Blocking protein degradation at the level of the proteasome leads to accumulation of intracellular protein that elicits ER stress and an unfolded protein response. Thus, ER stress may be functionally important for cell death induced by E1 inhibition. The next day, cells were treated with increasing concentrations of PYZD for 24 hours. Cell growth and viability was determined by the Alamar Blue assay. In cultured cells, inhibition of the E1 enzyme preferentially induced cell death in malignant cells over normal cells.
Therefore, we explored the effects of E1 inhibition in a mouse model of leukemia. Sixteen days after tumor implantation, the mice were killed, and the tumors excised and weighed. Compared with control-treated mice, treatment with PYZD delayed tumor growth and decreased tumor weight Figure 7 without untoward toxicity. Thus, inhibition of the E1 can achieve an antitumor effect in vivo. PYZD delays tumor growth in mouse model of leukemia. Tumor growth was monitored at least every other day by external calipers.
B At 16 days after tumor injection, mice were killed, and tumors were excised and weighed. Inhibition of this pathway with proteasome inhibitors is a well-established strategy for the treatment of malignancies such as multiple myeloma and mantle cell lymphoma. Here, we evaluated the effects of inhibiting the ubiquitination pathway in malignant cells genetically and chemically at the level of the E1 enzyme.
We demonstrated that leukemia cell lines and primary patient samples have increased levels of ubiquitinated proteins and that this increase could not be explained by a reduction in protein degradation.
To the best of our knowledge, the activity of the ubiquitination cascade has not been previously compared between normal and malignant hematopoietic cells. Potentially, the increased ubiquitination activity may reflect the higher metabolic rate required to support the growth of malignant cells. Because levels of the E1 protein did not differ between normal and malignant cells, it suggests that the E1 enzyme is more actively used in malignant cells. Thus, these observations suggest that malignant cells are more dependent on E1 activity and provide a rationale for targeting the E1 enzyme as an anticancer strategy.
Our studies also showed that malignant cells have increased activity of the proteasomal enzymes. This result is in keeping with prior studies that have shown increased abundance of proteasomal enzymes in malignant cells compared with normal cells, 23 , 24 thereby providing a biologic rationale for the development of proteasome inhibitors as anticancer agents.
Given that malignant cells may be more dependent on the E1 enzyme activity compared with normal cells, we examined genetic and chemical inhibition of this target. Knockdown of the E1 protein with shRNA induced cell death in malignant cell lines.
However, genetic knockout of the E1 protein was technically difficult in primary cells and mouse models of malignancy. Therefore, we developed PYZD as a chemical inhibitor of the E1 enzyme to better understand the effects of E1 inhibition in malignancy.
PYZD inhibited the activity of the E1 enzyme in cell-free systems and also blocked the E1 activity when added to cultured cells. PYZD is similar in structure to 4[4- 5-nitro-furanylmethylene -3,5-dioxo-pyrazolidinyl]-benzoic acid ethyl ester PYR that was recently identified as an E1 inhibitor in cell-free and cell-based assays.
That study also did not explore the effects of E1 inhibition on ER stress. Despite the similarities in structures, we cannot exclude that PYZD has additional targets beyond the E1 enzyme that may contribute to its cytotoxic effects. Although a useful chemical probe, the micromolar potency of PYZD is suboptimal and efforts are under way to identify chemical E1 inhibitors with improved potency. Inhibiting the E1 enzyme increased the abundance of short half-life proteins such as p However, cell death after E1 inhibition did not appear specifically dependent on the accumulation of a single protein such as p Rather, we demonstrated that E1 inhibition induced cell death through a mechanism linked to ER stress, suggesting a more global effect because of the general accumulation of intracellular proteins.
The specific effectors linking E1 inhibition to ER stress are uncertain and will be explored in future studies. However, there are probably similarities to proteasome inhibition. Several studies have observed ER stress and unfolded protein response after proteasome inhibition, but the mechanism is not fully understood.
Potentially, the accumulation of excess proteins in the cytoplasm inhibits retrograde protein translocation from the ER to the cytoplasm, 25 leading to ER stress and the unfolded protein response. BI-1 is a protein localized to the ER membrane and its overexpression protects cells from death by ER stress signals but not stimuli of the death receptor pathway of caspase activation.
These results coupled with the increase in ER stress proteins supports a mechanism of cell death linked to ER stress. However, additional studies to confirm this observation would be important. Further studies are also required to understand fully how ER stress after E1 inhibition leads to cell death. Proteins that are ubiquitinated with Klinked chains are specifically recognized by the 26S proteasome and subjected to degradation.
In contrast, proteins that are tagged with a single ubiquitin residue monoubiquitination or polyubiquitin chains formed by linking ubiquitin molecules through K residues K ubiquitination do not mark the protein for degradation.
Rather, K polyubiquitination or monoubiquitination alters protein localization and function. In our study, the effects of E1 inhibition that we observed appeared related to the accumulation of excess proteins.
However, we cannot exclude that inhibition of the E1 enzyme also leads to more subtle changes related to inhibition of monoubiquitination and K polyubiquitination.
Although the cellular effects of E1 inhibition and proteasome inhibition appear similar, the difference in targets suggests that E1 inhibition may overcome some forms of resistance to proteasome inhibitors. Thus, E1 inhibitors could be potentially useful therapeutic agents for some patients who are resistant to proteasome inhibitors. In summary, we demonstrated that malignant cell lines and primary patient samples have increased activity of the ubiquitination pathway and that blocking this pathway with chemical or genetic inhibitors of the E1 enzyme induces ER stress and is preferentially cytotoxic to malignant cells.
Thus, this work highlights the E1 enzyme as a new therapeutic target for the treatment of malignancy. The publication costs of this article were defrayed in part by page charge payment. Contribution: G. All authors reviewed and edited the paper. Correspondence: Aaron D. Sign In or Create an Account. Sign In. Skip Nav Destination Content Menu. Close Abstract. Article Navigation. The ubiquitin-activating enzyme E1 as a therapeutic target for the treatment of leukemia and multiple myeloma G.
Wei Xu , G. Wei Xu. This Site. Google Scholar. Mohsin Ali , Mohsin Ali. Tabitha E. Wood , Tabitha E. Derek Wong , Derek Wong.
Neil Maclean , Neil Maclean. Xiaoming Wang , Xiaoming Wang. Marcela Gronda , Marcela Gronda. Marko Skrtic , Marko Skrtic. Xiaoming Li , Xiaoming Li. Rose Hurren , Rose Hurren. Xinliang Mao , Xinliang Mao. The significance level was set at 0. Further information on research design is available in the Nature Research Reporting Summary linked to this article.
The source data underlying Figs. All other data supporting the findings of this study are available from the corresponding author upon reasonable request. Dutcher, S. The role of S. Cerevisiae cell division cycle genes in nuclear fusion.
Genetics , — Goebl, M. The yeast cell cycle gene CDC34 encodes a ubiquitin-conjugating enzyme. Science , — Haas, A. Ubiquitin-activating enzyme. CAS Google Scholar. Mathias, N. Cdc53p acts in concert with Cdc4p and Cdc34p to control the G1-to-S-phase transition and identifies a conserved family of proteins. Pagano, M. Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p Tam, S.
Kip1 degradation via the ubiquitin-proteasome pathway. Leukemia 11 , — PubMed Google Scholar. Verma, R. Cell 8 , — Henchoz, S. Phosphorylation- and ubiquitin-dependent degradation of the cyclin-dependent kinase inhibitor Far1p in budding yeast. Genes Dev. Tanaka, K. Acta BBA - Mol. Basis Dis. Takagi, K. Cell division cycle 34 is highly expressed in hepatitis C virus-positive hepatocellular carcinoma with favorable phenotypes.
Eliseeva, E. Expression and localization of the CDC34 ubiquitin-conjugating enzyme in pediatric acute lymphoblastic leukemia. Cell Growth Differ. Zeng, Y. Cancer 8 , 19 Article Google Scholar. Price, G. Phenotype-directed analysis of genotype in early-onset, familial breast cancers. Pathology 38 , — Macdonald, M. Control of cell cycle-dependent degradation of c-Ski proto-oncoprotein by Cdc Oncogene 23 , — Charrasse, S.
Oncogene 19 , — Chauhan, D. Wei, Y. Cdcmediated degradation of ATF5 is blocked by cisplatin. Ceccarelli, D. An allosteric inhibitor of the human Cdc34 ubiquitin-conjugating enzyme. Cell , — Wu, K. Natl Acad. Liu, Y. Small molecule therapeutics targeting F-box proteins in cancer. Cancer Biol. Hershko, A.
Immunochemical analysis of the turnover of ubiquitin-protein conjugates in intact cells. Relationship to the breakdown of abnormal proteins. Lv, Z. Domain alternation and active site remodeling are conserved structural features of ubiquitin E1. Lake, M. Mechanism of ubiquitin activation revealed by the structure of a bacterial MoeB-MoaD complex.
Nature , — Lois, L. EMBO J. Walden, H. Cell 12 , — Crystal structure of a human ubiquitin E1—ubiquitin complex reveals conserved functional elements essential for activity. Szczepanowski, R. Crystal structure of a fragment of mouse ubiquitin-activating enzyme.
Huang, D. Cell 17 , — Basis for a ubiquitin-like protein thioester switch toggling E1-E2 affinity. Tokgoz, Z. E1-E2 interactions in ubiquitin and Nedd8 ligation pathways. Olsen, S. Structure of a ubiquitin E1-E2 complex: insights to E1-E2 thioester transfer.
Cell 49 , — Cell 65 , — e6 Seol, J. Kamura, T. Schulman, B. Zheng, N. Hamilton, K. Structure of a conjugating enzyme-ubiquitin thiolester intermediate reveals a novel role for the ubiquitin tail. Structure 9 , — Kamadurai, H. Cell 36 , — Page, R. Biochemistry 51 , — Wiener, R. The mechanism of OTUB1-mediated inhibition of ubiquitination. Middleton, A. The molecular basis of lysine 48 ubiquitin chain synthesis by Ube2K.
Pruneda, J. Cell 47 , — Dou, H. Kleiger, G. The acidic tail of the Cdc34 ubiquitin-conjugating enzyme functions in both binding to and catalysis with ubiquitin ligase SCFCdc4. Ptak, C. Identification of a functional determinant within the tail that facilitates CDC34 self-association. Spratt, D. Association of the disordered C-terminus of CDC34 with a catalytically bound ubiquitin.
Choi, Y. The human Cdc34 carboxyl terminus contains a non-covalent ubiquitin binding activity that contributes to SCF-dependent ubiquitination. Huang, H. E2 enzyme inhibition by stabilization of a low-affinity interface with ubiquitin. Lee, I. Structural insights into E1-catalyzed ubiquitin activation and transfer to conjugating enzymes.
Pitluk, Z. Kolman, C. Identification of a portable determinant of cell cycle function within the carboxyl-terminal domain of the yeast CDC34 UBC3 ubiquitin conjugating E2 enzyme. Sandoval, D. Ubiquitin-conjugating enzyme Cdc34 and ubiquitin ligase Skp1-cullin-F-box ligase SCF interact through multiple conformations. Yuan, L. Patel, A. Wickliffe, K. The mechanism of linkage-specific ubiquitin chain elongation by a single-subunit E2.
Suryadinata, R. Molecular and structural insight into lysine selection on substrate and ubiquitin lysine 48 by the ubiquitin-conjugating enzyme Cdc Cell Cycle 12 , — Petroski, M.
Saha, A. Essential role for ubiquitin-ubiquitin-conjugating enzyme interaction in ubiquitin discharge from Cdc34 to substrate. Cell 42 , 75—83 Hill, S. However, the transport sulfation process is complex because both E1 and E2 enzymes form intermediate complexes, both of which undergo a series of conformational changes to bind to each other. UBA1 participates in ubiquitination and the NEDD8 pathway for protein folding and degradation, among many other biological processes.
This protein has been linked to X-linked spinal muscular atrophy type 2, neurodegenerative diseases, and cancers. NEDD8 is a ubiquitin-like protein, which regulates cell division, signaling and embryogenesis.
Ubiquitin-like modifier-activating enzyme 5 is a protein that in humans is encoded by the UBA5 gene. This gene encodes a member of the E1-like activating enzyme family. Two alternatively spliced transcript variants encoding distinct isoforms have been found for this gene. Ubiquitin-like modifier-activating enzyme 7 is a protein that in humans is encoded by the UBA7 gene. This gene encodes a member of the E1 ubiquitin-activating enzyme family. Autophagy-related protein 7 is a protein in humans encoded by ATG7 gene.
ATG7, present in both plant and animal genomes, acts as an essential protein for cell degradation and its recycling. The sequence associates with the ubiquitin- proteasome system, UPS, required for the unique development of an autophagosomal membrane and fusion within cells. The protein encoded by this gene binds to the beta-amyloid precursor protein.
0コメント