Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: UNIPROT:P01034 (cystatin C)
3,397 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mammalian intestinal apolipoprotein B (apoB) messenger RNA (mRNA) undergoes posttranscriptional editing, changing codon 2153 from CAA in apoB100 mRNA to an in-frame translational stop codon (UAA) in apoB48 mRNA. By contrast, chicken intestinal apoB cDNA contains a CAA codon at the corresponding site and apoB mRNA from chicken enterocytes, kidney, and liver is unedited. The cDNA sequence of chicken apoB spanning the edited base is divergent from mammalian apoB cDNA sequence, with 70% homology over the conserved 29-nucleotide sequence (6662-6690) flanking codon 2153. Efficient in vitro editing of both human and rat, but not chicken, synthetic apoB RNA was achieved using rat enterocyte S-100 extracts. By contrast, chicken enterocyte S-100 extracts failed to edit chicken, rat, or human synthetic apoB RNA. Mixing experiments, however, revealed that chicken enterocyte S-100 extracts enhance the in vitro editing activity of rat, pig, and human enterocyte S-100 extracts upon homologous RNAs. The editing enhancement activity of chicken enterocyte S-100 extracts is tissue-specific, heat-sensitive, substrate-saturable, and sensitive to proteinase K, but resistant to micrococcal nuclease. The activity was partially purified by Q-Sepharose chromatography and has an average molecular mass of 49 kDa when analyzed by gel filtration chromatography. We conclude that the evolutionary adaptation of intestinal apoB mRNA editing requires both a requisite RNA motif and tissue-specific factors which mediate the site-specific modification.
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PMID:Evolution of intestinal apolipoprotein B mRNA editing. Chicken apolipoprotein B mRNA is not edited, but chicken enterocytes contain in vitro editing enhancement factor(s). 140 Apr 37

Apolipoprotein (apo) B-100 mRNA is edited in the small intestine (in all mammals examined) and the liver (in mice and rats only) to produce apoB-48 mRNA. ApoB mRNA editing involves a C-->U conversion of the first base of the codon CAA for Gln-2153 in apoB-100, changing it to an in-frame stop codon (UAA). The edited mRNA encodes apoB-48, which is colinear with the N-terminal 48% of apoB-100. ApoB mRNA editing can be reproduced in vitro using cellular extracts from one species to edit synthetic apoB mRNA sequences from a different species. Editing of transcripts from transfected genes also appears not to be species-specific. We have produced transgenic mice that express full-length human apoB-100 mRNA at high levels in the liver and small intestine. Human apoB-100 (a 550-kDa protein) but not apoB-48 (a 260-kDa protein) is detected in total plasma (at approximately 22 mg/dl) and in very low density and low density lipoproteins. The endogenous mouse plasma apoB concentration is reduced by approximately 45% in the transgenic animals. Thus, the transgenic mice form an animal model for familial hyperapolipoprotein B, an inherited form of hyperlipidemia. To our surprise, we found that the full-length human apoB mRNA consists of > 99% apoB-100 mRNA in both the liver and small intestine; < 1% of edited (apoB-48) mRNA was detected. The proportions of endogenous mouse apoB-48 (edited) mRNA (60 and 90% in the liver and small intestine, respectively) were identical in transgenic mice and their nontransgenic littermates. Therefore, full-length human apoB mRNA is resistant to editing by the mouse editing enzyme in vivo; the unchanged proportion of endogenous mouse apoB-48 mRNA in the transgenic mice suggests that the human mRNA competes poorly with the mouse sequence for interacting with the editing enzyme. This observation has implications for the sequence specificity and mechanism of RNA editing. Furthermore, we should exercise caution in the interpretation of in vitro RNA-editing experiments.
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PMID:Transgenic mice expressing full-length human apolipoprotein B-100. Full-length human apolipoprotein B mRNA is essentially not edited in mouse intestine or liver. 835 69

Human livers produce apoB-100, a major protein of VLDL, while intestines produce apoB-48, the major protein of chylomicrons. ApoB-48 is translated from apoB-100 mRNAs that are post-transcriptionally edited at codon 2153, converting CAA (glutamine) to TAA, a stop codon. In contrast to humans, mouse and rat livers contain the apoB-100 mRNA editing mechanism. Because hormones and nutrients affect the metabolism of apoB containing lipoproteins, we studied the effects of sex hormones and diets on apoB mRNA editing. Groups of male and female C3H/HeJ mice were castrated and treated with 17 beta-estradiol at 0.16 (E2L) or at 5 micrograms (E2H), or with testosterone propionate at 1 microgram/g body weight/day for 14 days. Plasma apoB levels and ratios of apoB-100/apoB-48 both increased 2-fold, but only in the E2H group. To determine if the increased apoB-100/apoB-48 ratios were associated with altered levels of apoB-100 and apoB-48 mRNA, both forms of apoB mRNA were quantified. We found that indeed ApoB-100 mRNA increased 1.8-fold (p < 0.025) compared to apoB-48 mRNA only in the E2H group. Next, we studied the individual effects of dietary fatty acids and dietary cholesterol on the relative abundance of apoB-100 and apoB-48 mRNA. Contrary to the estrogen effect, the high fat-combination diet increased apoB-48 mRNA relative to apoB-100 mRNA. Total plasma apoB as well as apoB-48 synthesis in liver also increased. Our studies demonstrate that estrogens and high fat diet both modulate apoB editing in mouse liver, but that estrogens and fat diet affected apoB mRNA editing in opposite directions.
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PMID:Hormonal and nutritional stimuli modulate apolipoprotein B mRNA editing in mouse liver. 141 37

Apolipoprotein B (apoB) mRNA is edited in rat liver and intestine to convert a CAA glutamine codon to a UAA translational stop codon by the direct conversion of cytidine to uridine at nucleotide 6666. We have proposed the 'mooring sequence' model for apoB RNA editing, in which editing complexes (editosomes) assemble on specific apoB mRNA flanking sequences to direct this site-specific editing event. One sequence element (approx. nts 6671-81, the presumed 'mooring sequence') has been previously identified as necessary for editing. We have identified two additional sequence elements which are necessary for efficient editing: (1) a 5' 'Regulator' region which modulates editing efficiency and (2) a 'Spacer' region between the editing site and the 3' mooring sequence, whose distance is critical for efficient editing. Utilizing this data, we have induced editing at a cryptic site and have defined a 22 nucleotide 'cassette' of specific apoB sequence which is sufficient to support wild-type levels of editing in vitro in a background of distal apoB RNA sequence.
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PMID:Three distinct RNA sequence elements are required for efficient apolipoprotein B (apoB) RNA editing in vitro. 146 33

The solubilization and delivery of lipids in plasma rely on both forms of apolipoprotein B (apo B): apo B-100 and apo B-48. Apo B-48 is the translational product of apo B-100 mRNA that undergoes peritranscriptional conversion of C----U, replacing codon CAA (glutamine 2,153) with the inframe stop codon (UAA). We examined mRNA editing activity in the human and the rat by reverse transcription-polymerase chain reaction primer-extension analysis of intestine and liver total RNA. In rat intestine the percentage of apo B transcripts that undergo editing increases dramatically the day before birth (from approximately 1% to 80%), whereas the rat liver acquires an adult level of editing activity during the third postnatal week (rising from approximately 8% to 30%), when weaning is completed, bile acid composition matures, and plasma thyroid hormone levels peak. In contrast to the rat, the human intestine acquires adult levels of apo B mRNA editing relatively early in fetal development, rising from 10% at 10 weeks to approximately 80% by the end of the second trimester. Our results establish that apo B mRNA editing is 1) developmentally regulated in a tissue- and species-specific manner; 2) fully developed prenatally in both human and rat intestine, suggesting a crucial role of apo B-48 in mammalian fetal adaptation to extrauterine life; and 3) acquired early in human fetal intestine, implying a potential role for apo B-48 in prenatal lipid metabolism.
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PMID:Ontogenetic regulation of apolipoprotein B mRNA editing during human and rat development in vivo. 155 38

Two different molecular weight forms of apoB are produced from a common initial transcript via editing of a Gln codon (CAA) to a stop codon (UAA), leading to a truncated translation product (apo BS) that consists of the amino terminal half of the larger form (apoBL). Previous studies have shown that fasting coordinately decreases lipogenesis and the secretion of very low density lipoprotein (VLDL) lipids and apoBS. Secretion of the apoBL is unaffected by fasting. We studied whether editing of apoB RNA is repressed by fasting, thus accounting for the selective decreased secretion of apoBS. Column chromatography of [35S]methionine-labeled lipoproteins secreted by hepatocytes from fed rats showed that essentially all of apoBL is secreted in the VLDL fraction, whereas a significant amount (15%) of apoBS is secreted associated as lipoproteins eluting in the HDL fractions. Fasting decreased the relative amount of apoBS that eluted in the VLDL fractions and increased the amount secreted in the HDL fractions. Consistent with previous results, hepatocytes from fasted rats show a selective twofold decrease in apoBS secretion. Fasting did not affect the relative abundance of apoB RNA, determined by slot blot hybridization assays using two different 32P-labeled cDNA probes coding either for both molecular weight forms or for only the large molecular weight form. However, quantitative of the editing of apoB RNA showed that fasting caused a 60% decrease in the amount of apoB RNA possessing the stop codon. These data show that the editing of apoB RNA is sensitive to metabolic state (i.e., fasting) resulting in a selective decrease in the secretion of apoBS. However, since the total secretion of apoB was decreased by fasting, while apoB mRNA levels remained constant, additional (post-transcriptional) mechanisms play a role in regulating apoB secretion.
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PMID:Fasting decreases apolipoprotein B mRNA editing and the secretion of small molecular weight apoB by rat hepatocytes: evidence that the total amount of apoB secreted is regulated post-transcriptionally. 170 Oct 4

Rat hepatoma McA-RH7777 cell lines transfected with full-length human apolipoprotein (apo) B constructs produce mostly human apoB48 and only small amounts of apoB100, as a result of mRNA editing at codon 2153 (C to U conversion at nucleotide 6666). To abolish the formation of apoB48 and increase the yield of apoB100 and other forms of apoB longer than apoB48, site-specific mutations were introduced at or near the site of apoB mRNA editing. Among four mutations examined, only that in which codon 2153 was converted from CAA (Gln) to CTA (Leu) effectively precluded the formation of apoB48. In this mutant, a stop codon would not be generated even if the C to U conversion occurred. The three other mutations were introduced to disrupt the proposed stem-loop structure encompassing the editing site. Changes made in the third positions of five codons on the 5' side of the edited base or of four codons 3' of the edited base failed to eliminate the production of a protein with the approximate size of apoB48. A construct in which codon 2153 was changed from CAA to GAT (Asp) also failed to eliminate the production of a protein the size of apoB48. Analysis of the region between nucleotides 6200 and 6900 of the cDNA did not detect any prevalent alternate editing sites. Immunoblot analysis using polyclonal antibodies raised against synthetic peptides of human apoB100 indicated that the carboxyl terminus of the apoB48-like proteins probably resides between amino acid residues 2068 and 2129 of apoB100. These results provide some insight into the mechanism of apoB mRNA editing and will facilitate further studies on apoB-containing lipoproteins.
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PMID:Elimination of apolipoprotein B48 formation in rat hepatoma cell lines transfected with mutant human apolipoprotein B cDNA constructs. 173 Jun 41

Apolipoprotein (apo) B48 is produced in the mammalian intestine by a tissue-specific RNA-editing mechanism, which mediates a C to U conversion at position 6666 in apoB mRNA. This generates an inframe translation stop codon (UAA) in place of glutamine (CAA) at position 2153. To establish the sequences required for editing we have used an in vitro conversion assay to monitor the editing of synthetic RNAs by rat intestinal extracts. Transcripts containing 55 nucleotides (positions 6649-6703) or more of human apoB mRNA sequence were edited in vitro. Transcripts containing 42 nucleotides (positions 6648-6689) and 26 nucleotides (positions 6662-6687) were edited at 62 and 24% efficiency, respectively, of the 55-nucleotide sequence. To delineate the precise sequence requirements for editing, mutants were generated where 6-nucleotide sections of the 55-base region were changed to anti-sense sequence. Mutation of the 12-nucleotide region immediately downstream of C-6666 abolished editing, and mutation of 6-base sequences immediately 3' and 5' of this 12-nucleotide region significantly reduced editing. Having identified the key region of interest, a panel of 46 mutant RNAs carrying single base substitutions or deletions between nucleotide positions 6657 and 6685 was constructed. Mutagenesis in the sequence 5'-TGATCAGTATA-3' (positions 6671-6681) downstream of C-6666 had the most dramatic effect, since almost all mutations abolished or greatly reduced conversion in vitro. These results suggest that editing is a highly sequence-specific process. We propose that this downstream region is a recognition and/or binding site for the editing enzyme. A search for this sequence in other genes may help to reveal other RNAs that undergo editing.
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PMID:Sequence requirements for the editing of apolipoprotein B mRNA. 188 64

Apolipoprotein (apo) B-48 mRNA is the product of RNA editing which consists of a C----U conversion changing a CAA codon encoding Gln-2153 in apoB-100 mRNA to a UAA stop codon in apoB-48 mRNA. In the adult rat, RNA editing occurs both in the small intestine and the liver. We have studied the ability of rat liver nuclear extracts to bind to synthetic apoB mRNA segments spanning the editing site. Using an RNA gel mobility shift assay, we found the sequence-specific binding of a protein(s) to a 65-nucleotide apoB-100 mRNA. UV crosslinking followed by T1 ribonuclease digestion and SDS-polyacrylamide gel electrophoresis demonstrated the formation of a 40 kDa protein-RNA complex when 32P-labeled apoB-100 mRNA was incubated with a rat liver nuclear extract but not with HeLa nuclear extract. Binding was specific for the sense strand of apoB mRNA, and was not demonstrated with single-stranded apoB DNA, or antisense apoB RNA. The complex also failed to form if SDS was present during the UV light exposure. Binding experiments using synthetic apoB mRNAs indicate that the 40 kDa protein would also bind to apoB-48 mRNA but not apoA-I, apoA-IV, apoC-II or apoE mRNA. Experiments using deletion mutants of apoB-100 mRNA indicate efficient binding of wildtype 65-nucleotide (W65), 40-nucleotide (W40) and 26-nucleotide (W26) apoB-100 mRNA segments, but not 10-nucleotide (or smaller) segments of apoB-100 mRNA to the 40 kDa protein. In contrast, two other regions of apoB-100 mRNA, B-5' (bases 1128-3003) and B-3' (bases 11310-11390), failed to bind to the protein. The 40 kDa sequence-specific binding protein in rat liver nuclear extract may play a role in apoB-100 mRNA editing.
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PMID:A 40 kilodalton rat liver nuclear protein binds specifically to apolipoprotein B mRNA around the RNA editing site. 221 73

Apolipoprotein B (apoB) circulates in human plasma as two isoforms, apoB-100 (512 kDa) and apoB-48 (242 kDa). ApoB-48 is generated by a novel RNA editing mechanism which post-transcriptionally modifies apoB mRNA in the intestine by converting cytidine at nucleotide 6666 to uridine. This converts codon 2153 from glutamine (CAA) to a premature stop codon (UAA). To characterize the activity which edits apoB mRNA, extracts were prepared from enterocytes isolated from baboon small intestine. These extracts efficiently edit synthetic apoB RNA in vitro. Editing was detected by primer extension, and the specificity of the reaction was confirmed by DNA sequencing. Extracts prepared from other baboon tissues did not edit apoB RNA in vitro. The editing activity was partially purified by chromatography of the enterocyte extracts on DEAE-cellulose. The activity is sensitive to proteinase K but resistant to micrococcal nuclease and has an average molecular mass of 125 kDa when analyzed by gel filtration chromatography.
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PMID:Characterization of the apolipoprotein B mRNA editing activity in enterocyte extracts. 225


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