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Title: Biochemical and functional characterization of Golgi anti-apoptotic proteins (GAAP)
Author: Saraiva, Nuno Ricardo de Almeida
ISNI:       0000 0004 2715 2661
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2012
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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.
Supervisor: Smith, Geoffrey ; Johnson, Benjamin Sponsor: Portuguese Foundation for Science and Technology (FCT) ; Programa Operacional Potencial Humano (POPH)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral