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María Ángeles Pajares
Figure 4. Summary of the effects detected on S-adenosylmethionine synthesis in LEC rats. The different levels at which copper
accumulation alters SAM synthesis are shown, as well as the modifications prevented by neocuproine administration.
Further analyses of copper effects on MAT proteins
were carried out in vitro taking advantage of the refolding
protocol. These experiments showed effects on folding of
MATa1 and MATa2, the presence of copper reducing the
amount of soluble protein obtained, but having no effect in
the association state attained. Moreover, addition of copper
to correctly folded MATs had a direct effect on their
activity (20). MATa2 dimers were more susceptible to
inhibition than MAT I and MAT III under reducing
conditions, whereas this pattern was opposite when
reducing conditions were milder. This effect of copper on
activity correlated with the generation of high-Mr
aggregates, further supporting its role in the folding of
MATs. In this case, the residues affected by oxidative
stress remain unknown, and hence further studies should
be carried out to understand the mechanism of copper
effects on these proteins.
4.5. The affinity between MAT II subunits is increased by
NADP+ binding
Several years latter crystal structures of human
MATa2 homo-oligomers became available, monomers
showing the same organization than MATa1, including
three domains and a similar pattern for dimerization.
Following this achievement structures of MATß homo-
oligomers were solved and the presence of a Rossman-fold
including NADP+ demonstrated (77). However, no
explanation of the cofactor's role in the protein was
provided. Therefore, we adapted the refolding protocol of
the laboratory for human MATa2 production, and
improved the methods for recombinant human MATß
production and purification (46). Using isothermal titration
238 @Real Academia Nacional de Farmacia. Spain
calorimetry we were able to determine the affinity
parameters for NADP+ binding to MATß, to demonstrate
that the cofactor increases the affinity of MATß for
MATa2 subunits, and finally that MAT II is a hetero-
trimer composed by a MATa2 dimer and a single MATß
subunit (46).
The structural data available also allowed the
preparation of models for MAT II structure, the best
approach suggesting that the interaction between the
MATa2 dimer and MATß not only involves the NADP+
binding site, but takes place close to the protein active site.
The role of MATß residues postulated to bind the cofactor
was proven by site-directed mutagenesis, whereas
additional mutants lacking part of the N-terminal (similar
to the V2 splicing form) showed altered kinetic properties
upon their association with MATa2 dimers (46). Several
of our findings were confirmed by Murray et al. in 2014,
including the stoichiometry between MATa2 and MATß
subunits (2:1), and the fact that the N-terminal end of
MATß regulates the activity of MAT II (46,78). However,
their crystal structures of MAT II showed a different
interaction surface for the subunits, suggesting a role for
the C-terminal end of MATß (in this case the V2 splicing
form) and a tunnel at the interface of the MATa2 dimer
(78). Again, and in spite of the effort already performed,
more structural work is needed to clarify the disparities
reported to date.
4.6. MAT1A expression is not restricted to the liver
Northern blots carried out on samples from animal
models or human biopsies showed MAT1A expression in
liver, together with a switch towards MAT2A expression in