The contribution of surface residues to membrane binding and ligand transfer by the α-tocopherol transfer protein (α-TTP)

Previous work has shown that the α-tocopherol transfer protein (α-TTP) can bind to vesicular or immobilized phospholipid membranes. Revealing the molecular mechanisms by which α-TTP associates with membranes is thought to be critical to understanding its function and role in the secretion of tocopherol from hepatocytes into the circulation. Calculations presented in the Orientations of Proteins in Membranes database have provided a testable model for the spatial arrangement of α-TTP and other CRAL-TRIO family proteins with respect to the lipid bilayer. These calculations predicted that a hydrophobic surface mediates the interaction of α-TTP with lipid membranes. To test the validity of these predictions, we used site-directed mutagenesis and examined the substituted mutants with regard to intermembrane ligand transfer, association with lipid layers and biological activity in cultured hepatocytes. Substitution of residues in helices A8 (F165A and F169A) and A10 (I202A, V206A and M209A) decreased the rate of intermembrane ligand transfer as well as protein adsorption to phospholipid bilayers. The largest impairment was observed upon mutation of residues that are predicted to be fully immersed in the lipid bilayer in both apo (open) and holo (closed) conformations such as Phe165 and Phe169. Mutation F169A, and especially F169D, significantly impaired α-TTP-assisted secretion of α-tocopherol outside cultured hepatocytes. Mutation of selected basic residues (R192H, K211A, and K217A) had little effect on transfer rates, indicating no significant involvement of nonspecific electrostatic interactions with membranes.     

The role of α-tocopherol in motor hypofunction with aging in α-tocopherol transfer protein knockout mice as assessed by oxidative stress biomarkers

It has been hypothesized that oxidative stress plays a key role in aging. In order to elucidate the role of the antioxidant network — including α-tocopherol (αT) and αT transfer protein — in aging in vivo, α-tocopherol transfer protein knockout (αTTP^?/?) mice were fed a vitamin-E-depleted diet, and wild-type (WT) mice were fed a diet containing 0.002 wt.% αT from the age of 3 months to 1 1/2 years. The lipid oxidation markers total hydroxyoctadecadienoic acid (tHODE) and 8-iso-prostaglandin F₂α, and antioxidant levels in the blood, liver and brain were measured at 3, 6, 12 and 18 months. tHODE levels in the plasma of αTTP^?/? mice were elevated at 6 months compared to 3 months, and were significantly higher those in WT mice, although they decreased thereafter. On the other hand, tHODE levels in the liver and brain were constantly higher in αTTP^?/? mice than in WT mice. Motor activities decreased with aging in both mouse types; however, those in the αTTP^?/? mice were lower than those in the WT mice. It is intriguing to note that motor activities were significantly correlated with the stereoisomer ratio (Z,E/E,E) of HODE, which is a measure of antioxidant capacity in vivo, in the plasma, in the liver and even in the brain, but not with other factors such as antioxidant levels.In summary, using the biomarker tHODE and its stereoisomer ratio, we demonstrated that αT depletion was associated with a decrease in motor function, and that this may be primarily attributable to a decrease in the total antioxidant capacity in vivo.     

A1 toxicity in yeast. A role for Mg?

We have established conditions in which soluble Al is toxic to the yeast Saccharomyces cerevisiae. The major modifications to a standard synthetic medium were lowering the pH and the concentration of Mg ions. Alterations to the PO4, Ca, or K concentration had little effect on toxicity. Organic acids known to chelate Al reduced its toxicity, suggesting that Al3+ is the toxic Al species. The unique ability of Mg ions to ameliorate Al toxicity led us to investigate the hypothesis that Al inhibits Mg uptake by yeast. Yeast cells accumulate Mg, Co, Zn, Ni, and Mn ions via the same transport system (G.F. Fuhrmann, A. Rothstein [1968] Biochim Biophys Acta 163: 325-330). Al3+ inhibited the accumulation of 57Co2+ by yeast cells more effectively than Ga, La, or Mg. In addition, a mutant yeast strain with a defect in divalent cation uptake proved to be more sensitive to Al than a wild-type strain. Taken together, these results suggest that Al may cause Mg deficiency in yeast by blocking Mg transport. We discuss the relevance of yeast as a model for the study of Al toxicity in plant systems.     

A role for α-adducin (ADD-1) in nematode and human memory

Identifying molecular mechanisms that underlie learning and memory is one of the major challenges in neuroscience. Taken the advantages of the nematode Caenorhabditis elegans, we investigated α-adducin (add-1) in aversive olfactory associative learning and memory. Loss of add-1 function selectively impaired short- and long-term memory without causing acquisition, sensory, or motor deficits. We showed that α-adducin is required for consolidation of synaptic plasticity, for sustained synaptic increase of AMPA-type glutamate receptor (GLR-1) content and altered GLR-1 turnover dynamics. ADD-1, in a splice-form- and tissue-specific manner, controlled the storage of memories presumably through actin-capping activity. In support of the C. elegans results, genetic variability of the human ADD1 gene was significantly associated with episodic memory performance in healthy young subjects. Finally, human ADD1 expression in nematodes restored loss of C. elegans add-1 gene function. Taken together, our findings support a role for α-adducin in memory from nematodes to humans. Studying the molecular and genetic underpinnings of memory across distinct species may be helpful in the development of novel strategies to treat memory-related diseases.     

A role for protein kinase casein kinase2 α-subunits in the Arabidopsis circadian clock

Circadian rhythms are autoregulatory, endogenous rhythms with a period of approximately 24 h. A wide variety of physiological and molecular processes are regulated by the circadian clock in organisms ranging from bacteria to humans. Phosphorylation of clock proteins plays a critical role in generating proper circadian rhythms. Casein Kinase2 (CK2) is an evolutionarily conserved serine/threonine protein kinase composed of two catalytic α-subunits and two regulatory β-subunits. Although most of the molecular components responsible for circadian function are not conserved between kingdoms, CK2 is a well-conserved clock component modulating the stability and subcellular localization of essential clock proteins. Here, we examined the effects of a cka1a2a3 triple mutant on the Arabidopsis (Arabidopsis thaliana) circadian clock. Loss-of-function mutations in three nuclear-localized CK2α subunits result in period lengthening of various circadian output rhythms and central clock gene expression, demonstrating that the cka1a2a3 triple mutant affects the pace of the circadian clock. Additionally, the cka1a2a3 triple mutant has reduced levels of CK2 kinase activity and CIRCADIAN CLOCK ASSOCIATED1 phosphorylation in vitro. Finally, we found that the photoperiodic flowering response, which is regulated by circadian rhythms, was reduced in the cka1a2a3 triple mutant and that the plants flowered later under long-day conditions. These data demonstrate that CK2α subunits are important components of the Arabidopsis circadian system and their effects on rhythms are in part due to their phosphorylation of CIRCADIAN CLOCK ASSOCIATED1.     

A novel role for the apoptosis inhibitor ARC in suppressing TNFα-induced regulated necrosis

TNFα signaling can promote apoptosis or a regulated form of necrosis. ARC (apoptosis repressor with CARD (caspase recruitment domain)) is an endogenous inhibitor of apoptosis that antagonizes both the extrinsic (death receptor) and intrinsic (mitochondrial/ER) apoptosis pathways. We discovered that ARC blocks not only apoptosis but also necrosis. TNFα-induced necrosis was abrogated by overexpression of wild-type ARC but not by a CARD mutant that is also defective for inhibition of apoptosis. Conversely, knockdown of ARC exacerbated TNFα-induced necrosis, an effect that was rescued by reconstitution with wild-type, but not CARD-defective, ARC. Similarly, depletion of ARC in vivo exacerbated necrosis caused by infection with vaccinia virus, which elicits severe tissue damage through this pathway, and sensitized mice to TNFα-induced systemic inflammatory response syndrome. The mechanism underlying these effects is an interaction of ARC with TNF receptor 1 that interferes with recruitment of RIP1, a critical mediator of TNFα-induced regulated necrosis. These findings extend the role of ARC from an apoptosis inhibitor to a regulator of the TNFα pathway and an inhibitor of TNFα-mediated regulated necrosis.     

The effect of vitamin E analogues and long hydrocarbon chain compounds on calcium-induced muscle damage. A novel role for α-tocopherol?

Previous studies have demonstrated that supplemental α-tocopherol inhibited calcium-induced cytosolic enzyme efflux from normal rat skeletal muscles incubated in vitro and suggested that the protective action was mediated by the phytyl chain of α-tocopherol [1]. In order to investigate this further a number of hydrocarbon chain analogues of tocopherol (7.8-dimethyl tocol, 5,7-dimethyl tocol, tocol, α-tocotrienol, α-tocopherol [10], vitamin K1, vitamin K1 [10], vitamin K1 diacetate, vitamin K2 [20], phytyl ubiquinone and retinol) were tested for any ability to inhibit calcium ionophore, A23187, induced creatine kinase (CK) enzyme efflux. Some compounds were found to be very effective inhibitors and comparison of their structures and ability and to inhibit TBARS production in muscle homogenates revealed that the effects did not appear related to antioxidant capacity or chromanol methyl groups, but rather the length and structure of the hydrocarbon chain was the important mediator of the effects seen.     

A role for Rad23 proteins in 26S proteasome-dependent protein degradation?

Treatment of cells with genotoxic agents affects protein degradation in both positive and negative ways. Exposure of S. cerevisiae to the alkylating agent MMS resulted in activation of genes that are involved in ubiquitin- and 26S proteasome-dependent protein degradation. This process partially overlaps with the activation of the ER-associated protein degradation pathway. The DNA repair protein Rad23p and its mammalian homologues have been shown to inhibit degradation of specific substrates in response to DNA damage. Particularly the recently identified inhibition of degradation by mouse Rad23 protein (mHR23) of the associated nucleotide excision repair protein XPC was shown to stimulate DNA repair.Recently, it was shown that Rad23p and the mouse homologue mHR23B also associate with Png1p, a deglycosylation enzyme. Png1p-mediated deglycosylation plays a role in ER-associated protein degradation after accumulation of malfolded proteins in the endoplasmic reticulum. Thus, if stabilization of proteins that are associated with the C-terminus of Rad23p is a general phenomenon, then Rad23 might be implicated in the stimulation of ER-associated protein degradation as well. Interestingly, the recently identified HHR23-like protein Mif1 is also thought to play a role in ER-associated protein degradation. The MIF1 gene is strongly activated in response to ER-stress. Mif1 contains a ubiquitin-like domain which is most probably involved in binding to S5a, a subunit of the 19S regulatory complex of the 26S proteasome. On the basis of its localization in the ER-membrane, it is hypothesized that Mif1 could play a role in the translocation of the 26S proteasome towards the ER-membrane, thereby enhancing ER-associated protein degradation.     

Interaction studies between human α-tocopherol transfer protein and nitric oxide donor tocopherol analogues with LDL-protective activity

Nitric oxide-releasing α-tocopherol mimetics with LDL-protective activity were designed to maintain the tocopherol substructure necessary for its biochemical recognition by α-tocopherol transfer protein. In order to study the molecular interactions to α-TTP, theoretical binding studies by means of docking techniques and experimental binding assays, using a fluorescent probe, were performed. Furoxanyl-tocopherol-hybrid analogs 7 and 9 have the best ability to bind to α-TTP suggesting that they could be incorporated to LDL in vivo to further release nitric oxide and prevent oxidative modifications.     

The mechanism of apoptosis,cell membrane lipid peroxidation and a novel in vivo function for antioxidant vitamin E (α-tocopherol)

A critical novel function for the antioxidant vitamin E (α-tocopherol) may be its involvement in the control of apoptosis in which it acts as a ‘gate keeper’ in the regulation of membrane lipid peroxidation. The biochemical and biophysical antioxidant properties of the molecule are discussed together with recent evidence for its involvement in a possible model of the apoptotic mechanism. The original observation that led to the discovery of vitamin E (foetal resorption in pregnant rats fed on tocopherol-deficient diets) is considered as an unrecognised example of apoptosis.     

A novel autophagy modulator 6-Bio ameliorates SNCA/α-synuclein toxicity

Parkinson disease (PD) is a life-threatening neurodegenerative movement disorder with unmet therapeutic intervention. We have identified a small molecule autophagy modulator, 6-Bio that shows clearance of toxic SNCA/α-synuclein (a protein implicated in synucleopathies) aggregates in yeast and mammalian cell lines. 6-Bio induces autophagy and dramatically enhances autolysosome formation resulting in SNCA degradation. Importantly, neuroprotective function of 6-Bio as envisaged by immunohistology and behavior analyses in a preclinical model of PD where it induces autophagy in dopaminergic (DAergic) neurons of mice midbrain to clear toxic protein aggregates suggesting that it could be a potential therapeutic candidate for protein conformational disorders.     

Effects of oral micellized natural vitamin E (d-α-tocopherol) v. synthetic vitamin E (dl-α-tocopherol) in feed on α-tocopherol levels,stereoisomer distribution,oxidative stress and the immune response in piglets

This study evaluated the strategy of supplementing oral micellized natural vitamin E (d-α-tocopherol) to either piglets and/or sows on α-tocopherol concentrations in piglets serum and tissues after weaning. One first experiment tested the influence of the vitamin E supplementation source (natural form in water v. the synthetic form in feed) and dose administered to piglets and/or sows on serum α-tocopherol concentration, α-tocopherol stereoisomer accumulation, antioxidant capacity and immune response of weaned piglets. A second experiment studied the effect of sow source and dose vitamin E supplementation on some of these parameters in piglets. Oral supplementation to sows with natural vitamin E as a micellized form (d-α-tocopherol) at the lowest dose produced a similar concentration of α-tocopherol in serum at days 2, 14 and 28 postpartum to those supplemented with threefold higher dose of the synthetic form in feed. At day 39 of age, neither piglet supplementation source nor dose significantly affected α-tocopherol accumulation in the serum, muscle, subcutaneous fat or liver. Those piglets from sows supplemented with the micellized alcohol form had higher RRR-α-tocopherol stereoisomers (P<0.001) and lower (P<0.001) RRS- RSS- and RSR-α-tocopherol, at day 39 of age than those from sows supplemented with the synthetic form. A predominant importance of sow over piglet vitamin E supplementation was observed on stereoisomer distribution in piglets. Low doses of oral natural vitamin E supplementation to sows or piglets did not increase the oxidative stress of piglets when compared with the use of the synthetic form in feed. Immunoglobulin levels in piglet serum at day 39 were not affected by natural vitamin E supplementation at low doses in drinking water of piglets or sows when compared with the synthetic form in feed. IgA tended to be higher (P=0.145) at day 39 in piglets supplemented with natural vitamin E when compared with those supplemented with the synthetic form. Low doses of oral micellized natural vitamin E supplementation to sows is an interesting feeding strategy, when compared with the use of high doses of the synthetic form in feed, because it results in similar α-tocopherol concentrations, allows a predominant –R stereoisomer distribution in piglets and also maintains their oxidative status in vivo.     

Epstein-Barr virus in Burkitt’s lymphoma: A role for latent membrane protein 2A

Burkitt’s lymphoma (BL) is characterized by translocation of the MYC gene to an immunoglobulin locus. Transgenic mouse models have been used to study the molecular changes that are necessary to bypass tumor suppression in the presence of translocated MYC. Inactivation of the p53 pathway is a major step to tumor formation in mouse models that is also seen in human disease. Human BL is often highly associated with Epstein-Barr virus (EBV). The EBV latency protein latent membrane protein 2A (LMP2A) is known to promote B cell survival by affecting levels of pro-survival factors. Using LMP2A transgenic mouse models, we have identified a novel mechanism that permits lymphomagenesis in the presence of an intact p53 pathway. This work uncovers a contribution of EBV to molecular events that have documented importance in BL pathogenesis, and may underlie the poorly understood link between EBV and BL.     

Cytoprotective role of DL-α-Lipoic acid in cyclophosphamide induced myocardial toxicity

Cyclophosphamide (CP), a potent antitumor drug is known to cause severe cardiotoxicity. The present study is aimed at evaluating the cardioprotective role of lipoic acid in CP induced toxicity. Male albino rats of Wistar strain were divided into four groups and treated as follows: Group I served as control, Group II received a single dose of CP (200 mg/kg b.wt., i.p.), Group III received lipoic acid (25 mg/kg b.wt., orally) for 10 days, Group IV received CP immediately followed by lipoic acid for 10 days. In CP administered rats, the activities of tissue marker enzymes (creatine phosphokinase, lactate dehydrogenase, aspartate transaminase and alanine transaminase) were significantly (p<0.001) reduced, ATPases suffered loss in enzyme activity and thiols were depleted. Histopathological observations were also in agreement with the above abnormal changes. Lipoic acid effectively reverted these abnormal biochemical changes and minimized the histopathological lesions in heart. These observations highlight the protective role of lipoic acid in CP induced cardiac injury. (Mol Cell Biochem 276: 39–44, 2005)