As major nutrients, amino acids are requisites for maintaining normal cell functions and physiological activities1. The deregulated amino acids metabolism is commonly related to pathological states, such as aging-related disorders, metabolic diseases, and cancer2. Generally, the amino acids abundance is precisely regulated by complex components. The indirect mechanism for sensing general amino acids levels is associated with the release of tRNA molecules from the general control non-derepressible 2 (GCN2)3. Another important mechanism involves direct binding between amino acids and their specific sensors, such as CASTOR1/2 and SLC38A9 for arginine, and Sestrin2 and SAR1B for leucine4.
As an essential branched amino acid, valine is crucial for protein synthesis, neurological behavior, hematopoiesis5. However, the mechanism by which cellular valine abundancy is sensed for subsequent cellular functions remains undefined. In the current work published in Nature, valine specifically binds to the primate-specific SE14 repeat domain of HDAC6, retaining it in the cytoplasm. Valine deprivation promotes the nuclear translocation of HDAC6, which in turn increases active DNA demethylation and promotes DNA damage by deacetylating and activating TET2. Therapeutically, dietary valine restriction inhibits tumor growth, and enhances the therapeutic efficacy of PARP inhibitors.
Human HDAC6 serves as a primate-specific valine sensor
In this study, HDAC6, a microtubule-associated deacetylase, bound to valine, with the dissociation constant (Kd) around 1.95 mM. By mapping the valine-binding domain of HDAC6, the Ser-Glu-containing tetradecapeptide (SE14) repeat domain was identified with a Kd of around 26.5 μM, and its deletion abolished the binding of HDAC6 to valine.
An evolutionary difference in amino acids sensing may play important roles in highly sophisticated processes such as nervous function, distinguishing primates from other mammals. As the SE14 repeat domain is only found in HDAC6 proteins of primates, we evaluated the species-specific nature of valine binding to HDAC6. Mouse HDAC6, without the SE14 repeat domain, failed to bind valine, while a chimera of human SE14 with mouse HDAC6 and HDAC6 of primate Rhinopithecus bietirestored their binding to valine. These data indicate an evolutionary advance of HDAC6 in sensing valine.
Valine deprivation induces nuclear translocation of HDAC6
Since the SE14 repeat domain is responsible for the cytoplasmic localization of HDAC6, we investigated the role of valine binding in this process and found that HDAC6 was predominantly cytosolic in HCT116 cells. Valine deprivation induced the nuclear localization of HDAC6, while re-supplementation of valine restored its cytoplasmic localization.
As HDAC6 possesses one nuclear import (NLS) and two nuclear export sequences (NES), we evaluated their effects on valine deprivation-induced nuclear translocation of HDAC6. Treatment with importin inhibitor importazole or deletion of the NLS blocked valine deprivation-induced HDAC6 nuclear translocation. Consistently, binding of importin-α1 and importin-α7 to HDAC6 could be detected after valine deprivation, and this binding was blocked by both valine re-supplementation and deletion of the NLS or SE14 repeat domain.
Nuclear-localized HDAC6 binds and deacetylates TET2 to promote DNA damage
To investigate the biological function of valine deprivation-induced HDAC6 nuclear translocation, we identified 356 nuclear proteins specifically binding to HDAC6 in a valine-dependent manner. TET2, a dioxygenase converts 5mC into 5hmC to regulate gene expression, chromatin stability and tumorigenesis6, was in the first rank. Valine deprivation increased the cellular level of 5hmC in a HDAC6-dependent manner, as well as 5hmC, 5fC and 5caC levels in the valine deprivation-specific enhanced TET2-binding regions in a TET2-dependent manner.
Biologically, we found that valine deprivation induced DNA damage, and deletion of TET2 or HDAC6 blocked valine deprivation-induced DNA damage. As TET2-mediated DNA damage is mainly dependent on the formation of single-strand DNA breaks (SSBs) driven by thymine DNA glycosylase (TDG), we confirmed that valine deprivation upregulated the level of 5fC and 5caC, and induced SSBs formation, while TDG deficiency blocked the valine deprivation-induced DNA damage.
The SE14 repeat domain of HDAC6 was indeed required for the valine deprivation-induced DNA damage. The mutation of SE14 domain (SE-MUT HDAC6) could induce DNA demethylation and DNA damage, while the constructed nuclear-localized HDAC6 enhanced DNA hydroxymethylation and DNA damage in a TET2-dependent manner (Figure 1).
Figure 1: Illustration of the proposed mechanism of HDAC6 sensing valine deprivation via the domain of SE14 dictating the epigenetic modification of 5hmC.
Dietary valine restriction serves as a potential option for cancer treatment
Dietary modulation or pharmacologically targeting amino acids metabolism and sensing have emerged as adjuvant strategies for health span extension and disease treatment, especially cancer7. Given that valine deprivation promotes DNA damage, we investigated whether valine restriction worked in cancer treatment. An appropriate valine restriction diet strategy of 0.41% valine (w/w) was identified which significantly inhibited tumor growth with mild side effects in xenograft models. The effects were further validated by patient-derived xenografts in both prevention and treatment groups (Figure 2A).
Within tumor samples, a progressive reduction in valine levels was correlated with an incremental increase in the nuclear translocation of HDAC6, 5hmC levels and extent of DNA damage. As inducing DNA damage is an anticancer therapy, which can be achieved by inhibiting DNA repair clinically using PARP inhibitors, we used valine restriction and talazoparib, a PARP inhibitor. The combination therapy significantly enhanced the antitumor effects, providing evidence that it may serve as candidates for cancer treatment via inducing DNA damage (Figure 2B).
Figure 2: A. Schematic experimental design using colorectal PDXs. B. Valine-restricted diet combined with PARP inhibitors in the treatment of cancer.
In summary, our findings have identified a novel valine sensor, HDAC6, which possesses a primate-specific SE14 repeat domain that binds to valine, retaining it in the cytoplasm. This implies an evolutionary advancement of HDAC6 in primates. Nuclear HDAC6, in turn, activates DNA demethylation and DNA damage by deacetylating and activating TET2, indicating a new role of epigenetics in amino acids metabolism. Additionally, we noted that dietary valine restriction may induce DNA damage and inhibit the cancer growth and progression, providing a potential therapeutic option for cancer treatment.
References:
1 Lemos, H., Huang, L., Prendergast, G. C. & Mellor, A. L. Immune control by amino acid catabolism during tumorigenesis and therapy. Nat Rev Cancer. 2019 Mar;19(3):162-175..
2 Chantranupong, L., Wolfson, R. L. & Sabatini, D. M. Nutrient-sensing mechanisms across evolution. Cell. 2015 Mar 26;161(1):67-83.
3 He, X. D. et al. Sensing and Transmitting Intracellular Amino Acid Signals through Reversible Lysine Aminoacylations. Cell Metab. 2018 Jan 9;27(1):151-166.e6.
4 Chen, J. et al. SAR1B senses leucine levels to regulate mTORC1 signalling. Nature. 2021 Aug;596(7871):281-284..
5 Thandapani, P. et al. Valine tRNA levels and availability regulate complex I assembly in leukaemia. Nature. 2022 Jan;601(7893):428-433..
6 He, Y. F. et al. Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA. Science. 2011 Sep 2;333(6047):1303-7.
7 Kanarek, N., Petrova, B. & Sabatini, D. M. Dietary modifications for enhanced cancer therapy. Nature. 2020 Mar;579(7800):507-517.