Succinylation at a key residue of FEN1 is involved in the DNA damage response to maintain genome stability.

Human flap endonuclease 1 (FEN1) is a structure-specific, multifunctional endonuclease essential for DNA replication and repair. Our previous study showed that in response to DNA damage, FEN1 interacts with the PCNA-like Rad9-Rad1-Hus1 complex instead of PCNA to engage in DNA repair activities, such as stalled DNA replication fork repair, and undergoes SUMOylation by SUMO-1. Here, we report that succinylation of FEN1 was stimulated in response to DNA replication fork-stalling agents, such as ultraviolet (UV) irradiation, hydroxyurea, camptothecin, and mitomycin C. K200 is a key succinylation site of FEN1 that is essential for gap endonuclease activity and could be suppressed by methylation and stimulated by phosphorylation to promote SUMO-1 modification. Succinylation at K200 of FEN1 promoted the interaction of FEN1 with the Rad9-Rad1-Hus1 complex to rescue stalled replication forks. Impairment of FEN1 succinylation led to the accumulation of DNA damage and heightened sensitivity to fork-stalling agents. Altogether, our findings suggest an important role of FEN1 succinylation in regulating its roles in DNA replication and repair, thus maintaining genome stability.

Structural basis of 5' flap recognition and protein-protein interactions of human flap endonuclease 1.

Human flap endonuclease 1 (hFEN1) is a structure-specific nuclease essential for DNA replication and repair processes. hFEN1 has 5' flap removal activity, as well as gap endonuclease activity that is critical for restarting stalled replication forks. Here, we report the crystal structures of wild-type and mutant hFEN1 proteins in complex with DNA substrates, followed by mutagenesis studies that provide mechanistic insight into the protein-protein interactions of hFEN1. We found that in an α-helix forming the helical gateway of hFEN1 recognizes the 5' flap prior to its threading into the active site for cleavage. We also found that the ß-pin region is rigidified into a short helix in R192F hFEN1-DNA structures, suppressing its gap endonuclease activity and cycle-dependent kinase interactions. Our findings suggest that a single mutation at the primary methylation site can alter the function of hFEN1 and provide insight into the role of the ß-pin region in hFEN1 protein interactions that are essential for DNA replication and repair.

VEGF-induced Nrdp1 deficiency in vascular endothelial cells promotes cancer metastasis by degrading vascular basement membrane.

Vascular endothelial cells (VECs) are key players in the formation of neovessels and tumor metastasis, the ultimate cause of the majority of cancer-related human death. However, the crosstalk between VECs and metastasis remain greatly elusive. Based on our finding that tumor-associated VECs present significant decrease of Nrdp1 protein which is closely correlated with higher metastatic probability, herein we show that the conditional medium from hypoxia-incubated cancer cells induces extensive Nrdp1 downregulation in human and mouse VECs by vascular endothelial growth factor (VEGF), which activates CHIP, followed by Nrdp1 degradation in ubiquitin-proteasome-dependent way. More importantly, lung metastases of cancer cells significantly increase in conditional VECs Nrdp1 knockout mice. Mechanically, Nrdp1 promotes degradation of Fam20C, a secretory kinase involved in phosphorylating numerous secreted proteins. Reciprocally, deficiency of Nrdp1 in VECs (ecNrdp1) results in increased secretion of Fam20C, which induces degradation of extracellular matrix and disrupts integrity of vascular basement membrane, thus driving tumor metastatic dissemination. In addition, specific overexpression of ecNrdp1 by Nrdp1-carrying adeno-associated virus or chemical Nrdp1 activator ABPN efficiently mitigates tumor metastasis in mice. Collectively, we explore a new mechanism for VEGF to enhance metastasis and role of Nrdp1 in maintaining the integrity of vascular endothelium, suggesting that ecNrdp1-mediated signaling pathways might become potential target for anti-metastatic therapies.

Application of Natural Medicinal Plants Active Ingredients in Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is the most common malignant cancer of the head and neck, with high morbidity and mortality, ranking as the sixth most common cancer in the world. The treatment of OSCC is mainly radiotherapy, chemotherapy and surgery, however, the prognosis of patients is still poor and the recurrence rate is high. This paper reviews the range of effects of natural medicinal plant active ingredients (NMPAIs) on OSCC cancer, including the types of NMPAIs, anti-cancer mechanisms, involved signaling pathways, and clinical trials. The NMPAIs include terpenoids, phenols, flavonoids, glycosides, alkaloids, coumarins, and volatile oils. These active ingredients inhibit proliferation, induce apoptosis and autophagy, inhibit migration and invasion of OSCC cells, and regulate cancer immunity to exert anti-cancer effects. The mechanism involves signaling pathways such as mitogen-activated protein kinase, phosphatidylinositol 3 kinase/protein kinase B, nuclear factor kappa B, miR-22/WNT1/ß-catenin and Nrf2/Keap1. Clinically, NMPAIs can inhibit the growth of OSCC, and the combined drug is more effective. Natural medicinal plants are promising candidates for the treatment of OSCC.

Post-translational Modifications by Acyl Groups Regulate FEN1's Activities and Play Essential Roles in Cell Proliferation and DNA Repair.

BACKGROUND: Flap endonuclease 1 (FEN1), well known for its structural-specific nuclease, possessing 5'-flap endonuclease and 5'-3' exonuclease activities, is mainly involved in DNA replication and repair. Protein lysine acetylation is an important posttranslational modification that could regulate numerous proteins' activity, subcellular localization, protein-protein interaction etc., and influences many biological processes. Our previous studies on integrated succinylome profiles found that succinylation and acetylation levels of FEN1 would change under different conditions. Succinylation at FEN1 Lys200 site results in the accumulation of damaged DNA and increased susceptibility to fork-stalling agents. The interplay with other forms of modification could affects its protein interaction affinity and thus contribute to genome stability. OBJECTIVE: This article studied the biological role of FEN1 by acyl modification in HeLa cells. METHOD: In order to explore the function of FEN1 acylation in cells, we mimicked the presence or absence of acetylation or succinylation by mutating key amino acids to glutamic acid and glutamine. We carried out a series of experiments including cell cycle, MTS, enzyme kinetics measurements, immunofluorescence and so on. RESULTS: The absence of acylation of FEN1 leads to the blocked cell cycle process and the reduced efficiency of FEN1 on its DNA substrates, affecting the interaction of FEN1 with both repair and replication related proteins and thus its role in the repair of DNA damage. CONCLUSION: We have verified acyl groups could modify Lys125, Lys252 and Lys254 of FEN1. Acylation level of these three is important for enzyme activity, cell proliferation and DNA damage response, thus contributing to genome stability.

CDK7 Inhibitor THZ1 Induces the Cell Apoptosis of B-Cell Acute Lymphocytic Leukemia by Perturbing Cellular Metabolism.

B-cell acute lymphocytic leukemia (B-ALL) is a malignant blood cancer that develops in children and adults and leads to high mortality. THZ1, a covalent cyclin-dependent kinase 7 (CDK7) inhibitor, shows anti-tumor effects in various cancers by inhibiting cell proliferation and inducing apoptosis. However, whether THZ1 has an inhibitory effect on B-ALL cells and the underlying mechanism remains obscure. In this study, we showed that THZ1 arrested the cell cycle of B-ALL cells in vitro in a low concentration, while inducing the apoptosis of B-ALL cells in vitro in a high concentration by activating the apoptotic pathways. In addition, RNA-SEQ results revealed that THZ1 disrupted the cellular metabolic pathways of B-ALL cells. Moreover, THZ1 suppressed the cellular metabolism and blocked the production of cellular metabolic intermediates in B-ALL cells. Mechanistically, THZ1 inhibited the cellular metabolism of B-ALL by downregulating the expression of c-MYC-mediated metabolic enzymes. However, THZ1 treatment enhanced cell apoptosis in over-expressed c-MYC B-ALL cells, which was involved in the upregulation of p53 expression. Collectively, our data demonstrated that CDK7 inhibitor THZ1 induced the apoptosis of B-ALL cells by perturbing c-MYC-mediated cellular metabolism, thereby providing a novel treatment option for B-ALL.

CDK9 Inhibitor Induces the Apoptosis of B-Cell Acute Lymphocytic Leukemia by Inhibiting c-Myc-Mediated Glycolytic Metabolism.

B-cell acute lymphocytic leukemia (B-ALL), a common blood cancer in children, leads to high mortality. Cyclin-dependent kinase 9 inhibitor (CDK9i) effectively attenuates acute myeloid leukemia and chronic lymphoblastic leukemia by inducing apoptosis and inhibiting cell proliferation. However, the effect of CDK9i on B-ALL cells and the underlying mechanisms remain unclear. In this study, we showed that CDK9i induced the apoptosis of B-ALL cells in vitro by activating the apoptotic pathways. In addition, CDK9i restrained the glycolytic metabolism of B-ALL cells, and CDK9i-induced apoptosis was enhanced by co-treatment with glycolysis inhibitors. Furthermore, CDK9i restained the glycolysis of B-ALL cell lines by markedly downregulating the expression of glucose transporter type 1 (GLUT1) and the key rate-limiting enzymes of glycolysis, such as hexokinase 2 (HK2) and lactate dehydrogenase A (LDHA). Moreover, cell apoptosis was rescued in B-ALL cells with over-expressed c-Myc after treatment with CDK9i, which is involved in the enhancement of glycolytic metabolism. In summary, our findings suggest that CDK9 inhibitors induce the apoptosis of B-ALL cells by inhibiting c-Myc-mediated glycolytic metabolism, thus providing a new strategy for the treatment of B-ALL.

SUMO-1 modification of FEN1 facilitates its interaction with Rad9-Rad1-Hus1 to counteract DNA replication stress.

Human flap endonuclease 1 (FEN1) is a structure-specific, multi-functional endonuclease essential for DNA replication and repair. We and others have shown that during DNA replication, FEN1 processes Okazaki fragments via its interaction with the proliferating cell nuclear antigen (PCNA). Alternatively, in response to DNA damage, FEN1 interacts with the PCNA-like Rad9-Rad1-Hus1 complex instead of PCNA to engage in DNA repair activities, such as homology-directed repair of stalled DNA replication forks. However, it is unclear how FEN1 is able to switch between these interactions and its roles in DNA replication and DNA repair. Here, we report that FEN1 undergoes SUMOylation by SUMO-1 in response to DNA replication fork-stalling agents, such as UV irradiation, hydroxyurea, and mitomycin C. This DNA damage-induced SUMO-1 modification promotes the interaction of FEN1 with the Rad9-Rad1-Hus1 complex. Furthermore, we found that FEN1 mutations that prevent its SUMO-1 modification also impair its ability to interact with HUS1 and to rescue stalled replication forks. These impairments lead to the accumulation of DNA damage and heightened sensitivity to fork-stalling agents. Altogether, our findings suggest an important role of the SUMO-1 modification of FEN1 in regulating its roles in DNA replication and repair.

Mass spectrometry-based quantification of the cellular response to ultraviolet radiation in HeLa cells.

Ultraviolet (UV) irradiation is a common form of DNA damage that can cause pyrimidine dimers between DNA, which can cause gene mutations, even double-strand breaks and threaten genome stability. If DNA repair systems default their roles at this stage, the organism can be damaged and result in disease, especially cancer. To better understand the cellular response to this form of damage, we applied highly sensitive mass spectrometry to perform comparative proteomics of phosphorylation in HeLa cells. A total of 4367 phosphorylation sites in 2100 proteins were identified, many of which had not been reported previously. Comprehensive bioinformatics analysis revealed that these proteins were involved in many important biological processes, including signaling, localization and cell cycle regulation. The nuclear pore complex, which is very important for RNA transport, was changed significantly at phosphorylation level, indicating its important role in response to UV-induced cellular stress. Protein-protein interaction network analysis and DNA repair pathways crosstalk were also examined in this study. Proteins involved in base excision repair, nucleotide repair and mismatch repair changed their phosphorylation pattern in response to UV treatment, indicating the complexity of cellular events and the coordination of these pathways. These systematic analyses provided new clues of protein phosphorylation in response to specific DNA damage, which is very important for further investigation. And give macroscopic view on an overall phosphorylation situation under UV radiation.

Corrigendum: Lysine Acetylation and Succinylation in HeLa Cells and their Essential Roles in Response to UV-induced Stress.

This corrects the article DOI: 10.1038/srep30212.

Lysine Acetylation and Succinylation in HeLa Cells and their Essential Roles in Response to UV-induced Stress.

Lysine acetylation and succinylation are major types of protein acylation that are important in many cellular processes including gene transcription, cellular metabolism, DNA damage response. Malfunctions in these post-translational modifications are associated with genome instability and disease in higher organisms. In this study, we used high-resolution nano liquid chromatography-tandem mass spectrometry combined with affinity purification to quantify the dynamic changes of protein acetylation and succinylation in response to ultraviolet (UV)-induced cell stress. A total of 3345 acetylation sites in 1440 proteins and 567 succinylation sites in 246 proteins were identified, many of which have not been reported previously. Bioinformatics analysis revealed that these proteins are involved in many important biological processes, including cell signalling transduction, protein localization and cell metabolism. Crosstalk analysis between these two modifications indicated that modification switches might regulate protein function in response to UV-induced DNA damage. We further illustrated that FEN1 acetylation at different sites could lead to different cellular phenotypes, suggesting the multiple function involvement of FEN1 acetylation under DNA damage stress. These systematic analyses provided valuable resources and new insight into the potential role of lysine acetylation and succinylation under physiological and pathological conditions.