Patients with Alzheimer’s disease (AD) display amyloidopathy and tauopathy. In mouse models of AD, pharmacological inhibition using small molecule enzyme inhibitors, or genetic inactivation of Acyl-CoA: cholesterol acyltransferase 1 (ACAT1) diminished amyloidopathy and restored cognitive deficits. In microglia, ACAT1 blockage increases autophagosome formation and stimulates amyloid β peptide1–42 degradation. Here we hypothesize that in neurons ACAT1 blockage augments autophagy and increases autophagy-mediated degradation of P301L-tau protein. We tested this possibility in murine neuroblastoma cells ectopically expressing human tau, and in primary neurons isolated from triple transgenic AD (3XTg-AD) mice that express mutant forms of APP, PS1, and human tau. The results show that ACAT1 blockage increases autophagosome formation and decreases P301L-tau protein content without affecting endogenous mouse tau protein content. In vivo, lacking Acat1 decreases P301L-tau protein content in the brains of young 3XTg-AD mice but not in those of old mice, where extensive hyperphosphorylations and aggregation of P301L-tau take place. These results suggest that, in addition to ameliorating amyloidopathy in both young and old AD mice, ACAT1 blockage may benefit AD by reducing tauopathy at early stage.

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Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) is a membrane bound enzyme utilizing long-chain fatty acyl-coenzyme A and cholesterol to form cholesteryl esters and coenzyme A. Previously, we had expressed tagged human ACAT1 (hACAT1) in CHO cells and purified it to homogeneity; however, only a sparse amount of purified protein could be obtained. Here we report that the hACAT1 expression level in H293 cells is 18-fold higher than that in CHO cells. We have developed a milder purification procedure to purify the enzyme to homogeneity. The abundance of the purified protein enabled us to conduct difference intrinsic fluorescence spectroscopy to study the binding between the enzyme and its substrates in CHAPS/phospholipid mixed micelles. The results show that oleoyl CoA binds to ACAT1 with Kd=1.9 μM, and elicits significant structural changes of the protein as manifested by the significantly positive changes in its fluorescence spectrum; stearoyl CoA elicits a similar spectrum change with much lower in magnitude. Previously, kinetic studies had shown that cholesterol is an efficient substrate and an allosteric activator of ACAT1, while its diastereomer epicholesterol is neither a substrate nor an activator. Here we show that both cholesterol and epicholesterol induce positive changes in the ACAT1 fluorescence spectrum; however, the magnitude of spectrum changes induced by cholesterol is much larger than epicholesterol. These results show that stereospecificity, governed by the 3beta-OH moiety in steroid ring A, plays an important role in the binding of cholesterol to ACAT1.Acyl-coenzyme A:cholesterol acyltransferase utilizes long-chain fatty acyl-coenzyme A and cholesterol as its substrates, producing cholesteryl esters and coenzyme A as its products. In mammals, there are two ACAT genes that encode two similar but different proteins, ACAT1 (1) and ACAT2 (2), (3), (4). Tissue distribution studies show that ACAT1 is ubiquitously expressed, while ACAT2 is mainly expressed in intestinal enterocytes and in hepatocytes, as reviewed in (5). Both ACAT1 and ACAT2 are potential drug targets for therapeutic intervention of dyslipidemia and atherosclerosis (6), (7). ACAT1, ACAT2 and its close homolog diacylglycerol ayltransferase 1 (8) are founding members of the membrane bound acyltransferase (MBOAT) superfamily (9). The MBOATs are fatty acyltransferases with multiple transmembrane domains (TMD), and with an invariant histidine (His) embedded in † This work is supported by an NIH grant RO1 HL60306 to TYC and CCYC, and an NIH grant RO1 GM069818 to HNH. a long stretch of hydrophobic residues. There are more than a dozen MBOAT members that includes grehlin acyltransferase (10), (11) and certain lysophospholipid acyltransferases (12), (13)).ACAT1 is a resident enzyme in the ER (14), (15), (16); the enzyme is homotetrameric (17) and contains 9 TMDs, with the active site His located within TMD #7 (18). The recombinant human ACAT1 (hACAT1) expressed in Chinese hamster ovary (CHO) cells has been solubilized in zwitterion...

Background:Variations in the hepatic lipase (HL) gene are the potential candidate for coronary artery disease (CAD) especially in type 2 diabetes mellitus (T2DM) in diverse populations. We assessed the association of -514C/T and -250G/A polymorphisms in HL (LIPC) gene with CAD risk in Iranian population with type 2 diabetes.Materials and Methods:We evaluated 322 type 2 diabetic patients, 166 patients with normal angiograms as controls and 156 patients those identified with CAD undergoing their first coronary angiography as CAD cases. Genotyping of -514C/T and -250G/A polymorphisms in the promoter of the LIPC gene were studied by polymerase chain reaction (PCR)-restriction fragment length polymorphism technique.Results:Genotype distributions in CAD cases (73.7%, 20.5%, and 5.8% for −250G/A) and (62.2%, 32.7%, and 5.1% for -514C/T) were significantly different from those in controls (60.8%, 37.4%, and 1.8% for -250G/A) and (51.2%, 48.2%, and 0.6% for -514C/T). CAD cases had lower A-allele frequency than controls (0.131 vs. 0.196, P = 0.028). The odds ratio for the presence of -250 (GG + GA) genotype and A allele in CAD cases were 2.206 (95% confidence interval [CI] =1.33–3.65, P = 0.002) and 1.609 (95% CI = 1.051 −2.463, P = 0.029) respectively. Haplotype analysis demonstrated a significant association between especially LIPC double mutant (−250 A/-514 T) haplotype and presence of CAD.Conclusion:Our findings indicated that -250 G/A polymorphism rather than -514 C/T polymorphism of LIPC gene is more associated with the increased risk of CAD particularly in women with T2DM.

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Apolipoprotein (apo) A-I induces rapid translocation of protein kinase Ca and phospholipase Cc, and slow translocation of caveolin-1 and newly synthesized cholesterol to the cytosolic lipid-protein particle (CLPP) fraction in rat astrocytes. In order to understand the function of CLPP, we investigated the interaction with cytoskeletons of CLPP-related proteins such as caveolin-1 and protein kinase Ca and of CLPP-related lipids in rat astrocytes. Under the conditions that microtubules were depolymerized, association of cytosolic caveolin-1 with protein kinase Ca and a-tubulin was enhanced when the cells were treated with apoA-I for 5 min. This association was suppressed by a scaffolding domain-peptide of caveolin-1. Association with the microtubule-like filaments of cytosolic lipids, caveolin-1 and protein kinase Ca was also increased by the apoA-I treatment and inhibited by the scaffolding domain peptide. Paclitaxel (taxol), a compound to stabilize microtubules, suppressed the apoA-I-mediated intracellular translocation and release from the cells of the de novo synthesized cholesterol and phospholipid. The findings suggested that the association of CLPP with microtubules is mediated by a scaffolding domain of caveolin-1, induced by apoA-I and involved in regulation of intracellular cholesterol trafficking for assembly of cellular lipids to apoA-I-high-density lipoprotein (HDL).

Fibroblast growth factor-1 (FGF-1) is secreted by astrocytes and stimulates apolipoprotein E (apoE)-HDL biogenesis by an autocrine mechanism to help in recovery from brain injury. In apoE-deficient mouse astrocytes, FGF-1 stimulated cholesterol biosynthesis without enhancing its release, indicating a signaling pathway independent of apoE biosynthesis upregulation. SU5402, an inhibitor of FGF receptor, inhibited FGF-1-induced phosphorylation of MEK, ERK, and Akt, as well as all the apoE-HDL biogenesis-related events in rat astrocytes. LY294002, an inhibitor of phosphatidylinositide 3-OH kinase (PI3K) and of Akt phosphorylation, inhibited apoE-HDL secretion but not cholesterol biosynthesis, whereas U0126, an inhibitor of MEK and of ERK phosphorylation, inhibited cholesterol biosynthesis but not apoE-HDL secretion. Increase of apoE-mRNA by FGF-1 was not influenced by either inhibitor. When rat apoE/pcDNA3. his was transfected to transformed rat astrocyte GA-1 cells that otherwise do not synthesize apoE (GA-1/25), FGF-1 did not influence apoE-mRNA, but did increase the apoE secretion and Akt phosphorylation that were suppressed by LY294002. Lipid biosynthesis was increased by FGF-1 in GA-1/25 cells and suppressed by U0126. FGF-1 upregulates apoE-HDL biogenesis by three independent signaling pathways. The PI3K/Akt pathway upregulates secretion of apoE/apoE-HDL, the MEK/ERK pathway stimulates cholesterol biosynthesis, and an unknown pathway enhances apoE transcription.-Ito, J-i., Y. Nagayasu, K. Okumura-Noji, R. Lu, T. Nishida, Y. Miura, K. Asai, A. Kheirollah, S. Nakaya, and S. Yokoyama. Mechanism for FGF-1 to regulate biogenesis of apoE-HDL in astrocytes. J. Lipid Res. 2007Res. . 48: 2020Res. -2027. Supplementary key words astrocytesNeuronal cells in the central nervous system (CNS), such as neurons, oligodendroglias, and astrocytes, all appear in unique shapes, with large cell surfaces, and accordingly contain large amounts of cholesterol. Cholesterol content in the CNS could thus account for 25-30% of total body cholesterol in humans (1, 2). Cholesterol plays many key roles in the CNS, including roles in neurite outgrowth (3, 4) and synapse formation (5).Cholesterol homeostasis in animals is maintained by intra-and extracellular regulation of its metabolism (6), and extracellular transport of cholesterol in vertebrates is carried by the plasma lipoprotein system. However, the blood-brain barrier prevents the CNS from accessing this system, so the CNS operates as a unique and independent CNS-specific lipoprotein system for extracellular cholesterol transport. HDL is a lipoprotein found exclusively in cerebrospinal fluid that contains mainly apolipoprotein E (apoE) and apoA-I (7). Although apoA-I is not synthesized by neural cells, and its origin is uncertain (8, 9), apoE is known to be synthesized, at least in astrocytes and microglias, to generate apoE-HDL (10, 11). Many reports suggest that apoE-HDL is a key lipoprotein in the delivery of cholesterol to the neural cells, and this lipoprotein seems to ...

Expression of human epidermal growth factor receptor type 2 (HER2) in head and neck squamous cell carcinoma (HNSCC) cell line HN5 can be employed with great opportunities of success for specific targeting of anti-cancer chemotherapeutic agents. In the current study, HER2-specific affibody molecule, ZHER2:342 (an engineered protein with great affinity for HER2 receptors) was selected for conjugation to idarubicin (an anti-neoplastic antibiotic). ZHER2:342 affibody gene with one added cysteine code at the its 5' end was synthesized de novo and then inserted into pET302 plasmid and transferred to E. Coli BL21 hosting system. After induction of protein expression, the recombinant ZHER2 affibody molecules were purified using Ni-NTA resin and purity was analyzed through SDS-PAGE. Affinity-purified affibody molecules were conjugated to idarubicin through a heterobifunctional crosslinker, sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC). Specific toxicity of idarubicin-ZHER2 affibody conjugate against two HER2-positive cells, HN5 and MCF7 was assessed through MTT assay after an exposure time of 48 hours with different concentrations of conjugate. Idarubicin in the non-conjugated form showed potent toxic effects against both cell lines, while HN5 cells were significantly more sensitive compared to MCF-7 cells. Dimeric ZHER2 affibody showed a mild decreasing effect on growth of both HN5 and MCF-7 cells at optimum concentration. Idarubicin-ZHER2 affibody conjugate at an optimum concentration reduced viability of HN5 cell line more efficiently compared to MCF-7 cell line. In conclusion, idarubicin-ZHER2 affibody conjugate in optimum concentrations can be used for specific targeting and killing of HN5 cells.