👤 Anthony L Cook

Rates of overweight and obesity epidemic have risen significantly in the past few decades, and 34% of adults and 15-20% of children and adolescents in the United States are now obese. Melanocortin receptor 4 (MC4R), contributes to appetite control in hypothalamic neurons and is a target for future anti-obesity treatments (such as setmelanotide) or novel drug development effort. Proper MC4R trafficking regulation in hypothalamic neurons is crucial for normal neural control of homeostasis and is altered in obesity and in presence of lipids. The mechanisms underlying altered MC4R trafficking in the context of obesity is still unclear. Here, we discovered that C2CD5 expressed in the hypothalamus is involved in the regulation of MC4R endocytosis. This study unmasked a novel trafficking protein nutritionally regulated in the hypothalamus providing a novel target for MC4R dependent pathways involved in bodyweight homeostasis and Obesity. To evaluate the expression of C2cd5, we first used in situ hybridization and RNAscope technology in combination with electronic microscopy. For in vivo, we characterized the energy balance of wild type (WT) and C2CD5 whole-body knockout (C2CD5KO) mice fed normal chow (NC) and/or western-diet (high-fat/high-sucrose/cholesterol) (WD). To this end, we performed comprehensive longitudinal assessment of bodyweight, energy balance (food intake, energy expenditure, locomotor activity using TSE metabolic cages), and glucose homeostasis. In addition, we evaluated the consequence of loss of C2CD5 on feeding behavior changes normally induced by MC4R agonist (Melanotan, MTII) injection in the paraventricular hypothalamus (PVH). For in vitro approach, we tease out the role of C2CD5 and its calcium sensing domain C2 in MC4R trafficking. We focused on endocytosis of MC4R using an antibody feeding experiment (in a neuronal cell line - Neuro2A (N2A) stably expressing HA-MC4R-GFP; against HA-tag and analyzed by flux cytometry). We found that 1) the expression of hypothalamic C2CD5 is decreased in diet-induced obesity models compared to controls, 2) mice lacking C2CD5 exhibit an increase in food intake compared to WT mice, 3) C2CD5 interacts with endocytosis machinery in hypothalamus, 4) loss of functional C2CD5 (lacking C2 domain) blunts MC4R endocytosis in vitro and increases MC4R at the surface that fails to respond to MC4R ligand, and, 5) C2CD5KO mice exhibit decreased acute responses to MTII injection into the PVH. Based on these, we conclude that hypothalamic C2CD5 is involved in MC4R endocytosis and regulate bodyweight homeostasis. These studies suggest that C2CD5 represents a new protein regulated by metabolic cues and involved in metabolic receptor endocytosis. C2CD5 represent a new target and pathway that could be targeted in Obesity. Show less

The interplay between E2 and E3 enzymes regulates the polyubiquitination of substrates in eukaryotes. Among the several RING-domain E3 ligases in humans, many utilize two distinct E2s for polyubiquitination. For example, the cell cycle regulatory E3, human anaphase-promoting complex/cyclosome (APC/C), relies on UBE2C to prime substrates with ubiquitin (Ub) and on UBE2S to extend polyubiquitin chains. However, the potential coordination between these steps in ubiquitin chain formation remains undefined. While numerous studies have unveiled how RING E3s stimulate individual E2s for Ub transfer, here we change perspective to describe a case where the chain-elongating E2 UBE2S feeds back and directly stimulates the E3 APC/C to promote substrate priming and subsequent multiubiquitination by UBE2C. Our work reveals an unexpected model for the mechanisms of RING E3-dependent ubiquitination and for the diverse and complex interrelationship between components of the ubiquitination cascade. Show less

Carbamoyl phosphate synthetase 1 (CPS1) is a potential synthetic lethal target in LKB1-deficient nonsmall cell lung cancer, where its overexpression supports the production of pyrimidine synthesis. In other cancer types, CPS1 overexpression and activity may prevent the accumulation of toxic levels of intratumoral ammonia to support tumor growth. Herein we report the discovery of a novel series of potent and selective small-molecule inhibitors of CPS1. Piperazine Show less

Myoarchitectural disarray - the multiscalar disorganisation of myocytes, is a recognised histopathological hallmark of adult human hypertrophic cardiomyopathy (HCM). It occurs before the establishment of left ventricular hypertrophy (LVH) but its early origins and evolution around the time of birth are unknown. Our aim is to investigate whether myoarchitectural abnormalities in HCM are present in the fetal heart. We used wild-type, heterozygous and homozygous hearts (n = 56) from a Mybpc3-targeted knock-out HCM mouse model and imaged the 3D micro-structure by high-resolution episcopic microscopy. We developed a novel structure tensor approach to extract, display and quantify myocyte orientation and its local angular uniformity by helical angle, angle of intrusion and myoarchitectural disarray index, respectively, immediately before and after birth. In wild-type, we demonstrate uniformity of orientation of cardiomyocytes with smooth transitions of helical angle transmurally both before and after birth but with traces of disarray at the septal insertion points of the right ventricle. In comparison, heterozygous mice free of LVH, and homozygous mice showed not only loss of the normal linear helical angulation transmural profiles observed in wild-type but also fewer circumferentially arranged myocytes at birth. Heterozygous and homozygous showed more disarray with a wider distribution than in wild-type before birth. In heterozygous mice, disarray was seen in the anterior, septal and inferior walls irrespective of stage, whereas in homozygous mice it extended to the whole LV circumference including the lateral wall. In conclusion, myoarchitectural disarray is detectable in the fetal heart of an HCM mouse model before the development of LVH. Show less

Cardiac fibrosis is a final common pathology in inherited and acquired heart diseases that causes cardiac electrical and pump failure. Here, we use systems genetics to identify a pro-fibrotic gene network in the diseased heart and show that this network is regulated by the E3 ubiquitin ligase WWP2, specifically by the WWP2-N terminal isoform. Importantly, the WWP2-regulated pro-fibrotic gene network is conserved across different cardiac diseases characterized by fibrosis: human and murine dilated cardiomyopathy and repaired tetralogy of Fallot. Transgenic mice lacking the N-terminal region of the WWP2 protein show improved cardiac function and reduced myocardial fibrosis in response to pressure overload or myocardial infarction. In primary cardiac fibroblasts, WWP2 positively regulates the expression of pro-fibrotic markers and extracellular matrix genes. TGFβ1 stimulation promotes nuclear translocation of the WWP2 isoforms containing the N-terminal region and their interaction with SMAD2. WWP2 mediates the TGFβ1-induced nucleocytoplasmic shuttling and transcriptional activity of SMAD2. Show less

Genome-wide association studies (GWAS) have identified >250 loci for body mass index (BMI), implicating pathways related to neuronal biology. Most GWAS loci represent clusters of common, noncoding variants from which pinpointing causal genes remains challenging. Here we combined data from 718,734 individuals to discover rare and low-frequency (minor allele frequency (MAF) < 5%) coding variants associated with BMI. We identified 14 coding variants in 13 genes, of which 8 variants were in genes (ZBTB7B, ACHE, RAPGEF3, RAB21, ZFHX3, ENTPD6, ZFR2 and ZNF169) newly implicated in human obesity, 2 variants were in genes (MC4R and KSR2) previously observed to be mutated in extreme obesity and 2 variants were in GIPR. The effect sizes of rare variants are ~10 times larger than those of common variants, with the largest effect observed in carriers of an MC4R mutation introducing a stop codon (p.Tyr35Ter, MAF = 0.01%), who weighed ~7 kg more than non-carriers. Pathway analyses based on the variants associated with BMI confirm enrichment of neuronal genes and provide new evidence for adipocyte and energy expenditure biology, widening the potential of genetically supported therapeutic targets in obesity. Show less

Epithelial ovarian cancer (EOC) is the fifth leading cause of cancer mortality in American women. Normal ovarian physiology is intricately connected to small GTP binding proteins of the Ras superfamily (Ras, Rho, Rab, Arf, and Ran) which govern processes such as signal transduction, cell proliferation, cell motility, and vesicle transport. We hypothesized that common germline variation in genes encoding small GTPases is associated with EOC risk. We investigated 322 variants in 88 small GTPase genes in germline DNA of 18,736 EOC patients and 26,138 controls of European ancestry using a custom genotype array and logistic regression fitting log-additive models. Functional annotation was used to identify biofeatures and expression quantitative trait loci that intersect with risk variants. One variant, ARHGEF10L (Rho guanine nucleotide exchange factor 10 like) rs2256787, was associated with increased endometrioid EOC risk (OR = 1.33, p = 4.46 x 10-6). Other variants of interest included another in ARHGEF10L, rs10788679, which was associated with invasive serous EOC risk (OR = 1.07, p = 0.00026) and two variants in AKAP6 (A-kinase anchoring protein 6) which were associated with risk of invasive EOC (rs1955513, OR = 0.90, p = 0.00033; rs927062, OR = 0.94, p = 0.00059). Functional annotation revealed that the two ARHGEF10L variants were located in super-enhancer regions and that AKAP6 rs927062 was associated with expression of GTPase gene ARHGAP5 (Rho GTPase activating protein 5). Inherited variants in ARHGEF10L and AKAP6, with potential transcriptional regulatory function and association with EOC risk, warrant investigation in independent EOC study populations. Show less

Family- and population-based genetic studies have successfully identified multiple disease-susceptibility loci for Age-related macular degeneration (AMD), one of the first batch and most successful examples of genome-wide association study. However, most genetic studies to date have focused on case-control studies of late AMD (choroidal neovascularization or geographic atrophy). The genetic influences on disease progression are largely unexplored. We assembled unique resources to perform a genome-wide bivariate time-to-event analysis to test for association of time-to-late-AMD with ∼9 million variants on 2721 Caucasians from a large multi-center randomized clinical trial, the Age-Related Eye Disease Study. To our knowledge, this is the first genome-wide association study of disease progression (bivariate survival outcome) in AMD genetic studies, thus providing novel insights to AMD genetics. We used a robust Cox proportional hazards model to appropriately account for between-eye correlation when analyzing the progression time in the two eyes of each participant. We identified four previously reported susceptibility loci showing genome-wide significant association with AMD progression: ARMS2-HTRA1 (P = 8.1 × 10-43), CFH (P = 3.5 × 10-37), C2-CFB-SKIV2L (P = 8.1 × 10-10) and C3 (P = 1.2 × 10-9). Furthermore, we detected association of rs58978565 near TNR (P = 2.3 × 10-8), rs28368872 near ATF7IP2 (P = 2.9 × 10-8) and rs142450006 near MMP9 (P = 0.0006) with progression to choroidal neovascularization but not geographic atrophy. Secondary analysis limited to 34 reported risk variants revealed that LIPC and CTRB2-CTRB1 were also associated with AMD progression (P < 0.0015). Our genome-wide analysis thus expands the genetics in both development and progression of AMD and should assist in early identification of high risk individuals. Show less

The sterol-responsive nuclear receptors, liver X receptors α (LXRα, We systematically compared the global transcriptional response of macrophages to distinct classes of LXR ligands. This allowed us to identify both common and ligand-specific transcriptional responses in macrophages. Among these, we identified endonuclease-exonuclease-phosphatase family domain containing 1 ( In this study, we identify Show less

The strong male predominance in Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC) remains inadequately explained, but sex hormones might be involved. We hypothesized that single nucleotide polymorphisms (SNPs) in the androgen pathway influence risk of developing BE and EAC. This genetic-epidemiological analysis included 14 studies from Australia, Europe and North America. Polymorphisms in 16 genes coding for the androgen pathway were analyzed using a gene-based approach: versatile gene-based test association study. This method evaluates associations between a trait and all SNPs within a specific gene rather than each SNP marker individually as in a conventional GWAS. The data were stratified for sex, body-mass index, waist-to-hip ratio, tobacco smoking and gastroesophageal reflux status. Included were data from 1,508 EAC patients, 2,383 BE patients and 2,170 control participants. SNPs within the gene CYP17A1 were associated with risk of BE in the sexes combined (p = 0.002) and in males (p = 0.003), but not in females separately (p = 0.3). This association was found in tobacco smokers (p = 0.003) and in BE patients without reflux (p = 0.004), but not in nonsmokers (p = 0.2) or those with reflux (p = 0.036). SNPs within JMJD1C were associated with risk of EAC in females (p = 0.001). However, none of these associations replicated in a subsequent sample. Fourteen other genes studied did not reach statistically significant levels of association with BE, EAC or the combination of BE and EAC, after correcting for the number of genes included in the analysis. In conclusion, genetic variants in the androgen-related genes CYP17A1 and JMJD1C might be associated with risk of BE and EAC, respectively, but replication data with larger sample sizes are needed. Show less

Hypertrophic cardiomyopathy (HCM) is caused by mutations in sarcomeric proteins, the commonest being MYBPC3 encoding myosin-binding protein C. It is characterised by left ventricular hypertrophy but there is an important pre-hypertrophic phenotype with features including crypts, abnormal mitral leaflets and trabeculae. We investigated these during mouse cardiac development using high-resolution episcopic microscopy. In embryonic hearts from wildtype, homozygous (HO) and heterozygous (HET) Mybpc3-targeted knock-out (KO) mice we show that crypts (one or two) are a normal part of wildtype development but they almost all resolve by birth. By contrast, HO and HET embryos had increased crypt presence, abnormal mitral valve formation and alterations in the compaction process. In scarce normal human embryos, crypts were sometimes present. This study shows that features of the human pre-hypertrophic HCM phenotype occur in the mouse. In an animal model we demonstrate that there is an embryological HCM phenotype. Crypts are a normal part of cardiac development but, along with the mitral valve and trabeculae, their developmental trajectory is altered by the presence of HCM truncating Mybpc3 gene mutation. Show less

Understanding the genetic basis of airflow obstruction and smoking behaviour is key to determining the pathophysiology of chronic obstructive pulmonary disease (COPD). We used UK Biobank data to study the genetic causes of smoking behaviour and lung health. We sampled individuals of European ancestry from UK Biobank, from the middle and extremes of the forced expiratory volume in 1 s (FEV1) distribution among heavy smokers (mean 35 pack-years) and never smokers. We developed a custom array for UK Biobank to provide optimum genome-wide coverage of common and low-frequency variants, dense coverage of genomic regions already implicated in lung health and disease, and to assay rare coding variants relevant to the UK population. We investigated whether there were shared genetic causes between different phenotypes defined by extremes of FEV1. We also looked for novel variants associated with extremes of FEV1 and smoking behaviour and assessed regions of the genome that had already shown evidence for a role in lung health and disease. We set genome-wide significance at p<5 × 10(-8). UK Biobank participants were recruited from March 15, 2006, to July 7, 2010. Sample selection for the UK BiLEVE study started on Nov 22, 2012, and was completed on Dec 20, 2012. We selected 50,008 unique samples: 10,002 individuals with low FEV1, 10,000 with average FEV1, and 5002 with high FEV1 from each of the heavy smoker and never smoker groups. We noted a substantial sharing of genetic causes of low FEV1 between heavy smokers and never smokers (p=2.29 × 10(-16)) and between individuals with and without doctor-diagnosed asthma (p=6.06 × 10(-11)). We discovered six novel genome-wide significant signals of association with extremes of FEV1, including signals at four novel loci (KANSL1, TSEN54, TET2, and RBM19/TBX5) and independent signals at two previously reported loci (NPNT and HLA-DQB1/HLA-DQA2). These variants also showed association with COPD, including in individuals with no history of smoking. The number of copies of a 150 kb region containing the 5' end of KANSL1, a gene that is important for epigenetic gene regulation, was associated with extremes of FEV1. We also discovered five new genome-wide significant signals for smoking behaviour, including a variant in NCAM1 (chromosome 11) and a variant on chromosome 2 (between TEX41 and PABPC1P2) that has a trans effect on expression of NCAM1 in brain tissue. By sampling from the extremes of the lung function distribution in UK Biobank, we identified novel genetic causes of lung function and smoking behaviour. These results provide new insight into the specific mechanisms underlying airflow obstruction, COPD, and tobacco addiction, and show substantial shared genetic architecture underlying airflow obstruction across individuals, irrespective of smoking behaviour and other airway disease. Medical Research Council. Show less

The severity of sepsis is significantly affected by advanced age; however, age-dependent molecular mechanisms of this susceptibility are unknown. Nuclear liver X receptor-α (LXRα) is a regulator of lipid metabolism with associated anti-inflammatory properties. Here, we investigated the role of LXRα in age-dependent lung injury and outcome of sepsis. Male C57BL/6, LXRα-deficient (LXRα(-/-)) and wild type (WT) (LXRα(+/+)) mice of different ages were subjected to sepsis by cecal ligation and puncture (CLP). In pharmacological studies, treatment with the LXRα ligand T0901317 reduced lung neutrophil infiltration in C57BL/6 mice aged from 1 to 8 mo when compared with vehicle-treated animals subjected to CLP. The LXRα ligand improved survival in young mice (2-3 mo old) but did not affect survival or neutrophil infiltration in mature adult mice (11-13 mo old). Immunoblotting revealed an age-dependent decrease of lung LXRα levels. Young LXRα(-/-) mice (2-3 mo old) exhibited earlier mortality than age-matched WT mice after CLP. Lung damage and neutrophil infiltration, lung activation of the pro-inflammatory NF-κB and plasma IL-6 levels were higher in LXRα(-/-) mice 18 h after CLP compared with LXRα(+/+) mice. This study suggests that the anti-inflammatory properties of LXRα in sepsis are age-dependent and severely compromised in mature adult animals. Show less

The glycosyltransferase gene, Ext1, is essential for heparan sulfate production. Induced deletion of Ext1 selectively in Mx1-expressing bone marrow (BM) stromal cells, a known population of skeletal stem/progenitor cells, in adult mice resulted in marked changes in hematopoietic stem and progenitor cell (HSPC) localization. HSPC egressed from BM to spleen after Ext1 deletion. This was associated with altered signaling in the stromal cells and with reduced vascular cell adhesion molecule 1 production by them. Further, pharmacologic inhibition of heparan sulfate mobilized qualitatively more potent and quantitatively more HSPC from the BM than granulocyte colony-stimulating factor alone, including in a setting of granulocyte colony-stimulating factor resistance. The reduced presence of endogenous HSPC after Ext1 deletion was associated with engraftment of transfused HSPC without any toxic conditioning of the host. Therefore, inhibiting heparan sulfate production may provide a means for avoiding the toxicities of radiation or chemotherapy in HSPC transplantation for nonmalignant conditions. Show less

The MEK5 [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase 5]/ERK5 pathway is the least well studied MAPK signalling module. It has been proposed to play a role in the pathology of cancer. In the present paper, we review the role of the MEK5/ERK5 pathway using the 'hallmarks of cancer' as a framework and consider how this pathway is deregulated. As well as playing a key role in endothelial cell survival and tubular morphogenesis during tumour neovascularization, ERK5 is also emerging as a regulator of tumour cell invasion and migration. Several oncogenes can stimulate ERK5 activity, and protein levels are increased by a novel amplification at chromosome locus 17p11 and by down-regulation of the microRNAs miR-143 and miR-145. Together, these finding underscore the case for further investigation into understanding the role of ERK5 in cancer. Show less

Apolipoprotein A-IV (apoA-IV) is synthesized by intestinal enterocytes during lipid absorption and secreted into lymph on the surface of nascent chylomicrons. A compelling body of evidence supports a central role of apoA-IV in facilitating intestinal lipid absorption and in regulating satiety, yet a longstanding conundrum is that no abnormalities in fat absorption, feeding behavior, or weight gain were observed in chow-fed apoA-IV knockout (A4KO) mice. Herein we reevaluated the impact of apoA-IV expression in C57BL6 and A4KO mice fed a high-fat diet. Fat balance and lymph cannulation studies found no effect of intestinal apoA-IV gene expression on the efficiency of fatty acid absorption, but gut sac transport studies revealed that apoA-IV differentially modulates lipid transport and the number and size of secreted triglyceride-rich lipoproteins in different anatomic regions of the small bowel. ApoA-IV gene deletion increased expression of other genes involved in chylomicron assembly, impaired the ability of A4KO mice to gain weight and increase adipose tissue mass, and increased the distal gut hormone response to a high-fat diet. Together these findings suggest that apoA-IV may play a unique role in integrating feeding behavior, intestinal lipid absorption, and energy storage. Show less

The T347S polymorphism in the human apolipoprotein (apo) A-IV gene is present at high frequencies among all the world's populations. Carriers of a 347S allele exhibit faster clearance of triglyceride-rich lipoproteins, greater adiposity, and increased risk for developing atherosclerosis, which suggests that this conservative amino acid substitution alters the structure of apo A-IV. Herein we have used spectroscopic and surface chemistry techniques to examine the structure, stability, and interfacial properties of the apo A-IV 347S isoprotein. Circular dichroism spectroscopy revealed that the 347S isoprotein has similar alpha-helical structure but lower thermodynamic stability than the 347T isoprotein. Fluorescence spectroscopy found that the 347S isoprotein exhibits an enhanced tyrosine emission and reduced tyrosine-->tryptophan energy transfer, and second derivative UV absorption spectra noted increased tyrosine exposure, suggesting that the 347S isoprotein adopts a looser tertiary conformation. Surface chemistry studies found that although the 347S isoprotein bound rapidly to the lipid interface, it has a lower interfacial exclusion pressure and lower elastic modulus than the 347T isoprotein. Together, these observations establish that the T347S substitution alters the conformation of apo A-IV and lowers its interfacial activity-changes that could account for the effect of this polymorphism on postprandial lipid metabolism. Show less

Genome-wide genetic association analysis represents an opportunity for a comprehensive survey of the genes governing lipid metabolism, potentially revealing new insights or even therapeutic strategies for cardiovascular disease and related metabolic disorders. We have performed large-scale, genome-wide genetic analysis among 6382 white women with replication in 2 cohorts of 970 additional white men and women for associations between common single-nucleotide polymorphisms and low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglycerides, apolipoprotein(Apo) A1, and ApoB. Genome-wide associations (P < 5 x 10(-8)) were found at the PCSK9 gene, the APOB gene, theLPL gene, the APOA1-APOA5 locus, the LIPC gene, the CETP gene, the LDLR gene, and the APOE locus. In addition,genome-wide associations with triglycerides at the GCKR gene confirm and extend emerging links between glucose and lipid metabolism. Still other genome-wide associations at the 1p13.3 locus are consistent with emerging biological properties for a region of the genome, possibly related to the SORT1 gene. Below genome-wide significance, our study provides confirmatory evidence for associations at 5 novel loci with low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, or triglycerides reported recently in separate genome-wide association studies. The total proportion of variance explained by common variation at the genome-wide candidate loci ranges from 4.3% for triglycerides to 12.6% for ApoB. Genome-wide associations at the GCKR gene and near the SORT1 gene, as well as confirmatory associations at 5 additional novel loci, suggest emerging biological pathways for lipid metabolism among white women. Show less

Extracellular signal-regulated kinase-1 and -2 (ERK1/2) are activated by dual threonine and tyrosine phosphorylation of a TEY motif. The highly related kinase ERK5 is also activated by phosphorylation at a TEY motif. Inactivation of ERK1/2 is achieved by distinct members of the dual-specificity protein phosphatase (DUSP) family, which are responsible for the specific, regulated de-phosphorylation of the TEY motif. These include both nuclear (DUSP5) and cytoplasmic (DUSP6) enzymes. DUSP6, a candidate tumour suppressor gene, is thought to be highly specific for inactivation of ERK1/2 but several reports have suggested that it may also inactivate ERK5. Here we have compared the ability of DUSP6 to regulate the ERK1/2 and ERK5 protein kinases. We find that DUSP6 binds to ERK1/2 in both yeast and human cells but fails to bind to ERK5. Recombinant ERK2 can induce catalytic activation of DUSP6 whereas ERK5 cannot. Ectopic expression of DUSP6 can de-phosphorylate a co-expressed ERK2 construct but does not de-phosphorylate ERK5. Finally, expression of DUSP6 blocks the MEK1-driven activation of GAL4-ELK1, an ERK1/2-regulated transcription factor, but fails to block the MEK5-driven activation of GAL4-MEF2D, an ERK5-regulated transcription factor. These results demonstrate that even upon over-expression DUSP6 fails to inactivate ERK5, confirming that it is indeed an ERK1/2-specific DUSP. Show less

Human apolipoprotein A-V (apoA-V) is a potent modulator of plasma triacylglycerol (TG) levels. To probe different regions of this 343-amino-acid protein, four single Trp apoA-V variants were prepared. The variant with a Trp at position 325, distal to the tetraproline sequence at residues 293-296, displayed an 11-nm blue shift in wavelength of maximum fluorescence emission upon lipid association. To evaluate the structural and functional role of this C-terminal segment, a truncated apoA-V comprising amino acids 1-292 was generated. Far UV circular dichroism spectra of full-length apoA-V and apoA-V-(1-292) were similar, with approximately 50% alpha-helix content. In guanidine HCl denaturation experiments, both full-length and truncated apoA-V yielded biphasic profiles consistent with the presence of two structural domains. The denaturation profile of the lower stability component (but not the higher stability component) was affected by truncation. Truncated apoA-V displayed an attenuated ability to solubilize l-alpha-dimyristoylphosphatidylcholine phospholipid vesicles compared with full-length apoA-V, whereas a peptide corresponding to the deleted C-terminal segment displayed markedly enhanced kinetics. The data support the concept that the C-terminal region is not required for apoA-V to adopt a folded protein structure, yet functions to modulate apoA-V lipid-binding activity; therefore, this concept may be relevant to the mechanism whereby apoA-V influences plasma TG levels. Show less

Candidate genes in inflammation, thrombosis, coagulation, and lipid metabolism pathways have been implicated in venous thromboembolism (VTE). Using DNA samples collected at baseline in the Physicians' Health Study cohort, we genotyped 92 polymorphisms from 56 candidate genes among 304 individuals who subsequently developed VTE (144 idiopathic, 156 secondary cases) and among 2070 individuals who remained free of reported vascular disease over a mean follow-up of 13.2 years to prospectively determine whether these gene polymorphisms contribute to the risk of VTE. For idiopathic VTE, in addition to the factor V (Leiden) mutation (odds ratio [OR], 5.13; 95% confidence interval [CI], 3.24 to 8.14; P<0.0001; false discovery rate [FDR], P<0.0001), an N291S lipoprotein lipase gene polymorphism (OR, 3.09; 95% CI, 1.56 to 6.09; P=0.001; FDR, P=0.036) and a Q27E beta2-adrenergic receptor gene polymorphism (OR, 1.40; 95% CI, 1.09 to 1.79; P=0.006; FDR, P=0.036) were found to be significantly associated with increased risk. For secondary VTE, a Q360H apolipoprotein A4 gene polymorphism (OR, 0.34; 95% CI, 0.18 to 0.65; P=0.001; FDR, P=0.07) and an I50V interleukin-4 receptor polymorphism (OR, 0.66; 95% CI, 0.52 to 0.84; P=0.0009; FDR, P=0.07) were moderately, but not statistically and significantly, associated with reduced risk after adjustment for multiple comparisons. These present findings are hypothesis generating and require replication and confirmation in an independent investigation. Show less

The neuronal ceroid lipofuscinoses (NCLs, Batten disease) are a group of fatal recessively inherited neurodegenerative diseases of humans and animals characterised by common clinical signs and pathology. These include blindness, ataxia, dementia, behavioural changes, seizures, brain and retinal atrophy and accumulation of fluorescent lysosome derived organelles in most cells. A number of different variants have been suggested and seven different causative genes identified in humans (CLN1, CLN2, CLN3, CLN5, CLN6, CLN8 and CTSD). Animal models have played a central role in the investigation of this group of diseases and are extremely valuable for developing a better understanding of the disease mechanisms and possible therapeutic approaches. Ovine models include flocks of affected New Zealand South Hampshires and Borderdales and Australian Merinos. The ovine CLN6 gene has been sequenced in a representative selection of these sheep. These investigations unveiled the mutation responsible for the disease in Merino sheep (c.184C>T; p.Arg62Cys) and three common ovine allelic variants (c.56A>G, c.822G>A and c.933₉₃₄insCT). Linkage analysis established that CLN6 is the gene most likely to cause NCL in affected South Hampshire sheep, which do not have the c.184C>T mutation but show reduced expression of CLN6 mRNA in a range of tissues as determined by real-time PCR. Lack of linkage precludes CLN6 as a candidate for NCL in Borderdale sheep. Show less

Mitochondrial damage and dysfunction are thought to play an important role in the pathogenesis of sepsis-induced organ failures. Unfortunately, specific markers of mitochondrial damage in vital organs do not currently exist. Recently, carbomyl phosphate synthase (CPS)-1, a protein primarily localized to liver mitochondria, was found to be present in high concentrations in the plasma of septic humans. Thus, we hypothesized that the circulatory release of CPS-1 would correlate with mitochondrial damage or impaired mitochondrial function in the liver in a clinically relevant model of sepsis. Prospective, randomized, controlled animal study. University medical center research laboratory. Male, Balb/C mice, aged 10-12 wks. Animals were assigned to receive cecal ligation and puncture (CLP sepsis) or sham operation and compared with untreated controls. Plasma CPS-1 levels and liver mitochondrial variables, including morphology, respiratory activity, mass (i.e., cardiolipin content), and protein carbonylation, were assessed at various time points (8, 24, and 48 hrs and 6 days) after surgery. Oxidant stress (i.e., carbonylation) was detected within 8 hrs of CLP and persisted through 48 hrs. Plasma CPS-1 levels increased dramatically at 24 hrs, remained significantly elevated at 48 hrs, and normalized by 6 days in the sepsis group. Abnormalities of liver mitochondrial morphology and function coincided with increased plasma CPS-1 levels. Mitochondrial depletion in the liver was not due to cell death but was associated with increased lysosomal clearance. Increased expression of mitochondrial biogenesis factors preceded restoration of mitochondrial variables and normalization of CPS-1 levels by day 6. Circulating CPS-1 is a marker of mitochondrial damage and depletion in the liver during the subacute phase of CLP sepsis. From a mechanistic standpoint, mitochondrial depletion is not due to cell death but is apparently related to the removal of damaged mitochondria by lysosomes (i.e., autophagy), followed by repletion of mitochondrial populations. Further studies are needed to determine the clinical utility of CPS-1 as a marker of sepsis severity. Show less

Apolipoprotein A-V (apoA-V), the newest member of the plasma apolipoprotein family, was recently discovered by comparison of the mouse and human genomes. Studies in rodents and population surveys of human apoA-V polymorphisms have noted a strong effect of apoA-V on plasma triglyceride levels. Toward the elucidation of the biologic function of apoA-V, we used spectroscopic and surface chemistry techniques to probe its structure and interfacial activity. Computer-assisted sequence analysis of apoA-V predicts that it is very hydrophobic, contains a significant amount of alpha-helical secondary structure, and probably is composed of discrete structural regions with varying degrees of lipid affinity. Fluorescence spectroscopy of recombinant human apoA-V provided evidence of tertiary folding, and light scattering studies indicated that apoA-V transforms dimyristoylphosphatidylcholine vesicles into discoidal complexes with an efficiency similar to that of apoA-I. Surface chemistry techniques revealed that apoA-V displays high affinity, low elasticity, and slow binding kinetics at hydrophobic interfaces, properties we propose may retard triglyceride-rich particle assembly. Metabolic labeling and immunofluorescence studies of COS-1 cells transfected with human apoA-V demonstrated that apoA-V is poorly secreted, remains associated with the endoplasmic reticulum, and does not traffic to the Golgi. Given that overexpression of the apoA-V gene lowers plasma triglycerides in mice, these data together suggest that apoA-V may function intracellularly to modulate hepatic VLDL synthesis and/or secretion. Show less

We have examined gene expression in the fat tissue of normal mice at the onset of diet-induced obesity. Insulin-induced gene 1 (insig-1) mRNA rose progressively with a high-fat diet and declined on a restricted diet. Because insig-1 binds sterol regulatory element-binding protein cleavage-activating protein in the endoplasmic reticulum, thereby blocking proteolytic processing required for sterol regulatory element-binding protein activation, we tested its influence on lipogenesis. In differentiating 3T3-L1 cells, insig-1 and -2 rose in parallel with aP2 mRNA during differentiation. The mRNA of the lipogenic transcription factor, carbohydrate response element-binding protein, was undetectable in undifferentiated 3T3-L1 preadipocytes but rose dramatically during differentiation in 25 mM, but not in 5 mM, glucose. Transfection of mouse or human insig-1 into 3T3-L1 preadipocytes completely prevented oil red O staining and blocked upregulation of aP2, peroxisome proliferator-activated receptor gamma2, and carbohydrate response element-binding protein, while reducing down-regulation of preadipocyte factor 1. The results suggest that insig-1 expression restricts lipogenesis in mature adipocytes and blocks differentiation in preadipocytes. Show less

We used a panel of recombinant human apolipoprotein (apo) A-IV truncation mutants, in which pairs of 22-mer alpha-helices were sequentially deleted along the primary sequence, to examine the impact of protein structure and interfacial activity on the ability of apoA-IV to activate cholesterol ester transfer protein. Circular dichroism and fluorescence spectroscopy revealed that the secondary structure, conformation, and molecular stability of recombinant human apoA-IV were identical to the native protein. However, deletion of any of the alpha-helical domains in apoA-IV disrupted its tertiary structure and impaired its molecular stability. Surprisingly, determination of the water/phospholipid interfacial exclusion pressure of the apoA-IV truncation mutants revealed that, for most, deletion of amphipathic alpha-helical domains increased their affinity for phospholipid monolayers. All of the truncation mutants activated the transfer of fluorescent-labeled cholesterol esters between high and low density lipoproteins at a rate higher than native apoA-IV. There was a strong positive correlation (r = 0.790, p = 0.002) between the rate constant for cholesterol ester transfer and interfacial exclusion pressure. We conclude that molecular interfacial exclusion pressure, rather than specific helical domains, determines the degree to which apoA-IV, and likely other apolipoproteins, facilitate cholesterol ester transfer protein-mediated lipid exchange. Show less

Apolipoprotein (apo)A-IV is synthesized in the small intestine during fat absorption and is incorporated onto the surface of nascent chylomicrons. In circulation, apoA-IV is displaced from the chylomicron surface by high density lipoprotein-associated C and E apolipoproteins; this exchange is critical for activation of lipoprotein lipase and chylomicron remnant clearance. The variant allele A-IV-2 encodes a Q360H polymorphism that increases the lipid affinity of the apoA-IV-2 isoprotein. We hypothesized that this would impede the transfer of C and E apolipoproteins to chylomicrons, and thereby delay the clearance of postprandial triglyceride-rich lipoproteins. We therefore measured triglycerides in plasma, S(f) > 400 chylomicrons, and very low density lipoproteins (VLDL) in 14 subjects heterozygous for the A-IV-2 allele (1/2) and 14 subjects homozygous for the common allele (1/1) who were fed a standard meal containing 50 gm fat per m(2) body surface area. All subjects had the apoE-3/3 genotype. Postprandial triglyceride concentrations in the 1/2 subjects were significantly higher between 2;-5 h in plasma, chylomicrons, and VLDL, and peaked at 3 h versus 2 h for the 1/1 subjects. The area under the triglyceride time curves was greater in the 1/2 subjects (plasma, P = 0.045; chylomicrons, P = 0.027; VLDL, P = 0.063). A post-hoc analysis of the frequency of the apoA-IV T347S polymorphism suggested that it had an effect on triglyceride clearance antagonistic to that of the A-IV-2 allele. We conclude that individuals heterozygous for the A-IV-2 allele display delayed postprandial clearance of triglyceride-rich lipoproteins. Show less

The apolipoprotein C3-482C> T variant modulates insulin and glucose concentrations after an oral glucose tolerance test (OGTT) in young healthy white men. We evaluated the effect of this variant in different ethnic groups with different rates of Type II (non-insulin-dependent) diabetes mellitus and coronary heart disease. We investigated the -482C > T in a population-based cross-sectional study of white subjects (n = 462), South Asians (n = 442) and subjects of West African and Afro-Caribbean origin (n = 462), whose OGTT and fasting plasma triglyceride concentrations had been measured. The -482T allele frequency differed between the three groups: 0.25 (95 % CI 0.22-0.28) in white subjects, 0.44 (0.41-0.47) in South Asians and 0.71 (0.68-0.74) in black subjects (p < 0.0001). A positive association was found between body mass index and genotype in black women (p = 0.009) and in black men (p = 0.056) but not in white subjects or South Asians. Associations between -482C > T and fasting insulin were found in white subjects, where -482T allele carriers had higher concentrations (adjusted for age and sex, p = 0.007, also including smoking and body mass index, p = 0.038). Higher triglyceride concentrations (p = 0.004 and p = 0.007 in the two models) but not glucose concentrations were also associated with -482C > T. In black subjects, decreased fasting insulin (p = 0.04) and fasting glucose (p = 0.004) were associated with -482C > T. No relation was observed between genotype and any post-load measured. CONCLUSIONS/INTERPRETATION. Allele frequencies of the -482C > T and associations with insulin, glucose and triglyceride concentrations vary considerably among ethnic groups. Although the results are consistent among white subjects across different studies, the associations among black subjects and South Asians differ. Show less

Viscoelastic behavior of proteins at interfaces is a critical determinant of their ability to stabilize emulsions. We therefore used air bubble surfactometry and drop volume tensiometry to examine the dynamic interfacial properties of two plasma apolipoproteins involved in chylomicron assembly: apolipoprotein A-IV and apolipoprotein B-17, a recombinant, truncated apolipoprotein B. At the air/water interface apolipoproteins A-IV and B-17 displayed wide area - tension loops with positive phase angles indicative of viscoelastic behavior, and suggesting that they undergo rate-dependent changes in surface conformation in response to changes in interfacial area. At the triolein/water interface apolipoprotein A-IV displayed maximal surface activity only at long interface ages, with an adsorption rate constant of 1.0 3 10(-)(3) sec(-)(1), whereas apolipoprotein B-17 lowered interfacial tension even at the shortest interface ages, with an adsorption rate constant of 9.3 3 10(-)(3) sec(-)(1). Apolipoprotein A-IV displayed an expanded conformation at the air/water interface and a biphasic compression isotherm, suggesting that its hydrophilic amphipathic helices move in and out of the interface in response to changes in surface pressure. We conclude that apolipoproteins A-IV and B-17 display a combination of interfacial activity and elasticity particularly suited to stabilizing the surface of expanding triglyceride-rich particles. Show less