Human Insulin Growth Factor 2 mRNA Binding Protein 2 Increases MicroRNA 33a/b Inhibition of Liver ABCA1 Expression and Alters Low-Density Apolipoprotein Levels in Mice.

Genome-wide association studies (GWAS) have linked IGF2BP2 single-nucleotide polymorphisms (SNPs) with type 2 diabetes (T2D). Mice overexpressing mIGF2BP2 have elevated cholesterol levels when fed a diet that induces hepatic steatosis. These and other studies suggest an important role for insulin growth factor 2 mRNA binding protein 2 (IGF2BP2) in the initiation and progression of several metabolic disorders. The ATPase binding cassette protein ABCA1 initiates nascent high-density apolipoprotein (HDL) biogenesis by transferring phospholipid and cholesterol to delipidated apolipoprotein AI (ApoAI). Individuals with mutational ablation of ABCA1 have Tangier disease, which is characterized by a complete loss of HDL. MicroRNA 33a and 33b (miR-33a/b) bind to the 3' untranslated region (UTR) of ABCA1 and repress its posttranscriptional gene expression. Here, we show that IGF2BP2 works together with miR-33a/b in repressing ABCA1 expression. Our data suggest that IGF2BP2 is an accessory protein of the argonaute (AGO2)-miR-33a/b-RISC complex, as it directly binds to miR-33a/b, AGO2, and the 3' UTR of ABCA1 Finally, we show that mice overexpressing human IGF2BP2 have decreased ABCA1 expression, increased low-density lipoprotein-cholesterol (LDL-C) and cholesterol blood levels, and elevated SREBP-dependent signaling. Our data support the hypothesis that IGF2BP2 has an important role in maintaining lipid homeostasis through its modulation of ABCA1 expression, as its overexpression or loss leads to dyslipidemia.

Mice lacking ARV1 have reduced signs of metabolic syndrome and non-alcoholic fatty liver disease.

Metabolic syndrome (MetS) is a term used to characterize individuals having at least three of the following diseases: obesity, dyslipidemia, hyperglycemia, insulin resistance, hypertension, and nonalcoholic fatty liver disease (NAFLD). It is widespread, and the number of individuals with MetS is increasing. However, the events leading to the manifestation of MetS are not well-understood. Here, we show that loss of murine ARV1 (mARV1) results in resistance to acquiring diseases associated with MetS. Arv1-/- animals fed a high-fat diet were resistant to diet-induced obesity, had lower blood cholesterol and triglyceride levels, and retained glucose tolerance and insulin sensitivity. Livers showed no gross morphological changes, contained lower levels of cholesterol, triglycerides, and fatty acids, and showed fewer signs of NAFLD. Knockout animals had elevated levels of liver farnesol X receptor (FXR) protein and its target, small heterodimer protein (SHP). They also had decreased levels of CYP7α1, CYP8ß1, and mature SREBP1 protein, evidence suggesting that liver FXR signaling was activated. Strengthening this hypothesis was the fact that peroxisome proliferator-activating receptor α (PPARα) protein was elevated, along with its target, fibroblast growth factor 21 (FGF21). Arv1-/- animals excreted more fecal cholesterol, free fatty acids, and bile acids. Their small intestines had 1) changes in bile acid composition, 2) an increase in the level of the intestinal FXR antagonist, tauromuricholic acid, and 3) showed signs of attenuated FXR signaling. Overall, we believe that ARV1 function is deleterious when consuming a high-fat diet. We further hypothesize that ARV1 is critical for initiating events required for the progression of diseases associated with MetS and NAFLD.

Improving journal club: increasing student discussion and understanding of primary literature in molecular biology through the use of dialectical notes.

Reading the primary literature in a journal club format is an excellent practice where undergraduate students can develop their abilities in experimental data analysis and critical thinking, learn about new ideas and methods, and gain a foothold in scientific discourse. However, students are not familiar with the format, writing style, and depth of knowledge assumed when first reading journal articles. This can inhibit class discussion. To alleviate this problem, the author has instituted the use of dialectical notes in journal club, so that students fully engage the article. This novel use of a strategy borrowed from humanities has improved class participation.

A role for MGA2, but not SPT23, in activation of transcription of ERG1 in Saccharomyces cerevisiae.

The SaccharomycescerevisiaeMGA2 gene encodes an important regulator of unsaturated fatty acid production, by controlling transcription and mRNA stability of OLE1, the gene encoding the Δ9 fatty acid desaturase. Lipid composition studies indicated that the mga2Δ strain contains elevated relative amounts of squalene when compared to wild-type cells. The deletion of the MGA2 homologue SPT23 did not impact squalene levels. To explore the role of MGA2 in the regulation of sterol synthesis, the transcription of the ERG1 gene, which encodes squalene epoxidase, was studied using an ERG1 promoter-lacZ reporter gene construct. We report here that in addition to MGA2's role in regulation of unsaturated fatty acids, MGA2 is required for full basal expression of ERG1. Mga2p was found to be controlled by a novel regulator in its activation of ERG1, as neither unsaturated fatty acids nor cobalt affected ERG1 expression, as had previously been shown for Mga2p's regulation of OLE1. Further, response to miconazole treatment, which inhibits production of ergosterol at a later step in the sterol biosynthetic pathway and results in up-regulation of several genes in ergosterol synthesis, was not affected in the mga2Δ mutant. In each case, the spt23Δ mutant strain shows similar ERG1 expression to wild-type cells, while the mga2Δ/spt23Δ strain shows reduced ERG1 expression, comparable to the mga2Δ, suggesting that the role of regulation of ERG1 transcription is unique to Mga2p.

Substitution of DNA-contacting amino acids with functional variants in the Gata-1 zinc finger: a structurally and phylogenetically guided mutagenesis.

DNA-binding functionality among transcription factor proteins is afforded by a number of structural motifs, such as the helix-turn-helix, helix-loop-helix, and zinc finger domains. The common thread among these diverse structures is their sequence-specific binding to essential promoter or other genetic regulatory sequences with high selectivity and affinity. One such motif, present in a wide range of organisms from bacteria to vertebrates, is the Gata-type zinc finger. This family of DNA-binding proteins is characterized by the presence of one or two (Cys)(4) metal binding sites which recognize the protein's eponymous binding site, GATA. Unlike other conserved DNA-binding domains, Gata proteins appear to be restricted to binding consensus GATA sequences, or near variations, in DNA. Since the architecture of the Gata finger seems built around recognizing this particular sequence, we set out to define the allowable range of amino acid substitutions along the DNA-binding surface of a Gata finger that could continue to support sequence-specific DNA-binding activity. Accordingly, we set up a one-hybrid screen in yeast based on the chicken Gata-1 C-terminal zinc finger. Mutant libraries were generated at five amino acids identified in the Gata-DNA structure as likely to mediate sequence-specific contacts between the Gata finger and DNA. These libraries were designed to give as exhaustive amino acid coverage as possible such that almost all alternative amino acids were screened at each of the five probed positions. Screening and characterization of these libraries revealed several functional amino acid substitutions at two leucines which contact the DNA at the 3' and 5' flanks of the GATA binding site, but no functional substituents for amino acids near the core of the binding site. This pattern is consistent with amino acid sequences of known DNA-binding Gata fingers.

Expression of the Saccharomyces cerevisiae PIS1 gene is modulated by multiple ATGs in the promoter.

The PIS1 gene encodes a key branchpoint phospholipid biosynthetic enzyme, phosphatidylinositol synthase. The PIS1 promoter contains the unusual feature of three ATG codons (ATGs1, 2, and 3) in-frame with three stop codons, located just before the authentic start codon (ATG4). Using a PIS1(promoter)-lacZ reporter expression system and site-directed mutagenesis, we investigated the role the "upstream" ATG codons play in modulation of PIS1 expression. Of the single codon changes, mutation of the first ATG (ATG1) resulted in the largest increase of the reporter gene PIS1(promoter)-lacZ expression. All combinations of altered upstream ATG codons also resulted in greater reporter expression. Reverse transcription-PCR revealed that at least some PIS1 transcripts include all AUG codons, and their synthesis is probably directed by a second TATA box upstream of the putative TATA box. These results indicate that the multiple upstream AUG codons are present in at least some PIS1 transcripts and negatively impact PIS1 expression.

Arabidopsis thaliana expresses two functional isoforms of Arvp, a protein involved in the regulation of cellular lipid homeostasis.

Arv1p is involved in the regulation of cellular lipid homeostasis in the yeast Saccharomyces cerevisiae. Here, we report the characterization of the two Arabidopsis thaliana ARV genes and the encoded proteins, AtArv1p and AtArv2p. The functional identity of AtArv1p and AtArv2p was demonstrated by complementation of the thermosensitive phenotype of the arv1Delta yeast mutant strain YJN1756. Both A. thaliana proteins contain the bipartite Arv1 homology domain (AHD), which consists of an NH(2)-terminal cysteine-rich subdomain with a putative zinc-binding motif followed by a C-terminal subdomain of 33 amino acids. Removal of the cysteine-rich subdomain has no effect on Arvp activity, whereas the presence of the C-terminal subdomain of the AHD is critical for Arvp function. Localization experiments of AtArv1p and AtArv2p tagged with green fluorescent protein (GFP) and expressed in onion epidermal cells demonstrated that both proteins are exclusively targeted to the endoplasmic reticulum. Analysis of beta-glucuronidase (GUS) activity in transgenic A. thaliana plants carrying chimeric ARV1::GUS and ARV2::GUS genes showed that ARV gene promoters direct largely overlapping patterns of expression that are restricted to tissues in which cells are actively dividing or expanding. The results of this study support the notion that plants, yeast and mammals share common molecular mechanisms regulating intracellular lipid homeostasis.

Characterizing sterol defect suppressors uncovers a novel transcriptional signaling pathway regulating zymosterol biosynthesis.

erg26-1ts cells harbor defects in the 4alpha-carboxysterol-C3 dehydrogenase activity necessary for conversion of 4,4-dimethylzymosterol to zymosterol. Mutant cells accumulate toxic 4-carboxysterols and are inviable at high temperature. A genetic screen aimed at cloning recessive mutations remediating the temperature sensitive growth defect has resulted in the isolation of four complementation groups, ets1-4 (erg26-1ts temperature sensitive suppressor). We describe the characterization of ets1-1 and ets2-1. Gas chromatography/mass spectrometry analyses demonstrate that erg26-1ts ets1-1 and erg26-1ts ets2-1 cells do not accumulate 4-carboxysterols, rather these cells have increased levels of squalene and squalene epoxide, respectively. ets1-1 and ets2-1 cells accumulate these same sterol intermediates. Chromosomal integration of ERG1 ERG7 at their loci in erg26-1ts ets1-1 and erg26-1ts and ets2-1 mutants, respectively, results in the loss of accumulation of squalene and squalene epoxide, re-accumulation of 4-carboxysterols and cell inviability at high temperature. Enzymatic assays demonstrate that mutants harboring the ets1-1 allele have decreased squalene epoxidase activity, while those containing the ets2-1 allele show weakened oxidosqualene cyclase activity. Thus, ETS1 and ETS2 are allelic to ERG1 and ERG7, respectively. We have mapped mutations within the erg1-1/ets1-1 (G247D) and erg7-1/ets2-1 (D530N, V615E) alleles that suppress the inviability of erg26-1ts at high temperature, and cause accumulation of sterol intermediates and decreased enzymatic activities. Finally using erg1-1 and erg7-1 mutant strains, we demonstrate that the expression of the ERG25/26/27 genes required for zymosterol biosynthesis are coordinately transcriptionally regulated, along with ERG1 and ERG7, in response to blocks in sterol biosynthesis. Transcriptional regulation requires the transcription factors, Upc2p and Ecm22p.

Growth temperature affects accumulation of exogenous fatty acids and fatty acid composition in Schizosaccharomyces pombe.

The incorporation of exogenously supplied fatty acids, palmitic acid, palmitoleic acid, oleic acid and linoleic acid, was examined in the yeast Schizosaccharomyces pombe at two growth temperatures, 20 degrees C and 30 degrees C. Fatty acids supplied to S. pombe in the growth medium were found to be preferentially incorporated into the cells, becoming a dominant species. The relative increase in exogenous fatty acids in cells came at the expense of endogenous oleic acid as a proportion of total fatty acids. Lowering the temperature at which the yeast were grown resulted in decreased levels of incorporation of the fatty acids palmitic acid, palmitoleic acid and linoleic acid compared to cells supplemented at 30 degrees C. In addition, the relative amount of the endogenously produced unsaturated fatty acid oleic acid, while greatly reduced compared to unsupplemented cells, was increased in cells supplemented with fatty acids at 20 degrees C compared to supplemented cells at 30 degrees C. The differential production of oleic acid in S. pombe cells indicates that regulation of unsaturated fatty acid levels, possibly by control of the stearoyl-CoA desaturase, is an important control point in membrane composition in response to temperature and diet in this species.

Yeast cells lacking the ARV1 gene harbor defects in sphingolipid metabolism. Complementation by human ARV1.

arv1Delta mutant cells have an altered sterol distribution within cell membranes (Tinkelenberg, A.H., Liu, Y., Alcantara, F., Khan, S., Guo, Z., Bard, M., and Sturley, S. L. (2000) J. Biol. Chem. 275, 40667-40670), and thus it has been suggested that Arv1p may be involved in the trafficking of sterol in the yeast Saccharomyces cerevisiae and also in humans. Here we present data showing that arv1Delta mutants also harbor defects in sphingolipid metabolism. [(3)H]inositol and [(3)H]dihydrosphingosine radiolabeling studies demonstrated that mutant cells had reduced rates of biosynthesis and lower steady-state levels of complex sphingolipids while accumulating certain hydroxylated ceramide species. Phospholipid radiolabeling studies showed that arv1Delta cells harbored defects in the rates of biosynthesis and steady-state levels of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylglycerol. Neutral lipid radiolabeling studies indicated that the rate of biosynthesis and steady-state levels of sterol ester were increased in arv1Delta cells. Moreover, these same studies demonstrated that arv1Delta cells had decreased rates of biosynthesis and steady-state levels of total fatty acid and fatty acid alcohols. Gas chromatography/mass spectrometry analyses examining different fatty acid species showed that arv1Delta cells had decreased levels of C18:1 fatty acid. Additional gas chromatography/mass spectrometry analyses determining the levels of various molecular sterol species in arv1Delta cells showed that mutant cells accumulated early sterol intermediates. Using fluorescence microscopy we found that GFP-Arv1p localizes to the endoplasmic reticulum and Golgi. Interestingly, the heterologous expression of the human ARV1 cDNA suppressed the sphingolipid metabolic defects of arv1Delta cells. We hypothesize that in eukaryotic cells, Arv1p functions in the sphingolipid metabolic pathway perhaps as a transporter of ceramides between the endoplasmic reticulum and Golgi.

Mutations in erg4 affect the sensitivity of Saccharomyces cerevisiae to medium-chain fatty acids.

We have found that the medium-chain fatty acids (MCFAs) undecanoic acid (11:0), 10-undecenoic acid (11:1 Delta 10), and lauric acid (12:0) can affect the growth of Saccharomyces cerevisiae in a dose-dependent manner. The principal effect was a longer lag phase in MCFA-containing medium, although higher concentrations of 11:1 Delta 10 inhibited growth. Their relative order of inhibitory action was 11:1 Delta 10>11:0>12:0. Cellular content with MCFA supplementation was dependent on the concentration and the particular species of fatty acid, with 12:0 showing the highest relative accumulation and 11:1 Delta 10 the lowest at all concentrations. We have isolated and characterized a mutant that is hypersensitive to MCFA supplementation and is unable to grow at the normally permissive condition of 1 mM 11:1 Delta 10. However, it does not appear to accumulate higher relative levels of the fed MCFA compared to wild-type cells. Complementation of the mutant revealed that the ERG4 gene, encoding the enzyme that catalyzes the last step in ergosterol biosynthesis, had been mutated. The fatty acid composition of the erg4 Delta mutant differs only slightly from wild-type cells, mainly involving an increase in the relative amount of 12:0. These results indicate that yeast require ergosterol for optimal growth on certain MCFAs. We discuss the role ergosterol may have in cells responding to exogenous MCFAs and in supporting optimal cell growth.

Sterol-dependent regulation of sphingolipid metabolism in Saccharomyces cerevisiae.

We had previously isolated the temperature-sensitive erg26-1 mutant and characterized the sterol defects in erg26-1 cells (Baudry, K., Swain, E., Rahier, A., Germann, M., Batta, A., Rondet, S., Mandala, S., Henry, K., Tint, G. S., Edlind, T., Kurtz, M., and Nickels, J. T., Jr. (2001) J. Biol. Chem. 276, 12702-12711). We have now determined the defects in sphingolipid metabolism in erg26-1 cells, examined their effects on cell growth, and initiated studies designed to elucidate how might changes in sterol levels coordinately regulate sphingolipid metabolism in Saccharomyces cerevisiae. Using [(3)H]inositol radiolabeling studies, we found that the biosynthetic rate and steady-state levels of specific hydroxylated forms of inositolphosphorylceramides were decreased in erg26-1 cells when compared with wild type cells. [(3)H]Dihydrosphingosine radiolabeling studies demonstrated that erg26-1 cells had decreased levels of the phytosphingosine-derived ceramides that are the direct precursors of the specific hydroxylated inositol phosphorylceramides found to be lower in these cells. Gene dosage experiments using the sphingolipid long chain sphingoid base (LCB) hydroxylase gene, SUR2, suggest that erg26-1 cells may accumulate LCB, thus placing one point of sterol regulation of sphingolipid synthesis possibly at the level of ceramide metabolism. The results from additional genetic studies using the sphingolipid hydroxylase and copper transporter genes, SCS7 and CCC2, respectively, suggest a second possible point of sterol regulation at the level of complex sphingolipid hydroxylation. In addition, [(3)H]inositol radiolabeling of sterol biosynthesis inhibitor-treated wild type cells and late sterol pathway mutants showed that additional blocks in sterol biosynthesis have profound effects on sphingolipid metabolism, particularly sphingolipid hydroxylation state. Finally, our genetic studies in erg26-1 cells using the LCB phosphate phosphatase gene, LBP1, suggest that increasing the levels of the LCB sphingoid base phosphate can remediate the temperature-sensitive phenotype of erg26-1 cells.