Xianmei Yang

Nuclear Export of NF90 to Stabilize IL-2 mRNA Is Mediated by AKT-Dependent Phosphorylation at Ser647 in Response to CD28 Costimulation

IL-2 is one of the most important cytokines required for T cell-mediated immune responses. Costimulation of CD28 in T cells up-regulates IL-2 mRNA levels via transcription activation and mRNA stabilization. Upon T cell activation, NF90, an AU-rich element (ARE)-binding protein, translocates from the nucleus into the cytoplasm, where it binds to the ARE-containing 3′ untranslated regions of IL-2 mRNA and slows down degradation of IL-2 mRNA. The translocation of NF90 is mediated through a nuclear export signal at its N terminus, but how it is triggered is still unclear. Phosphorylation of ARE-binding proteins has been reported as a signal transduction pathway to stabilize ARE-containing transcripts. In this study, we demonstrate that AKT phosphorylates NF90 on Ser647 upon CD28 costimulation. This phosphorylation is necessary for nuclear export of NF90 and IL-2 mRNA stabilization by this protein, because a mutation at Ser647 abolished both functions. We observed that treatment of cells with CD28 costimulation induced distinct increase in phosphorylation of AKT and NF90 at Ser647 concomitantly. Phosphorylation at Ser647 of NF90 up-regulated IL-2 production in response to CD28 costimulation. In vivo and in vitro data support a model in which CD28 costimulation activates AKT to phosphorylate NF90 at Ser647 and phosphorylation triggers NF90 to relocate to the cytoplasm and stabilize IL-2 mRNA.

Phylogenetic and evolutionary analysis of the septin protein family in metazoan

Septins, a conserved family of cytoskeletal GTP‐binding proteins, were presented in diverse eukaryotes. Here, a comprehensive phylogenetic and evolutionary analysis for septin proteins in metazoan was carried out. First, we demonstrated that all septin proteins in metazoan could be clustered into four subgroups, and the representative homologue of every subgroup was presented in the non‐vertebrate chordate Ciona intestinalis, indicating that the emergence of the four septin subgroups should have occurred prior to divergence of vertebrates and invertebrates, and the expansion of the septin gene number in vertebrates was mainly by the duplication of pre‐existing genes rather than by the appearance of new septin subgroup. Second, the direct orthologues of most human septins existed in zebrafish, which suggested that human septin gene repertoire was mainly formed by as far as before the split between fishes and land vertebrates. Third, we found that the evolutionary rate within septin family in mammalian lineage varies significantly, human SEPT1, SEPT 10, SEPT 12, and SEPT 14 displayed a relative elevated evolutionary rate compared with other septin members. Our data will provide new insights for the further function study of this protein family.

Carbonyl reductase 1 as a novel target of (-)-epigallocatechin gallate against hepatocellular carcinoma.

Human carbonyl reductase 1 (CBR1) converts the antitumor drug and anthracycline daunorubicin (DNR) into the alcohol metabolite daunorubicinol (DNROL) with significantly reduced antitumor activity and cardiotoxicity, and this limits the clinical use of DNR. Inhibition of CBR1 can thus increase the efficacy and decrease the toxicity of DNR. Here we report that (−)‐epigallocatechin gallate (EGCG) from green tea is a promising inhibitor of CBR1. EGCG directly interacts with CBR1 and acts as a noncompetitive inhibitor with respect to the cofactor reduced nicotinamide adenine dinucleotide phosphate and the substrate isatin. The inhibition is dependent on the pH, and the gallate moiety of EGCG is required for activity. Molecular modeling has revealed that EGCG occupies the active site of CBR1. Furthermore, EGCG specifically enhanced the antitumor activity of DNR against hepatocellular carcinoma SMMC7721 cells expressing high levels of CBR1 and corresponding xenografts. We also demonstrated that EGCG could overcome the resistance to DNR by Hep3B cells stably expressing CBR1 but not by RNA interference of CBR1‐HepG2 cells. The level of the metabolite DNROL was negatively correlated with that of EGCG in the cell extracts. Finally, EGCG decreased the cardiotoxicity of DNR in a human carcinoma xenograft model with both SMMC7721 and Hep3B cells in mice. Conclusion: These results strongly suggest that EGCG can inhibit CBR1 activity and enhance the effectiveness and decrease the cardiotoxicity of the anticancer drug DNR. These findings also indicate that a combination of EGCG and DNR might represent a novel approach for hepatocellular carcinoma therapy or chemoprevention. (HEPATOLOGY 2010;)

Phylogenetic analysis of CDK and cyclin proteins in premetazoan lineages

BackgroundThe molecular history of animal evolution from single-celled ancestors remains a major question in biology, and little is known regarding the evolution of cell cycle regulation during animal emergence. In this study, we conducted a comprehensive evolutionary analysis of CDK and cyclin proteins in metazoans and their unicellular relatives.ResultsOur analysis divided the CDK family into eight subfamilies. Seven subfamilies (CDK1/2/3, CDK5, CDK7, CDK 20, CDK8/19, CDK9, and CDK10/11) are conserved in metazoans and fungi, with the remaining subfamily, CDK4/6, found only in eumetazoans. With respect to cyclins, cyclin C, H, L, Y subfamilies, and cyclin K and T as a whole subfamily, are generally conserved in animal, fungi, and amoeba Dictyostelium discoideum. In contrast, cyclin subfamilies B, A, E, and D, which are cell cycle-related, have distinct evolutionary histories. The cyclin B subfamily is generally conserved in D. discoideum, fungi, and animals, whereas cyclin A and E subfamilies are both present in animals and their unicellular relatives such as choanoflagellate Monosiga brevicollis and filasterean Capsaspora owczarzaki, but are absent in fungi and D. discoideum. Although absent in fungi and D. discoideum, cyclin D subfamily orthologs can be found in the early-emerging, non-opisthokont apusozoan Thecamonas trahens. Within opisthokonta, the cyclin D subfamily is conserved only in eumetazoans, and is absent in fungi, choanoflagellates, and the basal metazoan Amphimedon queenslandica.ConclusionsOur data indicate that the CDK4/6 subfamily and eumetazoans emerged simultaneously, with the evolutionary conservation of the cyclin D subfamily also tightly linked with eumetazoan appearance. Establishment of the CDK4/6-cyclin D complex may have been the key step in the evolution of cell cycle control during eumetazoan emergence.

IL-2 mRNA stabilization upon PMA stimulation is dependent on NF90-Ser 647 phosphorylation by protein kinase CβI

IL-2 is an important cytokine produced in T cells in response to Ag or mitogen stimulation. It is regulated at both transcriptional and posttranscriptional levels. One of the key regulators of IL-2 mRNA stability is NF90. Upon T cell activation, NF90 translocates from the nucleus into the cytoplasm, where it binds to the AU-rich element-containing 3′ untranslated regions of IL-2 mRNA and stabilizes it. Our previous work showed that CD28 costimulation of T cells activated AKT to phosphorylate NF90 at Ser647 and caused NF90 to undergo nuclear export and stabilize IL-2 mRNA. Phorbol ester (PMA) is a protein kinase C (PKC) activator. Through transcription activation and mRNA stabilization, IL-2 mRNA levels increase promptly when T cells are stimulated with PMA. However, how PMA stabilizes IL-2 mRNA was still unclear. In this study, we demonstrate that PMA stimulation led to phosphorylation of NF90 at Ser647 via PKCβI. This phosphorylation was necessary for nuclear export of NF90 in response to PMA and for IL-2 mRNA stabilization. We show that phosphorylation at NF90-Ser647 upregulated IL-2 production in response to PMA stimulation. Our results support a model in which PMA stimulation activates PKCβI to phosphorylate NF90-Ser647, and this phosphorylation triggers NF90 relocation to the cytoplasm and stabilize IL-2 mRNA. Thus, our study elucidates the mechanism by which PMA activates and stabilizes IL-2 expression in T cells.

The ancient function of RB-E2F pathway: insights from its evolutionary history.

BackgroundThe RB-E2F pathway is conserved in most eukaryotic lineages, including animals and plants. E2F and RB family proteins perform crucial functions in cycle controlling, differentiation, development and apoptosis. However, there are two kinds of E2Fs (repressive E2Fs and active E2Fs) and three RB family members in human. Till now, the detail evolutionary history of these protein families and how RB-E2F pathway evolved in different organisms remain poorly explored.ResultsWe performed a comprehensive evolutionary analysis of E2F, RB and DP (dimerization partners of E2Fs) protein family in representative eukaryotic organisms. Several interesting facts were revealed. First, orthologues of RB, E2F, and DP family are present in several representative unicellular organisms and all multicellular organisms we checked. Second, ancestral E2F, RB genes duplicated before placozoans and bilaterians diverged, thus E2F family was divided into E2F4/5 subgroup (including repressive E2Fs: E2F4 and E2F5) and E2F1/2/3 subgroup (including active E2Fs: E2F1, E2F2 and E2F3), RB family was divided into RB1 subgroup (including RB1) and RBL subgroup (including RBL1 and RBL2). Third, E2F4 and E2F5 share more sequence similarity with the predicted E2F ancestral sequence than E2F1, E2F2 and E2F3; E2F4 and E2F5 also possess lower evolutionary rates and higher purification selection pressures than E2F1, E2F2 and E2F3. Fourth, for RB family, the RBL subgroup proteins possess lower evolutionary rates and higher purification selection pressures compared with RB subgroup proteins in vertebrates,ConclusionsProtein evolutionary rates and purification selection pressures are usually linked with protein functions. We speculated that function conducted by E2F4/5 subgroup and RBL subgroup proteins might mainly represent the ancient function of RB-E2F pathway, and the E2F1/2/3 subgroup proteins and RB1 protein might contribute more to functional diversification in RB-E2F pathway. Our results will enhance the current understanding of RB-E2F pathway and will also be useful to further functional studies in human and other model organisms.ReviewersThis article was reviewed by Dr. Pierre Pontarotti, Dr. Arcady Mushegian and Dr. Zhenguo Lin (nominated by Dr. Neil Smalheiser).

Zinc finger transcription factor 191, directly binding to β‐catenin promoter, promotes cell proliferation of hepatocellular carcinoma

Activation of β‐catenin, the central effector of the canonical wingless‐type (Wnt) pathway, has been implicated in hepatocellular carcinoma (HCC). However, the transcription regulation mechanism of the β‐catenin gene in HCC remains unknown. Here we report that human zinc finger protein 191 (ZNF191) is a potential regulator of β‐catenin transcription. ZNF191, a Krüppel‐like protein, specifically interacts with the TCAT motif, which constitutes the HUMTH01 microsatellite in the tyrosine hydroxylase (TH) gene ex vivo. We demonstrate that ZNF191 is significantly overexpressed in human HCC specimens and is associated with growth of human HCC cells. Global profiling of gene expression in ZNF191 knockdown human hepatic L02 cells revealed that the important Wnt signal pathway genes β‐catenin and cyclin D1 messenger RNAs (mRNAs) are significantly down‐regulated. In agreement with transcription level, β‐catenin and cyclin D1 proteins are also down‐regulated in transient and stable ZNF191 knockdown L02 and hepatoma Hep3B cell lines. Moreover, significant correlation between ZNF191 and β‐catenin mRNA expression was detected in human HCCs. Promoter luciferase assay indicated that ZNF191 can increase transcription activity of the full‐length β‐catenin (CTNNB1) promoter, and nucleotide (nt)‐1407/‐907 of the CTNNB1 promoter exhibited the maximum transcriptional activity. Electrophoretic mobility shift assay showed that purified ZNF191 protein can directly bind to the CTNNB1 promoter, and the binding region is located at nt‐1254/‐1224. Finally, we demonstrate that the key binding sequence of ZNF191 in vivo is ATTAATT. Conclusion: ZNF191 can directly bind to the CTNNB1 promoter and activate the expression of β‐catenin and its downstream target genes such as cyclin D1 in hepatoma cell lines. This study uncovers a new molecular mechanism of transcription regulation of the β‐catenin gene in HCC. (HEPATOLOGY 2012;55:1830–1839)

Glyoxalase 1 is up-regulated in hepatocellular carcinoma and is essential for HCC cell proliferation

Glyoxalase 1 (Glo1), belonging to the glyoxalase system, participates in the detoxification of methylglyoxal (MG), a byproduct of glycolysis. Glo1 is associated with the progression of many human malignancies. However, the role of Glo1 in hepatocellular carcinoma (HCC) is unclear. We have discovered that the expression of Glo1 is up-regulated in HCC tissues compared with adjacent non-tumorous tissues, and knockdown of Glo1 expression by RNA interference significantly inhibited the proliferation of human HCC cell lines. Glo1 knockdown resulted in the accumulation of its cytotoxic substrate, MG. Overall, thus Glo1 might be essential for HCC progression and can be designated as a potential therapeutic target for HCC in the future.

E3 ubiquitin ligase Siah-1 facilitates poly-ubiquitylation and proteasomal degradation of the hepatitis B viral X protein

SIAH1 binds to HBx by pull down (View interaction)

Characterization and expression of HLysG2, a basic goose-type lysozyme from the human eye and testis.

Lysozyme plays an important role in human innate immunity by causing bacterial cell lysis. We describe for the first time, the actual performance of human lysozyme g-like 2 (HLysG2), a mammalian g-type lysozyme. RT-PCR revealed that the HLysG2 gene was transcribed in eye and testis tissues. A spot was detected from human tears using 2D gel electrophoresis and was identified as HLysG2 using MALDI-TOF/TOF MS and a MASCOT search with a matching score of 140 and 27% sequence coverage of the whole amino acid sequence. To gain insight into the in vitro antimicrobial activities of HLysG2, the mature peptide-coding region was cloned into Pichia pastoris for heterogeneous expression. Recombinant HLysG2, had an optimal at pH 6.0 and 30 °C, reached the peak activity of 1.2 × 10(4)U/mg at the sodium ion concentration of 75 mM and showed a higher salt tolerance than human c-type lysozyme (HLysC). Recombinant HlysG2 inhibited Gram-positive bacterial growth and did not inhibit Gram-negative bacterial and Candida albicans growth. Results indicated that HLysG2 is a potent antibacterial protein that may play a role in the innate immunity of the human eye.