The Mitochondrial Free Radical Theory of Aging - Supernova: Pliki
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The Mitochondrial Free Radical Theory of Aging - Supernova: Pliki

The Mitochondrial Free Radical Theory of Aging - Supernova: Pliki The Mitochondrial Free Radical Theory of Aging - Supernova: Pliki

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184 The Mitochondrial Free Radical Theory of Aging is compelling evidence that some mitochondrial protein import is similar: in yeast, some ribosomes engaged in translation are found bound to mitochondria. 24,25 This is actually no great surprise, since the signal that targets most proteins to mitochondria is at their N-terminus, which is synthesised first. Thus, this signal becomes “visible” to the targeting machinery before the protein synthesis is complete. In other words, contranslational import may not be obligatory; it may just happen by chance some of the time. This interpretation is supported by a number of other points. Firstly, it seems that only a minority of import is cotranslational: no transcripts have been found exclusively (or even predominantly) associated with mitochondria-bound ribosomes rather than free ones. 25 Secondly, the acceleration of import that is sometimes achieved by duplicating a protein’s leader sequence 18,11 (see Section 15.9) appears hard to explain on the basis of “more strenuous” import, since (based on our current understanding) the import machinery would not be expected to bind both sequences simultaneously; but it is easy to explain on the basis of more rapid targeting to mitochondria leading to more cotranslational (hence successful) import, since the targeting machinery will see a bigger signal more quickly on average. These considerations might suggest that it would be difficult to exploit cotranslational import for the present purpose. Since no protein is known which is predominantly imported cotranslationally, we must presume that there is nothing about the signal sequence (other than its size) which promotes such import, and we know (see Section 15.9) that bigger is better but not good enough. If contranslational import is a matter of chance, therefore, we would need to increase that chance. Pessimism may be premature, however. One approach that might possibly achieve this would be to exploit the nuclear genome’s codon bias.* Codon bias is thought to be self-sustaining, by virtue of rare codons being represented by small numbers of tRNA genes or by tRNAs with low efficiency. Thus, the idea is to give the transgenes deliberately terrible codon bias—to construct them with a large number of codons which are rare in human nuclear DNA, on the basis that they will typically be more slowly recognised and translated than normal, giving more time for import to begin (and, once begun, to keep up with translation). In bacteria, codon choice can alter translation rate by as much as sixfold, 26a so this approach has potential. Promotion of cotranslational import is a possible reason why overexpressing a protein involved in nucleocytoplasmic transport improves import of moderately hydrophic proteins. 26b The consideration of cotranslational import suggests another possible obstacle to mitochondrial gene therapy, however: it is quite conceivable that most mt-coded proteins are cotranslationally exported into the inner membrane. If they are, then the problem of folding (discussed in Section 15.7) becomes altogether more likely: it may very well be much easier for a protein to go straight into the membrane as it comes off a ribosome than as it comes through the Tim machinery, since the ribosome can face the membrane whereas the Tim machinery is facing the wrong way. * Codon bias is a numerical property of a collection of sequences of protein-coding genes—typically, of the set of all sequenced genes of a given species. Amino acids are encoded by triplets of nucleotides, so there are 64 possible triplets (codons), but there are only 20 amino acids. Thus, most amino acids are encoded by more than one codon—sometimes as many as six. One can therefore compare two synonymous codons (ones that translate to the same amino acid) with regard to how often they each appear in the collection of sequences. One will appear more often than the other: sometimes, it turns out, much more often. The difference between these pairs of numbers is the set's codon bias.

Transgenic Copies of mtDNA: Techniques and Hurdles Table 15.1. mtDNA gene complement in various species, relative to humans Species Nuclear in humans, Mitochondrial in Mitochondrial in mitochondrial here humans, absent here humans, nuclear here Most animals none none none Nematodes, Mytilus none ATP8? ATP8? Most fungi ATP9 none none S. cerevisiae, ATP9 NAD1-6, NAD4L none S. pombe Most plants RPs, SDH, etc. none none Beans RPs, SDH, etc. none COX2 e.g., V. radiata Selaginella elegans RPs, SDH, etc. none COX3 Chlamydomonas none none? COX2, 3, ATP6, 8 reinhardtii NAD3, 4L ??? 15.13. Semi-Import This is an approach to complementation of mtDNA mutations that has so far not been explored at all. It is inspired by the observation that the 13 mt-coded proteins are components of transmembrane enzymes, so might theoretically be able to sink into the membrane from the outside, rather than taking a detour into the matrix. They would still have to be transported across the outer membrane, so this could be called “semi-import”. Recall from Section 2.4.3 that this is exactly the route taken by some inner membrane proteins, particularly the anion carriers; 27,28 thus, there is no requirement for novel mechanisms to divert the proposed transgene-encoded proteins away from the Tim23-Tim17 complex (the system that transports proteins into the matrix). The signal sequences that direct these naturally semi-imported proteins to the mitochondrion are not N-terminal and are never removed, so incorporation of such a signal into the proposed transgenes faces the problem of potentially rendering the protein non-functional; but we do not yet know nearly enough about these signals to assess the true scale of that obstacle. Realistically, however, we probably cannot expect this approach to work in the general case. The OXPHOS enzymes are composed of so many subunits that their correct assembly is bound to be dependent on the arrival of each protein from the “expected” side of the membrane. Nevertheless, a few of the 13 may be able to find their correct juxtaposition despite arriving from the “wrong” side; for all we know, those may be the few whose import into the matrix turns out to be the hardest. Thus we should continue to bear this alternative seriously in mind. 185

Transgenic Copies <strong>of</strong> mtDNA: Techniques and Hurdles<br />

Table 15.1. mtDNA gene complement in various species, relative to humans<br />

Species Nuclear in humans, <strong>Mitochondrial</strong> in <strong>Mitochondrial</strong> in<br />

mitochondrial here humans, absent here humans, nuclear here<br />

Most animals none none none<br />

Nematodes, Mytilus none ATP8? ATP8?<br />

Most fungi ATP9 none none<br />

S. cerevisiae, ATP9 NAD1-6, NAD4L none<br />

S. pombe<br />

Most plants RPs, SDH, etc. none none<br />

Beans RPs, SDH, etc. none COX2<br />

e.g., V. radiata<br />

Selaginella elegans RPs, SDH, etc. none COX3<br />

Chlamydomonas none none? COX2, 3, ATP6, 8<br />

reinhardtii NAD3, 4L ???<br />

15.13. Semi-Import<br />

This is an approach to complementation <strong>of</strong> mtDNA mutations that has so far not been<br />

explored at all. It is inspired by the observation that the 13 mt-coded proteins are components<br />

<strong>of</strong> transmembrane enzymes, so might theoretically be able to sink into the membrane from<br />

the outside, rather than taking a detour into the matrix. <strong>The</strong>y would still have to be<br />

transported across the outer membrane, so this could be called “semi-import”. Recall from<br />

Section 2.4.3 that this is exactly the route taken by some inner membrane proteins, particularly<br />

the anion carriers; 27,28 thus, there is no requirement for novel mechanisms to divert the<br />

proposed transgene-encoded proteins away from the Tim23-Tim17 complex (the system<br />

that transports proteins into the matrix). <strong>The</strong> signal sequences that direct these naturally<br />

semi-imported proteins to the mitochondrion are not N-terminal and are never removed,<br />

so incorporation <strong>of</strong> such a signal into the proposed transgenes faces the problem <strong>of</strong> potentially<br />

rendering the protein non-functional; but we do not yet know nearly enough about these<br />

signals to assess the true scale <strong>of</strong> that obstacle.<br />

Realistically, however, we probably cannot expect this approach to work in the general<br />

case. <strong>The</strong> OXPHOS enzymes are composed <strong>of</strong> so many subunits that their correct assembly<br />

is bound to be dependent on the arrival <strong>of</strong> each protein from the “expected” side <strong>of</strong> the<br />

membrane. Nevertheless, a few <strong>of</strong> the 13 may be able to find their correct juxtaposition<br />

despite arriving from the “wrong” side; for all we know, those may be the few whose import<br />

into the matrix turns out to be the hardest. Thus we should continue to bear this alternative<br />

seriously in mind.<br />

185

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