A team led by Bettina Moser and Toru Nakamura, working with colleagues including Madeline Points, Sourav Agrawal, Adam Didier, Amanda Mennie, Ci Ji Lim and Yong-jie Xu, has assembled genetic and structural evidence for a three-part molecular machine in fission yeast: a complex containing the single-stranded DNA-binding protein RPA, the telomerase reverse transcriptase Trt1 (the yeast equivalent of human TERT), and Tpz1, the yeast counterpart of the mammalian telomere protein TPP1. Their findings, published under the title describing the RPA–TERT–Tpz1 complex as a promoter of telomere extension and a suppressor of telomere recombination, suggest that RPA is not merely a bystander at chromosome ends but an essential structural component of the active telomerase holoenzyme.

The significance of this work lies in how it reframes the telomerase activation problem. For years, researchers focused on the recruitment step: telomerase is delivered to telomeres through interactions between TERT and TPP1 in mammals, or between the telomerase subunit Est3 and the TPP1-like protein in budding yeast. But recruitment alone does not guarantee that telomerase will add DNA repeats. Recent studies in humans and budding yeast had hinted that Replication Protein A, better known for its role in DNA replication and repair, also stimulates telomerase by physically contacting TERT in human cells and the TPP1 ortholog in budding yeast. The new study takes that hint and turns it into a comprehensive molecular model.

The researchers began with a genetic screen in fission yeast, a single-celled organism that has long served as a powerful model for telomere biology because its telomere maintenance machinery closely mirrors that of humans. Mutations that disrupted telomere maintenance surfaced in the screen, and rather than stopping at the list of affected genes, the team turned to AlphaFold3, the artificial intelligence system that predicts protein structures and their interactions, to model how RPA, Trt1 and Tpz1 might fit together. The computational models then guided systematic mutagenesis: the researchers altered specific amino acids in each of the three proteins and asked how the changes affected telomere length and chromosome stability in living yeast cells.

The combined approach revealed four distinct protein-protein interfaces that together support telomerase function. Two of them connect RPA to Trt1: one involving the Ssb1 subunit of RPA (the equivalent of the human RPA1 subunit) and one involving the Ssb2 subunit (equivalent to human RPA2). A third interface links the Ssb2 subunit of RPA directly to Tpz1. The fourth is the previously characterized interaction between Trt1 and Tpz1, mediated by the so-called TEL patch—a cluster of acidic amino acids on TPP1-like proteins that is known to dock TERT. In other words, the active telomerase complex appears to be held together by a web of contacts in which RPA physically bridges the telomere-binding protein Tpz1 and the enzyme Trt1.

One of the most striking results concerns a specific amino acid in Tpz1, arginine 81. Earlier work had assigned this residue to the TEL patch, implying that it contacted Trt1 directly. The new structural modeling and mutagenesis data tell a different story: Tpz1-R81 instead reaches across to contact Ssb2, the RPA subunit. This reassignment matters because it changes the wiring diagram of the telomerase activation machine. Rather than a simple two-component handshake between the telomere anchor and the enzyme, the picture is now a ternary complex in which RPA occupies a central coordinating position, touching both partners simultaneously.

Genetic evidence backs up the structural model. Using epistasis analysis—a technique that tests whether mutations in different genes affect the same biological pathway—and suppressor analysis, in which a second mutation can be engineered to compensate for the first, the researchers showed that the newly identified RPA-Trt1 and RPA-Tpz1 interfaces collaborate with the Trt1-Tpz1 interface to enable telomerase activation after the enzyme has already been recruited to the telomere. Disrupting any one of these contacts compromises telomere extension, and the pattern of genetic interactions indicates that the interfaces function cooperatively rather than redundantly. The complex, in short, behaves like a multi-point scaffold that must be assembled correctly before telomerase can do productive work.

The study also uncovered a second, equally important function: keeping telomeres honest. When telomerase cannot extend chromosome ends, cells sometimes resort to recombination-based pathways that copy telomeric DNA from one chromosome to another. These recombination-based survival mechanisms are genomically destabilizing and, in human cells, are associated with the alternative lengthening of telomeres seen in many cancers. The fission yeast experiments showed that the RPA-Trt1-Tpz1 complex suppresses such recombination while promoting legitimate telomerase-mediated extension. The same molecular machine, therefore, performs a dual quality-control role: it turns on the proper lengthening enzyme and shuts down the error-prone backup pathway.

Perhaps the most consequential finding is evolutionary. Comparative AlphaFold3 analyses suggest that the interaction network identified in fission yeast is likely conserved in budding yeast and in humans. If the RPA-TERT-TPP1 ternary architecture holds across such distant branches of life, then RPA’s role as a core activator of telomerase is probably not a yeast quirk but a fundamental feature of eukaryotic chromosome-end maintenance. That has immediate implications for human biology: telomerase activation is a hallmark of roughly ninety percent of cancers, and drugs targeting the enzyme’s recruitment interface are under active investigation. If RPA contacts are required for productive telomerase engagement in human cells, those contacts represent an additional layer of the enzyme’s control system—and potentially an additional drug target.

The work also resolves a conceptual tension in the field. RPA binds single-stranded DNA and is abundant at telomeres, where the G-rich overhang provides an obvious binding substrate. Earlier models treated RPA’s presence at chromosome ends as incidental, or as a competitor that must be displaced for telomerase to act. The new data support the opposite view: RPA is an active participant, physically coordinating the telomere anchor and the enzyme so that recruitment is converted into extension. In this view, the telomerase holoenzyme at a chromosome end is a larger and more intricately assembled machine than the classic textbook depiction of TERT, its RNA template and a telomere anchor alone. As with many recent discoveries powered by AI-guided structural prediction combined with classical genetics, the lesson is that molecular machines often hide their most important players until someone looks at the whole assembly at once. With RPA now firmly installed at the heart of the active telomerase complex, the next challenge is to determine exactly how these interfaces are regulated through the cell cycle, and whether human cancers depend on the same contacts to keep their chromosomes endlessly young.

Subject of Research: Structure and function of the RPA–TERT–Tpz1 telomerase activation complex in fission yeast telomere maintenance

Article Title: Fission yeast RPA–TERT–Tpz1 TPP1 complex promotes telomere extension and suppresses telomere recombination

Article References: Fission yeast RPA–TERT–Tpz1 TPP1 complex promotes telomere extension and suppresses telomere recombination. (n.d.). https://doi.org/10.1371/journal.pgen.1012297

Image Credits: AI Generated

DOI: 10.1371/journal.pgen.1012297

Keywords: telomerase, telomeres, RPA, TERT, Tpz1, TPP1, fission yeast, telomere extension, telomere recombination, AlphaFold3, chromosome biology, PLOS Genetics

AMPK sits at the very heart of cellular energy bookkeeping. When energy levels run low, the enzyme springs into action, switching on energy-producing processes and shutting down energy-consuming ones such as protein synthesis and cell growth. It is a heterotrimer, built from a catalytic alpha subunit and two regulatory partners, beta and gamma, and its sequence is strikingly conserved from fungi to humans. Maximal activity requires phosphorylation of a conserved threonine in the alpha subunit, a molecular switch that has been preserved across hundreds of millions of years of evolution. Decades of work in fruit flies, nematode worms and fission yeast have shown that genetic manipulations or dietary interventions that boost AMPK activity reliably produce longer-lived animals, making the enzyme one of the most compelling therapeutic targets in the biology of ageing.

The problem, until now, has been specificity. Metformin, the most famous AMPK-linked drug, activates the enzyme only indirectly, by perturbing the cellular AMP-to-ATP ratio and hitting mitochondrial and lysosomal targets along the way. Its effects in vivo are so tangled that studies in worms and flies have shown the drug’s lifespan-extending power actually flows through the gut microbiota rather than the host itself, and even in mammals there is evidence that bacteria mediate part of the response. That complexity has left the field with an uncomfortable question: is AMPK itself the pro-longevity target, or just one bystander among many? To answer it, the research team turned to 991, a synthetic molecule that binds in the so-called ADaM site, a pocket wedged between the alpha and beta subunits of AMPK, and locks the enzyme into its active state.

The first challenge was evolutionary. 991 had only ever been tested in mammalian systems, so the researchers built homology models of the fly, worm and yeast AMPK complexes against the known human crystal structure. The modelling revealed a high degree of overlap in the drug-binding pocket, including a key serine residue in the beta subunit that coordinates 991 binding, and conserved hydrophobic residues that stabilise the interaction between the beta subunit and the kinase domain. In the laboratory, the predictions held up beautifully. 991 allosterically activated AMPK purified from fruit fly cells in a dose-dependent manner, protected the enzyme from dephosphorylation by phosphatases, and boosted AMPK activity in extracts from wild-type worms but not from mutants lacking the AMPK alpha gene aak-2. Micromolar concentrations of 991 achieved what required millimolar doses of metformin or phenformin, a thousand-fold potency advantage that underscores the difference between direct and indirect activation.

Getting the drug into living organisms required some pharmaceutical finesse. In flies, 991 activated AMPK in vivo only when delivered in a chemically defined diet, which offers better drug bioavailability than standard yeast-based food, a reminder that in ageing research the medium can matter as much as the molecule. Western blotting confirmed that dietary 991 raised levels of phosphorylated, active AMPK in flies, worms and fission yeast alike. With the target engagement established across three phyla, the team moved to the question everyone wanted answered: does directly activating AMPK actually make animals live longer?

The answer was yes, and in a remarkably consistent pattern. Flies fed 991 in defined medium lived significantly longer than controls, with the effect highly statistically significant at both low and moderate doses. Fission yeast treated with 10 micromolar 991 showed extended chronological lifespan, a measure of how long stationary-phase cells survive. Nematode worms gained longevity on two different bacterial diets, the standard OP50 strain and the alternative BW25113, and the drug did not substantially alter bacterial growth, indicating that, unlike metformin, 991 acts on the worm host directly rather than through the microbes in its gut. The drug also reshaped metabolism in both animals: flies showed dose-dependent reductions in whole-body triglyceride stores without any change in feeding rate or glycogen content, while worms displayed altered lipid staining patterns. Fecundity in the flies was untouched, ruling out the classic trade-off in which reproduction is sacrificed for survival.

The most convincing evidence came from the genetic controls. Because AMPK alpha-null flies do not survive to adulthood, the researchers tested the drug in yeast and worm mutants lacking the catalytic subunit. In both cases, the lifespan benefit vanished entirely. The ssp2 yeast mutant, which is hypersensitive to any stress, was actually harmed by the drug, and the aak-2 mutant worms showed no extension at all. That loss-of-effect in the absence of the target is exactly what a rigorous pharmacological study should show, and it distinguishes this work from much of the longevity literature, where compounds often extend lifespan through murky, multi-target mechanisms that resist genetic dissection. Higher doses of 991 shortened lifespan in flies and yeast, a dose-response caution that the authors emphasise as critical for any future therapeutic development.

To bridge the gap to mammals, the team delivered 991 to mice using biodegradable polymeric nanoparticles, injected intravenously three times a week for three weeks. Unbiased proteomic analysis of liver tissue from treated wild-type mice, and from a short-lived muscular dystrophy model, revealed a clear molecular signature of AMPK activation: increased ATP biosynthesis, inhibition of the pro-ageing mTOR pathway, and enhanced mitochondrial biogenesis. Phenotypic ontology analysis flagged survival-related processes as the most enriched category among the responding proteins. While these proteomic shifts fall short of a demonstrated lifespan extension in mammals, they are precisely the profile that pro-longevity interventions produce, and they suggest the machinery responds to direct AMPK activation in mice much as it does in the invertebrates.

There is also a pharmaceutical tailwind behind these findings. Several major companies, including Merck, Pfizer, Boehringer Ingelheim and Poxel, have developed second-generation direct AMPK activators that bind the same ADaM site as 991. These compounds have better bioavailability than 991, have activated AMPK in vivo across a range of rodent studies, improving glucose homeostasis, dyslipidemia and fatty liver disease, and have even been given to non-human primates. Most strikingly, PXL770 has already been through human clinical trials for metabolic liver disease, showing good safety and improved lipid profiles after just four weeks of treatment. The path from a worm surviving longer on a drug-laced agar plate to a human pill is long, but it is considerably shorter when the pill in question already has clinical safety data.

The authors are careful about the limits of their work. The mouse data are preliminary, delivered by injection rather than diet, and longer-term metabolic and longevity studies in mammals remain to be done, likely using the improved second-generation activators. The downstream mechanisms by which AMPK activation extends lifespan in vivo are still unknown, and additional healthspan measures will be needed to complement survival curves. Even so, the study delivers something the ageing field has rarely possessed: a direct activator of a single, evolutionarily conserved target that extends lifespan in three distantly related organisms, fails to do so when the target is genetically removed, and produces a pro-longevity molecular signature in mammals. If metformin’s tangled story taught researchers anything, it is that longevity drugs need clean mechanisms. With 991, they may finally have one.

Subject of Research: Direct pharmacological activation of AMP-activated protein kinase as a conserved mechanism for extending lifespan across species

Article Title: Direct Pharmacological Activation of AMPK Extends Lifespan in Yeast, Worms and Flies

Article References: dos Santos, E., Blickling, M., Leiper, F. C., Alao, J.-P., Lennicke, C., Foley, A., Wilson, J. R., Gamblin, S. J., Chazaud, B., Lollo, G., Mounier, R., Juban, G., Rallis, C., Carling, D., Cabreiro, F., & Cochemé, H. M. (2026). Direct Pharmacological Activation of AMPK Extends Lifespan in Yeast, Worms and Flies. Aging Cell, 25(10), Article e70721. https://doi.org/10.1111/acel.70721

Image Credits: AI Generated

DOI: 10.1111/acel.70721

Keywords: AMPK, ageing, lifespan extension, 991, metformin, Drosophila, C. elegans, fission yeast, ADaM site, geroprotectors, metabolism, proteomics