Solving Aging Series: Allotopic Gene Expression
Could this be the fountain of youth we’ve been looking for?
Had you been born 12,000 years ago, dying because of some hungry predator would be normal to you. Flash forward to only 150 years ago, death by tuberculosis would be your usual.
You might think you would never actually think of those horrible events as normal, no matter when you lived. But now, consider this. You were born in a time where aging is seen as normal. Like predation and pandemics, should the inevitability of aging not be questioned?
Aging is, after all, a process far from harmless. It is literally defined as “the life-long accumulation of ‘damage’ to the body that occurs as intrinsic side-effects of the body’s normal operations”, and the scientific community has many theories as to what causes it. In this article, we’ll concentrate on one of its main drivers: mitochondrial damage. The mitochondria is an organelle mostly known for its role in the production of ATP, the cell’s main source of energy. However, it is also crucial to many other vital cell functions, as we’ll see later on.
“Aging is the life-long accumulation of ‘damage’ to the body that occurs as intrinsic side-effects of the body’s normal operations” -Aubrey de Grey, biomedical gerontologist and Chief Science Officer of SENS Reseach Foundation
But first — How do mitochondria get damaged?
It has long been theorized that the main source of mitochondrial damage was free radicals. Considering the mechanics for energy production, it’s easy to see why. The mitochondria acts as a power plant and, like all power plants, it generates waste. In the case of the cells, the waste is made up of free radicals.
Free radicals are chemical compounds that lack an electron. To achieve greater stability, they try to “steal” an electron from another atom or molecule. This behavior led to the elaboration of the mitochondrial free radical damage theory. According to said theory, the free radicals by-produced by the mitochondria would be more susceptible to steal an electron coming from one of the building blocks of mitochondrial DNA (mtDNA) than from any other part of the cell, because they are generated so close to it. This would alter the mtDNA so that proteins necessary for ATP production would not be synthesized. However, the mitochondrial free radical damage theory is heavily debated for the following reasons.
- No difference in lifespan after modifying antioxidant production
The body produces its own antioxidants, and they do play a role in mitigating free radical damage. However, can a link be established between fewer oxidative damage and a slower rate of aging? Not according to many studies.
- A study conducted published by The Journals of Gerontology in 2009 has found that increasing the expression of different combinations of antioxidant enzymes did not increase lifespan in mice even though it did reduce oxidative damage.
- In another study from the same journal, published in 2007, mice with impaired production of GPx4, another antioxidant, actually lived longer.
- A study published by the Physiol Genomics in 2003 showed transgenic mice with impaired production of both SOD2 and GPx1, some of the aforementioned antioxidants, got higher oxidative damage but no signs of accelerated aging compared to their normal counterparts.
- An additional study on fruit flies, published by PNAS in 1990, has been disappointing as well. Researchers have prevented specimens from producing up to two antioxidants, Sod2 and Sod3, and all flies lived equally as long.
2. Oxidative stress could be a consequence rather than a cause
Reactive oxygen species (ROS) are free radicals that circulate in your body. Although free radicals may not be the cause for aging they were once deemed, this is not good news either. Indeed, ROS have been observed to act as crucial signaling molecules, indicating to the body that damage has occurred. Therefore, they help the cells adapt to stress by playing a role in triggering autophagy, immune cell function, and cell differentiation.
In light of that, it seems we might need another way of explaining mitochondrial damage. Such a way exists and consists of replication errors. It all starts during embryogenesis. You probably know that at this stage, the cells divide furiously. In order to do that, they have to replicate their DNA, but also their mtDNA. Since the mtDNA polymerase, the enzyme responsible for transcription of the genetic information of this organelle isn’t perfect, replication errors occur. Plus, there is no mechanism in place to ensure that each mtDNA molecule in a cell is replicated only once. That means that a cell with only one mutated mitochondria can give rise to daughter cells that mainly have mutated mitochondria. Furthermore, there seems to be a bias favoring the multiplication of these mutated organelles.
But no matter the cause of mitochondrial damage, the way of preventing it could be the same. Surprised? And yet it’s true: since treating mtDNA damage does not involve preventing the damage from happening, but rather enabling the mitochondria to deal with said damage, we should still be able to solve this problem even if the cause is not clearly known.
What happens when a mitochondria gets damaged?
If a mitochondrial mutation happens on a sequence that codes for a protein, the mitochondria will no longer be able to produce enough ATP to keep the cell functioning properly. The cell will then get into an abnormal state where it sends all remaining normal mitochondria to the recycling bin (the lysosome) to continue on a self-destructive path.
If you’re somewhat familiar with the main causes of aging, you may recognize the consequences of mitochondrial damage, namely cell loss and senescent cells. Indeed, a cell reaching the state described above is obviously no longer functional, and if it doesn’t die, which it may very well not, it will cause harm to surrounding cells by sending chemical signals that will trigger them into becoming senescent as well. This is likely because mitochondria plays a role in apoptosis, the process of controlled cell destruction.
Cell loss and senescent cells sometimes are themselves deemed as main pathways to aging, and treatments are being developed right now to tackle them separately. But since mitochondrial damage plays an important role in those deleterious processes, the question arises: would the treatments to those ailments still be necessary if mitochondrial damage weren’t a problem anymore?
Is it even possible for mtDNA mutation to be solved?
To allow the mitochondria to keep functioning even when damage arises, we could make use of a technique called allotopic gene expression. This method consists of providing the DNA with the genes that code for the proteins that the mitochondria needs to function. If, or rather when, the mtDNA gets damaged, it would still be able to access those proteins, not by creating them from its code, but because the nucleus would provide them for it.
The first step to achieving allotopic gene expression for all 13 proteins exclusive to the mitochondria is to synthesize those genes and adapt them so that the ribosomes can translate them properly.
The mitochondria has its own ribosomes, and that is for a good reason. Imagine the ribosomes as translators, either for the DNA language, or the mtDNA language. Both the languages use the same letters (A, C, G, T), and can thus form the same “words”. However, those identical “words” will not mean the same thing depending on the language they came from.
Imagine you provided a translator with the word “car” coming from english, but that translator only translates from french to, let’s say, Russian. It would then interpret that word as meaning “because” (“car” in French), and the output in in Russian would be incorrect. It’s the same principle with DNA and mtDNA, DNA being English, mtDNA being French and Russian being the amino acids.
Some identical codons mean one amino acid for the cytosolic ribosomes and another for the mitochondrial ribosomes. For example, met… is encoded by the codon AUA for the latter, while AUA means ile for the former. Scientists have to account for that fact when introducing the genes for mitochondrial proteins into the DNA, and modify them accordingly.
There is more to consider when synthesizing the genes for the proteins exclusive to the mitochondria. How will the preproteins reach their destination? The cell won’t just magically know where to send those preproteins, so we have to specify it. Some preproteins have on their NH2 terminal what is called a mitochondrial targeting sequence (MTS), a peptide chain ranging from 10 to 70 nucleotides in length that lead the transfer of a protein to the mitochondria. Thus, scientists would have to add a MTS to the synthethized code of the mitochondrial proteins.
What’s the next step for the preproteins?
They’d have to enter the mitochondria. There are four known pathways that preproteins go through, depending on where they have to end up. We’ll only look at two of them, because the 13 proteins encoded by the mtDNA are used for oxydative phosphorylation, the process of creating energy for the cell. This process takes place in the inner membrane. Hence, we’ll concentrate on pathways leading to that part of the mitochondria. The first one is called the presequence pathway.
When the preprotein arrives to the mitochondria’s outer membrane, it is first handled by “chaperone” proteins that ensure the preprotein is in a state where it can be imported into the mitochondria, which means being somewhat unfolded. The protein then passes through the TOM complex, short for translocase of the outer membrane. Translocase simply means the process of carrying a protein from one location to another. Three proteins in the TOM complex receive the preprotein: Tom20, Tom22 and Tom70. Those receptors recognize the protein and direct it towards the translocation pore formed by Tom40.
At this stage, the protein still bears a MTS that’ll be useful later on. Through this pore, the protein is passed over to the TIM complex, meaning translocase of the inner membrane. There, 3 proteins play a major role. First, there’s Tim23, that forms a pore the protein passes through. Second, there’s Tim 17, that regulates the pore. Last, there’s Tim44, which ensures the proteins are passed over to the mitochondrial matrix. Not all proteins have to go that way, though, and certainly not the ones exclusive to the mitochondria. If you remember, they have to get to the inner membrane. For these proteins, the translocation to the matrix is stopped by a hydrophobic sorting signal they carry in their cleavable MTS and they are routed to the inner membrane.
A second way preproteins can get into the inner membrane is the carrier pathway. When the preprotein arrives to the outer membrane, proteins such as Hsp70 and Hsp90 bind to them to prevent them from forming a cluster, which would be impossible to translocate. The assembly of the preprotein and and Hsp70 and Hsp90 is recognized by the TOM complex.
The proteins then get to the intermembrane, where the TOM and TIM complex work together to get the preproteins to the inner membrane. This is done through binding with the Tim9-Tim10 complex. When the preproteins reach the inner membrane, they are assembled into their functional form (which they were prevented from shaping into by Hsp70 and Hsp90) and can go accomplish their function.
So, is solving aging with allotopic gene expression possible?
We simply don’t know yet. The translocase of proteins into the mitochondria is a complex process we’ll need to understand better if we want to achieve successful allotopic expression of all 13 mitochondrial proteins. Many questions can be raised solely by looking at the simplified descriptions of the translocase processes above, such as:
- Do the Tom receptors recognize the inner membrane preproteins only because of their cleavable sequence, when they have one? If not, how to ensure our allotopically expressed preproteins get recognized as well?
- Will the Hsp70 and Hsp90 be able to bind to the allotopically proteins they have never been designed to deal with? If yes, will the TOM comlex still recognize this unusual combination?
- Will the mitochondria be able to assemble these inner proteins into their functional shape, considering it is unique?
In Conclusion…
While allotopic gene expression is a fascinating idea, it is to this day hard to determine whether it’s going to work and have a significant impact on the human body. Still, this is no reason to lose hope for rejuvenation. Remember how we asked ourselves if the treatments to cell loss and senescent cells would be necessary to heal us from aging? If the answer is yes, science has some good news for us. We are getting closer and closer to producing those treatments, and technologies such as tissue engineering and senolytics are just around the corner and very promising. It’s worth exploring if one day, soon maybe, it is those innovations who will free us from our 21st century killer, that predator we call aging, and allow us to live longer, healthier, and happier lives.
Sources
https://www.ncbi.nlm.nih.gov/books/NBK21471/
https://www.sciencedirect.com/science/article/pii/S2352304219300637
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3017613/
https://www.tempobioscience.com/blog/?p=617
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3675642/
http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/cytochromes.html
https://www.nature.com/scitable/topicpage/mtdna-and-mitochondrial-diseases-903/
https://www.sciencedirect.com/science/article/pii/S0167488911000176#bbb0250
