Study of the role of hydrophobic amino acids at the carboxyl-terminus of apolipoprotein A-I in the structure and function

Postgraduate Thesis uoadl:1318769 451 Read counter

Κατεύθυνση Βιοχημεία
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Γκολφινοπούλου Χριστίνα
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Μαίρη Μαυρή-Βαβαγιάννη Αναπλ. Καθηγήτρια ΕΚΠΑ
Original Title:
Μελέτη του ρόλου υδρόφοβων αμινοξέων στο καρβοξυ-τελικό άκρο της απολιποπρωτεΐνης Α-Ι στη δομή και τη λειτουργία της πρωτεΐνης
Translated title:
Study of the role of hydrophobic amino acids at the carboxyl-terminus of apolipoprotein A-I in the structure and function
Apolipoprotein A-I (apoA-I) is the major protein component of high-density
lipoprotein (HDL) and plays an essential role in the biogenesis, maturation,
and the functions of HDL. The biogenesis of HDL occurs extracellularly,
predominantly in the liver and, to a lesser extent, in peripheral tissues, and
requires the interaction of apoA-I with the cholesterol transporter ABCA1 and
the cholesterol esterifying enzyme LCAT. The crucial role of apoA-I, ABCA1, and
LCAT for the biogenesis of HDL has been established by functional cellular and
in vitro studies, animal studies and naturally occurring mutations in these
proteins in humans with low HDL levels. Previous studies showed the importance
of the C-terminal region for the structure and functions of apoA-I. More
specifically, it has been proposed that the 220-231 region of the C-terminal
site of apoA-I is important for the apoA-I/ABCA1 interactions and the
biogenesis of normal HDL particles, but the specific apoA-I residues involved
have not been identified. The aim of the current study is to examine how
structure, stability and functions of apoA-I are affected by point mutations in
the C-terminal region of the protein. For this purpose three sets of mutations
were introduced in the C-terminal region of apoA-I. Specifically, we
investigated the role of eight hydrophobic (L218, L219, V221, L222), (F225,
V227, F229, L230) and two charged (E223, K226) residues located within or in
the vicinity of the 220-231 region. The biophysical analysis revealed that the
three mutated proteins affect in a distinct matter the structural integrity and
plasticity of apoA-I which is necessary for the physiological functions of the
protein. The functional experiments demonstrated that the ability of
apoA-I[L218A/L219A/V221A/L222A] and apoA-I[F225A/V227A/F229A/L230A] to promote
ABCA1-mediated cholesterol efflux and to activate LCAT is 20-23% and 65-66%
respectively, compared with wild type apoA-I. ApoA-I[E223A/K226A] was found to
have slightly increased ability to promote ABCA1-mediated cholesterol efflux,
while its ability to activate LCAT is 66%, compared with wild type apoA-I.
These results are in accordance with in vivo experiments that showed that
expression of apoA-I[L218A/L219A/V221A/L222A] and
apoA-I[F225A/V227A/F229A/L230A] in apoA-I-/- or apoA-I- x apoE-/- mice prevents
the biogenesis of mature HDL, resulting in low levels of plasma apoA-I and HDL,
while the expression of apoA-I[E223A/K226A] caused only mild changes in HDL
biogenesis. In conclusion, our results shows the significance of the eight
hydrophobic amino acids present in the C-terminal 218-230 region of apoA-I for
the structure and interactions of apoA-I with other proteins participating in
the pathway of HDL biogenesis, as well as for its ability to form HDL. An
improved understanding of protein interactions involved in the biogenesis of
HDL will help us identify new targets for diagnosis, prognosis, therapy and
prevention of low HDL cholesterol and atherosclerosis.
ApoA-I mutations, Biogenesis of HDL, ABCA1, LCAT, Cholesterol efflux
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