saccharomyces cerevisiae deficient in CCC2 (Deltaccc2), the yeast MNK/WND homologue. Mutants (CXXC to SXXS) of MBS1, MBS6, and MBSs1-3 were able to complement Deltaccc2 while mutants of MBS4-6, MBS5-6 and all six MBS inactivated the protein. Each of the inactive mutants also failed to display Cu-induced trafficking suggesting a correlation between trafficking and transport activity. A similar correlation was found with mutants of MNK in which various MBSs were deleted, but two constructs with deletion of MBS5-6 were unable to traffic despite retaining 25% of copper transport activity. Chimeras in which the N-terminal MBSs of MNK were replaced with the corresponding MBSs of WND were used to investigate the region of the molecules that is responsible for the difference in Cu-trafficking of MNK and WND. The chimera which included the complete WND N-terminus localized to a vesicular compartment, similar to WND in elevated copper. Deletions of various MBSs of the WND N-terminus in the chimera indicate that a targeting signal in the region of MBS6 directs either WND/MNK or WND to a vesicular compartment of the cell.

Improved glusoce homeostasis and enhanced insulin signalling in Grb 14-deficient mice

Gene targeting was used to characterize the physiological role of growth factor receptor-bound (Grb)14, an adapter-type signalling protein that associates with the insulin receptor (IR). Adult male Grb14^-/- mice displayed improved glucose tolerance, lower circulating insulin levels, and increased incorporation of glucose into glycogen in the liver and skeletal muscle. In ex vivo studies, insulin-induced 2-deoxyglucose uptake was enhanced in soleus muscle, but not in epididymal adipose tissue. These metabolic effects correlated with tissue-specific alterations in insulin signalling. In the liver, despite lower IR autophosphorylation, enhanced insulin-induced tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and activation of protein kinase B (PKB) was observed. In skeletal muscle, IR tyrosine phosphorylation was normal, but signalling via IRS-1 and PKB was increased. Finally, no effect of Grb14 ablation was observed on insulin signalling in white adipose tissue. These findings demonstrate that Grb14 functions in vivo as a tissue-specific modulator of insulin action, most likely via repression of IR-mediated IRS-1 tyrosine phosphorylation, and highlight this protein as a potential target for therapeutic intervention.

Trafficking of the copper-ATPases, ATP7A and ATP7B: Role in copper homeostasis

Copper is essential for human health and copper imbalance is a key factor in the aetiology and pathology of several neurodegenerative diseases. The copper-transporting P-type ATPases, ATP7A and ATP7B are key molecules required for the regulation and maintenance of mammalian copper homeostasis. Their absence or malfunction leads to the genetically inherited disorders, Menkes and Wilson diseases, respectively. These proteins have a dual role in cells, namely to provide copper to essential cuproenzymes and to mediate the excretion of excess intracellular copper. A unique feature of ATP7A and ATP7B that is integral to these functions is their ability to sense and respond to intracellular copper levels, the latter manifested through their copper-regulated trafficking from the transGolgi network to the appropriate cellular membrane domain (basolateral or apical, respectively) to eliminate excess copper from the cell. Research over the last decade has yielded significant insight into the enzymatic properties and cell biology of the copper-ATPases. With recent advances in elucidating their localization and trafficking in human and animal tissues in response to physiological stimuli, we are progressing rapidly towards an integrated understanding of their physiological significance at the level of the whole animal. This knowledge in turn is helping to clarify the biochemical and cellular basis not only for the phenotypes conferred by individual Menkes and Wilson disease patient mutations, but also for the clinical variability of phenotypes associated with each of these diseases. Importantly, this information is also providing a rational basis for the applicability and appropriateness of certain diagnostic markers and therapeutic regimes. This overview will provide an update on the current state of our understanding of the localization and trafficking properties of the copper-ATPases in cells and tissues, the molecular signals and posttranslational interactions that govern their trafficking activities, and the cellular basis for the clinical phenotypes associated with disease-causing mutations.

Perinatal ω-3 polyunsaturated fatty acid supply modifies brain zinc homeostasis during adulthood

Dietary ω-3 polyunsaturated fatty acid (PUFA) influences the expression of a number of genes in the brain. Zinc transporter (ZnT) 3 has been identified as a putative transporter of zinc into synaptic vesicles of neurons and is found in brain areas such as hippocampus and cortex. Neuronal zinc is involved in the formation of amyloid plaques, a major characteristic of Alzheimer's disease. The present study evaluated the influence of dietary ω-3 PUFA on the expression of the ZnT3 gene in the brains of adult male Sprague-Dawley rats. The rats were raised and/or maintained on a control (CON) diet that contained ω-3 PUFA or a diet deficient (DEF) in ω-3 PUFA. ZnT3 gene expression was analyzed by using real-time PCR, free zinc in brain tissue was determined by zinquin staining, and total zinc concentrations in plasma and cerebrospinal fluid were determined by atomic absorption spectrophotometry. Compared with CON-raised animals, DEF-raised animals had increased expression of ZnT3 in the brain that was associated with an increased level of free zinc in the hippocampus. In addition, compared with CON-raised animals, DEF-raised animals had decreased plasma zinc level. No difference in cerebrospinal fluid zinc level was observed. The results suggest that overexpression of ZnT3 due to a perinatal ω-3 PUFA deficiency caused abnormal zinc metabolism in the brain. Conceivably, the influence of dietary ω-3 PUFA on brain zinc metabolism could explain the observation made in population studies that the consumption of fish is associated with a reduced risk of dementia and Alzheimer's disease.

Antioxidant defences and homeostasis of reactive oxygen species in different human mitochondrial DNA-depleted cell lines

Three pairs of parental (ρ+) and established mitochondrial DNA depleted (ρ0) cells, derived from bone, lung and muscle were used to verify the influence of the nuclear background and the lack of efficient mitochondrial respiratory chain on antioxidant defences and homeostasis of intracellular reactive oxygen species (ROS). Mitochondrial DNA depletion significantly lowered glutathione reductase activity, glutathione (GSH) content, and consistently altered the GSH2 : oxidized glutathione ratio in all of the ρ0 cell lines, albeit to differing extents, indicating the most oxidized redox state in bone ρ0 cells. Activity, as well as gene expression and protein content, of superoxide dismutase showed a decrease in bone and muscle ρ0 cell lines but not in lung ρ0 cells. GSH peroxidase activity was four times higher in all three ρ0 cell lines in comparison to the parental ρ+, suggesting that this may be a necessary adaptation for survival without a functional respiratory chain. Taken together, these data suggest that the lack of respiratory chain prompts the cells to reduce their need for antioxidant defences in a tissue-specific manner, exposing them to a major risk of oxidative injury. In fact bone-derived ρ0 cells displayed the highest steady-state level of intracellular ROS (measured directly by 2',7'-dichlorofluorescin, or indirectly by aconitase activity) compared to all the other ρ+ and ρ0 cells, both in the presence or absence of glucose. Analysis of mitochondrial and cytosolic/iron regulatory protein-1 aconitase indicated that most ROS of bone ρ0 cells originate from sources other than mitochondria.

Lipid, FA, and sterol composition of New Zealand green lipped mussel (Perna canaliculus) and Tasmanian blue mussel (Mytilus edulis)

The lipid, FA, and sterol composition of the New Zealand green lipped mussel (NZGLM, Perna canaliculus) and of the Tasmanian blue mussel (TBM, Mytilus edulis) were compared using TLC-FID and GC-MS. The respective mussel species were obtained from three different sites in both New Zealand (NZ) and Tasmania. Lipid class distribution of both mussel species was characterized by a high proportion of phospholipid (PL, 57–79%) and TG (10–25%), FFA (7–12%), and sterols (ST, 12–18%). The NZGLM had higher proportions of TG, FFA, and ST (P<0.01), whereas the TBM had a higher proportion of PL (P<0.01). There were higher proportions of total PUFA, saturated FA, n−3 FA, and hydroxy and nonmethyleneinterrupted FA (P<0.05) in the TBM compared with the NZGLM. The major FA in the NZGLM were 16∶0 (15–17%), 20∶5n-3 (14–20%), and 22∶6n-3 (11–17%). The same FA dominated lipids in the TBM, although there were significantly higher proportions of 16∶0 (P=0.000) and 22∶6 n−3 (P=0.003) and lower proportions of 20∶5n-3 (P=0.0072) in the TBM. A novel PUFA, 28∶8n-3, was detected in both mussels with higher amounts in the TBM, which probably reflects a greater dietary contribution of dinoflagellates for this species. Cholesterol was the dominant sterol in both mussels. Other major sterols included brassicasterol, 22-methylcholesterol, trans-22-dehydrocholesterol, and desmosterol. There were significant differences (P<0.05) between the NZGLM and TBM for 12 of the 20 sterols measured. Six sterols showed significant site differences for the NZGLM, and 10 for the TBM. The differences in the FA and sterol composition between the two species may be due to the diet of the NZGLM being more diatom-derived and the diet of the TBM having a greater dinoflagellate component.

Fatty acid and sterol composition of frozen and freeze-dried New Zealand green lipped mussel (Perna canaliculus) from three sites in New Zealand

In view of previously reported anti-inflammatory bioactivity of the New Zealand Green Lipped Mussel (NZGLM), the overall lipid profile and fatty acid and sterol composition of the NZGLM from various sites in New Zealand (Hallam Cove, Port Ligar, Little Nikau) were investigated using thin layer  chromatography (TLC) and gas liquid chromatography (GLC). Samples were either frozen (F) or freeze-dried (FD) soon after collection. It was also thought prior to the study, there may be differences in the dietary sources of phytoplankton between the sites, responsible for the bioactivity, however data collected in New Zealand reported no difference in the type of phytoplankton, but a difference in the quantity. There were no major significant differences in the major components of the lipid, fatty acid and sterol composition between FD or frozen samples, nor were there any significant differences in the major composition between sites. The only major difference was between total lipid composition of the freeze-dried and frozen samples due to the removal of water during freeze-drying. Total lipid content on a dry weight basis in FD samples was 8.4 g/100g tissue and was significantly higher than frozen samples (P < 0.05) and there was no significant site variation. The lipid class content between sites was also not significantly different as judged by TLC. Triglyceride (TG) lipid fraction appeared to be the most prominent in the frozen and FD samples. The free fatty acid (FFA) band was the next most prominent band and was visually more prominent in the frozen samples. Sterol esters (SE) were detected in higher amounts in the frozen samples compared with the FD samples. Phospholipid (PL) and sterols (ST) were distributed throughout all samples. Polyunsaturated fatty acids (PUFA) were the main group of fatty acids in both FD and frozen samples (45-46%), most of which were omega-3 (n-3) fatty acids (40-41%). Saturated fatty acids (SFA) accounted for approximately one quarter of total fatty acids, with little variation between FD and frozen samples. The major fatty acids of the NZGLM were docosahexaenoic acid (DHA; 22:6n-3) (19% in both FD and frozen samples), eicosapentaenoic acid (EPA; 20:5n-3) and palmitic acid (16:0) (15% in both FD and frozen samples). Cholesterol was the most prominent sterol (31% of total sterols). Other major sterols included desmosterol/ brassicasterol (co-eluting), 24-methylenecholesterol, trans-22-dehydrocholesterol, 24- nordehydrocholesterol and occelasterol. This study is unique as it compares the lipid composition of the NZGLM from three sites in New Zealand with the additional effect of processing. This is the second comparative study investigating the lipid, fatty acid and sterol composition of the NZGLM with added interest in the effect of freeze drying on the lipid content of the mussel. This study showed that there were no major significant differences in lipid, sterol and fatty acid composition between the FD and frozen samples of the NZGLM for three sites in New Zealand. Food chain studies and further research is warranted to investigate the presence and role of major and minor lipid.
components of the NZGLM.