Ba-Bie Teng

Mammalian apolipoprotein B (apo B) exists in two forms, each the product of a single gene. The shorter form, apo B48, arises by posttranscriptional RNA editing whereby cytidine deamination produces a UAA termination codon. A full-length complementary DNA clone encoding an apo B messenger RNA editing protein (REPR) was isolated from rat small intestine. The 229-residue protein contains consensus phosphorylation sites and leucine zipper domains. HepG2 cell extracts acquire editing activity when mixed with REPR from oocyte extracts. REPR is essential for apo B messenger RNA editing, and the isolation and characterization of REPR may lead to the identification of other eukaryotic RNA editing proteins.

The messenger RNA for apolipoprotein B undergoes a discrete and specific C to U editing of nucleotide 6666. This generates a stop translation codon and defines the carboxyl terminus of apolipoprotein B48. A 27-kDa rat intestinal protein that does not itself edit apolipoprotein B mRNA, but confers editing activity on chick intestinal extracts that do not have intrinsic editing activity, has recently been identified and its cDNA cloned (Teng, B., Burant, C. F., and Davidson, N. O. (1993) Science 260, 1816-1819). Here we show that p27 is homologous in the zinc coordinating region of the active site to cytidine deaminases from Escherichia coli, Bacillus subtilis, yeast, and man and to deoxycytidylate deaminases from T2 and T4 bacteriophages and man. p27 expressed in Xenopus laevis oocyte extracts has cytidine deaminase activity and specifically confers editing activity on chick intestinal extracts. The homologous E. coli cytidine deaminase does not confer editing activity. The zinc-specific chelating agent o-phenanthroline abolishes p27 activity and site-specific apolipoprotein B mRNA editing in rat enterocyte editing extracts. We conclude that p27 is the catalytic subunit of the apolipoprotein B mRNA editing enzyme and is a zinc-containing cytidine deaminase.

OBJECTIVE: proprotein convertase subtilisin/kexin type 9 (PCSK9) negatively regulates the low-density lipoprotein (LDL) receptor (LDLR) in hepatocytes and therefore plays an important role in controlling circulating levels of LDL-cholesterol. To date, the relationship between PCSK9 and metabolism of apolipoprotein B (apoB), the structural protein of LDL, has been controversial and remains to be clarified. METHODS AND RESULTS: We assessed the impact of PCSK9 overexpression (≈400-fold above baseline) on apoB synthesis and secretion in 3 mouse models: wild-type C57BL/6 mice and LDLR-null mice (Ldlr(-/-) and Ldlr(-/-)Apobec1(-/-)). Irrespective of LDLR expression, mice transduced with the PCSK9 gene invariably exhibited increased levels of plasma cholesterol, triacylglycerol, and apoB. Consistent with these findings, the levels of very-low-density lipoprotein and LDL were also increased whereas high-density lipoprotein levels were unchanged. Importantly, we demonstrated that endogenous PCSK9 interacted with apoB in hepatocytes. The PCSK9/apoB interaction resulted in increased production of apoB, possibly through the inhibition of intracellular apoB degradation via the autophagosome/lysosome pathway. CONCLUSIONS: We propose a new role for PCSK9 that involves shuttling between apoB and LDLR. The present study thus provides new insights into the action of PCSK9 in regulating apoB metabolism. Furthermore, our results indicate that targeting PCSK9 expression represents a new paradigm in therapeutic intervention against hyperlipidemia.

Apolipoprotein B (apoB) mRNA is modified by a posttranscriptional editing reaction in which a single base (C to U) change in apoB100 mRNA modifies a glutamine (CAA) to a translational stop codon (UAA), producing apoB48 mRNA in mammalian intestine. Rat liver normally contains both edited and unedited apoB mRNAs and previous work (Davidson, N. O., Powell, L. M., Wallis, S. C., and Scott, J. (1988) J. Biol. Chem. 263, 13482-13485) has demonstrated that the introduction of a translational stop codon can be modulated by thyroid hormone. In the current study, hepatic lipogenesis was modulated in vivo by fasting and refeeding a high carbohydrate diet, a maneuver which produced a 30-fold increase in hepatic triglyceride content. In this setting, hepatic apoB100 synthesis became undetectable in animals subjected to 48 h fasting and subsequently refed a high carbohydrate diet for either 24 or 48 h. This change was accountable for by an increase in the proportion of edited apoB mRNA, as determined by primer extension analysis, from 37% UAA in fasted animals to 79 and 91% UAA at 24 and 48 h of refeeding, respectively. The effect of this regimen on the expression of other hepatic apolipoprotein genes was less dramatic. ApoA-I and apoA-IV gene expression was modulated over a 2-fold range, in contrast to the (6-14-fold) pretranslational changes induced by thyroid hormone administration. ApoCIII mRNA abundance was unaltered in the setting of either fasting and refeeding or thyroid hormone administration, while apoE gene expression demonstrated a pretranslational increase following prolonged fasting. Taken together the data provide evidence that apoB mRNA editing is modulated by alterations in hepatic lipogenesis which additionally produce effects on the expression of other hepatic apolipoprotein genes suggesting that they are not coordinately regulated in vivo.