The Mitochondrial Free Radical Theory of Aging - Supernova: Pliki

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The Mitochondrial Free Radical Theory of Aging
imported from the nucleus, with the result that (as well as the stoichiometry still being
wrong) half the copies of that enzyme complex will be non-functioning.
Again, there are potential ways round this. One possibility is to insert into the nucleus
not only the genes for the mt-coded proteins, but also a gene whose product would enter
mitochondria and inhibit mtDNA transcription or translation. This is equivalent to
disrupting a nuclear-coded gene for mtDNA transcription or translation, but without the
drawback of potential damage to other genes (see Section 15.4), since it would act not on
the nuclear gene itself but on its protein product’s (mitochondrial) site of action.
15.6. The Inconvenience, but Irrelevance, of Mitochondrial RNAs
Some observers have suggested 7,9,10 that rescue of mtDNA mutations by importing
normally mt-coded proteins will not be any use in retarding aging, because many of the
mutations that are found to have occurred in anaerobic cells affect the mitochondrial
RNA-encoding genes, in particular the mt-coded tRNAs, and we have no idea how to get
cytoplasmic RNAs into mitochondria. This is a logically flawed objection, however. The
only thing a mitochondrial RNA does is to participate in the construction of mt-coded
proteins. Conversely, the only reason that a mutation in a mitochondrial tRNA gene is harmful
to OXPHOS is because it causes the absence of those proteins, which are integral components
of the OXPHOS machinery. Thus, successful import of all such proteins from the cytosol
will rescue not only mutations in their mt-coded genes but also mutations in tRNA (or
rRNA) genes—or, for that matter, any other mtDNA mutation such as a deletion.
However, it is a trifle inconvenient that tRNAs are such frequent victims of—“hotspots”
for—spontaneous mutations. What it means is that, even if the theory of aging presented in
this book is the truth, the whole truth and nothing but the truth about the aging process, we
are still rather unlikely to bring about more than a slight slowdown in aging until we get all
13 genes to work like clockwork from nuclear copies. If 12 of them are working, and one
not, and a tRNA gene goes down, that thirteenth protein will be lost and the cell will become
just as anaerobic as if the 12 were not working. This means that, initially, studies of the
efficacy of particular genes in their inserted copies will probably have to be done using
carefully predefined mutations in the mitochondrial copy of the relevant gene, in vitro.
Or will it? One highly ingenious way of studying each protein’s import in isolation
from the others has been developed by Claros and coworkers. 11,12 Rather than seeking
restoration of OXPHOS, they constructed “tribrid” proteins: they took the sequence of the
OXPHOS enzyme subunit, but then, as well as attaching a presequence to induce import
they also attached a “postsequence,” encoding a polypeptide that would thereby be attached
to the C-terminal end of the imported protein. This C-terminal polypeptide’s activity required
it to be imported into the matrix, but was not related to OXPHOS. Since the C-terminal end
of the protein is the last part to be imported, if this activity was seen then it indicated that
the whole protein must have been successfully imported. And since the function was
autonomous, not requiring cooperation from any mt-coded proteins, a mutation in a
mt-coded RNA would have no effect on the assay. Claros et al did this work in yeast, with a
protein that mammals do not actually have (because it is used to splice introns of some
mitochondrial genes, and mammalian mtDNA has no introns), but the principle can be
applied just as well to any nuclear-coded mitochondrial protein whose endogenous
chromosomal copy has been mutated.
15.7. Unfolding, Refolding and Prevention of Folding
It must also be borne in mind that the successful import of a nuclear-coded protein
into the mitochondrion is not the end of the incorporation process. Proteins derive their
specific properties from their stable three-dimensional structure, which is defined by their