Biochemical and Functional Characterization of Golgi Anti- Apoptotic Proteins (GAAP)
Author(s)
Saraiva, Nuno Ricardo de Almeida
Type
Thesis or dissertation
Abstract
Recently a new inhibitor of apoptosis, Golgi anti-apoptotic protein (GAAP),
was discovered in camelpox virus and in some vaccinia virus (VACV) strains. GAAP
is non-essential for VACV replication but affects virus virulence, is located in the
Golgi, inhibits apoptosis and modulates Ca2+ fluxes. The VACV GAAP (vGAAP)
shows high sequence identity with a previously uncharacterized human protein,
named human GAAP (hGAAP). GAAPs belong to the Bax inhibitor-1 family and are
highly conserved among eukaryotes, sharing a similar sequence, length and
hydrophobicity profile.
A yeast system was used to express and purify vGAAP and Arabidopsis
thaliana GAAP3 (AtGAAP3). Data from patch clamp experiments using reconstituted
purified protein in artificial lipid bilayers showed that vGAAP and AtGAAP3 allow the
passage of ions, suggesting that GAAPs might form ion channels or ion exchangers.
Amino acid sequence alignments of GAAP with other known ion channels or ion
exchangers allowed the identification of amino acid residues in GAAP that might be
involved in channel gating or ion selection. These residues were mutated in vGAAP
and the mutants screened for anti-apoptotic and Ca2+ modulation activity, and
purified to confirm and further characterize GAAP’s ion channel-like activity. Using
chemical crosslinking, FRET and cysteine mutagenesis it was shown that GAAPs are
able to form oligomers in a cysteine-dependent (vGAAP) or cysteine-independent
(hGAAP) manner.
Using detachment and attachment assays it was shown that hGAAP
overexpression is able to increase cell adhesion and, conversely hGAAP knock down
caused decreased adhesion and an elongated cell phenotype. Using a focal
adhesion (FA) marker it was observed that the FA turnover after GAAP knockdown is
reduced.
Taken together, these results suggest that GAAP modulation of apoptosis
and cell adhesion may be via its ion channel activity and subsequent modulation of
Ca2+ fluxes.
was discovered in camelpox virus and in some vaccinia virus (VACV) strains. GAAP
is non-essential for VACV replication but affects virus virulence, is located in the
Golgi, inhibits apoptosis and modulates Ca2+ fluxes. The VACV GAAP (vGAAP)
shows high sequence identity with a previously uncharacterized human protein,
named human GAAP (hGAAP). GAAPs belong to the Bax inhibitor-1 family and are
highly conserved among eukaryotes, sharing a similar sequence, length and
hydrophobicity profile.
A yeast system was used to express and purify vGAAP and Arabidopsis
thaliana GAAP3 (AtGAAP3). Data from patch clamp experiments using reconstituted
purified protein in artificial lipid bilayers showed that vGAAP and AtGAAP3 allow the
passage of ions, suggesting that GAAPs might form ion channels or ion exchangers.
Amino acid sequence alignments of GAAP with other known ion channels or ion
exchangers allowed the identification of amino acid residues in GAAP that might be
involved in channel gating or ion selection. These residues were mutated in vGAAP
and the mutants screened for anti-apoptotic and Ca2+ modulation activity, and
purified to confirm and further characterize GAAP’s ion channel-like activity. Using
chemical crosslinking, FRET and cysteine mutagenesis it was shown that GAAPs are
able to form oligomers in a cysteine-dependent (vGAAP) or cysteine-independent
(hGAAP) manner.
Using detachment and attachment assays it was shown that hGAAP
overexpression is able to increase cell adhesion and, conversely hGAAP knock down
caused decreased adhesion and an elongated cell phenotype. Using a focal
adhesion (FA) marker it was observed that the FA turnover after GAAP knockdown is
reduced.
Taken together, these results suggest that GAAP modulation of apoptosis
and cell adhesion may be via its ion channel activity and subsequent modulation of
Ca2+ fluxes.
Date Issued
2012
Date Awarded
2012-03
Advisor
Smith, Geoffrey
Johnson, Benjamin
Sponsor
Portuguese Foundation for Science and Technology (FCT)/ Programa Operacional Potencial Humano (POPH
Publisher Department
Medicine: Section of Virology
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)