Crystal structure of the human PRMT5:MEP50 complex Antonysamy, Stephen; Bonday, Zahid; Campbell, Robert M ...
Proceedings of the National Academy of Sciences,
10/2012, Letnik:
109, Številka:
44
Journal Article
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Protein arginine methyltransferases (PRMTs) play important roles in several cellular processes, including signaling, gene regulation, and transport of proteins and nucleic acids, to impact growth, ...differentiation, proliferation, and development. PRMT5 symmetrically di-methylates the two-terminal ω-guanidino nitrogens of arginine residues on substrate proteins. PRMT5 acts as part of a multimeric complex in concert with a variety of partner proteins that regulate its function and specificity. A core component of these complexes is the WD40 protein MEP50/WDR77/p44, which mediates interactions with binding partners and substrates. We have determined the crystal structure of human PRMT5 in complex with MEP50 (methylosome protein 50), bound to an S-adenosylmethionine analog and a peptide substrate derived from histone H4. The structure of the surprising hetero-octameric complex reveals the close interaction between the seven-bladed β-propeller MEP50 and the N-terminal domain of PRMT5, and delineates the structural elements of substrate recognition.
After feeding, adipose tissue lipoprotein lipase (LPL) activity should be maximized, therefore the potent LPL-inhibitory activity of angiopoietin-like protein 4 (ANGPTL4) must be blocked by ANGPTL8 ...through formation of ANGPTL4/8 complexes. ANGPTL4/8 tightly binds and protects LPL but also partially inhibits its activity. Recently, we demonstrated ANGPTL4/8 also binds tissue plasminogen activator (tPA) and plasminogen to generate plasmin that cleaves ANGPTL4/8 to restore LPL activity. Although fully active LPL in the fat postprandially is desirable, ANGPTL4/8 removal could subject LPL to profound inhibition by ANGPTL3/8 (the most potent circulating LPL inhibitor), inhibition by other LPL inhibitors like ANGPTL4, ANGPTL3, and ApoC3 or interfere with ApoC2-mediated LPL activation. To understand better these potential paradoxes, we examined LPL inhibition by ANGPTL3/8, ANGPTL4, ANGPTL3, and ApoC3 and LPL stimulation by ApoC2 in the presence of ANGPTL4/8 + tPA + plasminogen. Remarkably, ANGPTL3/8-mediated LPL inhibition was almost completely blocked, with the mechanism being cleavage of fibrinogen-like domain–containing ANGPTL3 present in the ANGPTL3/8 complex. The LPL-inhibitory effects of ANGPTL4, ANGPTL3, and ApoC3 were also largely reduced in the presence of ANGPTL4/8 + tPA + plasminogen. In contrast, the ability of ApoC2 to stimulate LPL activity was unaffected by ANGPTL4/8-mediated plasmin generation. Together, these results explain how plasmin generated by increased postprandial ANGPTL4/8 levels in adipose tissue enables maximal LPL activity by preventing ANGPTL3/8, ANGPTL4, ANGPTL3, and ApoC3 from inhibiting LPL, while permitting ApoC2-mediated LPL activation to occur.
The sirtuin SIRT1 is a NAD+-dependent histone deacetylase, a Sir2 family member, and one of seven human sirtuins. Sirtuins are conserved from archaea to mammals and regulate transcription, genome ...stability, longevity, and metabolism. SIRT1 regulates transcription via deacetylation of transcription factors such as PPARγ, NFκB, and the tumor suppressor protein p53. EX527 (27) is a nanomolar SIRT1 inhibitor and a micromolar SIRT2 inhibitor. To elucidate the mechanism of SIRT inhibition by 27, we determined the 2.5 Å crystal structure of the SIRT1 catalytic domain (residues 241–516) bound to NAD+ and the 27 analogue compound 35. 35 binds deep in the catalytic cleft, displacing the NAD+ nicotinamide and forcing the cofactor into an extended conformation. The extended NAD+ conformation sterically prevents substrate binding. The SIRT1/NAD+/35 crystal structure defines a novel mechanism of histone deacetylase inhibition and provides a basis for understanding, and rationally improving, inhibition of this therapeutically important target by drug-like molecules.
Posttranslational modifications of histone tails are a key contributor to epigenetic regulation. Histone H3 Arg26 and Lys27 are both modified by multiple enzymes, and their modifications have ...profound effects on gene expression. Citrullination of H3R26 by PAD2 and methylation of H3K27 by PRC2 have opposing downstream impacts on gene regulation; H3R26 citrullination activates gene expression, and H3K27 methylation represses gene expression. Both of these modifications are drivers of a variety of cancers, and their writer enzymes, PAD2 and EZH2, are the targets of drug therapies. After biochemical and cell-based analysis of these modifications, a negative crosstalk interaction is observed. Methylation of H3K27 slows citrullination of H3R26 30-fold, whereas citrullination of H3R26 slows methylation 30,000-fold. Examination of the mechanism of this crosstalk interaction uncovered a change in structure of the histone tail upon citrullination which prevents methylation by the PRC2 complex. This mechanism of crosstalk is reiterated in cell lines using knockdowns and inhibitors of both enzymes. Based our data, we propose a model in which, after H3 Cit26 formation, H3K27 demethylases are recruited to the chromatin to activate transcription. In total, our studies support the existence of crosstalk between citrullination of H3R26 and methylation of H3K27.
The present study aimed at making a rational usage for European eel bone by-products by preparing Europen eel bone peptide chelated calcium (EBPC-Ca). Nutritional properties and bioactivity of ...EBPC-Ca were evaluated. Results showed that nutritional properties of calcium ions will cause intra- and inter-molecular folding and aggregation of peptide to uniformly form EBPC-Ca chelate. The chelated compound of EBPC and calcium ion triggered a strong apoptosis in heterogeneous human epithelial colorectal adenocarcinoma (Caco-2) in concentration- and time-dependent manners. Western blot analysis revealed that the EBPC-Ca induced apoptosis may be the result of a blocked autophagy flux through mitochondrial-dependent pathway. Additionally, the increase in FGF-23 protein expression inhibited the absorption of calcium ions and alleviated cell apoptosis. It was also found that the cell apoptosis occurs with significant increases in the levels of reactive oxygen species (ROS) and Ca2+ in the cells, indicating the anti-tumor potential of EBPC-Ca may involve multiple channels.
Hundreds of target specific peptides are routinely discovered by peptide display platforms. However, due to the high cost of peptide synthesis only a limited number of peptides are chemically made ...for further analysis. Here we describe an accurate and cost effective method to bin peptides on-phage based on binding region(s), without any requirement for peptide or protein synthesis. This approach, which integrates phage and yeast display platforms, requires display of target and its alanine variants on yeast. Flow cytometry was used to detect binding of peptides on-phage to the target on yeast. Once hits were identified, they were synthesized to confirm their binding region(s) by HDX (Hydrogen deuterium exchange) and crystallography. Moreover, we have successfully shown that this approach can be implemented as part of a panning process to deplete non-functional peptides. This technique can be applied to any target that can be successfully displayed on yeast; it narrows down the number of peptides requiring synthesis; and its utilization during selection results in enrichment of peptide population against defined binding regions on the target.
DNA-dependent RNA polymerase II (RNAP II) largest subunit RPB1 C-terminal domain (CTD) kinases, including CDK9, are serine/threonine kinases known to regulate transcriptional initiation and ...elongation by phosphorylating Ser 2, 5, and 7 residues on CTD. Given the reported dysregulation of these kinases in some cancers, we asked whether inhibiting CDK9 may induce stress response and preferentially kill tumor cells. Herein, we describe a potent CDK9 inhibitor, LY2857785, that significantly reduces RNAP II CTD phosphorylation and dramatically decreases MCL1 protein levels to result in apoptosis in a variety of leukemia and solid tumor cell lines. This molecule inhibits the growth of a broad panel of cancer cell lines, and is particularly efficacious in leukemia cells, including orthotopic leukemia preclinical models as well as in ex vivo acute myeloid leukemia and chronic lymphocytic leukemia patient tumor samples. Thus, inhibition of CDK9 may represent an interesting approach as a cancer therapeutic target, especially in hematologic malignancies.
The extracts of S. oleraceus Linn (SOL) and its main phenolic compounds have shown anti-diabetic effects, but their underlying mechanisms for glucose homeostasis remain unclear. The aim of this study ...is to evaluate the anti-diabetic mechanism of SOL by using the streptozocin (STZ) induced diabetic rat model. When diabetic rats were fed with SOL at a dose of 400 mg/kg/day for 6 weeks, the concentrations of triglycerides (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) were reduced by 43%, 22%, and 16%, respectively. Meanwhile, it was also found that daily feeding of SOL to diabetic rats led to a decrease in plasma glucose level by approximately 23%. Positive effects were observed on glucose homeostasis due to the down-regulation of AMPK/Akt/GSK-3β pathway, as indicated by the suppressions of adenosine 5‘-monophosphate (AMP)-activated protein kinase (AMPK), protein kinase (Akt) phosphorylation, glycogen synthase kinase 3 beta (GSK-3β), and the hepatic insulin resistance. In HepG2 cells, AMPK, Akt and GSK-3β showed a consistent transcript regulation. SOL at dose of 400 mg/kg/day feeding for 6 weeks showed a positive effect comparable to metformin.
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•Major phenolics in S oleraceus Linn (SOL) was found to have intestinal stability.•SOL represented a very effective role in high blood glucose.•SOL regulated AMPK/Akt/GSK-3β signaling pathway in diabetic rat and HepG2.
•Lotus leaf oil could effectively inhibit melanin production.•Lotus leaf oil might be used as a potent tyrosinase inhibitor against melanoma.•Lotus leaf oil induces in B16 cells apoptosis.
This study ...was aimed at determining the influence of Folium nelumbinis (Lotus leaf) extracts on melanogenesis in vitro models of melanoma cell line. The anticancer activity of four fractions, including petroleum ether (PEE), n-hexane (HE), ethanol (EE), and ethyl acetate (EAE) from F. nelumbinis on B16 cell lines (C57BL/6J melanoma cell), were evaluated after 24 and 48 h treatment. Results showed that PEE as well as volatile-rich fractions of linolenic acid and linolenic acid ethyl ester significantly (p < 0.05) reduced tyrosinase activity and melanin content in B16 melanoma cells model. Meanwhile, PEE and its primarily contained compound triggered apoptosis of B16 cells in a dose-dependent way. These results demonstrated that PEE possessed effective activities against melanin and tyrosinase generations through the induction of apoptosis. Moreover, a relation between the volatile-rich fractions of F. nelumbinis and the anticancer effects was demonstrated as well.
Triglyceride (TG) metabolism is highly regulated by angiopoietin-like protein (ANGPTL) family members Y. Q. Chen
,
, 1203-1220 (2020). During feeding, ANGPTL8 forms complexes with the ...fibrinogen-like domain-containing protein ANGPTL4 in adipose tissue to decrease ANGPTL3/8- and ANGPTL4-mediated lipoprotein lipase (LPL)-inhibitory activity and promote TG hydrolysis and fatty acid (FA) uptake. The ANGPTL4/8 complex, however, tightly binds LPL and partially inhibits it in vitro. To try to reconcile the in vivo and in vitro data on ANGPTL4/8, we aimed to find novel binding partners of ANGPTL4/8. To that end, we performed pulldown experiments and found that ANGPTL4/8 bound both tissue plasminogen activator (tPA) and plasminogen, the precursor of the fibrinolytic enzyme plasmin. Remarkably, ANGPTL4/8 enhanced tPA activation of plasminogen to generate plasmin in a manner like that observed with fibrin, while minimal plasmin generation was observed with ANGPTL4 alone. The addition of tPA and plasminogen to LPL-bound ANGPTL4/8 caused rapid, complete ANGPTL4/8 cleavage and increased LPL activity. Restoration of LPL activity in the presence of ANGPTL4/8 was also achieved with plasmin but was blocked when catalytically inactive plasminogen (S760A) was added to tPA or when plasminogen activator inhibitor-1 was added to tPA + plasminogen, indicating that conversion of plasminogen to plasmin was essential. Together, these results suggest that LPL-bound ANGPTL4/8 mimics fibrin to recruit tPA and plasminogen to generate plasmin, which then cleaves ANGPTL4/8, enabling LPL activity to be increased. Our observations thus reveal a unique link between the ANGPTL4/8 complex and plasmin generation.
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