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Association of apolipoprotein E gene polymorphisms with blood lipids and their interaction with dietary factors
Several candidate genes have been identified in relation to lipid metabolism, and among these, lipoprotein lipase (LPL) and apolipoprotein E (APOE) gene polymorphisms are major sources of genetically determined variation in lipid concentrations. This study investigated the association of two single nucleotide polymorphisms (SNPs) at LPL, seven tagging SNPs at the APOE gene, and a common APOE haplotype (two SNPs) with blood lipids, and examined the interaction of these SNPs with dietary factors.
METHODS:
The population studied for this investigation included 660 individuals from the Prevention of Cancer by Intervention with Selenium (PRECISE) study who supplied baseline data. The findings of the PRECISE study were further replicated using 1238 individuals from the Caerphilly Prospective cohort (CaPS). Dietary intake was assessed using a validated food-frequency questionnaire (FFQ) in PRECISE and a validated semi-quantitative FFQ in the CaPS. Interaction analyses were performed by including the interaction term in the linear regression model adjusted for age, body mass index, sex and country.
RESULTS:
There was no association between dietary factors and blood lipids after Bonferroni correction and adjustment for confounding factors in either cohort. In the PRECISE study, after correction for multiple testing, there was a statistically significant association of the APOE haplotype (rs7412 and rs429358; E2, E3, and E4) and APOE tagSNP rs445925 with total cholesterol (P = 4 × 10- 4 and P = 0.003, respectively). Carriers of the E2 allele had lower total cholesterol concentration (5.54 ± 0.97 mmol/L) than those with the E3 (5.98 ± 1.05 mmol/L) (P = 0.001) and E4 (6.09 ± 1.06 mmol/L) (P = 2 × 10- 4) alleles. The association of APOE haplotype (E2, E3, and E4) and APOE SNP rs445925 with total cholesterol (P = 2 × 10- 6 and P = 3 × 10- 4, respectively) was further replicated in the CaPS. Additionally, significant association was found between APOE haplotype and APOE SNP rs445925 with low density lipoprotein cholesterol in CaPS (P = 4 × 10- 4 and P = 0.001, respectively). After Bonferroni correction, none of the cohorts showed a statistically significant SNP-diet interaction on lipid outcomes.
CONCLUSION:
In summary, our findings from the two cohorts confirm that genetic variations at the APOE locus influence plasma total cholesterol concentrations, however, the gene-diet interactions on lipids require further investigation in larger cohorts
Ras-association domain of sorting nexin 27 is critical for regulating expression of GIRK potassium channels
G protein-gated inwardly rectifying potassium (GIRK) channels play an important role in regulating neuronal excitability. Sorting nexin 27b (SNX27b), which reduces surface expression of GIRK channels through a PDZ domain interaction, contains a putative Ras-association (RA) domain with unknown function. Deleting the RA domain in SNX27b (SNX27b-DRA) prevents the down-regulation of GIRK2c/GIRK3 channels. Similarly, a point mutation (K305A) in the RA domain disrupts regulation of GIRK2c/GIRK3 channels and reduces H-Ras binding in vitro. Finally, the dominant-negative H-Ras (S17N) occludes the SNX27b-dependent decrease in surface expression of GIRK2c/GIRK3 channels. Thus, the presence of a functional RA domain and the interaction with Ras-like G proteins comprise a novel mechanism for modulating SNX27b control of GIRK channel surface expression and cellular excitability
Ras-association domain of sorting nexin 27 is critical for regulating expression of GIRK potassium channels
G protein-gated inwardly rectifying potassium (GIRK) channels play an important role in regulating neuronal excitability. Sorting nexin 27b (SNX27b), which reduces surface expression of GIRK channels through a PDZ domain interaction, contains a putative Ras-association (RA) domain with unknown function. Deleting the RA domain in SNX27b (SNX27b-DRA) prevents the down-regulation of GIRK2c/GIRK3 channels. Similarly, a point mutation (K305A) in the RA domain disrupts regulation of GIRK2c/GIRK3 channels and reduces H-Ras binding in vitro. Finally, the dominant-negative H-Ras (S17N) occludes the SNX27b-dependent decrease in surface expression of GIRK2c/GIRK3 channels. Thus, the presence of a functional RA domain and the interaction with Ras-like G proteins comprise a novel mechanism for modulating SNX27b control of GIRK channel surface expression and cellular excitability
Deletion of RA domain in SNX27b affects localization of GIRK2c/3 channels monitored with BiFC.
<p><b>A</b>, <i>Left</i>, Schematic shows placement of split YFP on GIRK2c and GIRK3. Note the C-terminal domains are free to interact with other proteins. <i>Right</i>, BiFC-tagged GIRK2c/3 channels are functional. Current-voltage plot is shown for <sup>CY</sup>GIRK2c/<sup>NY</sup>GIRK3 channels. Baclofen (100 µM) activates and Ba<sup>2+</sup> (1 mM) inhibits inwardly rectifying current. HEK293T cells were transfected with cDNA encoding GABA<sub>B1a</sub>, GABA<sub>B2</sub> and <sup>CY</sup>GIRK2c/<sup>NY</sup>GIRK3. Average baclofen-induced current densities were –13.2±6.0 pA⋅pF<sup>−1</sup> (n = 3) for <sup>CY</sup>GIRK2c/<sup>NY</sup>GIRK3. <b>B</b>, HEK293 cells were co-transfected with <sup>CY</sup>GIRK2c, <sup>NY</sup>GIRK3 and either control cDNA (<i>i</i>), wild-type SNX27b (<i>ii</i>), SNX27b-ΔRA (deletion of Asp272-Trp358) (<i>iii</i>) or SNX27b-Y51L (a PDZ mutation) (<i>iv</i>). Green fluorescence in images represents molecular recombination of <sup>CY</sup>GIRK2c/<sup>NY</sup>GIRK3 heterotetramers. Coexpression of wild-type SNX27b induced formation of puncta. By contrast, <sup>CY</sup>GIRK2c/<sup>NY</sup>GIRK3 fluorescence was diffuse in the cytoplasm for SNX27b-ΔRA and for SNX27b-Y51L, similar to control. Inset shows zoom of boxed area. <b>C,</b> SNX27b-ΔRA exhibited a pattern of punctate expression similar that of wild-type SNX27b. YFP was fused to the C-terminus of SNX27b or SNX27b-ΔRA to directly visualize expression. HEK293T cells were transfected with cDNA for SNX27b-YFP and SNX27bΔRA-YFP. Scale bar: 10 µm.</p
Deletion of RA domain in SNX27b impairs functional regulation of GIRK channels.
<p><b>A</b>, Cartoon shows model of GIRK channels regulation by SNX27b. GIRK channels recycle through endosomal compartments. SNX27b associating with GIRK2c/3 channels in the early endosome (EE) reduces plasma membrane expression (PM) by targeting some channels to the late endosome (LE). <b>B</b>, SNX27 contains three functional domains; PDZ, PX and RA. GIRK2c and GIRK3 contain a C-terminal PDZ binding motif (-E(S/N)ESKV). <b>C,</b> Examples of baclofen-induced (100 µM) and Ba<sup>2+</sup>-sensitive (1 mM Ba<sup>2+</sup>) currents in HEK293T cells transfected with cDNA for GABA<sub>B1a/B2</sub> receptors, GIRK2c/GIRK3 and either control vector, SNX27b or SNX27b-ΔRA. Agonist-independent basal currents are revealed by inhibition with 1 mM Ba<sup>2+</sup>. <b>D</b>, Average baclofen-induced current densities (I<sub>Baclofen</sub>) for control (–41.3±5.2 pA⋅pF<sup>−1</sup>, n = 8), SNX27b (–11.0±3.6 pA⋅pF<sup>−1</sup>, n = 8) and SNX27b- ΔRA (–55.9±8.2 pA⋅pF<sup>−1</sup>, n = 13) with GIRK2c/GIRK3. <b>E</b>, Average I<sub>Baclofen</sub> for control (–15.7±3.6 pA⋅pF<sup>−1</sup>, n = 6) and SNX27b-ΔRA (–18.6±4.9 pA⋅pF<sup>−1</sup>, n = 5) with GIRK1/GIRK3 (**P<0.05, one way ANOVA followed by Bonferroni <i>post hoc</i> test; n.s. – not significant).</p
Dominant-negative H-Ras (H-Ras<sub>S17N</sub>DN) prevents SNX27b-dependent down-regulation of GIRK channels.
<p><b>A</b>, Schematic shows GIRK2c and GIRK3 with PDZ-binding motif and GIRK2a and GIRK3-RR, which lack motifs that interact with SNX27-PDZ <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059800#pone.0059800-Balana1" target="_blank">[7]</a>. <b>B</b>, Examples of baclofen-induced (100 µM) and Ba<sup>2+</sup>-sensitive (1 mM Ba<sup>2+</sup>) currents in HEK293 cells transfected with cDNA for GABA<sub>B1a/B2</sub>, GIRK2c/GIRK3 and either empty vector (Control), H-Ras<sub>S17N</sub>DN or H-Ras<sub>S17N</sub>DN plus SNX27b. <b>C</b>, Bar graph shows average I<sub>Baclofen</sub> for GIRK2c/GIRK3 alone (–47.7±8.5 pA⋅pF<sup>−1</sup>, n = 6), GIRK2c/GIRK3 and H-Ras<sub>S17N</sub>DN (–22.0±3.8 pA⋅pF<sup>−1</sup>, n = 19) or GIRK2c/GIRK3, SNX27b and H-Ras<sub>S17N</sub>DN (–23.7±4.7 pA⋅pF<sup>−1</sup>, n = 16). <b>D</b>, Bar graph shows I<sub>Baclofen</sub> for control (GIRK2a/GIRK3-RR alone) (–18.8±3.0 pA⋅pF<sup>−1</sup>, n = 10) and GIRK2a/GIRK3-RR plus H-Ras<sub>S17N</sub>DN (–18.6±2.0 pA⋅pF<sup>−1</sup>, n = 11). <b>E</b>, Bar graph shows average I<sub>Barium</sub> for GIRK2c/GIRK3 alone (–27.6±9.8 pA⋅pF<sup>−1</sup>, n = 6), GIRK2c/GIRK3 and H-Ras<sub>S17N</sub>DN (–7.3±2.2 pA⋅pF<sup>−1</sup>, n = 18) or GIRK2c/GIRK3, SNX27b and H-Ras<sub>S17N</sub>DN (–7.1±1.5 pA⋅pF<sup>−1</sup>, n = 14). <b>F</b>, Bar graph shows average I<sub>Barium</sub> for GIRK2a/GIRK3-RR alone (−2.49±0.9 pA⋅pF<sup>−1</sup>, n = 10), GIRK2c/GIRK3 and H-Ras<sub>S17N</sub>DN (−1.84±0.4 pA⋅pF<sup>−1</sup>, n = 11). **P<0.05, one way ANOVA followed by Bonferroni post hoc test; n.s. – not significant.</p
K305A point mutation in SNX27b RA domain disrupts functional regulation of GIRK2c/GIRK3 channels.
<p><b>A</b>, Alignment of two different RA domains: a RID (Ras interacting domain) from RalGDS and a RA domain of RGL, with RA domain of SNX27. Residues implicated in Ras binding in Raf and RalGDS domains <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059800#pone.0059800-Block1" target="_blank">[28]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059800#pone.0059800-Vetter1" target="_blank">[30]</a> are highlighted in red. <b>B</b>, High-resolution structure shows R20, K32 and K52 at the binding interface of RID and Ras <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059800#pone.0059800-Huang1" target="_blank">[26]</a>. <b>C,</b> RA domain mutations impair the ability of SNX27b to induce formation of GIRK2c/3 puncta using BiFC. HEK293T cells were co-transfected with cDNA for <sup>CY</sup>GIRK2c/<sup>NY</sup>GIRK3 and either wild-type SNX27b (<b>i</b>), SNX27b-R276A (<b>ii</b>), SNX27b-R288A/K291A (<b>iii</b>) or SNX27b-K305A (<b>iv</b>). Green fluorescence indicates molecular complementation of <sup>CY</sup>GIRK2c/<sup>NY</sup>GIRK3. Scale bar: 10 µm. <b>D</b>, Colocalization of wild-type SNX27b (SNX27b-YFP; green) and SNX27b-K305A (anti-SNX27; green) with an early endosomal marker (anti-EEA1, green). Scale bar: 5 µm. <b>E</b>, Average I<sub>Baclofen</sub> currents for control (–53.4±9.4 pA⋅pF<sup>−1</sup>, n = 5), SNX27b (–8.05±3.44 pA⋅pF<sup>−1</sup>, n = 6) and SNX27b-K305A (–52.9±8.8 pA⋅pF<sup>−1</sup>, n = 10) with GIRK2c/GIRK3 channels.</p