RIG-I regulates myeloid differentiation by promoting TRIM25-mediated ISGylation.

Retinoic acid-inducible gene I (RIG-I) is up-regulated during granulocytic differentiation of acute promyelocytic leukemia (APL) cells induced by all-trans retinoic acid (ATRA). It has been reported that RIG-I recognizes virus-specific 5'-ppp-double-stranded RNA (dsRNA) and activates the type I interferons signaling pathways in innate immunity. However, the functions of RIG-I in hematopoiesis remain unclear, especially regarding its possible interaction with endogenous RNAs and the associated pathways that could contribute to the cellular differentiation and maturation. Herein, we identified a number of RIG-I-binding endogenous RNAs in APL cells following ATRA treatment, including the tripartite motif-containing protein 25 (TRIM25) messenger RNA (mRNA). TRIM25 encodes the protein known as an E3 ligase for ubiquitin/interferon (IFN)-induced 15-kDa protein (ISG15) that is involved in RIG-I-mediated antiviral signaling. We show that RIG-I could bind TRIM25 mRNA via its helicase domain and C-terminal regulatory domain, enhancing the stability of TRIM25 transcripts. RIG-I could increase the transcriptional expression of TRIM25 by caspase recruitment domain (CARD) domain through an IFN-stimulated response element. In addition, RIG-I activated other key genes in the ISGylation pathway by activating signal transducer and activator of transcription 1 (STAT1), including the modifier ISG15 and several enzymes responsible for the conjugation of ISG15 to protein substrates. RIG-I cooperated with STAT1/2 and interferon regulatory factor 1 (IRF1) to promote the activation of the ISGylation pathway. The integrity of ISGylation in ATRA or RIG-I-induced cell differentiation was essential given that knockdown of TRIM25 or ISG15 resulted in significant inhibition of this process. Our results provide insight into the role of the RIG-I-TRIM25-ISGylation axis in myeloid differentiation.

Systematic identification of arsenic-binding proteins reveals that hexokinase-2 is inhibited by arsenic.

Arsenic is highly effective for treating acute promyelocytic leukemia (APL) and has shown significant promise against many other tumors. However, although its mechanistic effects in APL are established, its broader anticancer mode of action is not understood. In this study, using a human proteome microarray, we identified 360 proteins that specifically bind arsenic. Among the most highly enriched proteins in this set are those in the glycolysis pathway, including the rate-limiting enzyme in glycolysis, hexokinase-1. Detailed biochemical and metabolomics analyses of the highly homologous hexokinase-2 (HK2), which is overexpressed in many cancers, revealed significant inhibition by arsenic. Furthermore, overexpression of HK2 rescued cells from arsenic-induced apoptosis. Our results thus strongly implicate glycolysis, and HK2 in particular, as a key target of arsenic. Moreover, the arsenic-binding proteins identified in this work are expected to serve as a valuable resource for the development of synergistic antitumor therapeutic strategies.

Bcl2-associated athanogene 3 interactome analysis reveals a new role in modulating proteasome activity.

Bcl2-associated athanogene 3 (BAG3), a member of the BAG family of co-chaperones, plays a critical role in regulating apoptosis, development, cell motility, autophagy, and tumor metastasis and in mediating cell adaptive responses to stressful stimuli. BAG3 carries a BAG domain, a WW domain, and a proline-rich repeat (PXXP), all of which mediate binding to different partners. To elucidate BAG3's interaction network at the molecular level, we employed quantitative immunoprecipitation combined with knockdown and human proteome microarrays to comprehensively profile the BAG3 interactome in humans. We identified a total of 382 BAG3-interacting proteins with diverse functions, including transferase activity, nucleic acid binding, transcription factors, proteases, and chaperones, suggesting that BAG3 is a critical regulator of diverse cellular functions. In addition, we characterized interactions between BAG3 and some of its newly identified partners in greater detail. In particular, bioinformatic analysis revealed that the BAG3 interactome is strongly enriched in proteins functioning within the proteasome-ubiquitination process and that compose the proteasome complex itself, suggesting that a critical biological function of BAG3 is associated with the proteasome. Functional studies demonstrated that BAG3 indeed interacts with the proteasome and modulates its activity, sustaining cell survival and underlying resistance to therapy through the down-modulation of apoptosis. Taken as a whole, this study expands our knowledge of the BAG3 interactome, provides a valuable resource for understanding how BAG3 affects different cellular functions, and demonstrates that biologically relevant data can be harvested using this kind of integrated approach.

Global identification of prokaryotic glycoproteins based on an Escherichia coli proteome microarray.

Glycosylation is one of the most abundant protein posttranslational modifications. Protein glycosylation plays important roles not only in eukaryotes but also in prokaryotes. To further understand the roles of protein glycosylation in prokaryotes, we developed a lectin binding assay to screen glycoproteins on an Escherichia coli proteome microarray containing 4,256 affinity-purified E.coli proteins. Twenty-three E.coli proteins that bound Wheat-Germ Agglutinin (WGA) were identified. PANTHER protein classification analysis showed that these glycoprotein candidates were highly enriched in metabolic process and catalytic activity classes. One sub-network centered on deoxyribonuclease I (sbcB) was identified. Bioinformatics analysis suggests that prokaryotic protein glycosylation may play roles in nucleotide and nucleic acid metabolism. Fifteen of the 23 glycoprotein candidates were validated by lectin (WGA) staining, thereby increasing the number of validated E. coli glycoproteins from 3 to 18. By cataloguing glycoproteins in E.coli, our study greatly extends our understanding of protein glycosylation in prokaryotes.

In vivo interstitial migration of primitive macrophages mediated by JNK-matrix metalloproteinase 13 signaling in response to acute injury.

Interstitial cell migration through extracellular matrix is a hallmark of the inflammation response, tumor invasion, and metastasis. We have established a stable zebrafish transgenic line expressing enhanced GFP under the lysozyme C promoter for visualizing and measuring primitive macrophage migration in vivo. We show that tissue-resident primitive macrophages migrate rapidly through extracellular matrix to the site of acute injury induced by tail transection. Mechanistically, the specific inhibition of JNK, but not p38 and ERK, dramatically abolished the chemotactic migration in a dose-dependent manner, suppressing the trauma-induced recruitment of phosphorylated C-Jun transcription factor to proximal AP-1 sites in the promoter of matrix metalloproteinase 13 (mmp13), a gene specifically expressed in primitive macrophages during embryogenesis and required for the interstitial migration. Furthermore, dexamethasone suppressed the trauma-induced JNK phosphorylation and macrophage migration accompanied by simultaneous up-regulation of mkp-1, a well-known phosphatase capable of inactivating phosphorylated JNK. The results indicate that the JNK-Mmp13 signaling pathway plays an essential role in regulating the innate immune cell migration in response to severe injury in vivo.

eena Promotes myeloid proliferation through stimulating ERK1/2 phosphorylation in zebrafish.

The EEN (extra eleven nineteen) gene is one of the fusion partners of mixed-lineage leukemia, located on chromosome 19p13. Here we cloned two een genes (designated as eena and eenb) in zebrafish, which are assigned to linkage groups 8 and 2, respectively. Whole-mount in situ hybridization assay showed that eena and eenb have overlapping but distinct expression patterns during embryogenesis. Ubiquitous or targeted overexpression of eena, but not eenb, into wild-type or transgenic embryos (green fluorescent protein-labeled myeloid progenitors) induced a significant proliferation and ectopic distribution of myeloid progenitors in the yolk sac. Using a morpholino antisense gene knockdown approach, we showed that the number of myeloid progenitors and their downstream mature myelomonocytic cells was significantly decreased in the eena- deficient embryos. Mechanistically, overexpression of eena selectively stimulated ERK phosphorylation and increased the level of transcription factor c-Fos in vitro and in vivo, whereas eena lacking the Src homology 3 domain completely abolished these effects. Furthermore, a MAPK/ERK kinase (MEK) inhibitor, PD98059, blocked the eena-induced cell proliferation and activation of ERK signaling. The results suggest that eena plays an important role in the development of the myeloid cell through activation of the ERK pathway and may provide a valuable reference for future studies of the role of EEN in leukemogenesis.

Aberrant transcriptional regulation of the MLL fusion partner EEN by AML1-ETO and its implication in leukemogenesis.

The EEN (extra eleven nineteen) gene, located on chromosome 19p13, was cloned as a fusion with MLL from a patient with acute myeloid leukemia (AML) with translocation t(11;19)(q23;p13). In this study, we characterized the genomic structure of the EEN gene, including its 5' regulatory region and transcription start site (TSS). We found that Sp1 could bind to the guanine-cytosine (GC)-stretch of the EEN promoter and was critical for the normal EEN expression, whereas the leukemia-associated fusion protein AML1-ETO could aberrantly transactivate the EEN gene through an AML1 binding site. Of note, overexpressed EEN showed oncogenic properties, such as transforming potential in NIH3T3 cells, stimulating cell proliferation, and increasing the activity of transcriptional factor AP-1. Retroviral transduction of EEN increased self-renewal and proliferation of murine hematopoietic progenitor cells. Moreover, Kasumi-1 and HL60-cell growth was inhibited with down-regulation of EEN by RNAi. These findings demonstrate that EEN might be a common target in 2 major types of AML associated with MLL or AML1 translocations, and overexpression of EEN may play an essential role in leukemogenesis.

Reversal of the phenotype by K-rasval12 silencing mediated by adenovirus-delivered siRNA in human pancreatic cancer cell line Panc-1.

AIM: To investigate the in vitro antitumor effect of adenovirus-mediated small interfering RNAs (siRNAs) on pancreatic cancer and the associated mechanism. METHODS: A 63-nucleotide (nt) oligonucleotide encoding K-ras(val12) and specific siRNA were introduced into pSilencer 3.1-H1, then the H1-RNA promoter and siRNA coding insert were subcloned into pAdTrack to get plasmid pAdTrackH1-K-ras(val12). After homologous recombination in bacteria and transfections of such plasmids into a mammalian packaging cell line 293, siRNA expressing adenovirus AdH1-K-ras(val12) was obtained. Stable suppression of K-ras(val12) was detected by Northern blot and Western blot. Apoptosis in Panc-1 cells was detected by flow cytometry. RESULTS: We obtained adenovirus AdH1-K-ras(val12) carrying the pSilencer 3.1-H1 cassette, which could mediate gene silencing. Through siRNA targeted K-ras(val12), the oncogenic phenotype of cancer cells was reversed. Flow cytometry showed that apoptotic index of Panc-1 cells was significantly higher in the AdH1-K-ras(val12)-treatment group (18.70% at 72 h post-infection, 49.55% at 96 h post-infection) compared to the control groups (3.47%, 3.98% at 72 and 96 h post-infection of AdH1-empty, respectively; 4.21%, 3.78% at 72 and 96 h post-infection of AdH1-p53, respectively) (P<0.05). CONCLUSION: These results demonstrate that adenoviral vectors can be used to mediate RNA interference (RNAi) to induce persistent loss of functional phenotypes. In gene therapy, the selective down-regulation of only the mutant version of a gene allows for highly specific effects on tumor cells, while leaving the normal cells untouched. In addition, the apoptosis of pancreatic cancer cell line Panc-1 can be induced after AdH1-K-ras(val12) infection. This kind of adenovirus based on RNAi might be a promising vector for cancer therapy.