N-3 polyunsaturated fatty acid, lipid microclusters, and Vit E Flashcards
lipid rafts
cholesterol-dependent signaling or sphingolipid-cholesterol-enriched microdomains that compartmentalize signalling proteins
cholesterol-dependent signaling or sphingolipid-cholesterol-enriched microdomains that compartmentalize signalling proteins
lipid rafts
lipid rafts are generally nanoscale, but can form micron scale domains upon ?
receptor ligation
N-3 PUFA directly binding G-protein coupled receptor (GPR) 120 results in ?
anti-inflammatory effects in macrophages important in regulating insulin signaling in obesity
N-3 PUFA acyl chains, upon enzymatic release from the membrane, regulate eicosanoid production and can serve as precursors for the synthesis of lipid mediators known as ?, ? and ?
resolvins, protectins, and maresins
downstream of the plasma membrane, n-3 PUFAs exert their effects by targeting ? to modify gene expression
transcription factors
presence of multiple double bonds in n-3 PUFA acyl chains gives a high/low degree of conformational freedom
high
Our understanding of DHAs structure comes from ? and ?
2H NMR (experimental) and MD simulations (computational)
In NMR studies, DHA is typically esterified to PC or PE in the ? position while palmitic or stearing acid occupies the ? position
DHA occupies sn-2. palmitic or stearing acid occupies the sn-1 position
the fundamental property that distinguishes n-3 PUFA from less unsaturated fatty acids
tremendously high disorder
origin of disorder in n-3 PUFAs
low energy barrier about C-C single bonds in the repeating units that link C=C double bonds
indicative of low microviscosity, order parameters measured by solid state 2H NMR throughout almost all of the DHA chain in the sn-2 position are large/small
small. S_CD ~ 0.01
order within the stearic acid chain at the sn-1 position is increased/decreased by the presence of DHA in sn-2 position
decreased order (~10%)
reduction in stearic acid order as a result of DHA is more significant in the upper/lower half
lower half
rapid isomerization of DHA chain. explores entire conformational space in ?
~ 10 nanoseconds
DHA MD simulations reveal bent configurations where ____
terminal methyl group approaches hydrophobic-philic and redistribution of the lower portion of DHA chains toward the interface
DHA acyl chains generally increase/decrease microviscosity (to different degrees in different cell types)
decrease
DHA effects on physical properties of bilayers
reduced membrane thickness, increased compressibility, permeability, fusion and flip-flop
DHA has high/low affinity for cholesterol
low affinity
rapid interconversion between torsional states undergone by PUFAs pushes the rigid steroid moiety away was shown by ?
low solubility and binding coefficients for cholesterol in polyunsaturated membranes
PUFA induced reduction in cholesterol affinity is more significant in PE or PC? why?
PE. tighter packing associated with smaller headgroup brings sterol and PUFA closer together
sphingolipid had high/low affinity for cholesterol. why?
high affinity. saturated and adopt largely linear conformation
in the extreme case of bilayers comprised of phospholipids with a PUFA chain at both sn-1 and sn-2 positions, sterol can be forced into an unusual position where?
deep within the interior of the bilayer
DHA remodels lipid raft-like domains that are nano-sized in a manner that is dependent upon the ____ headgroup. How was this observed?
phospholipid. observed using solid state 2H NMR, DSC, and detergent extraction experiments
Majority of PDPE was found to segregate into domains high/low in SM and cholesterol
low
introduction of cholesterol increased/decreased the order of PDPE more significantly than that seen with POPE. How was this observed?
increased. From 2H NMR spectra for analogs of PE perdeuterated in the sn-1 chain at 40 C
More than 3x as much PDPE (70%) as POPE (22%) was isolated in detergent soluble (nonraft) fractions while almost all of the cholesterol and SM (>90%) were isolated in detergent resistant (raft) fractions. How was this observed?
detergent extraction experiments
PDPC was shown to directly infiltrate into SM and cholesterol rich domains. This was observed using?
2H NMR (williams 2012)
what is the significance of the formation of inverted hexagonal (Hii) phase that becomes accentuated with increasing levels of acyl chain saturation
??? (Dekker, Kessel, Klomp 1983)
XX% of EPA containing PC was found in raft-like domains compared to 70% for DHA containing PC. Conclusion?
40% of EPA PC. DHA PC is more likely to incorporate into lipid rafts and disrupt organization
DRMs and DSMs are isolated from ____ and used to represent native rafts and nonrafts
sucrose gradient fractions of cells subjected to low levels of detergent at cold temperature
the most common approach to studying n-3 PUFAs and lipid rafts with DRM and DSM fractions is to either _____ or _____
either treat imortal cells with EPA or DHA or provide animals diets rich in EPA or DHA
Most prevalent change resulting from n-3 PUFA uptake? consequence?
arachidonic acid levels decrease. AA itself is a disordered PUFA and its loss could make membrane more packed. Also numerous lipid mediators are derived from AA.
Effect of n-3 PUFA uptake on cholesterol
cholesterol is displaced between DRM and DSM fractions. However, no clear consensus on direction of movement
In response to DHA treatment, cholesterol levels increase in ____ fractions and decrease in ____ fractions
DEPENDS! sometimes cholesterol moves between DRMs and DSMs in either direction, sometimes can selectively lower with no increase in the other
clustering of GM1 molecules is diminished in cell culture treated with DHA/EPA
DHA
what is cholera toxin subunit b used for in GM1 molecules
artificial means of inducing lipid rafts on a micron scale
translational studies with splenic B220+ B cells show that dietary administration of _____ to animals diminished GM1 clustering in a dose dependent manner
n-3 PUFAs
polarization microscopy experiments revelaed that n-3 PUFAs enhanced/diminished membrane packing in B and T lymphocytes
enhanced
fat-1 transgenic mice have high/low levels of endogenous n-3 PUFAs. why?
high levels due to a desaturase that converts n-6 to n-3
it was shown that lipid raft accumulation in the immunological synapse was high/low for fat-1 mice compared to control mice
accumulation high for fat-1 mice
consequence of accumulation in the immunological synapse
suppressed helper CD4+ Th1 cell activation (potential health benefits in murine models)
work in B cells and EL4 lymphomas showed that crosslinking of GM1 microdomains with cholera toxin subunit b had this effect on membrane packing?
enhanced membrane packing with dietary n-3 PUFAs compared to cells not cross-linked with cholera toxin
EPA and DHA ethyl esters (modeling prescription supplements) differentially enhanced membrane packing upon formation of lipid microdomains induced by ?
lowering the temperature of cells to 4 C
n-3 PUFAs MAY be redistributing cholesterol in a manner that increases/decreases formation of lipid rafts
increases
it is possible/not possible that the effects of n-3 PUFAs are independent of cholesterol
possible
disrupting lipid raft microdomains in B and T lymphocytes can displace key proteins and suppress function. This has potential health benefits for these types of diseases?
inflammatory diseases including rheumatoid arthritis, renal transplant, or metabolic diseases
In B cells, MHC II molecules are displaced from raft-enriched regions of the immunological synapse by n-3 PUFAs. This was observed using ?
confocal and acceptor photobleaching FRET studies. Similar observation for MHC I molecules.
In CD4+ T cells, protein kinase C and phospholipase C gamma-1 were displaced from raft-like membranes by n-3 PUFAs. This was observed using ?
biochemical DRM/DSM fractionations and confocal imaging
n-3 PUFAs generally have a preference for incorporation into ?
PE’s rather than PC’s
what might happen when n-3 PUFAs incorporate into PC’s
PC’s are abundant in lipid rafts. this may force cholesterol out of the lipid rafts and into non-raft regions making them more raft like
what might happen when n-3 PUFAs incorporate into PE’s
rafts are generally not enriched in PE’s. n-3 PUFA acyl chains may force cholesterol to move into rafts and make them more packed
n-3 PUFAs phase seperate from liquid-ordered lipid raft -like domains when incorporated into ?
incorporated into PE’s
n-3 PUFAs incorporate directly into liquid liquid-ordered rafts when esterified to ?
esterified to PC’s
aside from phospholipids, what else might n-3 PUFAs esterify to ?
SM (shown in murine) or cholesterol (shown in cholesterol esters. low membrane solubility)
the major lipid soluble antioxidant
Vitamin E (alpha tocopherol)
advantage of n-3 PUFA-vitamin E clusters?
could explain how low levels of Vit E can protect relatively large amounts of n-3 PUFA from oxidation
chromanol group of Vit E generally resides here?
near the hydrophobic-philic interface of bilayers
DMPC location in bilayer
in center at the water interface independent of headgroup or acyl chains
emerging evidence for sphingolipid enriched cholesterol-free domains comes from ?
high res mass spectrometry
emerging evidence for sphingolipid enriched cholesterol-free domains comes from ?. What was observed?
high res mass spectrometry in cell culture. labelled sphingolipids form micron size domains that are not disrupted by cholesterol depletion. However, they were effectd by disruption of the cytoskeleton.
helper t-cells isolated from fat-1 transgenic mice or from mice fed DHA displayed a 50% reduction in phosphatidylinositol 4,5-bisphosphate which contributed toward?
diminished actin remodeling and thereby T cell activation
___ and ___ delay opening of the mitochondrial permeability transition pore in the rat myocardium.
EPA and DHA
