👤 T R Graham

The aim of the present study was to establish mitochondrial cholesterol trafficking 18 kDa translocator protein (TSPO) as a potential therapeutic target, capable of increasing macrophage cholesterol efflux to (apo)lipoprotein acceptors. Expression and activity of TSPO in human (THP-1) macrophages were manipulated genetically and by the use of selective TSPO ligands. Cellular responses were analysed by quantitative PCR (Q-PCR), immunoblotting and radiolabelling, including [3H]cholesterol efflux to (apo)lipoprotein A-I (apoA-I), high-density lipoprotein (HDL) and human serum. Induction of macrophage cholesterol deposition by acetylated low-density lipoprotein (AcLDL) increased expression of TSPO mRNA and protein, reflecting findings in human carotid atherosclerosis. Transient overexpression of TSPO enhanced efflux (E%) of [3H]cholesterol to apoA-I, HDL and human serum compared with empty vector (EV) controls, whereas gene knockdown of TSPO achieved the converse. Ligation of TSPO (using PK11195, FGIN-1-27 and flunitrazepam) triggered increases in [3H]cholesterol efflux, an effect that was amplified in TSPO-overexpressing macrophages. Overexpression of TSPO induced the expression of genes [PPARA (peroxisome-proliferator-activated receptor α), NR1H3 (nuclear receptor 1H3/liver X receptor α), ABCA1 (ATP-binding cassette A1), ABCG4 (ATP-binding cassette G4) and APOE (apolipoprotein E)] and proteins (ABCA1 and PPARα) involved in cholesterol efflux, reduced macrophage neutral lipid mass and lipogenesis and limited cholesterol esterification following exposure to AcLDL. Thus, targeting TSPO reduces macrophage lipid content and prevents macrophage foam cell formation, via enhanced cholesterol efflux to (apo)lipoprotein acceptors. Show less

Wnt pathways control key biological processes that potentially impact on tumor progression and patient survival. We aimed to evaluate DNA methylation at promoter CpG islands (CGI) of Wnt pathway genes in ovarian tumors at presentation and identify biomarkers of patient progression-free survival (PFS). Epithelial ovarian tumors (screening study n = 120, validation study n = 61), prospectively collected through a cohort study, were analyzed by differential methylation hybridization at 302 loci spanning 189 promoter CGIs at 137 genes in Wnt pathways. The association of methylation and PFS was examined by Cox proportional hazards model. DNA methylation is associated with PFS at 20 of 302 loci (P < 0.05, n = 111), with 5 loci significant at false discovery rate (FDR) less than 10%. A total of 11 of 20 loci retain significance in an independent validation cohort (n = 48, P ≤ 0.05, FDR ≤ 10%), and 7 of these loci, at FZD4, DVL1, NFATC3, ROCK1, LRP5, AXIN1, and NKD1 genes, are independent from clinical parameters (adjusted P < 0.05). Increased methylation at these loci associates with increased hazard of disease progression. A multivariate Cox model incorporates only NKD1 and DVL1, identifying two groups differing in PFS [HR = 2.09; 95% CI (1.39-3.15); permutation test P < 0.005]. Methylation at DVL1 and NFATC3 show significant association with response. Consistent with their epigenetic regulation, reduced expression of FZD4, DVL1, and ROCK1 is an indicator of early-disease relapse in an independent ovarian tumor cohort (n = 311, adjusted P < 0.05). The data highlight the importance of epigenetic regulation of multiple promoter CGIs of Wnt pathway genes in ovarian cancer and identify methylation at NKD1 and DVL1 as independent predictors of PFS. Show less

Poly(A)-binding protein 1 (PABP1) has a fundamental role in the regulation of mRNA translation and stability, both of which are crucial for a wide variety of cellular processes. Although generally a diffuse cytoplasmic protein, it can be found in discrete foci such as stress and neuronal granules. Mammals encode several additional cytoplasmic PABPs that remain poorly characterised, and with the exception of PABP4, appear to be restricted in their expression to a small number of cell types. We have found that PABP4, similarly to PABP1, is a diffusely cytoplasmic protein that can be localised to stress granules. However, UV exposure unexpectedly relocalised both proteins to the nucleus. Nuclear relocalisation of PABPs was accompanied by a reduction in protein synthesis but was not linked to apoptosis. In examining the mechanism of PABP relocalisation, we found that it was related to a change in the distribution of poly(A) RNA within cells. Further investigation revealed that this change in RNA distribution was not affected by PABP knockdown but that perturbations that block mRNA export recapitulate PABP relocalisation. Our results support a model in which nuclear export of PABPs is dependent on ongoing mRNA export, and that a block in this process following UV exposure leads to accumulation of cytoplasmic PABPs in the nucleus. These data also provide mechanistic insight into reports that transcriptional inhibitors and expression of certain viral proteins cause relocation of PABP to the nucleus. Show less

Fasting and postprandial triacylglycerol (TAG) concentrations are risk factors for cardiovascular disease. This study evaluated whether interleukin-6 (IL-6) and incretin hormones [gastric inhibitory peptide (GIP) and glucagon-like peptide-1 (GLP-1) (active)] were associated with fasting and postprandial TAG in response to an oral lipid load, including very-low-density lipoprotein (VLDL) and chylomicron (CM) TAG, following one bout of exercise in nine men (age, 59 ± 2 years; body mass index, 34 ± 2 kg/m2; waist circumference, 113 ± 3 cm) with high fasting TAG (2.9 ± 0.2 mmol/L). Subjects completed two oral fat tolerance tests (OFTTs), randomized 1 week apart, that consisted of 1g fat/kg body weight emulsified lipids in the absence of carbohydrate and protein. Approximately 16 h prior to one OFTT, subjects completed 60 min of treadmill walking (estimated 55% VO2 peak; heart rate, 122 ± 4 beats/min). No exercise was performed on the day before the other OFTT. Fasted (0 h) and postprandial (1, 2, 3, 4, 5 and 6 h) blood samples were taken for analysis of TAG, IL-6 and incretins. Subcutaneous adipose tissue biopsies were taken at 0 and 6 h after OFTT ingestion for IL-6 and GIP receptor (GIPr) mRNA quantification. Exercise lowered fasting and postprandial TAG (P<.05) and VLDL TAG (P<.05), while postprandial CM TAG were similar in both OFTT trials (P>.05). Fasting and postprandial plasma IL-6, GIP and GLP-1 did not differ between rest and exercise OFTT trials (P>.05). Exercise reduced IL-6 and GIPr mRNA (P<.05) in adipose tissue. Our results suggest that the reduction in VLDL TAG following an acute bout of exercise is not associated with circulating IL-6 or incretin concentrations, despite reductions in the adipose tissue expression of IL-6 and GIPr. Show less

Serum triglyceride levels (TG) are important independent risk factors for coronary heart disease. The apolipoproteins C-III (apoCIII) and A-V (apoAV) are central to normal TG metabolism and the complete sequence analysis of these genes was carried out in severe cases (TG > 9 mmol/l) and controls (TG < 2 mmol/l). A total of 53 SNPs were identified in these genes with 17 being novel to this study. Further analysis defined four APOC3 SNPs and three APOA5 SNPs showing strong association with TG levels. Analysis of the two major SNPs from APOA5 [c.56C > G, c.-3A > G] and from APOC3 [c.102C > T, c.340C > G] using THESIAS has identified two major haplotypes relative to the most common CACC haplotype showing very strong association with hypertriglyceridaemia, CGTG and GATC (odds ratio 7.45 and 5.26). Logistic regression analysis of these four SNPs revealed that, carriage of the APOA5 c.56 G allele (odd ratios 4.49) and the APOA5 c.-3 G allele (odds ratio 3.23) were strong independent predictors of hypertriglyceridaemia (P < 0.001), whereas in contrast, carriage of the APOC3 c102 T allele (odds ratio 1.35) and the APOC3 c.340 G allele (odds ratio 1.37), did not show any significant effects that were independent of APOA5. Show less

The Wnt signaling pathway plays critical roles in embryonic development and tumorigenesis. Stimulation of the Wnt pathway results in the accumulation of a nuclear beta-catenin/Tcf complex, activating Wnt target genes. A crystal structure of beta-catenin bound to the beta-catenin binding domain of Tcf3 (Tcf3-CBD) has been determined. The Tcf3-CBD forms an elongated structure with three binding modules that runs antiparallel to beta-catenin along the positively charged groove formed by the armadillo repeats. Structure-based mutagenesis defines three sites in beta-catenin that are critical for binding the Tcf3-CBD and are differentially involved in binding APC, cadherin, and Axin. The structural and mutagenesis data reveal a potential target for molecular drug design studies. Show less

ADP-ribosylation factor (ARF) is a small GTP-binding protein that is thought to regulate the assembly of coat proteins on transport vesicles. To identify factors that functionally interact with ARF, we have performed a genetic screen in Saccharomyces cerevisiae for mutations that exhibit synthetic lethality with an arf1Delta allele and defined seven genes by complementation tests (SWA1-7 for synthetically lethal with arf1Delta). Most of the swa mutants exhibit phenotypes comparable to arf1Delta mutants such as temperature-conditional growth, hypersensitivity to fluoride ions, and partial protein transport and glycosylation defects. Here, we report that swa5-1 is a new temperature-sensitive allele of the clathrin heavy chain gene (chc1-5), which carries a frameshift mutation near the 3' end of the CHC1 open reading frame. This genetic interaction between arf1 and chc1 provides in vivo evidence for a role for ARF in clathrin coat assembly. Surprisingly, strains harboring chc1-5 exhibited a significant defect in transport of carboxypeptidase Y or carboxypeptidase S to the vacuole that was not observed in other chc1 ts mutants. The kinetics of invertase secretion or transport of alkaline phosphatase to the vacuole were not significantly affected in the chc1-5 mutant, further implicating clathrin specifically in the Golgi to vacuole transport pathway for carboxypeptidase Y. Show less