Dimerisation of APOBEC1 is dispensable for its RNA editing activity

ABSTRACTAmong the AID/APOBECs -a family of DNA and RNA deaminases-APOBEC1 physiologically partakes into a complex that edits a CAA codon into UAA Stop codon in the transcript of Apolipoprotein B (ApoB), a protein crucial in the transport of lipids in the blood. Catalytically inactive mutants of APOBEC1 have a dominant negative effect on its activity, as they compete for the targeting to the ApoB mRNA. Here we show that catalytically inactive chimeras of APOBEC1 restricted to different compartments of the cell present different abilities to titrate APOBEC1-mediated RNA editing, and that the ability of APOBEC1 to interact with these mutants is the main determinant for its activity. Our results demonstrate that dimerisation, a feature common to other APOBECs targeting DNA, is not required for APOBEC1 activity on mRNA. Furthermore, APOBEC1-mediated RNA editing is a dynamic process where interplay among the components of the editing complex is regulated through the balance between availability of A1CF, one of APOBEC1 cofactors, and nuclear degradation of APOBEC1.

Abstract 573: Human Microrna-548p Decreases apoB Secretion and Lipid Synthesis

Objective: MicroRNAs (miRs) play important regulatory roles in lipid and lipoprotein metabolism. ApoB, as the only essential scaffolding protein in the assembly of very low density lipoproteins, is a target to treat hyperlipidemia and atherosclerosis. We aimed to find out miRs that reduce apoB expression. Approach: Bioinformatics analyses predicted that hsa-miR-548p can interact with apoB mRNA.MiR-548p mimic and control were transfected in human and mouse hepatoma cell lines to test its role in regulating apoB secretion and mRNA expression levels. Site-directed mutagenesis was used to identify the interacting site of miR-548p in human apoB 3′-untranslated region. Fatty acid oxidation and lipid syntheses were examined in miR-548p overexpressing cells to investigate its function in lipid metabolism. Results: Experimentally, we observed that miR-548p significantly reduces apoB secretion from human hepatoma cells in time and dose dependent manner. Mechanistic studies showed that miR-548p interacts with the 3′-untranslated region of human apoB mRNA to enhance posttranscriptional degradation. Bioinformatics algorithms suggested two potential binding sites of miR-548p on human apoB mRNA. Site-directed mutagenesis studies revealed that miR-548p targets site II involving both seed and supplementary sequences. MiR-548p had no effect on fatty acid oxidation but significantly decreased lipid synthesis in human hepatoma cells by reducing the expression of HMGCR and ACSL4 enzymes involved in cholesterol and fatty acid synthesis. In summary, miR-548p reduces lipoprotein production and lipid synthesis by reducing expression of different genes in human hepatoma cells. Conclusion: These studies suggest that miR-548p could be useful in treating atherosclerosis, hyperlipidemia and hepatosteatosis.

Human MicroRNA-548p Decreases Hepatic Apolipoprotein B Secretion and Lipid Synthesis

Objective— MicroRNAs (miRs) play important regulatory roles in lipid metabolism. Apolipoprotein B (ApoB), as the only essential scaffolding protein in the assembly of very-low-density lipoproteins, is a target to treat hyperlipidemia and atherosclerosis. We aimed to find out miRs that reduce apoB expression. Approach and Results— Bioinformatic analyses predicted that hsa-miR-548p can interact with apoB mRNA. MiR-548p or control miR was transfected in human and mouse liver cells to test its role in regulating apoB secretion and mRNA expression levels. Site-directed mutagenesis was used to identify the interacting site of miR-548p in human apoB 3′-untranslated region. Fatty acid oxidation and lipid syntheses were examined in miR-548p overexpressing cells to investigate its function in lipid metabolism. We observed that miR-548p significantly reduces apoB secretion from human hepatoma cells and primary hepatocytes. Mechanistic studies showed that miR-548p interacts with the 3′-untranslated region of human apoB mRNA to enhance post-transcriptional degradation. Bioinformatic algorithms suggested 2 potential binding sites of miR-548p on human apoB mRNA. Site-directed mutagenesis studies revealed that miR-548p targets site I involving both seed and supplementary sequences. MiR-548p had no effect on fatty acid oxidation but significantly decreased lipid synthesis in human hepatoma cells by reducing HMGCR (3-hydroxy-3-methylglutaryl-coenzyme A reductase) and ACSL4 (Acyl-CoA synthetase long-chain family member 4) enzymes involved in cholesterol and fatty acid synthesis. In summary, miR-548p reduces lipoprotein production and lipid synthesis by reducing expression of different genes in human liver cells. Conclusions— These studies suggest that miR-548p regulates apoB secretion by targeting mRNA. It is likely that it could be useful in treating atherosclerosis, hyperlipidemia, and hepatosteatosis.

Abstract 202: MiR-1200 Differentially Modulates Plasma LDL and HDL-cholesterol Levels to Reduce Hyperlipidemia and Atherosclerosis in Mice

High LDL and low HDL are risk factors for heart disease. Drugs that can lower LDL and increase HDL might be ideal for the treatment of cardiovascular diseases. We performed a high throughput screening of human microRNA (miR) mimic library to identify miRs that regulate apoB (LDL scaffolding protein) and apoAI (major HDL protein) secretion in human hepatoma Huh-7 cells. MiR-1200 potently decreased apoB and increased apoAI secretion. We found that seed sequence of miR-1200 interacts with the 3’-untranslated region of apoB mRNA to enhance posttranscriptional degradation and reduce apoB secretion. In contrast, miR-1200 increased apoAI protein and mRNA levels by increasing transcription. Mechanistic studies revealed that miR-1200 reduced the expression of B-Cell Lymphoma 11B (BCL11B), a repressor of apoAI transcription, to increase apoAI expression. In vivo studies showed that overexpression of miR-1200 significantly reduced LDL-cholesterol and increased HDL-cholesterol levels without causing hepatic steatosis in Western diet fed C57Bl6J mice. Additionally, miR-1200 reduced atherosclerosis in Western diet fed Apoe -/- mice. Physiologic studies showed that miR-1200 reduced VLDL production. Further, HDL from miR-1200 injected mice showed increased cholesterol efflux capacity from lipid loaded macrophages suggesting increases in functionally adept HDL. In short, we have identified a novel miR-1200 that reduces dyslipidemia by reducing apoB-containing lipoproteins and increasing functional HDL and suggest that it can be useful in treating hyperlipidemia and atherosclerosis.

Abstract 380: Vascular Pcsk9: A Mediator for Atherogenesis Independent of LDL Receptor

PCSK9 (Proprotein convertase subtilisin/kexin type 9) increases the LDL levels by binding to hepatocyte LDL receptors (LDLR) and subjects it to degradation. We show that PCSK9 regulates apolipoprotein B (apoB) production by inhibiting its degradation process via the autophagic pathway, irrespective of the presence of LDLR. In addition to the role of PCSK9 in promoting hyperlipidemia, we hypothesized that vascular-PCSK9 in endothelial cells (EC) plays a role in initiating atherogenesis, irrespective of the presence of LDL receptor. Our laboratory has generated double knockout mice lacking both LDLR and Apobec1 (apoB mRNA editing enzyme), named LDb, Ldlr-/-Apobec1-/-. They have the lipoprotein phenotype mimics human with hyperlipidemia; elevated levels of VLDL and LDL with low levels of HDL. They develop atherosclerotic lesions spontaneously. To investigate the role of PCSK9 in atherogenesis, we deleted Pcsk9 gene from LDb mice to generate the triple knockout mice (named LTp, Ldlr-/-Apobec1-/-Pcsk9-/-). In comparison to LDb mice (n=14), the LTp mice (n=8) had significantly decreased levels of cholesterol (387±10 vs. 313±14 mg/dl; p<0.0008) and triglyceride (304±15 vs. 204±2.3 mg/dl; p<0.0002). However, despite their high cholesterol levels at over 300 mg/dl, the atherosclerotic lesions in LTp mice were significantly decreased in comparison to LDb mice (8.8%±3.5 vs. 24%±3.3, p=0.004, n=5 vs. 5). We hypothesized that vascular PCSK9 regulates the development of atherosclerosis. We incubated LDL containing PCSK9 (LDL/PCSK9) on primary aortic endothelial cells (EC) obtained from LDb or LTp to study the effects of LDL/PCSK9 on inflammation. We show that LDL/PCSK9 could not induce the expressions of Lox-1, TLR-2, or ICAM-1 in EC from LTp, resulting in absence responses on proinflammatory markers (CCL2 and CCL7) and autophagic molecules (p62 and TRAF6). In conclusion, our results suggest that vascular PCSK9 play an essential role in atherogenesis.

Hepatic mitochondrial and ER stress induced by defective PPARα signaling in the pathogenesis of hepatic steatosis

Emerging evidence demonstrates a close interplay between disturbances in mitochondrial function and ER homeostasis in the development of the metabolic syndrome. The present investigation sought to advance our understanding of the communication between mitochondrial dysfunction and ER stress in the onset of hepatic steatosis in male rodents with defective peroxisome proliferator-activated receptor-α (PPARα) signaling. Genetic depletion of PPARα or perturbation of PPARα signaling by high-fructose diet compromised the functional activity of metabolic enzymes involved in mitochondrial fatty acid β-oxidation and induced hepatic mitochondrial stress in rats and mice. Inhibition of PPARα activity further enhanced the expression of apolipoprotein B (apoB) mRNA and protein, which was associated with reduced mRNA expression of the sarco/endoplasmic reticulum calcium ATPase (SERCA), the induction of hepatic ER stress, and hepatic steatosis. Restoration of PPARα activity recovered the metabolic function of the mitochondria and ER, alleviated systemic hypertriglyceridemia, and improved hepatic steatosis. These findings unveil novel roles for PPARα in mediating stress signals between hepatic subcellular stress-responding machinery and in the onset of hepatic steatosis under conditions of metabolic stress.

Abstract 1: Role of MicroRNAs in Postranscriptional Regulation of Apolipoprotein B-100 mRNA

Hepatic apolipoprotein B-100 (apoB) synthesis and secretion appears to be regulated largely at the posttranscriptional and posttranslational levels. MicroRNAs (miRNAs) are among posttranscriptional regulators of gene expression that bind to complementary sequences on target messenger RNA (mRNA) transcripts, usually resulting in translational repression or degradation. It is unknown whether specific miRNAs are involved in posttranscriptional regulation of apoB mRNA. We performed bioinformatic analysis, showing that two specific miRNAs with satisfactory E-values level (with levels indicating greater similarity between the input and its match) namely, miR-544 (E-value = 0.91) and miR-1202 (E-value=0.86) have potential to interact with 3’ and 5’ UTR of apoB, respectively. We hypothesized that the interaction of these specific miRNAs (miR-544 and miR-1202) with the 3’ and 5’UTR of apoB mRNA leads to apoB mRNA translational repression and/or activation. Using a human hepatoma cell line model, HepG2, the effects of overexpressed miRNAs and inhibition of endogenous miRNAs on the expression of apoB mRNA and apoB protein synthesis were investigated. We further examined the effect of these miRNAs on apoB mRNA traffic into cytoplasmic P-bodies. Transfection of HepG2 cells with miR-544 led to a significant reduction in apoB mRNA expression and protein synthesis and induced an increase in the co-localization of apoB mRNA into P-bodies. The opposite effect was observed when anti-miR-544 was employed to inhibit the endogenous miR-544. Results from luciferase reporter assays indicated that the effects of miR-544 may be mediated via interaction with the 3’UTR of apoB mRNA. In contrast to miR-544, miR-1202 overexpression induced an increase in apoB mRNA expression and protein synthesis. Similarly, the opposite effect was observed when using anti-miR-1202. Data from luciferase reporter assays showed an increased expression of the reporter gene in constructs carrying 5’UTR of apoB mRNA suggesting that miR-1202 may function via the 5’UTR. In summary, these data demonstrate that specific miRNAs are involved in the regulation of expression and translational control of apoB mRNA in hepatocytes. However, these miRNAs do not appear to mediate insulin regulation.