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ANGELO BALDASSARE CEFALU'

FUNCTIONAL CHARACTERIZATION OF NOVEL AMINO ACID VARIANTS IN APOB IN FAMILIAL HYPOBETALIPOPROTEINEMIA

  • Autori: Magnolo, A.; Pinotti, E.; Di Leo, E.; Valenti, V.; Cefalu', A.; Yao, Z.; Averna, M.; Tarugi, P.
  • Anno di pubblicazione: 2013
  • Tipologia: Proceedings (TIPOLOGIA NON ATTIVA)
  • OA Link: http://hdl.handle.net/10447/104764

Abstract

Introduction. Familial Hypobetalipoproteinemia (FHBL) is a codominant disorder characterized by reduced levels of LDL and apolipoprotein B (apoB) in plasma. In approximately 50% of FHBL cases is due to mutations in APOB gene resulting in truncated apoBs of various size. Only a few missense mutations have been reported so far as the cause of FHBL. In vitro studies have shown that these mutations induce retention of the mutant apoB in the endoplasmic reticulum and impair the secretion of apoB-containing lipoproteins. We identi ed two novel amino acid variants (Thr26-27del and Tyr102Cys) located in the N-terminal 1000 amino acids of mature apoB in two hypocholesterolemic blood donors. Methods. To investigate the functional effect of these variants we constructed plasmids containing human apoB-48 cDNAs harbouring the novel mutations. Rat hepatoma cells (McA-RH7777) were transiently and stably transfected with wild-type or the mutant forms of human apoB-48. The secretion ef ciency of human apoB- 48 was determined by immunoblotting with human anti-apoB and the incorporation of apoB into medium lipoproteins. Immunocytochemistry was used to monitor the intracellular localization of the mutant proteins. The post-translational stability and the intracellular degradation pathways of mutant apoB-48 was evaluated.Results and Conclusions. The mutation Tyr102Cys had no effect on apoB-48 secretion. The mutant apoB-48-Thr26-27del almost entirely abolished the secretion of apoB-48 and apoB-containing lipoproteins in the medium, suggesting that the deletion of two amino acids alters the structure of the beta-barrel (the rst 267 amino acids) of N-terminal domain of apoB. This mutant apoB-48 appears to be retained in endoplasmic reticulum. The addiction of a proteasome inhibitor partially blocked the decay of cellular apoB-48-Thr26-27del suggesting that a signi cant proportion of the mutant protein was degraded by the proteasomal pathway. The role of autophagy in the degradation of the mutant apoB was excluded.