How an Amino Acid Affects a Key Cell-Growth Regulator

News & views dynamics of actin — a structural protein of signalling compared with wild-type aged in the development of age-related mammalian the cell skeleton. Knockdown of eps-8 in adult worms is consistent with previous work show- disorders that involve protein aggregation, worms prevented the destabilization of actin ing that, in the mutant worms, deubiquitin such as Parkinson’s disease and Alzheimer’s networks in muscles of the body wall and the ation is actively repressed and proteasome disease15. Therefore, understanding how the concomitant decrease in motility in old worms activity is increased8. Previous work also ubiquitination dynamics for different pro- (Fig. 1b). When eps-8 expression was reduced showed that the longevity of these animals teins change in different species with ageing specifically in the muscles or neuronal cells is, in part, supported by an increase in ubiq- could provide insight into potential strategies of adult worms, the worms lived longer than uitination9. for treating such disorders. normal. Concordantly, knockdown in adult- From a broader perspective, Koyuncu and hood of mig-2, one of the three C. elegans co-workers’ findings elegantly support the Bart P. Braeckman is in the Department of genes that encode RAC-like proteins, also antagonistic-pleiotropy hypothesis10 — an Biology, Ghent University, Ghent B-9000, extended lifespan. evolutionary explanation for why ageing is Belgium. In summary, Koyuncu et al. successfully link adaptive and therefore occurs. This hypoth- e-mail: [email protected] the age-related decrease in ubiquitination esis posits that genes that are beneficial for and subsequent increase in the abundance development or reproduction, but are detri- 1. Komander, D. Biochem. Soc. Trans. 37, 937–953 (2009). of two proteins, IFB-2 and EPS-8, to the loss of mental later in life, will be selected for during 2. Koyuncu, S. et al. Nature 596, 285–290 (2021). intestinal and muscular integrity — both prob- evolution. The study identifies two excellent 3. Greer, E. L. & Brunet, A. Aging Cell 8, 113–127 (2009). lems well known to be associated with age in examples of such genes — ifb-2 and eps-8. 4. Murphy, C. T. & Hu, P. J. in WormBook (eds the C. elegans Research Community) available at https://doi. 5,6 C. elegans . A key feature of this study is that A final and major question is whether or not org/10.1895/wormbook.1.164.1 (WormBook, 2013). it bridges the gap between molecular changes the age-associated deubiquitination that trig- 5. McGee, M. D. et al. Aging Cell 10, 699–710 (2011). that occur with ageing and their effects on gers late-life protein accumulation and aggre- 6. Herndon, L. A. et al. Nature 419, 808–814 (2002). 7. Lohr, J. N., Galimov, E. R. & Gems, D. Ageing Res. Rev. 50, tissue function and lifespan in the worm. gation in worms is also seen in more-complex 58–71 (2019). This work opens up several new research animals. In rats, protein ubiquitination seems 8. Matilainen, O., Arpalahti, L., Rantanen, V., Hautaniemi, S. 11–13 & Holmberg, C. I. Cell Rep. 3, 1980–1995 (2013). opportunities, yet also leaves open intrigu- to increase, rather than decrease, with age , 9. Ghazi, A., Henis-Korenblit, S. & Kenyon, C. Proc. Natl ing questions. One such question concerns suggesting that the dynamics of ubiquitin Acad. Sci. USA 104, 5947–5952 (2007). the potential for leveraging these findings to ation in worms are different from those in 10. Williams, G. C. Evolution 11, 398–411 (1957). 11. Li, F. et al. Mech. Ageing Dev. 129, 515–521 (2008). explore ways of extending lifespan. Koyuncu mammals. The distinctive ubiquitination 12. Zhang, L., Li, F., Dimayuga, E., Craddock, J. & Keller, J. N. et al. showed that knocking down individual pattern observed in C. elegans might relate FEBS Lett. 581, 5543–5547 (2007). genes that express proteins that tend to accu- to its peculiar ‘boom-and-bust’ life history 13. Altun, M. et al. J. Biol. Chem. 285, 39597–39608 (2010). 14. Ben-Zvi, A., Miller, E. A. & Morimoto, R. Proc. Natl Acad. mulate and aggregate with age can extend that favours both a programmed early col- Sci. USA 106, 14914–14919 (2009). 14 lifespan by a maximum of 21%. Given that lapse of the regulation of protein levels and 15. Lim, K.-H., Joo, J.-Y. & Baek, K.-H. Aging Res. Rev. 61, these genes probably act independently in ageing7. However, it is still possible that pro- 101088 (2020). different tissues, reducing the expression of teins for which proteasomal clearance is dys The author declares no competing interests. a combination of different genes in different regulated with ageing have an important role This article was published online on 28 July 2021. tissues might result in a much stronger effect on longevity. This strategy should not neces- Metabolism sarily be limited to the targets identified in this study. Treatment of old worms with the DUB inhibitor PR-619 generally returned ubiquitination How an amino acid affects in old worms to levels seen in younger worms, possibly by preventing or delaying age-related a key cell-growth regulator accumulation of protein aggregates in mul- tiple tissues simultaneously. However, given Tibor Vellai its broad activity, PR-619 might also interfere with crucial deubiquitination and therefore Cells can tailor their growth to current conditions by sensing disturb cellular homeostasis in a way that is nutrients. The protein complex mTORC1 enables cell growth to not compatible with longevity. be coordinated with the level of certain amino acids, and how it Another remaining pertinent question is, what causes the increase in DUB activity senses the amino acid leucine has now become clearer. See p.281 in old animals? The culprit could be sus- tained insulin/IGF-1 activity in the worms, because the authors found that its reduc- The protein mTOR is a type of enzyme called protein-synthesis machinery in the form of tion was sufficient to prevent upregulation a kinase, and it functions in two distinct ribosomes; lipid synthesis; and autophagy, a of all age-dysregulated DUBs. Moreover, complexes, termed mTORC1 and mTORC2. degradation process mediated by organelles insulin/IGF-1 activity has been proposed to mTORC1 boosts cell growth and prolifera- called lysosomes2. mTORC1 deficiency has promote senescence and age-related tissue tion in response to nutrient availability and been implicated in various human disorders, deterioration in C. elegans, thus causing older hormone-dependent signals that promote including cancer, neurodegeneration, meta- individuals to die, while increasing fitness of cell division. On page 281, Chen et al.1 provide bolic diseases and muscle atrophy, as well as the species at the colony level7. Hence, the insights into how mTORC1 is regulated by a being linked to changes involved in the ageing increase in DUB activity with age might be one specific amino acid — leucine. process3. Yet despite its major physiological of the mechanisms by which the insulin/IGF-1 mTORC1 carries out its role by regulating and clinical significance, how mTORC1 sig- signalling pathway controls lifespan. certain anabolic (synthetic) and catabolic nalling is influenced by upstream regulatory The increased ubiquitination of proteins (degradative) processes. These involve factors is not fully understood. in aged worms with reduced insulin/IGF-1 protein synthesis; production of the The amino acid leucine is a potent

The authors report that inhibition of SAR-1 a Low levels of amino acids b High levels of amino acids protein, the counterpart of human SAR1B in CASTOR1 Arg the nematode worm Caenorhabditis elegans, SAR1B Sestrin 2 Leu Leu also renders the worm version of mTORC1, called CeTORC1, insensitive to nutrient starvation in a GATOR2-dependent manner. When a human SAR1B gene is introduced into MIOS SEH1L nematodes defective in SAR-1 production, GATOR2 complex CeTORC1 sensitivity to nutrient restriction WDR59 WDR24 is restored. And, consistent with CeTORC1’s SEC13 function in ageing7, SAR-1 affects the lifespan Rheb of C. elegans under prolonged starvation. GATOR1 Inactive These results suggest that SAR1B’s role in mTORC1 complex the control of mTORC1 is evolutionarily Rag-mediated Activation conserved. recruitment Consistent with the association between RagA/B RagC/D Cell mTORC1 hyperactivity and cancer2, Chen growth Lysosome et al. report that the SAR1B gene is frequently Active mTORC1 deleted in lung tumours called squamous cell carcinoma and adenocarcinoma. The Figure 1 | A mechanism to activate the mTORC1 complex that depends on the level of specific amino acids. authors also find that treatment of cul- The complex mTORC1 regulates cell growth, and its activity is modulated by the intracellular availability tured SAR1B-deficient human cells with the 1 of amino acids. Chen et al. report their discovery that the protein SAR1B functions in this pathway. a, If mTORC1 inhibitor rapamycin significantly amino-acid levels are low, SAR1B and two other proteins, CASTOR1 and sestrin 2, inhibit the protein complex suppresses proliferation. Finally, eliminat- GATOR2 (which consists of the proteins MIOS, SEH1L, WDR24, SEC13 and WDR59). SAR1B inhibits MIOS and ing SAR1B activity promotes tumour growth sestrin 2 inhibits SEH1L. The complex GATOR1 (subunits not named here) inhibits the enzymes RagA, RagB, in mice given transplants of human tumour RagC and RagD, and the inactive mTORC1 complex fails to be recruited to an organelle called the lysosome. cells. These findings suggest that SAR1B b, High levels of the amino acid leucine (Leu) inhibit SAR1B and sestrin 2, and high levels of arginine (Arg) inhibit CASTOR1. As a result, GATOR2 is able to inhibit GATOR1. The Rag enzymes, which are no longer might serve as a potent target for antitumour inhibited, then recruit mTORC1 to the lysosome, and mTORC1 is activated by the protein Rheb (which can therapy. 8,9 sense aspects of cellular function such as energy levels). This pathway can thereby integrate information Arginine was previously shown to be about the cell to initiate growth when conditions are suitable. another essential amino acid that activates the mTORC1 pathway (Fig. 1). This amino acid is sensed by the CASTOR1 protein, which also stimulator of mTORC1: it blocks the inhibitory enables mTORC1 to localize to lysosomes, interacts directly with GATOR2 (refs 8, 9). Loss effect of the protein sestrin 2 on the GATOR2 even under conditions of leucine starvation. of CASTOR1 function or arginine abundance complex that activates mTORC1 (ref. 4). Such The authors found that SAR1B and sestrin 2 also leadS to mTORC1 activation. regulation links the cellular nutrient status — detect leucine by recognizing different parts Why do these two amino acids, leucine the level of intracellular amino acids — to the of the amino acid’s structure. They report that and arginine, seem to have a specific role control of cell growth (Fig. 1). SAR1B recognizes the amino group and side in controlling mTORC1 signalling, given Chen and colleagues show that leucine is chain of leucine, whereas it was known6 that that they are sensed4,9 by different GATOR2 also sensed by another regulatory factor in sestrin 2 identifies leucine’s amino and carboxyl modulators? An organism’s genetic code mammalian cells, the protein SAR1B, which groups. These two leucine sensors bind to the has redundancy because different nucleo- is a type of enzyme called a small GTPase. amino acid with different affinities: SAR1B has tide triplets (codons) can specify the same The authors report that reduced expression a higher binding affinity than does sestrin 2. amino acid. Methionine and tryptophan are of the SAR1B gene makes cells insensitive to each encoded by a single codon, whereas all leucine scarcity and activates the mTORC1 “These findings suggest the other amino acids are specified by mul- pathway. Similarly to the way in which ses- tiple codons. It is intriguing that leucine and trin 2 functions, SAR1B interacts physically that SAR1B might serve as a arginine are two of the three amino acids that with GATOR2 when leucine is removed, potent target for antitumour are determined by the highest number of affecting GATOR2’s function. However, the therapy.” codons — six in total. The relative frequency authors show that SAR1B binds to a different of a given amino acid in protein is affected GATOR2 subunit (the protein MIOS) from by the number of codons that can code for it sestrin 2 (which binds to the SEH1L protein). These features permit mTORC1 activation to (see go.nature.com/36xqd3l) When leucine levels in the cell are sufficiently occur over two steps by sequentially prompting Thus, it is possible that a system has high, the amino acid binds to SAR1B, caus- the dissociation of SAR1B and sestrin 2 from evolved in which the amino acids that are used ing a conformational change in the protein, GATOR2 at different intracellular leucine con- most frequently in proteins also function as which then dissociates from GATOR2. The centrations. Furthermore, the relative levels of signalling factors that determine the activ- liberated GATOR2 inhibits another protein SAR1B and sestrin 2 in a cell can vary in different ity of the major orchestrator of cell growth, complex, GATOR1, which leads four Rag tissues. At the subcellular level, some SAR1B mTORC1. Indeed, the third amino acid that is GTPase enzymes5 (RagA, RagB, RagC and localizes to lysosomes, and leucine stimu- specified by six codons is serine, and there is RagD) to recruit mTORC1 to the surface of lates its dissociation from both GATOR2 and evidence that this amino acid is also involved the lysosome, where the enzyme Rheb GTPase lysosomes. Together, the authors’ evidence in mTORC1 control10. The intracellular level activates mTOR (Fig. 1). Thus, the inhibi- indicates that SAR1B is a previously unknown of methionine, one of the amino acids that tory function of GATOR1 on Rag GTPases is leucine sensor that regulates mTORC1 activity occurs less frequently in proteins, is sensed SAR1B-dependent. Loss of SAR1B function through modulation of GATOR2. by the SAMTOR protein, which induces

News & views GATOR1 to repress mTORC1 signalling11. Eötvös Loránd University, Budapest H-1117, example, high-throughput single-molecule Together, these data trace out a model in Hungary. measurements have been developed in which the regulatory factors modulating e-mail: [email protected] ultrasmall containers, to detect individual mTORC1 activity can sense the intracellular biomolecules2. Extraordinary achievements level of specific amino acids that limit the rate 1. Chen, J. et al. Nature 596, 281–284 (2021). in single-molecule studies have also have of protein synthesis. Using this strategy, there 2. Saxton, R. A. & Sabatini, D. M. Cell 168, 960–976 (2017). been reported using scanning tunnelling 3. Liu, G. Y. & Sabatini, D. M. Nature Rev. Mol. Cell Biol. 21, 3 4 would be no need for the intracellular concen- 183–203 (2020). microscopy , nano-electrochemistry and tration of every amino acid to be monitored in 4. Wolfson, R. L. et al. Science 351, 43–48 (2016). an optical technique called super-resolution a cell; instead, only those used the most and 5. Bar-Peled, L. et al. Science 340, 1100–1106 (2013). localization microscopy (which achieves a res- 6. Saxton, R. A. et al. Science 351, 53–58 (2016). the least frequently in protein synthesis would 7. Vellai, T. et al. Nature 426, 620 (2003). olution higher than the diffraction limit — a need to be tracked. That would enable the evo- 8. Saxton, R. A., Chantranupong, L., Knockenhauer, K. E., fundamental restriction that usually limits the lution and operation of a relatively simple, Schwartz, T. U. & Sabatini, D. M. Nature 536, 229–233 resolution of optical imaging techniques)5. (2016). robust and economical regulatory network 9. Chantranupong, L. et al. Cell 165, 153–164 (2016). After decades of development, methods in for maintaining a balance between protein 10. Maddocks, O. D. K. et al. Nature 493, 542–546 (2013). which samples are irradiated with light have synthesis and degradation. 11. Gu, X. et al. Science 358, 813–818 (2017). arguably become the most useful means of

The author declares no competing interests. ‘seeing’ individual molecules — the 2014 Nobel Tibor Vellai is in the Department of Genetics, This article was published online on 21 July 2021. Prize in Chemistry was awarded for discover- ies in this field (see go.nature.com/3zs4aph). Physical chemistry Biologists, physicists and chemists quickly adopted these revolutionary microscopy techniques, all of which use clever arrange- ments of light and dark — similar to the chiaro Single reaction events scuro technique in art. However, because these methods require the use of short, high- imaged in total darkness power laser pulses, their performance is often limited by background scattered light and Frédéric Kanoufi & Neso Sojic photobleaching (the extinguishing of fluorescence as a result of the continuous excitation Single photons emitted from individual electrochemically of fluorescent molecules by light). In biologi- excited molecules in solution can now be detected. The cal applications, samples can also be damaged technique can be used to image cells at nanometre resolution, by the intense laser pulses, or produce their own fluorescence, obscuring the image of the without using an external light source. See p.244 fluorescent molecule of interest. Some organisms can produce light in situ using biochemical reactions, rather than rely- If you want to admire the Milky Way in all unknown. This issue can be partly overcome ing on external light sources — such biolumi- its splendour, you should choose a moon- by restricting the volume of the solution, or nescence illuminates the darkest regions of less (and possibly romantic) night, away by separating events in space and/or time, the oceans, for example. Dong and colleagues’ from urban light pollution. Exactly the allowing each one to be detected alone. For approach to single-molecule imaging takes a same principle applies in microscopy, at the other end of the observational size scale: it is much easier to observe faint objects in complete darkness. On page 244, Dong et al.1 Fast photon emission report their use of this simple but powerful idea in a direct optical method for imag- Excited ing single photons generated by individual dye chemical-reaction events in solution. The Solution Rare authors’ method is conceptually different from the fluorescence-based approaches used TPA TPA• Oxidized Dye in conventional single-molecule microscopy, Cell dye because the excitation process that leads to photon emission is controlled by electro- e– e– chemistry and chemical reactivity, and does Electrode not require irradiation with light. The authors Electrode demonstrate that their imaging method can be used for super-resolution microscopy of living cells. Figure 1 | A microscopy technique involving single-molecule imaging of electrochemical reactions. Dong et al.1 have produced images of cells attached to the surface of electrodes using a phenomenon Chemical reactions typically involve the called electrochemiluminescence. The electrode is immersed in a solution of dye molecules (a ruthenium collisions of individual molecules in solution. complex) and a co-reactant (tripropylamine, TPA). TPA is oxidized (loses an electron, e–) at an electrode, and However, because measurements of reactions produces a radical (TPA•). The radicals have an extremely short lifetime, and are therefore found only at very usually record the average parameters of high dilution in a thin region above the electrode surface. The radicals thus encounter dye molecules only ensembles of these molecular events, the rarely. If dye molecules are oxidized at the electrode and encounter a radical, the dye enters its excited state. characteristics of individual events are usually The excited dye molecules almost instantaneously emit a photon, revealing the location and time of the obscured, and the precise location and time reaction event. By imaging photons over time, an image of the electrode surface is produced. Photons are of each reacting molecule in the solution is not produced at regions where the cell is attached, thus creating a negative image of the cell.

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