Browsing by Author "Cui, Liwang"

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    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.
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    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.
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    Extensive lysine acetylation occurs in evolutionarily conserved metabolic pathways and parasite-specific functions during Plasmodium falciparum intraerythrocytic development
    (Wiley, 2013) Miao, Jun; Lawrence, Matthew; Jeffers, Victoria; Zhao, Fangqing; Parker, Daniel; Ge, Ying; Sullivan, William J., Jr.; Cui, Liwang; Pharmacology and Toxicology, School of Medicine
    Lysine acetylation has emerged as a major post-translational modification involved in diverse cellular functions. Using a combination of immunoisolation and liquid chromatography coupled to accurate mass spectrometry, we determined the first acetylome of the human malaria parasite Plasmodium falciparum during its active proliferation in erythrocytes with 421 acetylation sites identified in 230 proteins. Lysine-acetylated proteins are distributed in the nucleus, cytoplasm, mitochondrion and apicoplast. Whereas occurrence of lysine acetylation in a similarly wide range of cellular functions suggests conservation of lysine acetylation through evolution, the Plasmodium acetylome also revealed significant divergence from those of other eukaryotes and even the closely related parasite Toxoplasma. This divergence is reflected in the acetylation of a large number of Plasmodium-specific proteins and different acetylation sites in evolutionarily conserved acetylated proteins. A prominent example is the abundant acetylation of proteins in the glycolysis pathway but relatively deficient acetylation of enzymes in the citrate cycle. Using specific transgenic lines and inhibitors, we determined that the acetyltransferase PfMYST and lysine deacetylases play important roles in regulating the dynamics of cytoplasmic protein acetylation. The Plasmodium acetylome provides an exciting start point for further exploration of functions of acetylation in the biology of malaria parasites.