Abstract
Cullin-RING E3 ubiquitin ligases (CRLs), the largest family of multi-subunit E3 ubiquitin ligases in eukaryotic cells, represent core cellular machinery for executing protein degradation and maintaining proteostasis. Here, we asked what roles Cullin proteins play in human mesenchymal stem cell (hMSC) homeostasis and senescence. To this end, we conducted a comparative aging phenotype analysis by individually knocking down Cullin members in three senescence models: replicative senescent hMSCs, Hutchinson-Gilford Progeria Syndrome hMSCs, and Werner syndrome hMSCs. Among all family members, we found that CUL2 deficiency rendered hMSCs the most susceptible to senescence. To investigate CUL2-specific underlying mechanisms, we then applied CRISPR/Cas9-mediated gene editing technology to generate CUL2-deficient human embryonic stem cells (hESCs). When we differentiated these into hMSCs, we found that CUL2 deletion markedly accelerates hMSC senescence. Importantly, we identified that CUL2 targets and promotes ubiquitin proteasome-mediated degradation of TSPYL2 (a known negative regulator of proliferation) through the substrate receptor protein APPBP2, which in turn down-regulates one of the canonical aging marker-P21waf1/cip1, and thereby delays senescence. Our work provides important insights into how CRL2APPBP2-mediated TSPYL2 degradation counteracts hMSC senescence, providing a molecular basis for directing intervention strategies against aging and aging-related diseases.
This is a preview of subscription content, log in via an institution to check access.
Data availability
The transcriptomic and whole genome sequencing data generated in this study have been deposited in the Genome Sequence Archive (Chen et al., 2021) at the National Genomics Data Center, China National Center for Bioinformation, Chinese Academy of Sciences, with accession number HRA004808. The proteomic data obtained in this study have been deposited in the iProX partner repository (Chen et al., 2022) and are available through the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) under the accession numbers PXD042924 and PXD043036. The differentially expressed genes identified in CUL2−/− hMSCs have been submitted and deposited in the Aging Atlas (AA, https://ngdc.cncb.ac.cn/aging/index) databases (Atlas, 2021).
References
Atlas, A. (2021). Aging Atlas: a multi-omics database for aging biology. Nucleic Acids Res 49, D825–D830.
Bao, H., Cao, J., Chen, M., Chen, M., Chen, W., Chen, X., Chen, Y., Chen, Y., Chen, Y., Chen, Z., et al. (2023). Biomarkers of aging. Sci China Life Sci 66, 893–1066.
Bi, S., Liu, Z., Wu, Z., Wang, Z., Liu, X., Wang, S., Ren, J., Yao, Y., Zhang, W., Song, M., et al. (2020). SIRT7 antagonizes human stem cell aging as a heterochromatin stabilizer. Protein Cell 11, 483–504.
Cai, N., Li, M., Qu, J., Liu, G.H., and Izpisua Belmonte, J.C. (2012). Post-translational modulation of pluripotency. J Mol Cell Biol 4, 262–265.
Cai, W., and Yang, H. (2016). The structure and regulation of Cullin 2 based E3 ubiquitin ligases and their biological functions. Cell Div 11, 7.
Cai, Y., Song, W., Li, J., Jing, Y., Liang, C., Zhang, L., Zhang, X., Zhang, W., Liu, B., An, Y., et al. (2022). The landscape of aging. Sci China Life Sci 65, 2354–2454.
Chai, Z., Sarcevic, B., Mawson, A., and Toh, B.H. (2001). SET-related cell division autoantigen-1 (CDA1) arrests cell growth. J Biol Chem 276, 33665–33674.
Chen, J., Ou, Y., Yang, Y., Li, W., Xu, Y., Xie, Y., and Liu, Y. (2018). KLHL22 activates amino-acid-dependent mTORC1 signalling to promote tumorigenesis and ageing. Nature 557, 585–589.
Chen, T., Chen, X., Zhang, S., Zhu, J., Tang, B., Wang, A., Dong, L., Zhang, Z., Yu, C., Sun, Y., et al. (2021). The genome sequence archive family: toward explosive data growth and diverse data types. Genomics Proteomics Bioinformatics 19, 578–583.
Chen, T., Ma, J., Liu, Y., Chen, Z., Xiao, N., Lu, Y., Fu, Y., Yang, C., Li, M., Wu, S., et al. (2022). iProX in 2021: connecting proteomics data sharing with big data. Nucleic Acids Res 50, D1522–D1527.
Choppara, S., Ganga, S., Manne, R., Dutta, P., Singh, S., and Santra, M.K. (2018). The SCFFBXO46 ubiquitin ligase complex mediates degradation of the tumor suppressor FBXO31 and thereby prevents premature cellular senescence. J Biol Chem 293, 16291–16306.
Consortium, A.B., Zhang, L., Guo, J., Liu, Y., Sun, S., Liu, B., Yang, Q., Tao, J., Tian, X. L., Pu, J., et al. (2023). A framework of biomarkers for vascular aging: A consensus statement by the Aging Biomarker Consortium. Life Med doi: https://doi.org/10.1093/lifemedi/lnad033.
Deng, L., Ren, R., Liu, Z., Song, M., Li, J., Wu, Z., Ren, X., Fu, L., Li, W., Zhang, W., et al. (2019). Stabilizing heterochromatin by DGCR8 alleviates senescence and osteoarthritis. Nat Commun 10, 3329.
Dikic, I. (2017). Proteasomal and autophagic degradation systems. Annu Rev Biochem 86, 193–224.
Dobin, A., Davis, C.A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., Batut, P., Chaisson, M., and Gingeras, T.R. (2013). STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21.
Duda, D.M., Borg, L.A., Scott, D.C., Hunt, H.W., Hammel, M., and Schulman, B.A. (2008). Structural insights into NEDD8 activation of Cullin-RING ligases: conformational control of conjugation. Cell 134, 995–1006.
Epping, M.T., Lunardi, A., Nachmani, D., Castillo-Martin, M., Thin, T.H., Cordon-Cardo, C., and Pandolfi, P.P. (2015). TSPYL2 is an essential component of the REST/NRSF transcriptional complex for TGFβ signaling activation. Cell Death Differ 22, 1353–1362.
Fu, L., Hu, Y., Song, M., Liu, Z., Zhang, W., Yu, F.X., Wu, J., Wang, S., Izpisua Belmonte, J.C., Chan, P., et al. (2019). Up-regulation of FOXD1 by YAP alleviates senescence and osteoarthritis. PLoS Biol 17, e3000201.
Geng, L., Zhang, B., Liu, H., Wang, S., Cai, Y., Yang, K., Zou, Z., Jiang, X., Liu, Z., Li, W., et al. (2023). A comparative study of metformin and nicotinamide riboside in alleviating tissue aging in rats. Life Med 2, lnac045.
He, Y., Ji, Q., Wu, Z., Cai, Y., Yin, J., Zhang, Y., Zhang, S., Liu, X., Zhang, W., Liu, G. H., et al. (2023). 4E-BP1 counteracts human mesenchymal stem cell senescence via maintaining mitochondrial homeostasis. Protein Cell 14, 202–216.
Hershko, A., and Ciechanover, A. (1992). The ubiquitin system for protein degradation. Annu Rev Biochem 61, 761–807.
Hipp, M.S., Kasturi, P., and Hartl, F.U. (2019). The proteostasis network and its decline in ageing. Nat Rev Mol Cell Biol 20, 421–435.
Hua, Z., and Vierstra, R.D. (2011). The Cullin-RING ubiquitin-protein ligases. Annu Rev Plant Biol 62, 299–334.
Huang, D., Zuo, Y., Zhang, C., Sun, G., Jing, Y., Lei, J., Ma, S., Sun, S., Lu, H., Zhang, X., et al. (2022). A single-nucleus transcriptomic atlas of primate testicular aging reveals exhaustion of the spermatogonial stem cell reservoir and loss of Sertoli cell homeostasis. Protein Cell doi: https://doi.org/10.1093/procel/pwac057.
Jia, L., Li, H., and Sun, Y. (2011). Induction of p21-dependent senescence by an NAE inhibitor, MLN4924, as a mechanism of growth suppression. Neoplasia 13, 561–569.
Johmura, Y., Harris, A.S., Ohta, T., and Nakanishi, M. (2020). FBXO22, an epigenetic multiplayer coordinating senescence, hormone signaling, and metastasis. Cancer Sci 111, 2718–2725.
Kubben, N., Zhang, W., Wang, L., Voss, T.C., Yang, J., Qu, J., Liu, G.H., and Misteli, T. (2016). Repression of the antioxidant NRF2 pathway in premature aging. Cell 165, 1361–1374.
Langmead, B., and Salzberg, S.L. (2012). Fast gapped-read alignment with Bowtie 2. Nat Methods 9, 357–359.
Lei, J., Jiang, X., Huang, D., Jing, Y., Yang, S., Geng, L., Yan, Y., Zheng, F., Cheng, F., Zhang, W., et al. (2023). Human ESC-derived vascular cells promote vascular regeneration in a HIF-1α dependent manner. Protein Cell doi: https://doi.org/10.1093/procel/pwad027.
Li, L.Z., Yang, K., Jing, Y., Fan, Y., Jiang, X., Wang, S., Liu, G.H., Qu, J., Ma, S., and Zhang, W. (2023). CRISPR-based screening identifies XPO7 as a positive regulator of senescence. Protein Cell 14, 623–628.
Li, Z., and Xiong, Y. (2017). Cytoplasmic E3 ubiquitin ligase CUL9 controls cell proliferation, senescence, apoptosis and genome integrity through p53. Oncogene 36, 5212–5218.
Liang, C., Liu, Z., Song, M., Li, W., Wu, Z., Wang, Z., Wang, Q., Wang, S., Yan, K., Sun, L., et al. (2021). Stabilization of heterochromatin by CLOCK promotes stem cell rejuvenation and cartilage regeneration. Cell Res 31, 187–205.
Liao, Y., Smyth, G.K., and Shi, W. (2014). featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930.
Lin, H.C., Yeh, C.W., Chen, Y.F., Lee, T.T., Hsieh, P.Y., Rusnac, D.V., Lin, S.Y., Elledge, S.J., Zheng, N., and Yen, H.C.S. (2018). C-terminal end-directed protein elimination by CRL2 ubiquitin ligases. Mol Cell 70, 602–613.e3.
Lin, J.J., Milhollen, M.A., Smith, P.G., Narayanan, U., and Dutta, A. (2010). NEDD8-targeting drug MLN4924 elicits DNA rereplication by stabilizing Cdt1 in S phase, triggering checkpoint activation, apoptosis, and senescence in cancer cells. Cancer Res 70, 10310–10320.
Liu, H., Peng, L., So, J., Tsang, K.H., Chong, C.H., Mak, P.H.S., Chan, K.M., and Chan, S.Y. (2019). TSPYL2 regulates the expression of EZH2 target genes in neurons. Mol Neurobiol 56, 2640–2652.
Liu, X., Liu, Z., Wu, Z., Ren, J., Fan, Y., Sun, L., Cao, G., Niu, Y., Zhang, B., Ji, Q., et al. (2023). Resurrection of endogenous retroviruses during aging reinforces senescence. Cell 186, 287–304.e26.
López-Otín, C., Blasco, M.A., Partridge, L., Serrano, M., and Kroemer, G. (2023). Hallmarks of aging: An expanding universe. Cell 186, 243–278.
Love, M.I., Huber, W., and Anders, S. (2014). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15, 550.
Magni, M., Buscemi, G., Maita, L., Peng, L., Chan, S.Y., Montecucco, A., Delia, D., and Zannini, L. (2019). TSPYL2 is a novel regulator of SIRT1 and p300 activity in response to DNA damage. Cell Death Differ 26, 918–931.
Noormohammadi, A., Calculli, G., Gutierrez-Garcia, R., Khodakarami, A., Koyuncu, S., and Vilchez, D. (2018). Mechanisms of protein homeostasis (proteostasis) maintain stem cell identity in mammalian pluripotent stem cells. Cell Mol Life Sci 75, 275–290.
Okumura, F., Matsuzaki, M., Nakatsukasa, K., and Kamura, T. (2012). The role of elongin BC-containing ubiquitin ligases. Front Oncol 2, 10.
Pan, H., Cai, N., Li, M., Liu, G., and Izpisua Belmonte, J.C. (2013). Autophagic control of cell ‘sternness’. EMBO Mol Med 5, 327–331.
Pan, H., Guan, D., Liu, X., Li, J., Wang, L., Wu, J., Zhou, J., Zhang, W., Ren, R., Zhang, W., et al. (2016). SIRT6 safeguards human mesenchymal stem cells from oxidative stress by coactivating NRF2. Cell Res 26, 190–205.
Petroski, M.D., and Deshaies, R.J. (2005). Function and regulation of Cullin-RING ubiquitin ligases. Nat Rev Mol Cell Biol 6, 9–20.
Ren, X., Hu, B., Song, M., Ding, Z., Dang, Y., Liu, Z., Zhang, W., Ji, Q., Ren, R., Ding, J., et al. (2019). Maintenance of nucleolar homeostasis by CBX4 alleviates senescence and osteoarthritis. Cell Rep 26, 3643–3656.e7.
Sarikas, A., Hartmann, T., and Pan, Z.Q. (2011). The cullin protein family. Genome Biol 12, 220.
Scheffner, M., Nuber, U., and Huibregtse, J.M. (1995). Protein ubiquitination involving an E1-E2-E3 enzyme ubiquitin thioester cascade. Nature 373, 81–83.
Soucy, T.A., Smith, P.G., Milhollen, M.A., Berger, A.J., Gavin, J.M., Adhikari, S., Brownell, J.E., Burke, K.E., Cardin, D.P., Critchley, S., et al. (2009). An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer. Nature 458, 732–736.
Tao, K.P., Fong, S.W., Lu, Z., Ching, Y.P., Chan, K.W., and Chan, S.Y. (2011). TSPYL2 is important for G1 checkpoint maintenance upon DNA damage. PLoS ONE 6, e21602.
Toh, B.H., Tu, Y., Cao, Z., Cooper, M.E., and Chai, Z. (2010). Role of cell division autoantigen 1 (CDA1) in cell proliferation and fibrosis. Genes 1, 335–348.
Tsang, K.H., Lai, S.K., Li, Q., Yung, W.H., Liu, H., Mak, P.H.S., Ng, C.C.P., McAlonan, G., Chan, Y.S., and Chan, S.Y. (2014). The nucleosome assembly protein TSPYL2 regulates the expression of NMDA receptor subunits GluN2A and GluN2B. Sci Rep 4, 3654.
Tu, Y., Wu, T., Dai, A., Pham, Y., Chew, P., de Haan, J.B., Wang, Y., Toh, B.H., Zhu, H., Cao, Z., et al. (2011). Cell division autoantigen 1 enhances signaling and the profibrotic effects of transforming growth factor-β in diabetic nephropathy. Kidney Int 79, 199–209.
Tu, Y., Wu, W., Wu, T., Cao, Z., Wilkins, R., Toh, B.H., Cooper, M.E., and Chai, Z. (2007). Antiproliferative autoantigen CDA1 transcriptionally up-regulates p21Waf1/Cip1 by activating p53 and MEK/ERK1/2 MAPK pathways. J Biol Chem 282, 11722–11731.
Wang, C., Yang, K., Liu, X., Wang, S., Song, M., Belmonte, J.C.I., Qu, J., Liu, G.H., and Zhang, W. (2023). MAVS antagonizes human stem cell senescence as a mitochondrial stabilizer. Research 6, 0192.
Wang, K., and Liu, X. (2022). Determining the effects of neddylation on Cullin-RING ligase-dependent protein ubiquitination. Curr Protocols 2, e401.
Wang, Q., Li, H., Tajima, K., Verkerke, A.R.P., Taxin, Z.H., Hou, Z., Cole, J.B., Li, F., Wong, J., Abe, I., et al. (2022a). Post-translational control of beige fat biogenesis by PRDM16 stabilization. Nature 609, 151–158.
Wang, S., Wang, G., Lu, S., Zhang, J., Zhang, W., Han, Y., Cai, X., Zhuang, Y., Pu, F., Yan, X., et al. (2022b). Proteome expression profiling of red blood cells during the tumorigenesis of hepatocellular carcinoma. PLoS ONE 17, e0276904.
Wisniewski, J.R., Zougman, A., Nagaraj, N., and Mann, M. (2009). Universal sample preparation method for proteome analysis. Nat Methods 6, 359–362.
Xu, X., Sarikas, A., Dias-Santagata, D.C., Dolios, G., Lafontant, P.J., Tsai, S.C., Zhu, W., Nakajima, H., Nakajima, H.O., Field, L.J., et al. (2008). The CUL7 E3 ubiquitin ligase targets insulin receptor substrate 1 for ubiquitin-dependent degradation. Mol Cell 30, 403–414.
Yan, P., Li, Q., Wang, L., Lu, P., Suzuki, K., Liu, Z., Lei, J., Li, W., He, X., Wang, S., et al. (2019). FOXO3-engineered human ESC-derived vascular cells promote vascular protection and regeneration. Cell Stem Cell 24, 447–461.e8.
Yan, P., Ren, J., Zhang, W., Qu, J., and Liu, G.H. (2020). Protein quality control of cell stemness. Cell Regen 9, 22.
Zhang, B., Yan, H., Liu, X., Sun, L., Ma, S., Wang, S., Qu, J., Liu, G.H., and Zhang, W. (2023). SenoIndex: S100A8/S100A9 as a novel aging biomarker. Life Med 2, lnad022.
Zhang, S., Wu, Z., Shi, Y., Wang, S., Ren, J., Yu, Z., Huang, D., Yan, K., He, Y., Liu, X., et al. (2022a). FTO stabilizes MIS12 and counteracts senescence. Protein Cell 13, 954–960.
Zhang, Y., Liu, X., Klionsky, D.J., Lu, B., and Zhong, Q. (2022b). Manipulating autophagic degradation in human diseases: from mechanisms to interventions. Life Med 1, 120–148.
Zhao, H., Ji, Q., Wu, Z., Wang, S., Ren, J., Yan, K., Wang, Z., Hu, J., Chu, Q., Hu, H., et al. (2022). Destabilizing heterochromatin by APOE mediates senescence. Nat Aging 2, 303–316.
Zhou, Y., Zhou, B., Pache, L., Chang, M., Khodabakhshi, A.H., Tanaseichuk, O., Benner, C., and Chanda, S.K. (2019). Metascape provides a biologist-oriented resource for the analysis of systems-level datasets. Nat Commun 10, 1523.
Zhu, J., An, Y., Wang, X., Huang, L., Kong, W., Gao, M., Wang, J., Sun, X., Zhu, S., and Xie, Z. (2022). The natural product rotundic acid treats both aging and obesity by inhibiting PTP1B. Life Med 1, 372–386.
Zimmerman, E.S., Schulman, B.A., and Zheng, N. (2010). Structural assembly of cullin-RING ubiquitin ligase complexes. Curr Opin Struct Biol 20, 714–721.
Acknowledgement
This work was supported by the National Key Research and Development Program of China (2020YFA0804000, 2022YFA1103700, 2020YFA0112200, 2021YFF1201000, the STI2030-Major Projects-2021ZD0202400, 2022YFA1103800), the National Natural Science Foundation of China (82201714, 81921006, 82125011, 92149301, 92168201, 91949209, 92049304, 92049116, 32121001, 82192863, 82122024, 82071588, 32000500, 82271600, 82001477, 82201727), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16000000), CAS Project for Young Scientists in Basic Research (YSBR-076, YSBR-012), the Program of the Beijing Natural Science Foundation (Z190019), the Fellowship of China Postdoctoral Science Foundation (2022M712216), the Project for Technology Development of Beijing-affiliated Medical Research Institutes (11000023T000002036310), the Pilot Project for Public Welfare Development and Reform of Beijing-affiliated Medical Research Institutes (11000022T000000461062), Youth Innovation Promotion Association of CAS (E1CAZW0401, 2022083, 2023092), Young Elite Scientists Sponsorship Program by CAST (YESS20200012, YESS20210002), the Informatization Plan of Chinese Academy of Sciences (CAS-WX2021SF-0301, CAS-WX2022SDC-XK14, CAS-WX2021SF-0101), New Cornerstone Science Foundation through the XPLORER PRIZE (2021-1045), Excellent Young Talents Program of Capital Medical University (12300927), Excellent Young Talents Training Program for the Construction of Beijing Municipal University Teacher Team (BPHR202203105), and Beijing Hospitals Authority Youth Programme (QML20230806). We thank L. Bai, R. Bai, J. Lu, J. Chen, Y. Yang, and X. Li for their administrative assistance, Y. Jing and C. Wang for their help in cell culture, J. Jia and F. Liu for their help in animal experiments, J. Jia and Y. Deng for their help with FACS experiments. J. Wang for his help in LC-MS/MS.
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
The author(s) declare that they have no conflict of interest.
Electronic supplementary material
Rights and permissions
About this article
Cite this article
Huang, D., Zhao, Q., Yang, K. et al. CRL2APPBP2-mediated TSPYL2 degradation counteracts human mesenchymal stem cell senescence. Sci. China Life Sci. 67, 460–474 (2024). https://doi.org/10.1007/s11427-023-2451-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11427-023-2451-3