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Next generation mitochondrial antioxidant


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#1

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Posted 08 August 2004 - 11:50 PM


J. Biol. Chem., Vol. 279, Issue 33, 34682-34690, August 13, 2004
Cell-permeable Peptide Antioxidants Targeted to Inner Mitochondrial Membrane inhibit Mitochondrial Swelling, Oxidative Cell Death, and Reperfusion Injury
 
Reactive oxygen species (ROS) play a key role in promoting mitochondrial cytochrome c release and induction of apoptosis. ROS induce dissociation of cytochrome c from cardiolipin on the inner mitochondrial membrane (IMM), and cytochrome c may then be released via mitochondrial permeability transition (MPT)-dependent or MPT-independent mechanisms. We have developed peptide antioxidants that target the IMM, and we used them to investigate the role of ROS and MPT in cell death caused by t-butylhydroperoxide (tBHP) and 3-nitropropionic acid (3NP). The structural motif of these peptides centers on alternating aromatic and basic amino acid residues, with dimethyltyrosine providing scavenging properties. These peptide antioxidants are cell-permeable and concentrate 1000-fold in the IMM. They potently reduced intracellular ROS and cell death caused by tBHP in neuronal N2A cells (EC50 in nM range). They also decreased mitochondrial ROS production, inhibited MPT and swelling, and prevented cytochrome c release induced by Ca2+ in isolated mitochondria. In addition, they inhibited 3NP-induced MPT in isolated mitochondria and prevented mitochondrial depolarization in cells treated with 3NP. ROS and MPT have been implicated in myocardial stunning associated with reperfusion in ischemic hearts, and these peptide antioxidants potently improved contractile force in an ex vivo heart model. It is noteworthy that peptide analogs without dimethyltyrosine did not inhibit mitochondrial ROS generation or swelling and failed to prevent myocardial stunning. These results clearly demonstrate that overproduction of ROS underlies the cellular toxicity of tBHP and 3NP, and ROS mediate cytochrome c release via MPT. These IMM-targeted antioxidants may be very beneficial in the treatment of aging and diseases associated with oxidative stress

From the discussion:
 
These SS peptides (SS-02 and SS-31) are the first antioxidants that selectively target and concentrate in the IMM, thereby enabling scavenging of ROS at the site of production. Using these peptide antioxidants, we were able to show that overproduction of ROS underlies the cellular toxicity of tBHP and 3NP. Our studies with isolated mitochondria also demonstrated that ROS mediate cytochrome c release via MPT and rupture of the OMM. By reducing ROS production, these peptide antioxidants were able to prevent mitochondrial depolarization in cells exposed to 3NP. Finally, these peptide antioxidants were able to prevent myocardial stunning associated with reperfusion in the ischemic heart in an ex vivo model. The inability of SS-20, which does not have antioxidant ability, to prevent Ca2+-mediated mitochondrial swelling or reperfusion injury confirms that the protective actions of these peptides are mediated via their antioxidant actions.
...
In summary, we have designed cell-permeable peptide antioxidants that target the site of ROS generation and protect mitochondrial function. Our results demonstrate that ROS play a major role in mediating mitochondrial dysfunction induced by tBHP, Ca2+, and 3NP. These antioxidant peptides may be beneficial in the treatment of aging and diseases associated with oxidative damage such as ischemia-reperfusion injury and neurodegeneration.


The peptides: tetrapeptides with alternating aromatic residues and basic amino acids. SS-02 (Dmt-D-Arg-Phe-Lys-NH2; Dmt = 2',6'-dimethyltyrosine), SS-20 (Phe-D-Arg-Phe-Lys-NH2), SS-31 (D-Arg-Dmt-Lys-Phe-NH2)

Delivery of peptide: Continuous cardiac perfusion with Krebs-Henseleit solution containing various SS peptides.


Key points to take away:
1. the antioxidant localizes in the IMM
2. it is a simple tetrapeptide (much easier to manufacture and create a delivery system for)

#2 jaydfox

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Posted 09 August 2004 - 12:03 AM

How would this play into discussions of genetic vs. pharmaceutical means of retarding the aging process? You have been a big proponent of focusing on genetic solutions to this problem.

While I agree that genetic manipulations will be a key mid-term solution--with nanomedicine providing the long-term solution--offering 30-150 years of extended lifespan, I still feel that pharmaceuticals will have an important part to play in the short-term in providing substantional extension of the healthy lifespan--on the order of 10-25 years. While most people can get this same benefit by eating a healthy diet and exercising, 10-25 years is still significant.

Jay Fox

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#3

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Posted 09 August 2004 - 03:11 AM

Pharmacological interventions are good short term solutions whilst we clarify our understanding of specific mechanisms and refine gene construct delivery methods. This is a significant short term intervention provided a viable delivery method can be found. If it could be delivered orally it's the sort of thing everyone over the age of 30 should be compelled to go on for the rest of their life and anyone over the age of 20 should be thinking about (just because we have a high degree of cellular redundancy does not mean that cellular damage does not begin at an early age).

A note on nanotechnology Jay - what is the difference between, say, engineering proteins and their delivery in cells and nanotechnology?

#4 Lazarus Long

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Posted 09 August 2004 - 05:59 PM

A note on nanotechnology Jay - what is the difference between, say, engineering proteins and their delivery in cells and nanotechnology?


You know Prometheus, for the longest time I have been arguing that for all practical purposes genetics should be seen as a specific *Organic* category of nanotech and perhaps it would be wiser to see the rest of nanotech as encompassing the *inorganic* models as they are both derived of the same basic molecular science that must be resolved through a form of unified theory of *language* to accompany the principles of physics.

I basically see genetics as practical (natural) nanotech and I suspect as we better understand *why* genetics works we will gain a fuller understanding of the primary language of *intelligent* matter that will *translate* over into more complex *inorganic* applications.

#5 jaydfox

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Posted 12 August 2004 - 03:00 AM

A note on nanotechnology Jay - what is the difference between, say, engineering proteins and their delivery in cells and nanotechnology?

I fully concede that life, genetics, proteomics, etc., are all forms of nanotechnology. However, as Lazarus points out, they are a subset of what is possible.

Materials stronger and more resistant to damage will one day be created at a size scale to match that of what genetics alone is capable of (ignoring the case where we engineer an organelle's genome from scratch, rather than tweaking what's already there, so that it can "grow" these stronger alloys, or indeed CNTs or diamonds).

In addition to strength and resistance to many forms of molecular damage, nanotechnology will allow incorporated nanoprocessors: simple ones, to be sure, but there's a lot you can do with a few tens of thousands of molecular-scale transistors.

Again, though, this is a long-term project. Genetics will probably get us to the point of actuarial escape velocity in the next 15-30 years. However, achieving AEV isn't enough: if the progress is not sustained, we'll fall behind the curve again. At some point, inorganic nanotech, to borrow the phrase from Lazarus, will get us the rest of the way to seeing a large fraction of a millenium.

Jay Fox

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Posted 12 August 2004 - 04:47 AM

So your definitions are:

organic nanotech = molecular biology
inorganic nanotech = molecular engineering

And, inorganic nanotech will take over from organic nanotech eventually - yes?

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#7 jaydfox

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Posted 12 August 2004 - 09:42 PM

So your definitions are:

organic nanotech = molecular biology
inorganic nanotech = molecular engineering

And, inorganic nanotech will take over from organic nanotech eventually - yes?

Not in all aspects, but it will certainly complement it. Inorganic is not always better than organic. After all, wood was a much more flexible building material than stone and brick. Not just because it was less dense and therefore easier to hoist into place. But it allowed a new paradigm in building: supporting weight and stress with tension, rather than with compression alone (what stone offers). Man, in this case, expanded his capabilities by embracing something natural. Eventually, however, steel, aluminum, carbon fiber, and now non-crystalline alloys are pushing the limits of what can be built. CNT-based fibers will push those limits further still.

It's a poor analogy, but I foresee something similar happening with nanotech. Genetics, protein delivery systems, and a dozen other forms of biological methods (organic nanotech) will vastly improve upon what we have at our disposal now: drugs, surgeries, supplements, etc. (and ironically enough, drugs and supplements are often "organicly" based).

However, eventually our engineered nanotech will far exceed what could be accomplished though "organic" nanotech. On some fundamental level, it's all the same technology. But when you're doing it with precisely shaped nanoscopic diamonds, CNTs, nanoprocessors, and electric circuits (powered in vivo with an analog of glucose metabolism, ironically) powering motors with a million-fold higher power output potential (limited really only by heat dissipation rate), it's not really the same anymore. It'll probably be blended with organics, to blend in to the living environment better, so it's not like it will be only "inorganic" nanotech.

But it's more than just genetics. It's this nebulous "nanotech" that so many have discussed. Some have discussed it in great detail, using what we know today. Some of the machines are already designed, at least at the structural if not the molecular and atomic level. Just because we can't build them yet, that doesn't mean we can't study them and prove (in simulation) their *potential* efficacy.

Sure, their designs might not work, or at least they might not be as effective as hoped. But they're designing these devices using the knowledge we have today. By the time we can assemble structures atom by atom, be it 10 or 100 years from now, we'll have the knowledge to make those devices work.

So yes, I eventually see inorganic nanotech "taking over", but only in the sense that whatever jobs cannot be accomplished using organic tech will be supplemented with inorganic. For creating a building, the difference is moot: just use what's strongest, lightest, and most resistant to fatigue. For enhancing our fundamental body chemistry, inorganic is only really suited to cleaning up the messes that we can't solve genetically, etc. Hence, I assume that genetics will extend the human lifespan to at least 150 years, and more likely to 200. I even see 300 as a small possibility, and 500 as a very remote possibility. But there will always be damage that takes more than just a switch, even an analog switch, to fix. There will be problems that require intelligent intervention. Some of those problems will be at the macroscopic level, amenable to surgery, stem cell therapy, etc. Some of those problems will be at the intracellular level, and will require inorganic nanotech. Short of "uploading" ourselves, which doesn't sit well with me at the moment, the only way I foresee living for millenia would be nanotech-based techniques.

Luckily, if genetics does hold the key of extending the human lifespan to 200 years, and if stem cell therapies hold the key of reversing most age-related damage in the elderly to give them that same chance with late intervention, then nanotech won't be needed for the majority of us alive today until well after genetic manipulations are in place. That buys us a few more decades. By which point, this question will hopefully have been answered.

Jay Fox

#8 olaf.larsson

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Posted 13 August 2004 - 11:44 AM

I have not seen any test that this compounds acctually make any creature to live longer It would be nice to see some evidence for this.

#9 jaydfox

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Posted 13 August 2004 - 04:53 PM

I have not seen any test that this compounds acctually make any creature to live longer It would be nice to see some evidence for this.

True.

Though if A is known to lead to B, and B is known to lead to C, and C is believed to lead to D, then A should lead to D, in theory.

This peptide (A) acts as a highly effective and targetted mitochondrial antioxidant (B); mitochondrial oxidation byproducts (ROS is the common term) are known to cause protein, RNA, DNA, and other molecular damage, most especially within the mitochondria themselves, but also to a lesser degree throughout the cell ©; and molecular damage at the very least is highly correlative with senescense, if not causal (D), so... In theory, this compound should have a non-negligible positive impact on lifespan. Whether that impact results in a 15% increase in lifespan, or a 100% increase, remains to be seen. But there is very little chance this compound will have negligible or negative impact on lifespan.

But admittedly, there is a chance this compound won't extend lifespan. I suspect that if such were found to be the case, it wouldn't be because A didn't lead to D. It would be because at some step (probably B), the compound affected another mechanism in addition to senescence, which on its own negatively impacts lifespan, and together the two or more effects cancelled each other to some degree.

Another possible reason that A might not lead to D, is if C doesn't lead to D. If so, a compound like this could help cast doubt on that causal link. I doubt it will happen, but there are scientists out there who don't buy into mitochondrial ROS-production as a cause of senescence, let alone the chief cause.

Jay Fox

#10

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Posted 14 August 2004 - 12:34 AM

Wolfram, as Jay reasoned, anything that keeps mitochondria from being damaged by reducing the oxidative damage would putatively keep them functional for a longer period, allowing the cell to survive longer, etc. Note the localization of the compound - the inner mitochondrial membrane and note the apoptotic trigger pathway associated with the membrane.

In a previous post I talked about oxidative damage to the TOM complex that would impact on the transport of proteins targeted to the mitochondrion from the ER. This experiment suggests that the compound would ameliorate this type damage and the physiological consequences, even though it has not been shown.

However, the localization is a double edged sword as the anti-oxidant function does not enter the mitochondrion and soak up excess oxidants where they can do other types of damage such as in mtDNA.

This presents an interesting scenario:
1. apoptotic pathway activation rate - down
2. TOM damage rate - down
3. mtDNA damage rate - no change

What do you think would be the consequences of such conditions?

#11 jaydfox

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Posted 14 August 2004 - 01:20 AM

This presents an interesting scenario:
1. apoptotic pathway activation rate - down
2. TOM damage rate - down
3. mtDNA damage rate - no change

What do you think would be the consequences of such conditions?

Worst case scenario? The cell, seeing a lower damage profile from the outside, might not upregulate DNA repair factors sufficiently within the mitochondria, leading to an increased mtDNA damage rate.

But I'm not an expert, so I don't know to what degree the cell can affect gene expression and protein levels within the mitochondria, especially those related to DNA repair factors.

Jay Fox

#12 jaydfox

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Posted 14 August 2004 - 05:29 AM

These SS peptides (SS-02 and SS-31) are the first antioxidants that selectively target and concentrate in the IMM, thereby enabling scavenging of ROS at the site of production.
...
These peptide antioxidants are cell-permeable and concentrate 1000-fold in the IMM.
...
In summary, we have designed cell-permeable peptide antioxidants that target the site of ROS generation and protect mitochondrial function. Our results demonstrate that ROS play a major role in mediating mitochondrial dysfunction induced by tBHP, Ca2+, and 3NP. These antioxidant peptides may be beneficial in the treatment of aging and diseases associated with oxidative damage such as ischemia-reperfusion injury and neurodegeneration.

If this new peptide does indeed increase lifespan, whether by 10% or 30% or 50%, how would this stack with CR?

Results of stacking CR-mimetics with CR have shown negligible, and sometimes negative, effects in regards to age-retardation. Stacking dwarfism with CR has shown smaller absolute benefits, and thus significantly smaller relative benefits, as compared to CR alone.

The problems in these cases seem to be linked to gene expression profiles. The profiles induced, while not identical, are closely related.

However, this new peptide doesn't get it's job done by upregulating DNA-repair factors, etc. While it might affect gene expression is some small way, that effect most likely will not correlate at all with the profile of CR.

So, I'm hypothesizing here that the two could stack additively. E.g., a 20% lifespan increase due to this peptide, stacked with a level of CR that by itself would produce a 30% increase in lifespan, would result in a 50% increase. The best case scenario is that they could stack multiplicatively: 1.2 times longer lifespan, and 1.3 times longer still, results in 1.2*1.3=1.56 times longer lifespan. In reality, it will probably be less than either, but we could hope for 45%, right?

And what if this peptide is found to extend remaining lifespan 40%. Combine that with a 42% increase found with CR in 19-month-old mice, and you might have a formula for making a serious attempt the Reversal Prize. 80% isn't enough to double remaining lifespan, but it makes the odds of getting a statistical fluke to beat the current record (set by another statistical fluke) even better than with any other technique known yet.

Jay Fox

#13 olaf.larsson

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Posted 14 August 2004 - 09:40 PM

If A, B, C, D, and E are lifespan limiting factors. Interfering with only E doesn´t cause any dramaticaly increased lifespan. In my local popular science magazine there were claims that antioxidants could increase lifespan by 35 years.

Development of this compounds are not very advanced or complicated compaired with the some of the other speculative treatments proposed here. Introduction of the first real antiaging medicine is within reach and will cause an explosion in antiaging reasearch and an enormous wealth for the inventor(s).

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#14 Lazarus Long

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Posted 16 August 2004 - 12:51 AM

I just came across an interesting tid bit of news to support the basic outline on the developing taxonomy for Nanotech. It also happens to overlap this topic.

http://story.news.ya...notechnology_dc

Genetic Material May Help Make Nano-Devices: Study
Thu Aug 12,10:18 AM ET  Science - Reuters

WASHINGTON (Reuters) - The genetic building blocks that form the basis for life may also be used to build the tiny machines of nanotechnology, U.S. researchers said on Thursday.

A team at Purdue University said they had used ribonucleic acid, or RNA, to build microscopic structures such as spirals, triangles, rods and hairpins, that could serve as components of nanotechnology devices.
(excerpt)


I intend to post the full text and some followup in the Nanotech forum




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