ISG15 is critical in the control of Chikungunya virus infection independent of UbE1L mediated conjugation

Chikungunya virus (CHIKV) is a re-emerging alphavirus that has caused significant disease in the Indian Ocean region since 2005. During this outbreak, in addition to fever, rash and arthritis, severe cases of CHIKV infection have been observed in infants. Challenging the notion that the innate immune response in infants is immature or defective, we demonstrate that both human infants and neonatal mice generate a robust type I interferon (IFN) response during CHIKV infection that contributes to, but is insufficient for, the complete control of infection. To characterize the mechanism by which type I IFNs control CHIKV infection, we evaluated the role of ISG15 and defined it as a central player in the host response, as neonatal mice lacking ISG15 were profoundly susceptible to CHIKV infection. Surprisingly, UbE1L^−/− mice, which lack the ISG15 E1 enzyme and therefore are unable to form ISG15 conjugates, displayed no increase in lethality following CHIKV infection, thus pointing to a non-classical role for ISG15. No differences in viral loads were observed between wild-type (WT) and ISG15^−/− mice, however, a dramatic increase in proinflammatory cytokines and chemokines was observed in ISG15^−/− mice, suggesting that the innate immune response to CHIKV contributes to their lethality. This study provides new insight into the control of CHIKV infection, and establishes a new model for how ISG15 functions as an immunomodulatory molecule in the blunting of potentially pathologic levels of innate effector molecules during the host response to viral infection.     

The basis for selective E1-E2 interactions in the ISG15 conjugation system

E1 and E2 enzymes coordinate the first steps in conjugation of ubiquitin (Ub) and ubiquitin-like proteins (Ubls). ISG15 is an interferon-alpha/beta-induced Ubl, and the E1 and E2 enzymes for ISG15 conjugation are Ube1L and UbcH8, respectively. UbcH7 is the most closely related E2 to UbcH8, yet it does not function in ISG15 conjugation in vivo, while both UbcH7 and UbcH8 have been reported to function in Ub conjugation. Kinetic analyses of wild-type and chimeric E2s were performed to determine the basis for preferential activation of UbcH8 by Ube1L and to determine whether UbcH8 is activated equally well by Ube1L and E1(Ub) (Ube1). K(m) determinations confirmed the strong preference of Ube1L for UbcH8 over UbcH7 (a 29-fold K(m) difference), similar to the preference of E1(Ub) for UbcH7 over UbcH8 (a 36-fold K(m) difference). Thioester assays of chimeric E2s identified two structural elements within residues 1-39 of UbcH8 that play a major role in defining Ube1L-UbcH8 specificity: the alpha1-helix and the beta1-beta2 region. The C-terminal ubiquitin fold domain (UFD) of Ube1L was required for transfer of ISG15 to UbcH8 and for binding of Ube1L to UbcH8. Replacement of the Ube1L UFD with that from E1(Ub) resulted in preferential transfer of ISG15 to UbcH7. Together, these results indicate that Ube1L discriminates between UbcH8 and closely related Ub E2s based on specific interactions between the Ube1L UFD and determinants within the N-terminal region of UbcH8.     

Negative regulation of protein phosphatase 2Cbeta by ISG15 conjugation

ISG15, an interferon-upregulated ubiquitin-like protein, is covalently conjugated to various cellular proteins (ISGylation). In this study, we found that protein phosphatase 2Cbeta (PP2Cbeta), which functions in the nuclear factor kappaB (NF-kappaB) pathway via dephosphorylation of TGF-beta-activated kinase, was ISGylated, and analysis by NF-kappaB luciferase reporter assay revealed that PP2Cbeta activity was suppressed by co-expression of ISG15, UBE1L, and UbcH8. We determined the ISGylation sites of PP2Cbeta and constructed its ISGylation-resistant mutant. In contrast to the wild type, this mutant suppressed the NF-kappaB pathway even in the presence of ISG15, UBE1L, and UbcH8. Thus, we propose that ISGylation negatively regulates PP2Cbeta activity.     

Isolation and sequence of an interferon-tau-inducible, pregnancy- and bovine interferon-stimulated gene product 15 (ISG15)-specific, bovine ubiquitin-activating E1-like (UBE1L) enzyme

Bovine (bov) interferon-stimulated gene product 15 (ISG15) is produced in the endometrium in response to conceptus-secreted interferon (IFN)-tau. ISG15 conjugates to endometrial proteins through an enzymatic pathway that is similar to ubiquitinylation. Ubiquitin-activating enzyme 1-like protein (UBE1L) initiates enzymatic conjugation by forming a thioester bond with ISG15, thus preparing it for transfer to the next series of enzymes. The bovUBE1L has not been described. We hypothesized that bovUBE1L was induced by pregnancy and IFN-tau in the endometrium. A 110-kDa protein was purified from bovine endometrial (BEND) cells based on affinity with recombinant (r) glutathione S-transferase (GST)-ISG15. This protein was digested in gel with trypsin. Seven peptides were purified using HPLC, sequenced using liquid chromatography-mass spectroscopy-mass spectroscopy and found to share 43-100% identity with human UBE1L. The full-length bovUBE1L cDNA was isolated from a BEND cell cDNA library, sequenced, and found to share 83% identity with human UBE1L cDNA. Northern blot revealed two mRNAs that were detected in greater (P<0.05) concentrations in endometrium from Day 17-21 pregnant versus nonpregnant cows. Western blots using antihuman UBE1L antibody revealed a similar pattern of pregnancy-associated expression of UBE1L protein in the uterus. The bovUBE1L mRNA was localized, using in situ hybridization, primarily to glandular and luminal epithelium, with more diffuse localization to stroma of the endometrium from pregnant cows. Because bovUBE1L was purified through its interaction with rGST-ISG15 and shares significant amino acid and cDNA sequence identity with human UBE1L, it is concluded that it mediates conjugation of ISG15 to uterine proteins in response to the developing and attaching conceptus.     

ISG15 Arg151 and the ISG15-Conjugating Enzyme UbE1L Are Important for Innate Immune Control of Sindbis Virus

Interferon (IFN)-stimulated gene 15 (ISG15) is a ubiquitin-like molecule that conjugates to target proteins via a C-terminal LRLRGG motif and has antiviral function in vivo. We used structural modeling to predict human ISG15 (hISG15) residues important for interacting with its E1 enzyme, UbE1L. Kinetic analysis revealed that mutation of arginine 153 to alanine (R153A) ablated hISG15-hUbE1L binding and transthiolation of UbcH8. Mutation of other predicted UbE1L-interacting residues had minimal effects on the transfer of ISG15 from UbE1L to UbcH8. The capacity of hISG15 R153A to form protein conjugates in 293T cells was markedly diminished. Mutation of the homologous residue in mouse ISG15 (mISG15), arginine 151, to alanine (R151A) also attenuated protein ISGylation following transfection into 293T cells. We assessed the role of ISG15-UbE1L interactions in control of virus infection by constructing double subgenomic Sindbis viruses that expressed the mISG15 R151A mutant. While expression of mISG15 protected alpha/beta-IFN-receptor-deficient (IFN-αβR^−/−) mice from lethality following Sindbis virus infection, expression of mISG15 R151A conferred no survival benefit. The R151A mutation also attenuated ISG15's ability to decrease Sindbis virus replication in IFN-αβR^−/− mice or prolong survival of ISG15^−/− mice. The importance of UbE1L was confirmed by demonstrating that mice lacking this ISG15 E1 enzyme were highly susceptible to Sindbis virus infection. Together, these data support a role for protein conjugation in the antiviral effects of ISG15.     

Study of the mechanisms of crocetin-induced differentiation and apoptosis in human acute promyelocytic leukemia cells

Crocetin, the major carotenoid in saffron, exhibits potent anticancer effects. However, the antileukemic effects of crocetin are still unclear, especially in primary acute promyelocytic leukemia (APL) cells. In the current study, the potential antipromyelocytic leukemia activity of crocetin and the underlying molecular mechanisms were investigated. Crocetin (100 µM), like standard anti-APL drugs, all-trans retinoic acid (ATRA, 10 µM) and As₂O ₃ (arsenic trioxide, 50 µM), significantly inhibited proliferation and induced apoptosis in primary APL cells, as well as NB4 and HL60 cells. The effect was associated with the decreased expressions of prosurvival genes Akt and BCL2, the multidrug resistance (MDR) proteins, ABCB1 and ABCC1 and the inhibition of tyrosyl-DNA phosphodiesterase 1 (TDP1), while the expressions of proapoptotic genes CASP3, CASP9, and BAX/BCL2 ratio were significantly increased. In contrast, crocetin at relatively low concentration (10 µM), like ATRA (1 µM) and As ₂O ₃ (0.5 µM), induced differentiation of leukemic cells toward granulocytic pattern, and increased the number of differentiated cells expressing CD11b and CD14, while the number of the immature cells expressing CD34 or CD33 was decreased. Furthermore, crocetin suppressed the expression of clinical marker promyelocytic leukemia/retinoic acid receptor-α ( PML/RARα) in NB4 and primary APL cells, and reduced the expression of histone deacetylase 1 ( HDAC1) in all leukemic cells. The results suggested that crocetin can be considered as a candidate for future preclinical and clinical trials of complementary APL treatment.     

Development of capping ability during differentiation of HL-60 human promyelocytic leukemia cells

Developmental changes in cell surface and cytoskeletal elements have been studied in human promyelocytic leukemia cells (line HL-60) which differentiate into functionally mature myeloid cells when grown in dimethyl sulfoxide (DMSO)-supplemented medium. Both differentiated and undifferentiated HL-60 cells bind fluorescent concanavalin A (F-Con A) in a diffuse pattern over the entire cell surface. As with normal neutrophils, pretreatment of the differentiated HL-60 cells with colchicine before incubation with Con A causes the formation of large cytoplasmic protrusions over which the lectin associates into a cap. On the other hand, similarly treated undifferentiated HL-60 cells do not form the cytoplasmic protuberances and are unable to cap the Con A. Transmission electron microscopy reveals that the number and distribution of microtubules and microfilaments change during differentiation. Thus, developing myeloid cells undergo important alterations in the structure and function of the cytoskeleton as they differentiate into mature phagocytes.     

Expression of leukocyte adherence-related glycoproteins during differentiation of HL-60 promyelocytic leukemia cells

We used the HL-60 human promyelocytic leukemia cell line to analyze the surface expression of a family of adherence-related leukocyte surface antigens during myeloid differentiation. These antigens are composed of discrete alpha subunits, designated alpha L, alpha M, and alpha X, that are each noncovalently associated with a common beta subunit. Monoclonal antibodies directed against the individual subunits served as markers in both indirect immunofluorescence studies and immunoprecipitations from HL-60 cells differentiated preferentially towards mature granulocytes (DMSO, retinoic acid) or monocyte/macrophages (PMA, vitamin D3). In undifferentiated HL-60 cells, the alpha L and alpha X subunits were constitutively expressed, whereas the alpha M subunit was not. Differentiation of HL-60 cells along the granulocytic pathway with DMSO resulted in a marked increase in alpha M and minimal increases in alpha L and alpha X. The phenotypic expression of these antigens on DMSO-treated HL-60 cells closely resembled that on normal circulating PMN. Differentiation along the monocyte/macrophage pathway when using PMA or vitamin D3 resulted in major increases in alpha L and alpha X expression, as well as alpha M. These changes resulted in a surface phenotype characteristic of that present on human monocyte-derived macrophages. Triggering of undifferentiated HL-60 cells with PMA caused no increase in subunit expression, whereas stimulation of DMSO-differentiated HL-60 cells with PMA produced more than a 1.5-fold enhancement of both the alpha M and alpha X subunits, and stimulation of human PMN with PMA increased the surface expression of alpha M more than fourfold and alpha X subunit twofold. Stimulation with PMA produced no change in expression of the alpha L subunit in any of the three cell populations. These results indicate that the alpha subunits of this glycoprotein family can be selectively regulated during in vitro differentiation of a human promyelocytic leukemia cell line. Second, DMSO-differentiated HL-60 cells and human PMN possessed an intracellular pool of alpha M and alpha X, but not alpha L, that could be translocated to the surface. Thus, despite structural and functional relationships among the alpha subunits in this glycoprotein family, they undergo disparate surface expression and intracellular regulation during differentiation.     

Over-expression of Mcl-1 impairs the ability of ATRA to induce growth arrest and differentiation in acute promyelocytic leukemia cells

Exposure of acute promyelocytic leukemia (APL) cells to all-trans retinoic acid (ATRA) increases levels of Mcl-1, however, the implication of ATRA-mediated expressions of Mcl-1 in these cells remains to be fully elucidated. This study found that exposure of NB4 and PL-21 cells to ATRA increased levels of Mcl-1 in association with phosphorylation of c-jun N-terminus kinases. Down-regulation of Mcl-1 by a small interfering (siRNA) or an inhibitor of JNK significantly potentiated the ability of ATRA to induce differentiation and apoptosis in these cells. On the other hand, the anti-leukemia effects of ATRA were blunted when Mcl-1 was forced expressed in NB4 and PL-21 cells as well as leukemia cells isolated from individuals with APL. Furthermore, down-regulation of Mcl-1 by an siRNA sensitized non-APL U937 and KG-1 leukemia cells to ATRA-mediated differentiation and apoptosis. Taken together, inhibition of Mcl-1 might be useful to potentiate the action of ATRA in APL as well as non-APL AML cells.     

Metformin induces differentiation in acute promyelocytic leukemia by activating the MEK/ERK signaling pathway

Recent studies have shown that metformin, a widely used antidiabetic agent, may reduce the risk of cancer development. In this study, we investigated the antitumoral effect of metformin on both acute myeloid leukemia (AML) and acute promyelocytic leukemia (APL) cells. Metformin induced apoptosis with partial differentiation in an APL cell line, NB4, but only displayed a proapoptotic effect on several non-M3 AML cell lines. Further analysis revealed that a strong synergistic effect existed between metformin and all-trans retinoic acid (ATRA) during APL cell maturation and that metformin induced the hyperphosphorylation of extracellular signal-regulated kinase (ERK) in APL cells. U0126, a specific MEK/ERK activation inhibitor, abrogated metformin-induced differentiation. Finally, we found that metformin induced the degradation of the oncoproteins PML-RARα and c-Myc and activated caspase-3. In conclusion, these results suggest that metformin treatment may contribute to the enhancement of ATRA-induced differentiation in APL, which may deepen the understanding of APL maturation and thus provide insight for new therapy strategies.     

Influenza B virus NS1 protein inhibits conjugation of the interferon (IFN)-induced ubiquitin-like ISG15 protein

Of the several hundred proteins induced by interferon (IFN) alpha/beta, the ubiquitin-like ISG15 protein is one of the most predominant. We demonstrate the novel way in which the function of the ISG15 protein is inhibited by influenza B virus, which strongly induces the ISG15 protein: a specific region of the influenza B virus NS1 protein, which includes part of its effector domain, blocks the covalent linkage of ISG15 to its target proteins both in vitro and in infected cells. We identify UBE1L as the E1 enzyme that catalyzes the first activation step in the conjugation of ISG15, and show that the NS1B protein inhibits this activation step in vitro. Influenza A virus employs a different strategy: its NS1 protein does not bind the ISG15 protein, but little or no ISG15 protein is produced during infection. We discuss the likely basis for these different strategies.     

15/17 Translocation in acute promyelocytic leukaemia

Summary A translocation between a chromosome 15 and a chromosome 17 was found in the bone marrow of a 14-year-old boy who had clinical and laboratory symptoms of acute promyelocytic leukemia (APL). As far as we know, this is the sixth case of APL with 15/17 translocation to be reported in the literature. This observation gives further support to the hypothesis that there is an association between this chromosomal rearrangement and APL.     

Spatial distribution patterns of interphase centromeres during retinoic acid-induced differentiation of promyelocytic leukemia cells

BACKGROUND: The pericentromeric heterochromatin is an important element for the regulation of gene silencing. Its spatial distribution during interphase appears to be cell-type specific. This study analyzes three-dimensional (3D) centromere distribution patterns during cellular differentiation along the neutrophil pathway. METHODS: Differentiation of the promyelocytic leukemia cell line NB4 was induced by retinoic acid. Centromeres in interphase nuclei were visualized by immunofluorescence staining of centromere-associated proteins with CREST serum. 3D images of nuclei were obtained by confocal microscopy. Automated methods for the segmentation of point-like objects in 3D images were implemented to detect the position of centromeres. Features of centromere localization patterns were determined by constructing the minimal spanning tree of the centromere distribution. RESULTS: In differentiated NB4 cells, the number of centromere conglomerates (chromocenters) was decreased and the distance between chromocenters was increased as compared with untreated controls. The nuclear volume did not differ between the two groups. CONCLUSIONS: The measured rearrangement of centromeres indicates a progressive clustering of heterochromatin and a global remodeling of interphase chromosome territories during differentiation of NB4 cells. The developed methods for the analysis of 3D centromere distribution patterns provide the opportunity for a fast and objective analysis of heterochromatin remodeling.