The Mitochondrial Free Radical Theory of Aging - Supernova: Pliki

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The Mitochondrial Free Radical Theory of Aging
machinery. In Section 2.4.3 it was explained that, since nearly all mitochondrial proteins are
already encoded in the nucleus, a system exists to transport them into mitochondria. This
system is now fairly well understood; in particular, we know that the addition of a particular
type of sequence at the beginning of such a protein causes it to be imported all the way into
the matrix. 5 Therefore, if we simply prepend the nucleotide sequence of such a signal onto
each of the inserted genes, there is a chance that the encoded proteins will thereby be
transported to mitochondria. Once inside, the presequence is removed and the protein will
be indistinguishable (in theory) from what it would have been if it had been constructed in
the mitochondrion in the first place. Thus it should be incorporated correctly into the
OXPHOS machinery.
This sounds straightforward in principle, but would it work? We certainly have reason
for hope, since the yeast gene that had been recoded in 1986 1 was properly imported and
incorporated, so that it really worked, as long ago as 1988. 6 Other proteins have so far proved
harder (see Section 15.9), but there are after all only 13 proteins, 13 problems.
15.3. Regulation of Expression; Copy Number
It may have caught the reader’s notice that the proposal discussed above is to incorporate
“copies” of the mt-coded genes into the nucleus, but that no mention has yet been made of
how many copies. This is indeed an issue of central importance to the proposed treatment,
because the mt-coded proteins are all subunits of enzyme complexes, of which other subunits
are already nuclear-coded. If the mitochondrion has too much of one subunit relative to
another, it may fail to assemble any complexes correctly. This obstacle has been recognised
ever since protein import was first proposed as a treatment for aging. 3
In fact, however, we can turn the above observation to our advantage. Since all 13
mt-coded proteins do their work in direct physical combination with nuclear-coded ones,
they must normally be generated in exactly the same stoichiometry, relative to those
nuclear-coded ones, as they exist in the complete complex—usually 1:1. So, for example, in
order to get the right amount of cytochrome b, we could embed our nuclear copy in the
same regulatory DNA that naturally surrounds the Rieske protein. In this way we should
ensure that exactly two copies of the inserted gene will do the job, since that is the number
of copies that cells have of their nuclear-coded genes.
How can this be correct, since cells have so many mitochondria, each of which has its
own DNA (and in several copies, at that)? Surely that means that we must insert thousands
of copies into the nucleus in order to achieve correct levels of expression? Well, we cannot
escape the fact that cells manage with two copies of the gene for cytochrome c, etc., compared
to thousands of copies of that for cytochrome b etc. Therefore, for some reason the
mitochondrially-encoded genes must be transcribed and/or translated thousands of times
more slowly than the nuclear-coded ones. The question is, why? Conceptually, there are two
possible answers: either there is an intrinsic feature of the genes’ DNA sequence that makes
the nuclear ones much easier to translate, or else the transcription and/or translation
machinery in the mitochondrion is simply much less efficient than that in the cell. The
former alternative is biologically implausible, since the mtDNA is made up of the same
chemical constituents as the nuclear DNA. The latter must therefore be correct. But then, if
we insert a gene into the nucleus, it will promptly benefit from the more efficient machinery,
so it need only be present in the same copy number—two—as the naturally nuclear-coded
genes.
15.4. Stoichiometry: Interference by Endogenous Wild-Type mtDNA
Germ-line transformation gives us no choice about which cells include the inserted
DNA: they all do. Gene therapy may in time be more selective, but probably not at first. This