Metabolic regulation of ApoB mRNA editing is associated with phosphorylation of APOBEC-1 complementation factor

Apolipoprotein B (apoB) mRNA editing is a nuclear event that minimally requires the RNA substrate, APOBEC-1 and APOBEC-1 Complementation Factor (ACF). The co-localization of these macro-molecules within the nucleus and the modulation of hepatic apoB mRNA editing activity have been described following a variety of metabolic perturbations, but the mechanism that regulates editosome assembly is unknown. APOBEC-1 was effectively co-immunoprecipitated with ACF from nuclear, but not cytoplasmic extracts. Moreover, alkaline phosphatase treatment of nuclear extracts reduced the amount of APOBEC-1 co-immunoprecipitated with ACF and inhibited in vitro editing activity. Ethanol stimulated apoB mRNA editing was associated with a 2- to 3-fold increase in ACF phosphorylation relative to that in control primary hepatocytes. Significantly, phosphorylated ACF was restricted to nuclear extracts where it co-sedimented with 27S editing competent complexes. Two-dimensional phosphoamino acid analysis of ACF immunopurified from hepatocyte nuclear extracts demonstrated phosphorylation of serine residues that was increased by ethanol treatment. Inhibition of protein phosphatase I, but not PPIIA or IIB, stimulated apoB mRNA editing activity coincident with enhanced ACF phosphorylation in vivo. These data demonstrate that ACF is a metabolically regulated phosphoprotein and suggest that this post-translational modification increases hepatic apoB mRNA editing activity by enhancing ACF nuclear localization/retention, facilitating the interaction of ACF with APOBEC-1 and thereby increasing the probability of editosome assembly and activity

Metabolic regulation of APOBEC-1 Complementation Factor trafficking in mouse models of obesity and its positive correlation with the expression of ApoB protein in hepatocytes

AbstractAPOBEC-1 Complementation Factor (ACF) is an RNA-binding protein that interacts with apoB mRNA to support RNA editing. ACF traffics between the cytoplasm and nucleus. It is retained in the nucleus in response to elevated serum insulin levels where it supports enhanced apoB mRNA editing. In this report we tested whether ACF may have the ability to regulate nuclear export of apoB mRNA to the sites of translation in the cytoplasm. Using mouse models of obesity-induced insulin resistance and primary hepatocyte cultures we demonstrated that both nuclear retention of ACF and apoB mRNA editing were reduced in the livers of hyperinsulinemic obese mice relative to lean controls. Coincident with an increase in the recovery of ACF in the cytoplasm was an increase in the proportion of total cellular apoB mRNA recovered in cytoplasmic extracts. Cytoplasmic ACF from both lean controls and obese mouse livers was enriched in endosomal fractions associated with apoB mRNA translation and ApoB lipoprotein assembly. Inhibition of ACF export to the cytoplasm resulted in nuclear retention of apoB mRNA and reduced both intracellular and secreted ApoB protein in primary hepatocytes. The importance of ACF for modulating ApoB was supported by the finding that RNAi knockdown of ACF reduced ApoB secretion. An additional discovery from this study was the finding that leptin is a suppressor ACF expression. Dyslipidemia is a common pathology associated with insulin resistance that is in part due to the loss of insulin controlled secretion of lipid in ApoB-containing very low density lipoproteins. The data from animal models suggested that loss of insulin regulated ACF trafficking and leptin regulated ACF expression may make an early contribution to the overall pathology associated with very low density lipoprotein secretion from the liver in obese individuals

Control of Mitochondrial Morphology Through Differential Interactions of Mitochondrial Fusion and Fission Proteins

Mitochondria in mammals are organized into tubular networks that undergo frequent shape change. Mitochondrial fission and fusion are the main components mediating the mitochondrial shape change. Perturbation of the fission/fusion balance is associated with many disease conditions. However, underlying mechanisms of the fission/fusion balance are not well understood. Mitochondrial fission in mammals requires the dynamin-like protein DLP1/Drp1 that is recruited to the mitochondrial surface, possibly through the membrane-anchored protein Fis1 or Mff. Additional dynamin-related GTPases, mitofusin (Mfn) and OPA1, are associated with the outer and inner mitochondrial membranes, respectively, and mediate fusion of the respective membranes. In this study, we found that two heptad-repeat regions (HR1 and HR2) of Mfn2 interact with each other, and that Mfn2 also interacts with the fission protein DLP1. The association of the two heptad-repeats of Mfn2 is fusion inhibitory whereas a positive role of the Mfn2/DLP1 interaction in mitochondrial fusion is suggested. Our results imply that the differential binding of Mfn2-HR1 to HR2 and DLP1 regulates mitochondrial fusion and that DLP1 may act as a regulatory factor for efficient execution of both fusion and fission of mitochondria

Metabolic regulation and structural evaluation of APOBEC‐1 complementation factor

Thesis (Ph. D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Biochemistry & Biophysics, 2009.APOBEC‐1 Complementation Factor (ACF) is an AU‐rich RNA binding protein discovered through its requirement to properly target cytidine to uridine deamination of C6666 in apoB mRNA in the intestinal tissue of all mammals and the livers of select species. Metabolic perturbations that enhance editing efficiency in hepatic tissue, specifically insulin and ethanol stimulation, were correlated with the accumulation of phosphorylated nuclear ACF. Through ACF purification and phosphoamino acid analysis I determined serine phosphorylation to be the metabolically regulated site(s). Inhibition of protein phosphatase 1 by cantharidin resulted in the nuclear retention of ACF and suggested ACF’s nuclear export required its de‐phosphorylation. Treatment of primary rat hepatocytes with protein kinase activators suggested PKC as the kinase active on ACF and in vitro phosphorylation with recombinant PKC demonstrated effective phosphorylation of recombinant ACF, while PKA could not phosphorylate ACF. Metabolic labeling of primary hepatocytes from APOBEC‐1 knockout mice and human primary hepatocytes demonstrated ACF phosphorylation was not dependent on its interaction with APOBEC‐1 (the cytidine deaminase it targets to apoB mRNA for editing) nor was this post‐translational modification of ACF species specific. Significantly, in apoB editing incompetent tissue, human liver, the phosphorylation of ACF was on serine suggestive of a general role of phosphorylation in ACF regulation. Cumulative evidence supported an interaction of ACF with apoB mRNA in both the nucleus and cytoplasm. The hypothesis tested in this thesis is that ACF may have a function in the export of apoB mRNA from the nucleus to the cytoplasm. In this regard, ACF trafficking to the nucleus and back out to the cytoplasm may modulate the availability of apoB mRNA for ApoB protein translation. Evaluation of the hypothesis in the leptin deficient Ob/Ob mouse model of obesity and insulin‐resistance demonstrated leptin regulation of hepatic ACF expression. In this animal model, ACF nuclear retention was not responsive to insulin though ACF phosphorylation was compartmentalized to the nucleus. The accumulation of cytoplasmic ACF in Ob/Ob mice correlated with an increased proportion of cytoplasmic apoB mRNA, relative to lean controls. In the cytoplasm, ACF was localized to the low density microsomal (LDM) fraction (the site of apoB translation) in both mice with elevated levels observed in the Ob/Ob mice. This supported the hypothesis that ACF association with apoB mRNA may be functionally significant to apoB mRNA translation. I propose the interactions of ACF with apoB mRNA and its role in hepatic trafficking of this mRNA may serve as a paradigm for a more global function of ACF. Similar to other members of the ELAV/Hu family of RNA binding proteins, ACF may control the stability and trafficking of other transcripts and this might explain the ubiquitous tissue expression of ACF and its requirement for embryonic development. In light of this I have defined a minimal functional portion of ACF, comprised of amino acids 1‐320 (ACF320), capable of specifically binding apoB RNA and APOBEC‐1. As such, ACF320 was sufficient to complement editing. Purification of recombinant ACF320 and analytical ultracentrifugation suggested the protein existed as a monomer in the absence of RNA. However, in the presence of RNA, multimeric complexes of ACF320 were observed. This observation was confirmed in vivo where ACF320 self associated with the ACF subunits oriented with N‐termini in close proximity as determined FqRET analyses. The findings in this thesis will serve to lead investigation on the structure and function of ACF and facilitate future research to identify new mRNA targets of ACF, its role in post‐transcriptional control of gene expression and its regulation of development

Mitochondrial Dynamics in Diabetes

Mitochondria are at the center of cellular energy metabolism and regulate cell life and death. The cell biological aspect of mitochondria, especially mitochondrial dynamics, has drawn much attention through implications in human pathology, including neurological disorders and metabolic diseases. Mitochondrial fission and fusion are the main processes governing the morphological plasticity and are controlled by multiple factors, including mechanochemical enzymes and accessory proteins. Emerging evidence suggests that mitochondrial dynamics plays an important role in metabolism–secretion coupling in pancreatic β-cells as well as complications of diabetes. This review describes an overview of mechanistic and functional aspects of mitochondrial fission and fusion, and comments on the recent advances connecting mitochondrial dynamics with diabetes and diabetic complications. Antioxid. Redox Signal. 14, 439–457