Utility of a Fluorescent Vitamin E Analogue as a Probe for Tocopherol Transfer Protein Activity

Morley, Samantha; Cross, Valerie; Cecchini, Matt; Nava, Phil; Atkinson, Jeffrey; Manor, Danny

doi:10.1021/bi052271y

Cited by 31 publications

(27 citation statements)

References 35 publications

(52 reference statements)

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“…The hope is that a-tocohexaenol will be able to be used in a similar fashion as cis-parinaric acid, but be able to report more specifically on the location of tocopherol in a membrane. Other fluorescent analogues of tocopherol including C9-NBD-a-tocopherol [87] and C8-BODIPY-a-tocopherol (West, R., submitted) have been useful in determining the cellular location and transfer of tocopherol [88][89][90][91][92], but do not change fluorescence on oxidation.…”

Section: Do We Know Where Tocopherol Does Its Most Important Job?mentioning

confidence: 99%

The location and behavior of α‐tocopherol in membranes

Atkinson

Harroun

Wassall

et al. 2010

Molecular Nutrition Food Res

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167

127

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Vitamin E (a-tocopherol) has long been recognized as the major antioxidant in biological membranes, and yet many structurally related questions persist of how the vitamin functions. For example, the very low levels of a-tocopherol reported for whole cell extracts question how this molecule can successfully protect the comparatively enormous quantities of PUFAcontaining phospholipids found in membranes that are highly susceptible to oxidative attack. The contemporary realization that membranes laterally segregate into regions of distinct lipid composition (domains), we propose, provides the answer. We hypothesize a-tocopherol partitions into domains that are enriched in polyunsaturated phospholipids, amplifying the concentration of the vitamin in the place where it is most needed. These highly disordered domains depleted in cholesterol are analogous, but organizationally antithetical, to the wellstudied lipid rafts. We review here the ideas that led to our hypothesis. Experimental evidence in support of the formation of PUFA-rich domains in model membranes is presented, focusing upon docosahexaenoic acid that is the most unsaturated fatty acid commonly found. Physical methodologies are then described to elucidate the nature of the interaction of a-tocopherol with PUFA and to establish that the vitamin and PUFA-containing phospholipids co-localize in non-raft domains.

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Section: Do We Know Where Tocopherol Does Its Most Important Job?mentioning

confidence: 99%

The location and behavior of α‐tocopherol in membranes

Atkinson

Harroun

Wassall

et al. 2010

Molecular Nutrition Food Res

Self Cite

167

127

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“…NBD-cholesterol (Invitrogen) and NBD-tocopherol ( 42,43 ) were complexed to serum lipoprotein as described earlier ( 35,44 ) and added to the culture media to a fi nal concentration of 20 M and incubated for 17 h at 37°C. The fl uorescent lipid was "chased" by incubation in normal media for 3 h more.…”

Section: Fluorescence Microscopymentioning

confidence: 99%

“…Given that sphingomyelin, glycosphingolipids, and phospholipids also accumulate in NPC-affected lysosomes ( 2, 48-51 ), we hypothesized that NPC1/2 proteins participate in the endocytic processing of the lipid-soluble antioxidant ␣ -tocopherol (vitamin E). To visualize the intracellular traffi cking of ␣ -tocopherol, we utilized NBD-tocopherol, a fl uorescent analog that we previously characterized in vitro ( 42,43,52,53 ) and in vivo ( 35,54 ). Using fl uorescence microscopy, we visualized the accumulation of NBD-tocopherol in cultured fi broblasts isolated from an NPC-affected patient (harboring the c.709C>T and c.3182T>C substitutions in the NPC1 gene) and control fi broblasts.…”

Section: ␣ -Tocopherol Accumulates In Npc-affected Fi Broblastsmentioning

confidence: 99%

Altered vitamin E status in Niemann-Pick type C disease

Ulatowski

Parker

Davidson

et al. 2011

Journal of Lipid Research

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“…Uptake of ␣-Tocopherol in Cultured Hepatocytes-To examine the uptake of ␣-tocopherol into hepatocytes with high spatial resolution, we utilized NBD-␣-tocopherol, a fluorescent analog that we have previously shown to be a reliable reporter of the vitamin's location in cells and when bound to TTP (19,30,31,33,(42)(43)(44)(45)(46). To examine the time course of vitamin E uptake, cells were incubated with the serum⅐NBD-␣-tocopherol complex at 4°C, and the unbound vitamin was washed with serumfree media and uptake initiated by switching the cells back to 37°C.…”

Section: Resultsmentioning

confidence: 99%

“…The next day, DMEM was added to achieve a final serum concentration of 20% (v/v). Concentration of NBD-␣-tocopherol was determined spectrophotometrically, using an extinction coefficient ⑀ 466 nm ϭ 22,000 M Ϫ1 cm Ϫ1 (30,42). ␣-Tocopherol Treatment-We employed two separate protocols for treating vitamin E-depleted cells with ␣-tocopherol or NBD-␣-tocopherol.…”

Section: Methodsmentioning

confidence: 99%

Vitamin E and Phosphoinositides Regulate the Intracellular Localization of the Hepatic α-Tocopherol Transfer Protein

Chung¹,

Ghelfi

Atkinson

et al. 2016

Journal of Biological Chemistry

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␣-Tocopherol (vitamin E) is an essential nutrient for all vertebrates. From the eight naturally occurring members of the vitamin E family, ␣-tocopherol is the most biologically active species and is selectively retained in tissues. The hepatic ␣-tocopherol transfer protein (TTP) preferentially selects dietary ␣-tocopherol and facilitates its transport through the hepatocyte and its secretion to the circulation. In doing so, TTP regulates body-wide levels of ␣-tocopherol. The mechanisms by which TTP facilitates ␣-tocopherol trafficking in hepatocytes are poorly understood. We found that the intracellular localization of TTP in hepatocytes is dynamic and responds to the presence of ␣-tocopherol. In the absence of the vitamin, TTP is localized to perinuclear vesicles that harbor CD71, transferrin, and Rab8, markers of the recycling endosomes. Upon treatment with ␣-tocopherol, TTP-and ␣-tocopherol-containing vesicles translocate to the plasma membrane, prior to secretion of the vitamin to the exterior of the cells. The change in TTP localization is specific to ␣-tocopherol and is time-and dose-dependent. The aberrant intracellular localization patterns of lipid binding-defective TTP mutants highlight the importance of protein-lipid interaction in the transport of ␣-tocopherol. These findings provide the basis for a proposed mechanistic model that describes TTP-facilitated trafficking of ␣-tocopherol through hepatocytes.Vitamin E is a plant-derived lipid that was discovered as a dietary component vital for female fertility in rodents (1) and for neuronal health in humans (2). Eight vitamin E forms are synthesized by plants, differing in the degree of methylation of the chromanol ring and the saturation of the isoprenoid side chain (3). Of the eight naturally occurring forms of vitamin E, ␣-tocopherol exhibits the most potent biological activity in preventing deficiency-induced reproductive failure in rodents (4, 5). ␣-Tocopherol's efficacy in scavenging free radicals (i.e. functioning as an antioxidant) is thought to underlie its critical roles in health (6 -8), but additional redox-independent actions have been recently proposed (9).The major regulator of vitamin E status is the ␣-tocopherol transfer protein (TTP), 2 and mutations in the TTPA gene cause heritable vitamin E deficiency. In humans, mutations in the TTPA gene cause systemic vitamin E deficiency accompanied by neurological compromise, primarily spinocerebellar ataxia (10 -17). Patients afflicted with this disorder (ataxia with vitamin E deficiency; OMIM 277460) present with debilitating neurodegeneration, the severity of which depends on the specific mutation's effect on TTP's biochemical activity (18,19). Lifelong vitamin E supplementation can delay or reverse disease progression, especially when patients are treated in earlier stages (20,21). Disruption of the TtpA gene in mice recapitulated the human ataxia with vitamin E deficiency disorder (22).TTP is expressed at high levels in parenchymal cells of the liver (23, 24), but low level expression has b...

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Utility of a Fluorescent Vitamin E Analogue as a Probe for Tocopherol Transfer Protein Activity

Cited by 31 publications

References 35 publications

The location and behavior of α‐tocopherol in membranes

The location and behavior of α‐tocopherol in membranes

Altered vitamin E status in Niemann-Pick type C disease

Vitamin E and Phosphoinositides Regulate the Intracellular Localization of the Hepatic α-Tocopherol Transfer Protein

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