AI tools are designing entirely new proteins that could transform medicine (proteins in aging)

Alex K Chen · July 13, 2023

https://www.nature.com/articles/d41586-023-02227-y

I remember the "Future of Aging" book that Greg Fahy once edited. This could be the way to find new enzymes to break down lipofuscin and crosslinks... [i know one suggestion was to use microbial hydrolases...]

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The proposed SENS strategy (de Grey et al. 2005; Methuselah Foundation 2007; de Grey 2006c) is to give all cells extra enzymes (such as microbial hydrolases found in natural soil bacteria and fungi) that can degrade the relevant biomaterial, or other accessory microbial proteins such as transporters to restore lysosomal acid- ity. The lack of such exogenous enzymes can be regarded as a genetic deficiency that results in pathological intracellular storage disease (Section 23.6.4.5), so the SENS treatment would be analogous to replacing a natural lysosomal enzyme for which patients are congenitally deficient as in enzyme replacement therapies (ERT). The ERT treatment can be directed to all cells as a complete whole-body gene therapy, or it can be directed only to modified stem cells via a bone marrow trans- plant that produces enhanced macrophages (Section 23.7.1.1), a stopgap approach that still allows the intracellular storage disease to progress to full senescence in somatic cells which are then removed and successfully digested by the enhanced macrophages. Possible difficulties with both approaches include: (1) inactivity or toxicity of microbial genes introduced into mammalian cells, (2) rapid degradation of the new microbial enzymes by lysosomal proteases whose normal function is to destroy other proteins, (3) immune rejection of microbial enzymes or proteins when cells expressing or containing them are attacked by lymphocytes, and (4) the inabil- ity of therapeutic enzymes in ERT to cross the blood-brain barrier in patients with cerebral neuropathies; though it is believed that further research can overcome all these problems (de Grey et al. 2005).

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Novel lysosomal hydrolases (“LysoSENS”)

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SENS research proposals include: (1) finding new or synthetic deglycating enzymes that can couple the link- breakage to the hydrolysis of ATP to ADP (the most common power source inside cells), requiring the enzyme to shuttle back and forth across the cell membrane to acquire fresh ATP for each link-breakage cycle as there is very little ATP in the extracellular matrix; (2) engineering single-use link-breaking molecules analogous in action to the DNA repair protein MGMT which reacts with a stable molecule (DNA) but thereby inactivates itself (by transferring methyl and alkyl lesions from the O6 position of guanine on damaged DNA to a cysteine in its own structure (Pieper 1997)); or (3) increasing the rate of natural ECM turnover, taking care to avoid “dire side-effects such as hemorrhage from leaky blood vessels as collagen molecules are removed and replaced” (Furber 2006).

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In the SENS strategy (de Grey 2003, 2006b), it is theoretically possible to iden- tify chemicals that can selectively dissociate crosslink bonds without breaking any other bonds, because many crosslink bonds have unusual chemical structures not found in proteins or other natural biomolecules. Some of these crosslink bonds may be unstable enough to be readily breakable by drugs, such as alagebrium chloride (aka. PMTC, ALT-711) which appeared to break one subset of glucose crosslinks

768 R.A. Freitas Jr.

(sugar-derived alpha-diketone bridges) in clinical trials (Bakris et al. 2004), but other crosslink bonds (e.g., acid-labile glucosepane (Lederer and Bühler 1999) and K2P (Cheng et al. 2004), and the highly stable pentosidine (Sell et al. 1991)) are probably too stable to be breakable by simple catalysis

https://www.nature.com/articles/s41598-018-19991-x

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Amadoriases, also known as fructosyl amine oxidases (FAOX), are enzymes that catalyze the de-glycosylation of fructosyl amino acids. As such, they are excellent candidates for the development of enzyme-based diagnostic and therapeutic tools against age- and diabetes-induced protein glycation

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Two types of enzymes, fructosyl lysine oxidase and fructose lysine 3-phosphokinase, catalyze the deglycation reaction and generate free amine groups

Edited July 18, 2023 by InquilineKea

Alex K Chen · July 24, 2023

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Dr. Aubrey de Grey, an anti-aging pioneer, first introduced me to the concept of lipofuscin through his 2005 TED talk and book that he co-authored with Michael Rae, “Ending Aging: The Rejuvenation Breakthroughs That Could Reverse Human Aging in Our Lifetime [lxi].” He proposed a way of eliminating it from the body involving “xeno- catabolism”. It is based on his realization that lipofuscin, which is typically fluorescent, must be eliminated from human corpses by microbial enzymes, or else graveyards would glow in the dark. He postulated that we can co-opt enzymes from soil bacteria and fungi and install them in our cells to degrade our lipofuscin [lxii]. However, I do not believe this will be feasible in our lifetimes at least, as lipofuscin is quite heterogeneous - it varies widely in composition between cell types and even cells of the same type, potentially [lxiii]. It also varies in composition between different individuals, probably in part due to differ- ences in diet.

Thus, it would likely require an inordinate number of microbial enzymes to degrade the majority of our lipofuscin, almost all of which would still have to be discovered, evolved, or rationally-designed - and many of them may be toxic to our cells. There are

Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 20 March 2023 doi:10.20944/preprints202208.0229.v6

A.

exocytosis - even in youthful cells [ , ]. Perhaps over time, TFEB could work well enough to exocytose all a post-mitotic cell’s lipofuscin, but other cellular mechanisms may need to be combined with TFEB for truly effective exocytosis - and there may be cell type differences in what machinery is required. A small molecule was shown to induce some lipofuscin exocytosis in monkey retinal pigment epithelial cells, which are important in macular degeneration [xxiv]. A final issue is that with lysosomal exocytosis, lipofuscin granules may break apart and/or get caught in various parts of the extracellular matrix - causing a problem by being there. The lipofuscin could also at least eventually be taken up by the same cells that ejected it, neighboring cells that need to be cleared of garbage themselves, or tissue-resident macrophages. It’s probably better for the lipofuscin to be inside an intact vesicle that can prevent re-uptake - only to be picked up by bioengineered macrophages.

Lipofuscin could also theoretically be exported from post-mitotic cells through mi- crovesicular secretion of lysosomes or by transferring lysosomes through tunneling nano- tubes (TNTs) to bioengineered macrophages delivered to their locale. (However, the di- ameter of some lipofuscin-laden lysosomes may be too great for them to be transferred through TNTs.) Secretory autophagy may also be relevant here [lxix, lxx]. Secretory au- tophagy utilizes autophagosomes, which are double-membrane vesicles. Perhaps a pathway involving the exocytosis of lysosomes engulfed in single-membrane vesicles could also be possible [lxxi].

some molecular species that seem to be major contributors to lipofuscin or lipofuscin’s toxic effects at least in some cell types, like the fluorophore A2E (retina) [lxiv] and the oxysterol 7-ketocholesterol (throughout the body - especially atherosclerotic plaques and the retina) [lxv], but we cannot neglect other organs or tissues, or they will fail and we will die anyway. It is worth looking into to see if there are major lipofuscin constituents present across a wide variety of cell types that would be very good targets. But it is un- likely, at least in my mind, that a small number of enzymes will be sufficient to degrade the majority of our lipofuscin deposits.

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