Browsing by Author "Liu, Min"

Now showing 1 - 5 of 5
  • Thumbnail Image
    Item
    Characterization of protein arginine methyltransferase of TgPRMT5 in Toxoplasma gondii
    (Springer Nature, 2019-05-08) Liu, Min; Li, Fen-Xiang; Li, Chun-Yuan; Li, Xiao-Cong; Chen, Long-Fei; Wu, Kun; Yang, Pei-Liang; Lai, Zhi-Fa; Liu, Ting-kai; Sullivan, William J., Jr.; Cui, Liwang; Chen, Xiao-Guang; Pharmacology and Toxicology, School of Medicine
    BACKGROUND: Protein arginine methylation is a prevalent post-translational modification. The protein arginine methyltransferase family (PRMT) is involved in many cellular processes in eukaryotes, including transcriptional regulation, epigenetic regulation, RNA metabolism, and DNA damage repair. Toxoplasma gondii, an opportunistic protozoan parasite, encodes five conserved PRMTs. PRMT5 is thought to be responsible for substantial PRMT activity in T. gondii; however, it has not yet been characterized. METHODS: We tagged the 3' end of the endogenous TgPRMT5 genomic locus with sequence encoding a 3X hemagglutinin (HA) epitope. IFA and WB were performed to check the expression and subcellular localization of TgPRMT5 in tachyzoites and bradyzoites. In vitro methylation assays were performed to determine whether endogenous TgPRMT5 has arginine methyltransferase activity. RESULTS: IFA and WB results showed that T. gondii PRMT5 (TgPRMT5) was localized in the cytoplasm in the tachyzoite stage; however, it shifts largely to the nuclear compartment in the bradyzoite stage. The in vitro methylation showed that TgPRMT5 has authentic type II PRMT activity and forms monomethylarginines and symmetric dimethylarginines. CONCLUSIONS: We determined the expression and cellular localization of TgPRMT5 in tachyzoites and bradyzoites and confirmed its type II PRMT activity. We demonstrated the major changes in expression and cellular localization of TgPRMT5 during the tachyzoite and bradyzoite stages in T. gondii. Our findings suggest that TgPRMT5 protein may be involved in tachyzoite-bradyzoite transformation.
  • Thumbnail Image
    Item
    Characterization of TgPuf1, a member of the Puf family RNA-binding proteins from Toxoplasma gondii
    (Springer Nature, 2014-03-31) Liu, Min; Miao, Jun; Liu, Tingkai; Sullivan, William J., Jr.; Cui, Liwang; Chen, Xiaoguang; Pharmacology and Toxicology, School of Medicine
    Background: Puf proteins act as translational regulators and affect many cellular processes in a wide range of eukaryotic organisms. Although Puf proteins have been well characterized in many model systems, little is known about the structural and functional characteristics of Puf proteins in the parasite Toxoplasma gondii. Methods: Using a combination of conventional molecular approaches, we generated endogenous TgPuf1 tagged with hemagglutinin (HA) epitope and investigated the TgPuf1 expression levels and localization in the tachyzoites and bradyzoites. We used RNA Electrophoretic Mobility Shfit Assay (EMSA) to determine whether the recombination TgPuf1 has conserverd RNA binding activity and specificity. Results: TgPuf1 was expressed at a significantly higher level in bradyzoites than in tachyzoites. TgPuf1 protein was predominantly localized within the cytoplasm and showed a much more granular cytoplasmic staining pattern in bradyzoites. The recombinant Puf domain of TgPuf1 showed strong binding affinity to two RNA fragments containing Puf-binding motifs from other organisms as artificial target sequences. However, two point mutations in the core Puf-binding motif resulted in a significant reduction in binding affinity, indicating that TgPuf1 also binds to conserved Puf-binding motif. Conclusions: TgPuf1 appears to exhibit different expression levels in the tachyzoites and bradyzoites, suggesting that TgPuf1 may function in regulating the proliferation or/and differentiation that are important in providing parasites with the ability to respond rapidly to changes in environmental conditions. This study provides a starting point for elucidating the function of TgPuf1 during parasite development.
  • Thumbnail Image
    Item
    The protective role of DOT1L in UV-induced melanomagenesis
    (Nature Publishing Group, 2018-01-17) Zhu, Bo; Chen, Shuyang; Wang, Hongshen; Yin, Chengqian; Han, Changpeng; Peng, Cong; Liu, Zhaoqian; Wan, Lixin; Zhang, Zhang; Zhang, Jie; Lian, Christine G.; Ma, Peilin; Xu, Zhi-xiang; Prince, Sharon; Wang, Tao; Gao, Xiumei; Shi, Yujiang; Liu, Dali; Liu, Min; Wei, Wenyi; Wei, Zhi; Pan, Jingxuan; Wang, Yongjun; Xuan, Zhenyu; Hess, Jay L.; Hayward, Nicholas K.; Goding, Colin R.; Chen, Xiang; Zhou, Jun; Cui, Rutao; Pathology and Laboratory Medicine, School of Medicine
    The DOT1L histone H3 lysine 79 (H3K79) methyltransferase plays an oncogenic role in MLL-rearranged leukemogenesis. Here, we demonstrate that, in contrast to MLL-rearranged leukemia, DOT1L plays a protective role in ultraviolet radiation (UVR)-induced melanoma development. Specifically, the DOT1L gene is located in a frequently deleted region and undergoes somatic mutation in human melanoma. Specific mutations functionally compromise DOT1L methyltransferase enzyme activity leading to reduced H3K79 methylation. Importantly, in the absence of DOT1L, UVR-induced DNA damage is inefficiently repaired, so that DOT1L loss promotes melanoma development in mice after exposure to UVR. Mechanistically, DOT1L facilitates DNA damage repair, with DOT1L-methylated H3K79 involvement in binding and recruiting XPC to the DNA damage site for nucleotide excision repair (NER). This study indicates that DOT1L plays a protective role in UVR-induced melanomagenesis.
  • Thumbnail Image
    Item
    Small Molecules Engage Hot Spots through Cooperative Binding To Inhibit a Tight Protein–Protein Interaction
    (ACS, 2017-03) Liu, Degang; Xu, David; Liu, Min; Knabe, William Eric; Yuan, Cai; Zhou, Donghui; Huang, Mingdong; Meroueh, Samy O.; Biochemistry and Molecular Biology, School of Medicine
    Protein–protein interactions drive every aspect of cell signaling, yet only a few small-molecule inhibitors of these interactions exist. Despite our ability to identify critical residues known as hot spots, little is known about how to effectively engage them to disrupt protein–protein interactions. Here, we take advantage of the ease of preparation and stability of pyrrolinone 1, a small-molecule inhibitor of the tight interaction between the urokinase receptor (uPAR) and its binding partner, the urokinase-type plasminogen activator uPA, to synthesize more than 40 derivatives and explore their effect on the protein–protein interaction. We report the crystal structure of uPAR bound to previously discovered pyrazole 3 and to pyrrolinone 12. While both 3 and 12 bind to uPAR and compete with a fluorescently labeled peptide probe, only 12 and its derivatives inhibit the full uPAR·uPA interaction. Compounds 3 and 12 mimic and engage different hot-spot residues on uPA and uPAR, respectively. Interestingly, 12 is involved in a π–cation interaction with Arg-53, which is not considered a hot spot. Explicit-solvent molecular dynamics simulations reveal that 3 and 12 exhibit dramatically different correlations of motion with residues on uPAR. Free energy calculations for the wild-type and mutant uPAR bound to uPA or 12 show that Arg-53 interacts with uPA or with 12 in a highly cooperative manner, thereby altering the contributions of hot spots to uPAR binding. The direct engagement of peripheral residues not considered hot spots through π–cation or salt-bridge interactions could provide new opportunities for enhanced small-molecule engagement of hot spots to disrupt challenging protein–protein interactions.
  • Thumbnail Image
    Item
    Transforming Growth Factor-induced Protein Promotes NF-κB-mediated Angiogenesis during Postnatal Lung Development
    (American Thoracic Society, 2021) Liu, Min; Iosef, Cristiana; Rao, Shailaja; Domingo-Gonzalez, Racquel; Fu, Sha; Snider, Paige; Conway, Simon J.; Umbach, Gray S.; Heilshorn, Sarah C.; Dewi, Ruby E.; Dahl, Mar J.; Null, Donald M.; Albertine, Kurt H.; Alvira, Cristina M.; Pediatrics, School of Medicine
    Pulmonary angiogenesis is a key driver of alveolarization. Our prior studies showed that NF-κB promotes pulmonary angiogenesis during early alveolarization. However, the mechanisms regulating temporal-specific NF-κB activation in the pulmonary vasculature are unknown. To identify mechanisms that activate proangiogenic NF-κB signaling in the developing pulmonary vasculature, proteomic analysis of the lung secretome was performed using two-dimensional difference gel electrophoresis. NF-κB activation and angiogenic function was assessed in primary pulmonary endothelial cells (PECs) and TGFBI (transforming growth factor-β-induced protein)-regulated genes identified using RNA sequencing. Alveolarization and pulmonary angiogenesis was assessed in wild-type and Tgfbi null mice exposed to normoxia or hyperoxia. Lung TGFBI expression was determined in premature lambs supported by invasive and noninvasive respiratory support. Secreted factors from the early alveolar, but not the late alveolar or adult lung, promoted proliferation and migration in quiescent, adult PECs. Proteomic analysis identified TGFBI as one protein highly expressed by the early alveolar lung that promoted PEC migration by activating NF-κB via αvβ3 integrins. RNA sequencing identified Csf3 as a TGFBI-regulated gene that enhances nitric oxide production in PECs. Loss of TGFBI in mice exaggerated the impaired pulmonary angiogenesis induced by chronic hyperoxia, and TGFBI expression was disrupted in premature lambs with impaired alveolarization. Our studies identify TGFBI as a developmentally regulated protein that promotes NF-κB-mediated angiogenesis during early alveolarization by enhancing nitric oxide production. We speculate that dysregulation of TGFBI expression may contribute to diseases marked by impaired alveolar and vascular growth.