The mechanistic (formerly mammalian) target of rapamycin (mTOR) is an atypical serine/threonine protein kinase of the phosphatidylinositol 3-kinase-related kinase superfamily. mTOR coordinates several signaling networks that promote anabolic processes and control cellular and organismal growth (1, 2). In mammals, there are two distinct, but potentially complementary, catalytic complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (3). Numerous extracellular stimuli such as growth factors and mitogens can initiate mTOCR1 activation by inhibiting tuberous sclerosis complex (TSC), which functions as another pleiotropic hub integrating subcellular energy and amino acid availability (4). Besides growth factors, cellular amino acid pool can also activate mTORC1, which affects global translation not only as a rate-limiting reactant, but also as an upstream regulator of signaling pathways.
In the presence of amino acids, mTORC1 is activated, leading to the activation of downstream effectors to utilize amino acids via upregulation of global translation (5, 6). mTORC2 is not activated by amino acids (7). Under normal conditions, the ubiquitin-proteasome system (UPS) is the primary catabolic system that degrades proteins and provides cells with amino acids for new protein synthesis (8). If the amino acid supply is limited, bulk autophagy, another cellular degradation system, is induced to provide additional anabolic intermediates (9). For example, the inhibition of UPS rapidly depletes available amino acids, leading to compensatory induction of autophagy to restore cellular proteolytic capacity, while the activation of proteasome results in the suppression of autophagy (10-14). These two pathways appear to be connected via a negative feedback mechanism. As a whole, they coordinate their functions to maintain the cellular amino acid pool.
By inhibiting the early stage of cellular autophagy flux, the mTOR pathway can prevent futile cycles of continuous proteolysis and translation. mTORC1 can directly phosphorylate and suppress autophagy-initiating kinase ULK1 (15, 16). Inhibition of mTOR mimics nutrient-depleted conditions, subsequently inducing autophagy. mTOR inhibitors such as rapamycin and Torin1 are widely used both
Although the link between mTOR and autophagy regulation has been established, the biochemical connection between the mTOR pathway and proteasomal protein degradation remains unclear. Furthermore, even though the regulation of 26S proteasome synthesis is well studied, much less is known about the role of mTOR in this process. In this review, we will focus on the effect of mTOR inhibition on proteasome homeostasis and overall proteolysis without providing a comprehensive synopsis of signaling networks. We will summarize key findings from three essential articles (21-23) as well as the latest research from our group.
Only a few studies so far have investigated the connection between the mTOR pathway and 26S proteasome. Zhang
The coupled pathways presented in Case #1 (abundant amino acids → mTORC1 activation → 1) ribosome/translation factor, 2) NRF1 → 1) global translation, 2) proteasome biogenesis → 1) larger proteome, 2) proteasome activity → adequate amino acid pool and protein quality) as a possible adaptive response mechanism have been challenged by many groups. Zhao
It has been reported that selective inhibition (using 0.2 μg/ml rapamycin for 3 h) of TORC1 in yeast can facilitate 19S proteasome assembly and increase cellular proteasome levels/activity more than 2-fold (30). Treatment of yeast with ER stress inducers such as tunicamycin can result in similar outcomes (30). In general, ER stress antagonizes cellular anabolism. However, it can either enhance or suppress mTORC1 signaling (23). The coordinated regulation between mTOR and ER stress is not fully understood yet. Rousseau and Bertolotti (30, 31) have reported that the increased proteasome abundance after TORC1 inhibition is mediated by the mitogen-activated protein kinase Mpk1/ERK5 and Adc17 which is an inducible chaperone for proteasome assembly. This might contribute to the rapid and reversible elimination of misfolded proteins during ER stress.
We have also investigated the effect of mTORC1 inhibition on proteasome activity and function using purified proteasomes from HEK293 cells (32-34). We found that mTORC1 inhibition with 250 nM Torin1 did not significantly affect proteasome content or direct phosphorylation on proteasome (Fig. 1A-C). When purified proteasomes were analyzed using non-denaturing (native) polyacrylamide gel electrophoresis and subsequent in-gel activity analysis, Torin1-treated proteasomes showed structural integrity and activity similar to control 26S proteasomes (Fig. 1D). We did not find any significant differences in chymotrypsin- like β5 proteasome activity (measured by suc-LLCY-AMC hydro-lysis) or other catalytic activities (such as caspase-like β1 and trypsin-like β2 activities) either in whole-cell lysates upon Torin1 treatment (Fig. 1E and data not shown). Taken together, these results indicated that inhibition of mTORC1 did not appear to rapidly alter cellular proteasome levels or activity in HEK293 cells.
Currently, reports investigating effects of mTORC1 inhibition on the synthesis of 26S proteasomes have presented contradictory results. Cells have numerous adaptive strategies to cope with an increased number of target substrates either at the translational level or the posttranslational level. Considering that the proteasome is long-lived (half-life > ∼10 days in mammals (35-37) and present in excess (more than 1% of yeast proteome (38), its spatiotemporal regulation could be more adaptable to efficient proteolytic responses to stress than homeostatic regulation. For example, instead of energy-costly degradation and synthesis, proteasomes could be sequestered into cytoplasmic granules during quiescence, nuclear foci under hypertonic stress, or aggresomes when catalytically inhibited (39-41). The spatiotemporal sequestration of proteasomes appears to be mediated by reversible liquid-liquid or liquid-gel phase separation. Proteasomes in liquid droplets can recover to be a functional enzyme in the absence of cellular stress, while proteasomes in less soluble aggregates might have already lost their structural integrity and thus they are destined for autophagic degradation.
Despite tremendous progress in mTOR biology (an anabolic process), many questions regarding its role in the UPS (a catabolic process) remain to be addressed. The molecular mechanisms regulated by mTOR as an amino acid sensor and cellular autophagy suppressor have been thoroughly investigated. However, our understanding of the role of the proteasome in the mTOR signaling cascade is far from complete. It is essential to confirm the effect of mTORC1 inhibition on 26S proteasome biogenesis by independent groups using standardized protocols. In addition, quantification of cellular amino acid pool, NRF1 activation, and polyubiquitin linkages in response to either chronic or acute mTORC1 inhibition is necessary. These results will provide mechanistic clues to clarify the remaining pathways of this complex signaling circuit. Considering that dysregulation of cellular amino acid pool is implicated in a diverse array of human diseases, the ability to regulate mTORC1 and proteasome activity levels could further extend therapeutic applications of mTORC1 inhibitors.
This work was supported by grants from the NRF (2020R1A5A 1019023 and 2021R1A2C2008023 to M.J.L.), the Korea Health Industry Development Institute and Korea Dementia Research Center (HU21C0071 to M.J.L.), and the Creative-Pioneering Researchers Program through Seoul National University, and the BK21 FOUR programs (W.H.C. and S.H.P.).
The authors have no conflicting interests.