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Prometheus vs. SENS


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#1 John Schloendorn

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Posted 04 February 2005 - 02:36 PM


Hi Prometheus,
Since you attack SENS at every mention of Aubrey's name lately, and I think these criticisms should be pursued and / or pried apart, I would like to focus this discussion into a distinct thread. For a start (from the reason = crackpot thread):

The proviso, as Estep mentioned, is whether we can develop the technology to direct stem cells to repopulate and regenerate specific tissues. In due course I am confident this will be achieved and that such approaches will form the core of the next evolution of medicine which is directed regeneration.
Is this the best solution for the problem? And is such a solution in keeping with Aubrey's self-proclaimed "engineering approach" of finding the shortest distance between two points? Not necessarily when a far more rapid and implementable solution of dealing with the root of the problem - DNA damage - is available.

But I can at least see that the DNA maintenance approach is far less expensive to research

What is such a solution, how can it be implemented at a comparatively small cost, and why is this forseable?

#2 jaydfox

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Posted 04 February 2005 - 03:21 PM

Hey cool, good idea to centralize this thread, at is it somewhat spread out across the website. I suppose we could have a navigator move the relevant posts to this thread, to facilitate a starting point for discussion.

#3 caliban

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Posted 04 February 2005 - 03:25 PM

Joern, I don't want to preempt prometheus but I'd guess this comes down to the ancient question (that has been discussed here time and again as well) whether "aging" is essentially a genetic phenomenon and if it was, whether its genetic components are sufficiently limited to be identifiable and become the subject of intervention.

Based on this hypothesis, it has been suggested that part of the "solution" you quote could be endorsement of research on fruit flies similar to the MMP.

I'd just like you warn you that prometheus can get very excited about this topic. If you are lucky he could call you a "closet immortophobe" should you happen to disagree. ;))

#4 jaydfox

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Posted 04 February 2005 - 03:43 PM

But I can at least see that the DNA maintenance approach is far less expensive to research

What is such a solution, how can it be implemented at a comparatively small cost, and why is this forseable?

While I look into what it will take to track down and reference/quote, if not move, the relevant posts to this thread, I should point out that the discussions on the merits of a Fly Prize that took place last July/August laid out the basic case that DNA maintenance would be relatively cheap to research. Summed in a sentence:

Drosophila provides a means to very cheaply and very rapidly test tens or hundreds of thousands of genetics modulations, singly and in pairs and triples, etc., to find a subset of such genetic modulations to validate in mice models.

The size of this subset can be arbitrarily large, to compensate for the occasional (or even common) non-correlation of orthologs in flies and mice. If we came up with 100 highly successful genetic modulations in flies, which would require tens of millions of dollars to validate in mice, it would still be cheaper than testing 500 genetic modulations in mice by blind (semi-educated) guessing, and with better results being nearly a given (or so Prometheus and the relevant literature suggest). Or we could wait a few decades for the basic science to be researched by the universities.

#5 jaydfox

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Posted 04 February 2005 - 03:53 PM

Note that the DNA maintenance approach is not necessarily viewed as a way to cure aging. You can't stop all DNA damage. Well, some day maybe, but not soon.

I personally don't think it will do better than triple human lifespan, at best, and I'm really only hoping for an extra 50%-100%. But that would basically double the time between WILT treatments, providing the dual benefit of reduced medical infrastructure requirements (only need half the extra personnel and facilities) and reduced philosophical objections to WILT.

As de Grey admitted, his 10 year figure was rather aribitrary, and could be extended to perhaps 20 years, as long as it's considerably less than the 40 years it takes for cancer to become prevalent. With 50%-100% extended lifespan due to DNA maintenance, that 20 years could be turned into 30-40 years between WILT treatments. That's still not acceptable as a long term solution, but it certainly is acceptable in getting escape velocity under way.

And that's what SENS is all about: not curing aging and death, so much as bringing humanity to the cusp of escape velocity (shameless plug). DNA maintenance should be one of the pillars of that plan, not treated like a nuisance. I think de Grey should embrace it, not apologize for omitting it. But I have no personal beef with him, if he's reading this.

#6 ag24

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Posted 04 February 2005 - 05:13 PM

I'm very busy today so just a few words. If I have appeared negative about any ideas of others (whether they be alternative ways to do as well as the therapies I currently advocate or whether they be "sub-SENS" therapies that are not claimed to be able to get us to escape velocity), that's only because I find it a little demotivating to be accused of myopia and dubious "tactics" when it should be plain to everyone that I've consistently done my level best here and everywhere else to answer questions and suggestions totally earnestly. As to what should be included in SENS in the sense of being advertised on my site and promoted in my publications, I'm always quick to incorporate the former of the above categories (new ways that I think could be good enough to help us reach escape velocity), but I regard "sub-SENS" therapies as too many and varied to be given thorough (or even cursory) coverage without distracting attention from the SENS stuff. If someone thinks a particular therapy is promising enough to be a contender for SENS proper but I disagree, well, that's a matter of scientific judgement. My judgement is that therapies which aim only to slow down the accuulation of damage, rather than to repair it, are sub-SENS virtually by definition. Sure, when combined with bona fide repair therapies they make those repair therapies less frequently needed; but that's true for lots of other sub-SENS things -- I'd need to incorporate half of LEF's website.

#7 jaydfox

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Posted 04 February 2005 - 07:00 PM

But what if the only way to make SENS economically viable is to push the time between WILT treatments to 40 or 50 years, the region of high cancer risk? 100 to 125 million WILT treatments worldwide a year is an incredibly ambitious goal for such a large gap, but 250 (or 500) million treatments a year for smaller gaps may be undoable.

I disagree that robust DNA maintenance is sub-SENS, in the sense that good nutrition, supplementation, and exercise are sub-SENS. Besides which, most of those aim to square the mortality curve, or at best add a few percent to the tail end, as they tend only to affect the current mortality rate, with little impact on the doubling period. DNA maintenance could very well add several decades to the top decile (or percentile), by increasing the mortality doubling period itself. But as I must sometimes remind myself, I am not a biologist...

Take your time, anyway, I don't want to take up too much of it (though perhaps I already have). I've made my point and am about ready to let things go as they go, for a while. Prometheus will probably see this thread soon and respond, however.

#8 reason

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Posted 04 February 2005 - 09:45 PM

I think you have to draw the line somewhere - and plenty of time and energy (far too much time and energy in my opinion) goes towards very subobtimal attempts to tinker ones way to the upper bound of currently possibly healthy life span.

Serious efforts towards radical life extension comprise such a small fraction of total efforts - this is the real problem we must address. Until such time as radical life extension efforts are well funded, then I think it's a bad choice to focus any of our efforts on the tinkering side of things...

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Posted 05 February 2005 - 03:29 AM

ag24:

If I have appeared negative about any ideas of others (whether they be alternative ways to do as well as the therapies I currently advocate or whether they be "sub-SENS" therapies that are not claimed to be able to get us to escape velocity), that's only because I find it a little demotivating to be accused of myopia and dubious "tactics" when it should be plain to everyone that I've consistently done my level best here and everywhere else to answer questions and suggestions totally earnestly.


I haven't been keeping up with these discussions entirely, but I think the last thing anyone intends is to demotivate you and others who are pursuing means of extending human life indefinitely.

I'm quite sure you and those like you have influenced many more people than you realize. Everytime I see someone ask in the "Introduce yourself" forum about what they can do, what they should study, to contribute to life extension I'm reminded of that.

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Posted 05 February 2005 - 04:17 AM

Firstly, I should confirm that I have nothing but the greatest respect for Aubrey's tireless efforts and for the man himself. His courage, competence and compassion is admirable. I could fill pages on why I think he is a terrific guy.

Secondly, I am not against SENS - on the contrary - I am completely for it. As I am for the Methuselah Prize. These efforts will go down in history, as part of the spark that ignited the realization that mankind need not be bound by a mere 80+ years of life.

What I am also for is accelerating progress which is why I criticized the MPrize for such things as the choice of model organism, the diffuse research direction and poor web presence. At all times this critique has gone hand in hand with offering solutions: drosophila as a more suitable start-up organism, a more structured research competition model and improvements to the website.

With SENS I see similar opportunities for improvement - namely the incorporation of a new research imperative that would take precedence over the present solutions for mutations.

The present SENS solutions for mutations are:

1. Mitochondrial Genome Mutations

Strategy: Transfer the "last" 13 mitochondrial (mt) genes into the nucleus.

Rationale: The mt genome suffers as many as 100 times more DNA damage than the nuclear genome. Reduce damage to the mt genome by transferring it into the relatively safer environment of the nucleus Evolutionary processes have systematically "transferred" the majority of mt genes into the nucleus - let's get the rest across.

Challenging considerations:
1.1 regulation of mt proteins produced - there can be as few a tens or as many as hundreds or thousands of mt in a single cell at any time so an enormous variability in the quantity of protein that has to be expressed in controlled fashion based on cell type requirements.
1.2 transporting mt proteins encoded in nucleus to mt - the hydrophobic nature of certain mt proteins (COX-1, COB) prevents them from being directly transported into mitochndria (1). A possible mechanism could involve sending the protein in a less hydrophobic pre-peptide form into mt and then via a peptidase have it assume its native form. But such a solution would require also sending the corresponding peptidase in to the mt.
1.3 the mt genome is non-standard (2) - a nuclear analogue would require resequencing to be compatible with nuclear polymerases
1.4 toxicity of mt proteins - some mt proteins could be toxic if they found themselves in cytosol in native form (3). But a solution as for 1.2 could be effected.

2. Nuclear Genome Mutations

Strategy: Whole-body Interdiction of Lengthening of Telomere (WILT) which is the total deletion of telomere elongation capacity throughout every cell in the body including stem cells and germ line cells. This is supported by a periodic repopulation of "fresh" stem cells from an external source.

Rationale: Most cancers express telomerase reverse transcriptase which enables them to pass the division limit of somatic cells and proliferate indefinately (or until the host dies). Prevent telomerase expression and you prevent tumor proliferation. Replenish stem cell reservoirs with fresh, DNA damage free, telomerase negative stem cells periodically to compensate for non-endogenous production.

Challenging considerations:
2.1 Repopulation frequency - some cells, such as those in the lining of the stomach divide every 24 hours. These cells would require repopulation every 1-2 months.
2.2 Germline cells - not only will there have to be an off-vivo depository for stem cells, but since the host cannot carry germline cells there will have to be a bank for sperm and eggs.
2.3 Telomerase does more than maintain telomere length - it is also responsible for DNA repair (4) so DNA repair rates would have to be increased to compensate for the loss of this function.
2.4 Stem cell localization - we do not yet know how to get stem cells to localize into specific tissue reservoirs so the repopulation would require surgical procedures.
2.5 Nuclear DNA damage contributes to more than cancer - preventing telomerase expression is not going to prevent DNA damage to continue to occur and induce other gene expression changes
2.6 The stem cell/germline bank - there are many technological and logistical issues with setting up such a facility in order that every individual have suffcient haplocompatible stem/germline cells
2.7 Telomerase may not be the key to cancer - removing telomerase function in mice does not decrease the incidence of cancer (5) even though it reduces their lifespan when some DNA repair function is also decreased


An Alternative

Both nuclear and mitochondrial mutations are a function of a cells inate ability to deal with DNA damage.

A solution that is based on increasing the rate and responsiveness of DNA repair to damage can be implemented by identifying the upstream regulators responsible for DNA maintenance and selectively overexpressing in mutagenic conditions. This enables a lab to rapidly screen for the best performing genomic maintenance candidates - doable science using technology that is widely available. With a number of suitable canditates in place, therapeutic regimens for delivering DNA maintenance enhancement genes using conventional gene therapy techniques can be made available.

Living in a world that exposes us to more carcinogens than ever before provides a strong impetus for such a project. DNA maintenance enhancement can easily be folded into a cancer treatment/precautionary therapeutic without the stigma of "anti-aging" being associated. This also means easier funding than more controversial research.

A review of the literature will reveal very little has been done in the area of DNA maintenance/repair factor overexpression studies. This is a field ripe for harvesting.


On Stem Cell Replenishing

Stem cell replenishing is an area in which I completely agree, and in my view should be separated from the telomerase ablation of WILT - which is a putative treatment for cancer and not a true SENS. The mode of delivery and implementation, however, is a topic that needs further thought.


On Replacing WILT

WILT without the stem cell replenishment can be very generally described as another form of chemo or radiotherapy which similarly targets rapidly dividing cells. As I have mentioned in the past, a genetic construct deliverable by an engineered adenovirus and consisting of a tumor specific promoter controlling the expression of a suicide gene would provide a superior solution in terms of development and patient administration.


References:

(1) Limitations to in vivo import of hydrophobic proteins into yeast mitochondria. The case of a cytoplasmically synthesized apocytochrome b, Eur. J. Biochem. 228 (1995)762–771.

(2) Animal mitochondrial genomes, Nucl. Acids Res. 27 (1999)1767–1780.

(3) The evolution of the Calvin cycle from prokaryotic to eukaryotic chromosomes: a case study of functional redundancy in ancient pathways through endosymbiosis, Curr. Genet. 32(1997)1–18

(4) Introduction of human telomerase reverse transcriptase to normal human fibroblasts enhances DNA repair capacity. Clin Cancer Res. 2004 Apr 1;10(7):2551-60.

(5) Impact of telomerase ablation on organismal viability, aging, and tumorigenesis in mice lacking the DNA repair proteins PARP-1, Ku86, or DNA-PKcs.
J Cell Biol 167:4, 627-38 (2004)

#11

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Posted 05 February 2005 - 05:29 AM

My judgement is that therapies which aim only to slow down the accumulation of damage, rather than to repair it, are sub-SENS virtually by definition.


It seems to me that it is only by experiment that we can determine if the problem of DNA damage is better resolved by WILT/stem cell replenishment and mitochondrial genome to nuclear relocation than a strategy whose aim is to prevent senescence and its primary inducer, DNA damage, by a more direct approach.

From a theoretical perspective a means of enhancing genome maintenance is no less or more SENSworthy than any other of the existing SENS proposals.

It is, however, far easier to validate.

#12 ag24

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Posted 05 February 2005 - 07:39 PM

Many thanks Prometheus. Let me try to explain (a) why I think that the challenges to allotopic expression and WILT that you mention are less daunting than you suggest, and why the challenges to the alternatives you mention are more daunting than you suggest. For more details, see the WILT paper in ANYAS 1019 and my Trends Biotech paper on allotopic expression, both available at my site.

First allotopic expression. Regulation: we're lucky there, because every one of the 13 mt-coded proteins is a subunit of a complex that also has nuclear-coded ones in 1:1 stoichiometry. So even if it turns out to be necessary to mimic existing expression patterns really well (and in fact there is some evidence that this is not actually very crucial, e.g. heterozygous deletion of nuclear-coded OXPHOS complex subunits never has a phenotype in flies), we can just slap our coding region into the regulatory DNA of such a nuclear gene. Hydrophobicity: the main problem, but now seems very likely to be solvable just by judicious amno-acid substitutions that marginally reduce hydrophobicity without affecting function. Candidates can be found by looking in other species. The post-import modification idea is also plausible, and indeed a version of it, using inteins, was the main novel component of my TibTech paper mentioned above. But your concern is invalid, because (a) some such post-import modifications, inteins among them, do not require a peptidase but rearrange purely autocatalytically, and (b) even if a peptidase is needed, it will be a soluble protein so not hydrophobic, so its import will be easy. Resequencing: clearly necessary but equally clearly trivial - can be done for the whole mt genome by a grad student in a week. Toxicity: has been raised in the past but currently no evidence for it. If it were to occur, though, solutions include a long leader sequence that acts as a cis-chaperone to prevent folding (this happens for one of the nuclear-coded ATPase subunits in fact).

Now WILT. Frequency: the gut numbers you quote are the dogma but are almost certainly completely wrong, as if they were right we would see gut dysfunction in telomerase-negative mice long before we see skin and blood dysfunction, and that's not the case. If you go back to the Potten papers that came up with these rates you will see they're built on very fragile assumptions, some actually now known to be wrong uch as that each stem cell in a crypt does its own thing (most crypts are in fact monoclonal). Germline - yeah who cares, we'll be giving up procreation anyway. (I jest of course - but the option you give is fine.) Telomerase - be very careful - in early-generation KO mice no one has reported any phenotype. Zero. Also, all the evidence for DNA maintenance function for telomerase is for the catalytic subunit, so deleting the RNA subunit would not have any side-effect on current evidence. Localisation - discussed in detail in the WILT paper, but basically already being done for blood (bone marrow transplant) and skin (cosmetic surgery and burns therapy) and gut's been done in mice, though that did involve slicing them open and we would use some kind of endoscopy approach presumably. Non-DNA damage: see my previous answers, they will matter in the end but I claim not for many tiimes a currently normal lifetime. Telomerase-independent cancers: indeed, and in mice they don't even need ALT because the telomeres are so long and mice are so small that a small tumour is lethal. In humans we've no problem of cancer without telomere maintenance, but we've certainly got to worry about ALT, as extensively discussed; it's no accident that I have two top ALT researchers coming to SENS 2 (and I may get a third).

Now, DNA repair/maintenance improvements. I confess that the ultimate reason I'm pessimistic about this is that it's so obvious. You remark that "A review of the literature will reveal very little has been done" -- but unfortunately no, what a review of the literature reveals is that very little has been published. I think we all know the fate of negative results in contemporary science. Nonetheless, it is without question that some people are a great deal less cancer-prone than others and that there is a substantial heritable component in that variation, so I anticipate that DNA maintenance/repair can indeed be improved to the point that cancer is delayed by say 30 years in terms of average age of onset. I just have a bad feeling that that will be a tough project because it will take a lot of genes being changed. But as previously said, I'm all for all such experiments being done. I just don't prioritise them above hastening WILT.

Finally, suicide genes. The weak link in "a genetic construct deliverable by an engineered adenovirus and consisting of a tumor specific promoter controlling the expression of a suicide gene" is the phrase "tumour-specific promoter". Viruses are smart at getting into cells, but the immune system is even smarter at killing cells that are expressing foreign antigens, so cancer immunotherapy should work very well indeed if it were this easy, just by directing it against telomerase for example. Indeed, this is a very reasonable and promising cancer immunotherapy approach, as PubMed will reveal. But the reality is not encouraging. I had high hopes for this area for a while in 2001, which were more or less vaporised when I went to a cancer immunotherapy meeting and saw how pitifully patchy the results were. Two basic problems: (1) when a tumour finds that its cells die when they express a given gene, it often just finds a way to stop expressing that gene. (2) it finds ways to confuse the immune system generically. Typical tumours are shot through with cytotoxic T cells, but they're just sitting there like lemons not doing their job. Cells can confuse/repel viruses a lot more easily than that. The virus approach is worth pushing too, though, don't get me wrong - again, see the SENS 2 program (Meruelo). The most promising type of immunotherapy I know of is autologous (don't just use one antigen, but make antibodies against everything unusual in the tumour). David berd (SENS2 speaker) is the leading light in this.

I mention all these speakers not just to attract people to the meeting (though that would be great) but to emphasise that I take what I call "sub-SENS" therapies very seriously indeed when I can draw the line more widely than in my own work (i.e. when I can have 50 speakers).

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Posted 06 February 2005 - 02:36 AM

You're welcome and I as well as other readers of this forum, I am sure, appreciate you taking the time to clear up the finer points of SENS. More importantly, the biogerontology community should take the time to look over your responses.

When I went over the material in composing the previous post it was not with the intention of demonstrating that allotopic expression of the mitochondrial genome is impossible - in fact I found myself coming up with possible solutions to each of the hurdles as I considered them. I did want to point out, however, that there are technical challenges that when compared against the technical challenges of implementing an enhanced in-mitochondrial genome maintenance system were as you said, more daunting. I still maintain that to be the case although I can now say with confidence that there is no reason why allotopic expression cannot be implemented and that such an implementation would have broad anti-senescence benefits due to decreased DNA damage of the mt genome. I still have some more questions pertaining to regulation and implementation:

On regulation

1. The number of mitochondria in a cell at any time are in a constant flux balanced by fission and fusion. This mechanism determines their activity including the total amount of mt proteins produced per cell as encoded by the mt genome. Some extracellular signals, such thyroid hormone will induce an increase in mt number and consequently increase mt protein production. How would such signaling be coupled to the now nuclear residing mt genome so that the total amount of protein produced corresponds and meets with the needs of the total volume of mt organelle?

2. Mitochondria are involved in calcium homeostasis and have an extensive system of cytosolic calcium sensing. What effect does Ca++ have on the mt genome and how can that be replicated in the nucleus?

3. A possible p53 binding sequence was recently reported to exist in the mt genome (1). It is likely that more such sequences exist that modulate transcription of mt DNA within the mt organelle. Are you aware of other similar sequences and how would this type of regulation also be transferred to the nucleus?

Essentially my point is that transferring the 13 mt genes into the nucleus is not enough. Nor is it enough to "slap our coding region into the regulatory DNA" since the intracellular signaling from a mt perspective will always be different to a nuclear one. The intracellular signaling and regulatory system would also have to transferred to the nucleus - once it has been elucidated. Certainly not impossible - but definitely more difficult than overexpressing key DNA repair factors. It would mean that the mt regulatory networks would have to be mapped out - reasonably easy compared to the nuclear genome - and then reworked to fucntion from the nucleus where the mt genes will now be residing. Once more I underline - a task more complex and involved than mere overexpression of key DNA repair factors.

On implementation.

The only way I can envisage delivering the allotopic modification to cells is via application to stem cells which are then transplanted or directly to embryos (which is fine if you are not born yet). This means that such treatments have to wait until the delivery of such engineered stem cells becomes available. I don't know about you but I would like to take a shot that infects as many cells as possible with a DNA repair enhancement virus as soon as it becomes available. And the clock is ticking.


Back to WILT.

I cannot help but see WILT as an evolution of the existing way of treating advanced cancer which is to wipe out all rapidly dividing cells using chemo or radiotherapy (broadly equivalent to targeting all telomerase positive cells) and reconstituting the eradicated stem cell population using bone marrow or cord blood transplants (equivalent to the periodic stem cell replacement).

I also still see telomerase as a "friend" to be cajoled to work in our favor to keep cells alive as long as possible and not an enemy to be targeted since other targets are available (see below). I think you would have great difficulty growing sufficient stem cell in culture with telomerase switched off.

I still maintain that stem cell replacement is unequivocally required by any consideration and should be a separate SENS objective and that the method for cancer cell targeting remain open. After all if we wanted to perform the telomerase ablation component of WILT today we could easily do it using RNAi technology!


On cancer vaccines

V-Max, like other cancer vaccines rely on sensitizing the immune response to the tumor which is a fine approach but still vulnerable to the ability of a cancer cell to alter its expression profile and evade detection. Also do not forget that some cancer evade the immune response by preventing MHCI expression, thereby making themselves invisible to T cells (still vulnerable to NK cells but still seem to survive though). The approach which I suggested does not require the immune system to be programmed to target a particular antigen but relies instead on the particular transcriptional expression profiles of cancer cells. Indeed "tumor specific promoter" is the link on which the success of such a treatment hinges on but there is plenty of scope for improvement. Note that the technology for combining multiple promoter systems to express the lethal genes as well as tissue specific conditionally replicating adenoviruses exists.


DNA repair/maintenance improvements

Can you explain what you mean by: "I confess that the ultimate reason I'm pessimistic about this is that it is so obvious"?

You say it's a tough project. C'mon, do you realize how silly a statement that is coming from de Grey. ;)



Refs

(1) Identification of a putative p53 binding sequence within the human mitochondrial genome.
FEBS Lett. 2004 Dec 3;578(1-2):198-202.

Edited by prometheus, 06 February 2005 - 02:59 AM.


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Posted 06 February 2005 - 03:26 AM

Now WILT.  Frequency: the gut numbers you quote are the dogma but are almost certainly completely wrong, as if they were right we would see gut dysfunction in telomerase-negative mice long before we see skin and blood dysfunction, and that's not the case.  If you go back to the Potten papers that came up with these rates you will see they're built on very fragile assumptions, some actually now known to be wrong such as that each stem cell in a crypt does its own thing (most crypts are in fact monoclonal). 


Incidentally, the mouse/telomerase model is not a particularly good biological comparator since in mice, telomere shortening appears not to cause aging. Even in telomerase knockout mice, it takes several telomerase-deficient generations of interbred mice for the accelerating aging syndrome to manifest. Mice have longer telomeres and more abundant telomerase than humans and mouse WRN knockouts are not affected in the same way.

In any case are you saying that a telomerase -ve cell can divide beyond the Hayflick limit or that there are so many crypts it is possible to do without a stem cell replacement for 10 years (according to WILT)?

#15 ag24

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Posted 06 February 2005 - 01:02 PM

> When I went over the material in composing the previous post it was not
> with the intention of demonstrating that allotopic expression of the
> mitochondrial genome is impossible - in fact I found myself coming up
> with possible solutions to each of the hurdles as I considered them

Well, please remember how busy I am. If you can save me time by just writing something down instead of making me write it down, please do so.

Mitochondrial regulation -- you seem to have missed my main point. In complex III (for example), there is one mt-coded subunit (cytochrome b) and a dozen nuclear-coded ones (eg the Rieske protein) in 1:1 ratio. The complex doesn't work if there is anything other than a 1:1 ratio. So, whenever the cell needs more complex III, it has to express more of each subunit. It's very impressive that cells can express Rieske and cytochrome b to the same extent and vary that extent in synch depending on need, because (as you say) there are huge differences in the context in which transcription and translation occurs (not to mention the machinery that performs it). But we don't need to know **anything** about how it does that in order for what I said ("slap our coding region [of cytochrome b] into the regulatory DNA [of Rieske]") to work -- if we do that, then cytochrome b will be expressed at the same levels in response to the same signals as Rieske, which is what we need. Intracellular signaling from a mt perspective will always be different to a nuclear one, certainly, but that makes no difference.

Aside: This is a great example of what I say about the difference between the creativity of basic scientists and the creativity of engineers. For a scientist it is just really difficult to think in terms of factoring out unknowns, i.e. finding solutions that don't depend on those unknowns, because finding things out is the whole deal. For an engineer it's central to factor unknowns out, because finding things out is just a means to an end.

Implementation: no, gene therapy can deliver allotopic expression just as easily (more or les) as it can deliver other genes. There are only about 12,000 coding nucleotides in the mtDNA, so they can be got in in one or two viruses. This can be reduced even more if Yagi's NDI1 results pan out - one gene replaces seven.

WILT - yes it's an extension of chemo plus bone marrow transplant. I don't know why you think exogenous telomerase-mediated stem cell expansion in culture is impractical. RNAi unfortunately is no easier to make sufficiently comprehensive than suicide adenoviruses.

Cancer vaccines: exactly, some cancer cells avoid the immune response. But my point was that they can also avoid, rather more easily, being infected by a virus, because a virus uses particular cell characteristics to get in and get itself expressed, and those characteristics can be altered by the cancer cell to avoid being infected.

By "I confess that the ultimate reason I'm pessimistic about this is that it is so obvious" I simply meant that I bet lots of people have tried to overexpress all manner of DNA repair/maintenance enzymes and have failed to obtain results interesting enough to publish.

When I say it's a tough project I mean there is a whole lot more to find out about which genes we need to overexpress (or modify) and it's not clear to me that there is a way forward that factors out much of the stuff we don't know. Telomerase-negative mice -- quite, my point entirely. If telomerase really had important telomere-extension-independent functions, early generation knockouts should have a phenotype, and they don't.

Crypts - no, I'm not saying that telomeraseless cells can exceed the Hayflick limit, nor that the fact that there are so many crypts is of any help. I'm saying that the true stem cells that give rise to crypt cells must actually divide only about as often as blood and skin stem cells. (One possibility that has yet to be excluded is that crypts are in fact maintained by the occassional sequestration of a blood stem cell from the circulation.) The frequencies of cell division **must** be about the same, or the late-generation telomerase-negative mice would get gut problems at an earlier age than blood and skin problems, which they don't.

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Posted 07 February 2005 - 01:28 AM

Back to allotopic regulation one more time:

In fact I did not miss your point, I wanted to illustrate other regulatory and signaling concerns that emerge eg. Ca++, mt DNA transcription factor binding sites. I appreciate the 1:1 of a nuclear DNA encoded component to mt DNA encoded component and also read,

But we don't need to know **anything** about how it does that


(ouch!) meaning we can just ignore an entire regulatory system. How can you possibly say that when the presence of signaling ligands is going to be different in the nucleus than in mitochondria. You can only try it and see what happens, I suppose.

Then I am dismayed by your effortless dismissal, obviously designed as a riposte for any such concerns:

For a scientist it is just really difficult to think in terms of factoring out unknowns, i.e. finding solutions that don't depend on those unknowns, because finding things out is the whole deal. For an engineer it's central to factor unknowns out, because finding things out is just a means to an end.


Whilst it may ring well for the average reader - it is really not good enough - it sounds arrogant will be your greatest impediment, in getting mainstream scientists to follow. At the very least a mention is deserved that one component of the SENS objectives is going to be the determination of whether such regulatory networks will impact on the allotopic solution and if so, a strategy to emulate their function from the nuclear perspective. That's all it would take to give SENS more credibility with mainstream science.

So to put this issue to rest I take it that at this stage no there is no plan for dealing with the regulatory network of mt gene expression once it has been transplanted to the nucleus.

On telomerase & Hayflick limit
You said: "I don't know why you think exogenous telomerase-mediated stem cell expansion in culture is impractical." I don't. So this is how you're going to get around it.

So the highest frequency of cell division in humans, you say must be, as per blood cells. This is a tenuous argument if it is going to be based on the mouse studies alone for reasons I mentioned previously. So the point of contention is what are the most rapidly dividing cells in humans and which tissues do they predominate in, as this is going to determine the required rate of stem cell replenishment and ultimately determine the strength of entire foundation that WILT rests upon. In certain epithelial cells, particularly mucosal types in the GI where damage and replacement is occurring daily at very high rate suggests a similar frequency of cell division.

On WILT
You said: "RNAi unfortunately is no easier to make sufficiently comprehensive than suicide adenoviruses" At present it has room for improvement but that is no reason to delay using it for the purposes of validating your theory.

On cancer vaccines
You said: "my point was that they can also avoid, rather more easily, being infected by a virus, because a virus uses particular cell characteristics to get in and get itself expressed, and those characteristics can be altered by the cancer cell to avoid being infected" Of course, it is an evolving therapeutical strategy and a legitimate one until something better can be developed. There are delivery alternatives, however, such as liposomes that can be used to complement such treatments.

On DNA repair enhancement
There is some literature on a positive effect (1), nil effect (2) and a system (3) of XP overexpression; on the naturally induced overexpression of Rad50 and DNA topoisomerase as protection against cardiac ischemia (4); on the effects of imbalanced DNA repair factor overexpression (5); on reduction of mt DNA damage via hOGG1 overexpression (6); the protective effects of overexpression of SIR2 alpha in cardiac myocytes (7).

The list goes on and supports the view that overexpresison of key DNA repair/maintenance (kDRM) factors are of benefit in the reduction of DNA damage and its phenotypic effects.

What we do not but need to see is a study on the effects of overexpression of kDRM factors in aging. The ideal experiment would involve a short lived species treated with various combinations of overexpressed kDRM factors using gene therapy as means of introducing the foreign genes.

Refs

(1) Overexpression of the XPA repair gene increases resistance to ultraviolet radiation in human cells by selective repair of DNA damage.
Cancer Res 55:24, 6152-60 (1995)

(2) Low amounts of the DNA repair XPA protein are sufficient to recover UV-resistance.
Carcinogenesis 23:6, 1039-46 (2002)

(3) Overexpression and purification of human XPA using a baculovirus expression system.
Protein Expr Purif 19:1, 1-11 (2000)

(4) Antibody-array technique reveals overexpression of important DNA-repair proteins during cardiac ischemic preconditioning.
J Mol Cell Cardiol 38:1, 99-102 (2005)

(5) APE1 overexpression in XRCC1-deficient cells complements the defective repair of oxidative single strand breaks but increases genomic instability.
Nucleic Acids Res 33:1, 298-306 (2005)

(6) MITOCHONDRIAL DNA DAMAGE TRIGGERS MITOCHONDRIAL DYSFUNCTION AND APOPTOSIS IN OXIDANT-CHALLENGED LUNG ENDOTHELIAL CELLS.
Am J Physiol Lung Cell Mol Physiol , (2004)

(7) Silent information regulator 2alpha, a longevity factor and class III histone deacetylase, is an essential endogenous apoptosis inhibitor in cardiac myocytes.
Circ Res 95:10, 971-80 (2004)

#17

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Posted 07 February 2005 - 07:45 AM

John, since you're the one who got this topic going, would you care to start structuring the debate (and pry the points apart) so that some value can come out of it?

#18 ag24

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Posted 07 February 2005 - 12:20 PM

> Back to allotopic regulation one more time:
>
> In fact I did not miss your point

You quite clearly did, since you still do. Yes, we can just ignore an entire regulatory system in this case. One more time: what matters is that the normally mt-coded proteins are expressed in the same pattern and at roughly the same level as the naturally nuclear-coded proteins of the same complex. It doesn't matter how we make that so, only that we do make it so. Allotopic expression allows us to make it so irrespective of how mtDNA expression works. I don't believe there is anything confusing in this, so I infer that you're just not bothering to think about what I'm saying. That's not arrogance, it's the conclusion I'm forced to.

> So the highest frequency of cell division in humans, you say must be,
> as per blood cells. This is a tenuous argument if it is going to be
> based on the mouse studies alone for reasons I mentioned previously.

Here too you demonstrate that you're just not bothering to think about what I'm saying. If gut stem cells in mice divide ten times faster than blood or skin stem cell, then telomerase knockout mice of a generation late enough to get blood and skin problems should get gut problems at a considerably earlier age than the blood and skin problems. That is not a tenuous argument, it is a cast-iron argument. I don't believe there is any lack of clarity in how I've described it, either.

#19 jaydfox

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Posted 07 February 2005 - 01:18 PM

John, since you're the one who got this topic going, would you care to start structuring the debate (and pry the points apart) so that some value can come out of it?

John, while you're at it, do you think we should designate a cheering/commentator section that doesn't interfere with the flow of the debate? In effect, sidelines? I have my own comments to interject for the broader audience, but I've already butted into the debate enough as it is.

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Posted 07 February 2005 - 04:07 PM

> Back to allotopic regulation one more time:
>
> In fact I did not miss your point

You quite clearly did, since you still do.  Yes, we can just ignore an entire regulatory system in this case.  One more time: what matters is that the normally mt-coded proteins are expressed in the same pattern and at roughly the same level as the naturally nuclear-coded proteins of the same complex.  It doesn't matter how we make that so, only that we do make it so.  Allotopic expression allows us to make it so irrespective of how mtDNA expression works.  I don't believe there is anything confusing in this, so I infer that you're just not bothering to think about what I'm saying.  That's not arrogance, it's the conclusion I'm forced to.


If I am yet to be convinced does it mean I miss your point? I think not. I just can't see it and I am merely pointing out not only the technical challenges but the void in knowledge that exists about the regulation of gene expression in mitochondria. One more time from this end too Aubrey: if you have a system [in mitochondria] whose regulation is based on things such as Ca++ concentration and binding sites for transcription factors in the mitochondrial genome whose very entry through the mitochondrial membrane could well be limited by receptors and ligand gated channels unique to mitochondria you would have to admit it is not as easy a task as you make it out to be, this uprooting of an entire regulatory system from mitochondria to nucleus. But I am really fascinated by your proposed methodology (It doesn't matter how we make that so, only that we do make it so). Of course if you can get the mitochondrial proteins to be expressed similarly from the nucleus is all that matters. My point is how are you planning to achieve that in light of what I mentioned above? That is what is confusing - the methodology.

> So the highest frequency of cell division in humans, you say must be,
> as per blood cells. This is a tenuous argument if it is going to be
> based on the mouse studies alone for reasons I mentioned previously.

Here too you demonstrate that you're just not bothering to think about what I'm saying.  If gut stem cells in mice divide ten times faster than blood or skin stem cell, then telomerase knockout mice of a generation late enough to get blood and skin problems should get gut problems at a considerably earlier age than the blood and skin problems.  That is not a tenuous argument, it is a cast-iron argument.  I don't believe there is any lack of clarity in how I've described it, either.


I don't know what I must be demonstrating but lets stick to the issues if we could, I really don't want this to get personal. I respect you as a committed and creative scientist and my only interest is to look at the science and in doing so explore potential solutions and engage others into similar participation.

The reason I suggest that using mice as a comparator with humans in the context of stem cell biology may be tenuous is because of studies that have indicated there are substantial differences in telomerase and other gene expression function associated with stem cell proliferation between different species and particularly between mice and humans. For example we know that in humans replicative cell senescence results from telomere shortening whereas in mice senescence is not a function of telomere shortening and must be due to other reasons. We also know that mice have very long telomeres compared to humans. You're saying that this does not matter because once the telomeres shorten sufficiently in telomerase negative mice then the there is very little difference in the time where the various tissues start to fail - gut fails at the same time as other cells therefore the rate of stem cell division must be the same for the gut as for other tissues.

But one can interpret the data differently: all the mouse tissues are failing because the telomeres have finally "worn out" and mouse stem cells have a different telomere biology to what we have observed in humans so that they are dying for reasons other than cell division rate, or, all of the mouse stem cells in all tissues are dividing at the rate of 1 per day.

In any case, surely you have more supporting data than the telomerase knockout mice experiment to refute Potten's estimate of 1 stem cell division per day.

#21

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Posted 07 February 2005 - 04:24 PM

Potten's paper for those interested in stem cell division rates in the gut..

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#22

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Posted 07 February 2005 - 04:34 PM

Hornsby's paper on the differences between human and mouse senescence and telomerase influence..

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#23 ag24

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Posted 07 February 2005 - 06:59 PM

> Of course if you can get the mitochondrial proteins to be expressed
> similarly from the nucleus is all that matters. My point is how are you
> planning to achieve that in light of what I mentioned above?

I'm at a loss to see what part of this you don't understand, and I have a pretty good track record at explaining biology to people of all levels of knowledge, so maybe it would be best if someone else tried to explain it. Let me try one last form of words. We don't need to reproduce the mitochondrial expression system in the nucleus, because it has already *been* reproduced for us, by evolution, in order to get the nuclear-coded subunits of these complexes into mitochondria in the right amounts at the right times. The Rieske protein is expressed (from a nuclear gene) in the pattern that we want to express cytochrome b. We don't need to know how that nuclear regulation (for Rieske) works, we only need to use it for cytochrome b too.

> You're saying that this does not matter because once the telomeres
> shorten sufficiently in telomerase negative mice then the there is very
> little difference in the time where the various tissues start to fail -
> gut fails at the same time as other cells therefore the rate of stem
> cell division must be the same for the gut as for other tissues.

Right.

> But one can interpret the data differently: all the mouse tissues are
> failing because the telomeres have finally "worn out" and mouse stem
> cells have a different telomere biology to what we have observed in
> humans so that they are dying for reasons other than cell division
> rate, or, all of the mouse stem cells in all tissues are dividing at
> the rate of 1 per day.

No, because there is plenty of data showing that mouse blood stem cells divide only once or twice a month.

> In any case, surely you have more supporting data than the telomerase
> knockout mice experiment to refute Potten's estimate of 1 stem cell
> division per day.

Yes -- humans with telomere maintenance defects get a disease called dyskeratosis congenita, and generally they too get problems in all the highly proliferative tissues at about the same age. I find this data less cast-iron than the mose data though, because in DC the telomere elongation defect is not total and one can postulate that gut stem cells are somehow compensating better than blood. But if you find it more persuasive than the mice, no problem. I must repeat however that this is all in my published paper on WILT, available on my site, so you can answer this and plenty of other questions yourself.

#24

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Posted 08 February 2005 - 04:17 AM

I'm at a loss to see what part of this you don't understand, and I have a pretty good track record at explaining biology to people of all levels of knowledge, so maybe it would be best if someone else tried to explain it.  Let me try one last form of words.  We don't need to reproduce the mitochondrial expression system in the nucleus, because it has already *been* reproduced for us, by evolution, in order to get the nuclear-coded subunits of these complexes into mitochondria in the right amounts at the right times.  The Rieske protein is expressed (from a nuclear gene) in the pattern that we want to express cytochrome b.  We don't need to know how that nuclear regulation (for Rieske) works, we only need to use it for cytochrome b too.


I have no issue with these facts:

1. The Rieske FeS protein is a component of the cytochrome b complex
2. Rieske Fes protein is encoded by the nuclear gene RIP1
2. The rest of the cytochrome b complex proteins are encoded by genes in the mitochondrial genome
4. The Rieske protein is produced at a ratio of 1:1 in the nucleus as per the rest of the subunits for cytochrome b which are produced in mitochondria.

In a broader scope here are some important mitochondrial complexes in terms of where the genes are presently encoded (mt vs nuclear):

NADH - 7 subunits encoded in mt out of 46 subunits
succinate dehydrogenase - 1 of 4 subunits
cytochrome c oxidoreductase - 1 of 11
cytochrome c oxidase - 3 of 13
ATP synthase - 2 of 17
pyruvate dehydrogenase - nuclear
TIM/TOM complex - nuclear

From this perspective one can agree with your view since most proteins are nucleus encoded and whatever regulatory system is operating it is sufficient to maintain the required proteins synthesis of the subunits encoded in mitochondria.

However, would you agree that permeability to transcription factors and other signaling molecules is different between the mitochondrial membrane and the nuclear membrane?

And if such permeability is different then it would imply that the regulation of the mitochondrial genome in terms of gene expression activity resulting from said transcription factors and other signaling molecules would also be altered if it were transferred to the nucleus.

Therefore if regulation is altered due to allotopic expression then it has to be accounted for (more work).

Finally, how do we introduce these new cells with allotopic expression of all mt genes into an adult without resorting to presently non-existent delivery methods? The only foreseeable way is via stem cell transplantation of highly engineered stem cells and that is a long time coming. What do we do for the next 10 - 20 years?

The most rapid solution is to increase DNA repair, don't you think?





** Note also that I am not opposed to allotopic expression in the way I am to WILT. I do agree it could be of huge benefit. I am making a point of the technical challenges of implementing it in contrast to dealing with the DNA damage problem more directly.

#25

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Posted 08 February 2005 - 05:11 AM

In relation to rates of cell division/WILT viability.

As you say in your WILT article

For WILT, however, the implication is inescapable that the gut should survive as long between transplants as the blood.


So I would like to get one fact straight before progressing further on this point: do you dispute that the mucosal surface of the GI tract turns over with a frequency of 24 - 72 hours?

Edited by prometheus, 08 February 2005 - 05:44 AM.


#26

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Posted 08 February 2005 - 05:25 AM

A recent review on GI stem cells by Brittan & Wright..

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#27 ag24

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Posted 08 February 2005 - 07:59 AM

> The rest of the cytochrome b complex proteins are encoded by genes in the mitochondrial genome

It's the ubiquinol-cytochrome c oxidoreductase complex (complex III). Cytochrome b is the one mt-coded subunit of the 11 (see your list).

> NADH [dehydrogenase] - 7 subunits encoded in mt out of 46 subunits
> succinate dehydrogenase - 1 of 4 subunits
> cytochrome c oxidoreductase - 1 of 11
> cytochrome c oxidase - 3 of 13
> ATP synthase - 2 of 17
> pyruvate dehydrogenase - nuclear
> TIM/TOM complex - nuclear

Mostly right - SDH is actually 0 out of 4

> Would you agree that permeability to transcription factors and other
> signaling molecules is different between the mitochondrial membrane and
> the nuclear membrane?

Yes, of course.

> And if such permeability is different then it would imply that the
> regulation of the mitochondrial genome in terms of gene expression
> activity resulting from said transcription factors and other signaling
> molecules would also be altered if it were transferred to the nucleus.

Yes, of course. But the proposal is not to put the mtDNA in the nucleus, complete with all the non-coding DNA (the D loop) that is necessary for its appropriate transcription in mitochondria. The proposal is to put the coding regions into the nucleus in the regulatory context of other, naturally nuclear genes. Transcription of the mtDNA is of the whole circle as one message in each direction, starting at the D loop. We do not need to copy that.

> Finally, how do we introduce these new cells with allotopic expression
> of all mt genes into an adult without resorting to presently
> non-existent delivery methods?

Of course we need better delivery. But we also need the exact same better delivery to increase DNA repair.

> do you dispute that the mucosal surface of the GI tract turns over with a
> frequency of 24 - 72 hours?

If you mean the cell division rate of the stem cells that give rise to the mucosal surface (which is the relevant rate), yes I do.

Note: I am on a plane for the next while and very busy for the next week, so I may not be able to post again here for a while.

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Posted 08 February 2005 - 11:39 AM

Take your time and take care. Hopefully we can sort through the chaff in the postings by the time you return.

Cheers.

PS With SDH there is a b cytochrome peripheral group which I am assuming also has a Rieske protein.

#29

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Posted 08 February 2005 - 12:04 PM

Now John, how about some objective sorting out here. Perhaps Jay could help too. He has done a great job in the past of summarizing the essential elements of conflicting positions.

#30 jaydfox

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Posted 10 February 2005 - 05:16 PM

Well, hopefully John can be a little more objective. At any rate, I suspect he's more in support of de Grey's view, and I'm more in support of Prometheus's view, so if we both comment, then it should end up somewhat balanced in the end.

There's too much to sift through in one response, so I'll take it piecewise, and not necessarily in the best order.

First, the issue of mtDNA expression rates. Prometheus originally challenged that regulating the expression of mtDNA from the nucleus would be too complex, owing the the different chemistries. de Grey countered that for each complex, the ratios of the individual genes for the complex are typically 1:1.

In layman's terms, if Complex Q has three genes, Qa, Qb, and Qc, then for each Qa that gets produced, there should be 1 Qb and 1 Qc. If I'm interpreting this wrong, then de Grey needs to be more clear.

Okay, with this as a given, if we know that the right amounts of Qa are already being transcribed in the nucleus and transported to where they are needed, then we can assume that all we need to do is transcribe Qb and Qc at the same time. Hence, de Grey assumes that we can insert the coding regions for Qb and Qc, from the mtDNA, into the coding region for Qa in the nuclear genome. When the region coding Qa is transcribed, a copy of Qb and Qc will be transcribed as well. (If I am reading this wrong, then a better explanation is required, at least for us laymen.)

The same logic would hold for the other complexes.

Prometheus initially challenged this concept, then partially yeilded:

I have no issue with these facts:

1. The Rieske FeS protein is a component of the cytochrome b complex
2. Rieske Fes protein is encoded by the nuclear gene RIP1
2. The rest of the cytochrome b complex proteins are encoded by genes in the mitochondrial genome
4. The Rieske protein is produced at a ratio of 1:1 in the nucleus as per the rest of the subunits for cytochrome b which are produced in mitochondria.

...

From this perspective one can agree with your view since most proteins are nucleus encoded and whatever regulatory system is operating it is sufficient to maintain the required proteins synthesis of the subunits encoded in mitochondria.

However, I say partially yielded, because an interesting point remains:

However, would you agree that permeability to transcription factors and other signaling molecules is different between the mitochondrial membrane and the nuclear membrane?

And if such permeability is different then it would imply that the regulation of the mitochondrial genome in terms of gene expression activity resulting from said transcription factors and other signaling molecules would also be altered if it were transferred to the nucleus.

Therefore if regulation is altered due to allotopic expression then it has to be accounted for (more work).

(emphasis original)

de Grey has responded to this concern, although his responses not seem to obviate the concern, merely point out that it might not be a show-stopper. Sample:

Hydrophobicity: the main problem, but now seems very likely to be solvable just by judicious amno-acid substitutions that marginally reduce hydrophobicity without affecting function. Candidates can be found by looking in other species. The post-import modification idea is also plausible, and indeed a version of it, using inteins, was the main novel component of my TibTech paper mentioned above. But your concern is invalid, because (a) some such post-import modifications, inteins among them, do not require a peptidase but rearrange purely autocatalytically, and (b) even if a peptidase is needed, it will be a soluble protein so not hydrophobic, so its import will be easy. Resequencing: clearly necessary but equally clearly trivial - can be done for the whole mt genome by a grad student in a week. Toxicity: has been raised in the past but currently no evidence for it. If it were to occur, though, solutions include a long leader sequence that acts as a cis-chaperone to prevent folding (this happens for one of the nuclear-coded ATPase subunits in fact).

(my emphasis added)

This particular exchange is somewhat beyond me, but it did remind me of a question that I have. Assuming a cell has hundreds or even thousands of mitochondria, we can assume that we can produce the right amount of each protein in each complex, based on the 1:1 ratios we already discussed. However, how do we ensure that those proteins go to the correct mitochondria?

Presumably, the mtDNA-coded proteins will be produced in the proper amounts (ignoring dysfynction for now). The nDNA-coded proteins must have some existing, complex machinery that directs them to where they are needed.

Now de Grey's solution for transcibing the relevant genes in the proper ratios depends on the regulatory machinery that operates in the nucleus, no? Correct me if I am misunderstanding where this initial transcription takes place.

So we are talking about one location. However, the machinery that transports the nuclear-coded proteins to the correct mitochondria is in the cytoplasm, or at least partially in the cytoplasm, right? Dr. de Grey, can this machinery be as easily coaxed into operating on the mtDNA-coded genes that we just moved to the nuclear genome? Is it as easy as inserting genes between coding region markers (am I overstepping my limited knowledge by assuming that the "coding region" is defined by markers?)?

Can we ensure that each mitochondrion will get the proper amounts of the proteins that we are moving out of the mtDNA? It does us no good to produce the proteins in the proper ratios if we don't have the machinery to get them where they are needed. This is in addition to addressing the issue of hydrophobicity that Prometheus has brought up. Again, more work.




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