Expansion of interferon inducible gene pool via USP18 inhibition promotes cancer cell pyroptosis.

While immunotherapy has emerged as a breakthrough cancer therapy, it is only effective in some patients, indicating the need of alternative therapeutic strategies. Induction of cancer immunogenic cell death (ICD) is one promising way to elicit potent adaptive immune responses against tumor-associated antigens. Type I interferon (IFN) is well known to play important roles in different aspects of immune responses, including modulating ICD in anti-tumor action. However, how to expand IFN effect in promoting ICD responses has not been addressed. Here we show that depletion of ubiquitin specific protease 18 (USP18), a negative regulator of IFN signaling, selectively induces cancer cell ICD. Lower USP18 expression correlates with better survival across human selected cancer types and delays cancer progression in mouse models. Mechanistically, nuclear USP18 controls the enhancer landscape of cancer cells and diminishes STAT2-mediated transcription complex binding to IFN-responsive elements. Consequently, USP18 suppression not only enhances expression of canonical IFN-stimulated genes (ISGs), but also activates the expression of a set of atypical ISGs and NF-κB target genes, including genes such as Polo like kinase 2 (PLK2), that induce cancer pyroptosis. These findings may support the use of targeting USP18 as a potential cancer immunotherapy.

Type I Interferon Regulates a Coordinated Gene Network to Enhance Cytotoxic T Cell-Mediated Tumor Killing.

Type I interferons (IFN), which activate many IFN-stimulated genes (ISG), are known to regulate tumorigenesis. However, little is known regarding how various ISGs coordinate with one another in developing antitumor effects. Here, we report that the ISG UBA7 is a tumor suppressor in breast cancer. UBA7 encodes an enzyme that catalyzes the covalent conjugation of the ubiquitin-like protein product of another ISG (ISG15) to cellular proteins in a process known as "ISGylation." ISGylation of other ISGs, including STAT1 and STAT2, synergistically facilitates production of chemokine-receptor ligands to attract cytotoxic T cells. These gene-activation events are further linked to clustering and nuclear relocalization of STAT1/2 within IFN-induced promyelocytic leukemia (PML) bodies. Importantly, this coordinated ISG-ISGylation network plays a central role in suppressing murine breast cancer growth and metastasis, which parallels improved survival in patients with breast cancer. These findings reveal a cooperative IFN-inducible gene network in orchestrating a tumor-suppressive microenvironment. SIGNIFICANCE: We report a highly cooperative ISG network, in which UBA7-mediated ISGylation facilitates clustering of transcription factors and activates an antitumor gene-expression program. These findings provide mechanistic insights into immune evasion in breast cancer associated with UBA7 loss, emphasizing the importance of a functional ISG-ISGylation network in tumor suppression.This article is highlighted in the In This Issue feature, p. 327.

Identification of Compounds That Prolong Type I Interferon Signaling as Potential Vaccine Adjuvants.

Vaccines are reliant on adjuvants to enhance the immune stimulus, and type I interferons (IFNs) have been shown to be beneficial in augmenting this response. We were interested in identifying compounds that would sustain activation of an endogenous type I IFN response as a co-adjuvant. We began with generation of a human monocytic THP-1 cell line with an IFN-stimulated response element (ISRE)-ß-lactamase reporter construct for high-throughput screening. Pilot studies were performed to optimize the parameters and conditions for this cell-based Förster resonance energy transfer (FRET) reporter assay for sustaining an IFN-α-induced ISRE activation signal. These conditions were confirmed in an initial pilot screen, followed by the main screen for evaluating prolongation of an IFN-α-induced ISRE activation signal at 16 h. Hit compounds were identified using a structure enrichment strategy based on chemoinformatic clustering and a naïve "Top X" approach. A select list of confirmed hits was then evaluated for toxicity and the ability to sustain IFN activity by gene and protein expression. Finally, for proof of concept, a panel of compounds was used to immunize mice as co-adjuvant with a model antigen and an IFN-inducing Toll-like receptor 4 agonist, lipopolysaccharide, as an adjuvant. Selected compounds significantly augmented antigen-specific immunoglobulin responses.

Negative regulation of type I IFN signaling.

Type I IFNs (α, ß, and others) are a family of cytokines that are produced in physiological conditions as well as in response to the activation of pattern recognition receptors. They are critically important in controlling the host innate and adaptive immune response to viral and some bacterial infections, cancer, and other inflammatory stimuli. However, dysregulation of type I IFN production or response can contribute to immune pathologies termed "interferonopathies", pointing to the importance of balanced activating signals with tightly regulated mechanisms of tuning this signaling. Here, we summarize the recent advances of how type I IFN production and response are controlled at multiple levels of the type I IFN signaling cascade.

STAT2 is an essential adaptor in USP18-mediated suppression of type I interferon signaling.

Type I interferons (IFNs) are multifunctional cytokines that regulate immune responses and cellular functions but also can have detrimental effects on human health. A tight regulatory network therefore controls IFN signaling, which in turn may interfere with medical interventions. The JAK-STAT signaling pathway transmits the IFN extracellular signal to the nucleus, thus resulting in alterations in gene expression. STAT2 is a well-known essential and specific positive effector of type I IFN signaling. Here, we report that STAT2 is also a previously unrecognized, crucial component of the USP18-mediated negative-feedback control in both human and mouse cells. We found that STAT2 recruits USP18 to the type I IFN receptor subunit IFNAR2 via its constitutive membrane-distal STAT2-binding site. This mechanistic coupling of effector and negative-feedback functions of STAT2 may provide novel strategies for treatment of IFN-signaling-related human diseases.

Type I IFN induces protein ISGylation to enhance cytokine expression and augments colonic inflammation.

Type I IFNs have broad activity in tissue inflammation and malignant progression that depends on the expression of IFN-stimulated genes (ISGs). ISG15, one such ISG, can form covalent conjugates to many cellular proteins, a process termed "protein ISGylation." Although type I IFNs are involved in multiple inflammatory disorders, the role of protein ISGylation during inflammation has not been evaluated. Here we report that protein ISGylation exacerbates intestinal inflammation and colitis-associated colon cancer in mice. Mechanistically, we demonstrate that protein ISGylation negatively regulates the ubiquitin-proteasome system, leading to increased production of IFN-induced reactive oxygen species (ROS). The increased cellular ROS then enhances LPS-induced activation of p38 MAP kinase and the expression of inflammation-related cytokines in macrophages. Thus our studies reveal a regulatory role for protein ISGylation in colonic inflammation and its related malignant progression, indicating that targeting ubiquitin-activating enzyme E1 homolog has therapeutic potential in treating inflammatory diseases.

Identification and characterization of a novel ISG15-ubiquitin mixed chain and its role in regulating protein homeostasis.

As a ubiquitin-like modifier, ISG15 is conjugated to many cellular proteins in a process termed protein ISGylation. However, the crosstalk between protein ISGylation and the ubiquitin proteasome system is not fully understood. Here, we report that cellular ubiquitin is a substrate of ISG15 and Lys 29 on ubiquitin is the major ISG15 acceptor site. Using a model substrate, we demonstrate that ISG15 can modify ubiquitin, which is immobilized on its substrate, to form ISG15-ubiquitin mixed chains. Furthermore, our results indicate that ISG15-ubiquitin mixed chains do not serve as degradation signals for a ubiquitin fusion degradation substrate. Accordingly, an ISG15-ubiquitin fusion protein, which mimics an ISG15-ubiquitin mixed chain, negatively regulates cellular turnover of ubiquitylated proteins. In addition, ISG15-ubiquitin mixed chains, which are detectable on endogenously ubiquitylated proteins, dampen cellular turnover of these proteins. Thus, our studies unveil an unanticipated interplay between two protein modification systems and highlight its role in coordinating protein homeostasis.

Murine Herc6 Plays a Critical Role in Protein ISGylation In Vivo and Has an ISGylation-Independent Function in Seminal Vesicles.

ISG15 conjugation (ISGylation) to proteins is a multistep process involving interferon (IFN)-inducible UBE1L (E1), UbcH8 (E2), and ISG15 E3 ligases (E3s). Studies performed over the past several years have shown that ISGylation plays a pivotal role in the host antiviral response against certain viruses. Recent in vitro studies revealed that human Herc5 and mouse Herc6 are major ISG15 E3 ligases, respectively. However, the global function of Herc5/6 proteins in vivo still remains unclear. Here, we report generation and initial characterization of Herc6 knockout mice. Substantial reductions of ISGylation were observed in Herc6-deficient cells after polyinosinic-polycytidylic acid double-stranded RNA injection of mice or IFN treatment of cells. On the other hand, Herc6-deficient cells and wild-type (WT) cells had similar responses to IFN stimulation, Sendai virus (Z strain) infection, and vesicular stomatitis virus infection. These results indicate that Herc6 does not play a critical role in antiviral defense of these viral infections in mice. Interestingly, male Herc6-deficient mice showed seminal vesicle hypertrophy. No such problem was detected in WT and ISG15 activating enzyme Ube1L-deficient mice. These results suggest that in addition to promoting protein ISGylation, Herc6 has a novel and protein ISGylation-independent function in the male reproductive system.

RUNX1a enhances hematopoietic lineage commitment from human embryonic stem cells and inducible pluripotent stem cells.

Advancements in human pluripotent stem cell (hPSC) research have potential to revolutionize therapeutic transplantation. It has been demonstrated that transcription factors may play key roles in regulating maintenance, expansion, and differentiation of hPSCs. In addition to its regulatory functions in hematopoiesis and blood-related disorders, the transcription factor RUNX1 is also required for the formation of definitive blood stem cells. In this study, we demonstrated that expression of endogenous RUNX1a, an isoform of RUNX1, parallels with lineage commitment and hematopoietic emergence from hPSCs, including both human embryonic stem cells and inducible pluripotent stem cells. In a defined hematopoietic differentiation system, ectopic expression of RUNX1a facilitates emergence of hematopoietic progenitor cells (HPCs) and positively regulates expression of mesoderm and hematopoietic differentiation-related factors, including Brachyury, KDR, SCL, GATA2, and PU.1. HPCs derived from RUNX1a hPSCs show enhanced expansion ability, and the ex vivo-expanded cells are capable of differentiating into multiple lineages. Expression of RUNX1a in embryoid bodies (EBs) promotes definitive hematopoiesis that generates erythrocytes with ß-globin production. Moreover, HPCs generated from RUNX1a EBs possess ≥9-week repopulation ability and show multilineage hematopoietic reconstitution in vivo. Together, our results suggest that RUNX1a facilitates the process of producing therapeutic HPCs from hPSCs.

ISG15 regulates IFN-γ immunity in human mycobacterial disease.

Interferon-gamma (IFN-γ) is crucial for immunity against different pathogens due to its broad effects on the multiple arms of the immune system. The regulation of IFN-γ immunity is of extensive interest to research as well as practical activity for drug discovery. New evidence supports previous findings that ubiquitin-like protein ISG15 acts as an extracellular cytokine and promotes IFN-γ production, providing intriguing insights of the importance of ISG15 into the control of human mycobacterial disease.

The contrasting effect of macromolecular crowding on amyloid fibril formation.

BACKGROUND: Amyloid fibrils associated with neurodegenerative diseases can be considered biologically relevant failures of cellular quality control mechanisms. It is known that in vivo human Tau protein, human prion protein, and human copper, zinc superoxide dismutase (SOD1) have the tendency to form fibril deposits in a variety of tissues and they are associated with different neurodegenerative diseases, while rabbit prion protein and hen egg white lysozyme do not readily form fibrils and are unlikely to cause neurodegenerative diseases. In this study, we have investigated the contrasting effect of macromolecular crowding on fibril formation of different proteins. METHODOLOGY/PRINCIPAL FINDINGS: As revealed by assays based on thioflavin T binding and turbidity, human Tau fragments, when phosphorylated by glycogen synthase kinase-3ß, do not form filaments in the absence of a crowding agent but do form fibrils in the presence of a crowding agent, and the presence of a strong crowding agent dramatically promotes amyloid fibril formation of human prion protein and its two pathogenic mutants E196K and D178N. Such an enhancing effect of macromolecular crowding on fibril formation is also observed for a pathological human SOD1 mutant A4V. On the other hand, rabbit prion protein and hen lysozyme do not form amyloid fibrils when a crowding agent at 300 g/l is used but do form fibrils in the absence of a crowding agent. Furthermore, aggregation of these two proteins is remarkably inhibited by Ficoll 70 and dextran 70 at 200 g/l. CONCLUSIONS/SIGNIFICANCE: We suggest that proteins associated with neurodegenerative diseases are more likely to form amyloid fibrils under crowded conditions than in dilute solutions. By contrast, some of the proteins that are not neurodegenerative disease-associated are unlikely to misfold in crowded physiological environments. A possible explanation for the contrasting effect of macromolecular crowding on these two sets of proteins (amyloidogenic proteins and non-amyloidogenic proteins) has been proposed.

Usp18 promotes conventional CD11b+ dendritic cell development.

Dendritic cells (DCs) represent the key cells linking innate and adaptive immune responses. It is critical to understand the molecular factors regulating DC differentiation. Usp18 is an IFN-inducible member of the ubiquitin-specific protease family, which deconjugates ubiquitin-like modifier ISG15 from target proteins and competitively inhibits IFN-α/ß-induced JAK/STAT activation. This study demonstrates that the frequency of conventional CD11b(+) DCs in the spleen of Usp18(-/-) mice was significantly reduced, whereas the frequencies of conventional CD8(+) DCs and plasmacytoid DCs remained normal. In addition, Usp18(-/-) bone marrow (BM) cells generate DCs less efficiently in GM-CSF-supplemented culture, demonstrating a fundamental defect throughout the DC differentiation pathway. Usp18(-/-) BM cells were rescued by exogenous expression of either wild-type or deconjugation-inactive Usp18, and superimposition of an IFN-α/ß receptor knockout returned in vivo DC populations to normal, clearly showing that the defect seen is due solely to Usp18's effect on IFN signaling. Finally, Usp18(-/-) BM-derived DCs expressed high levels of SOCS1/SOCS3, known inhibitors of GM-CSF signaling, providing a mechanistic explanation for the phenotype. In conclusion, we have identified a novel role of Usp18 in modulating conventional CD11b(+) DC development via its inhibitory effect on type I IFN signaling.

Two independent mechanisms promote expression of an N-terminal truncated USP18 isoform with higher DeISGylation activity in the nucleus.

Expression of the ISG15 specific protease USP18 is highly induced by type I interferons. The two main functions of USP18, i.e. its enzymatic activity and down-regulation of type I interferon signaling, are well characterized. However, to date all functional studies focused on full-length USP18. Here, we report that translation of human USP18 is initiated by a rare start codon (CUG). Usage of this non-canonical initiation site with its weak translation initiation efficiency promotes expression of an N-terminal truncated isoform (USP18-sf). In addition, an internal ribosome entry site (IRES) located in the 5'-coding region of USP18 also contributes to translation of USP18-sf. Functionally, both isoforms exhibit enzymatic activity and interfere with type I interferon signaling. However, USP18-sf shows different subcellular distribution compared with the full-length protein and enhanced deISGylation activity in the nucleus. Taken together, we report the existence of an N-terminal truncated isoform of USP18, whose expression is controlled on translational level by two independent mechanisms providing translational flexibility as well as cell type-specific resistance to inhibition of cap-dependent translation.

Parkinson disease drug screening based on the interaction between D(2) dopamine receptor and beta-arrestin 2 detected by capillary zone electrophoresis.

Parkinson's disease is the second most common neurodegenerative disease in the world. Beta-arrestin-2 has been reported to be an important protein involved in D(2) dopamine receptor desensitization, which is essential to Parkinson's disease. Moreover, the potential value of pharmacological inactivation of G protein-coupled receptor kinase or arrestin in the treatment of patients with Parkinson's disease has recently been shown. We studied the interaction between D(2) dopamine receptor and beta-arrestin-2 and the pharmacological regulation of chemical compounds on such interaction using capillary zone electrophoresis. The results from screening more than 40 compounds revealed three compounds that remarkably inhibit the beta-arrestin-2/D(2) dopamine receptor interaction among them. These compounds are promising therapies for Parkinson's disease, and the method used in this study has great potential for application in large-scale drug screening and evaluation.

Fibrillization of human tau is accelerated by exposure to lead via interaction with His-330 and His-362.

BACKGROUND: Neurofibrillary tangles, mainly consisted of bundles of filaments formed by the microtubule-associated protein Tau, are a hallmark of Alzheimer disease. Lead is a potent neurotoxin for human being especially for the developing children, and Pb(2+) at high concentrations is found in the brains of patients with Alzheimer disease. However, it has not been reported so far whether Pb(2+) plays a role in the pathology of Alzheimer disease through interaction with human Tau protein and thereby mediates Tau filament formation. In this study, we have investigated the effect of Pb(2+) on fibril formation of recombinant human Tau fragment Tau(244-372) and its mutants at physiological pH. METHODOLOGY/PRINCIPAL FINDINGS: As revealed by thioflavin T and 8-anilino-1-naphthalene sulfonic acid fluorescence, the addition of 5-40 µM Pb(2+) significantly accelerates the exposure of hydrophobic region and filament formation of wild-type Tau(244-372) on the investigated time scale. As evidenced by circular dichroism and Fourier transform infrared spectroscopy, fibrils formed by wild-type Tau(244-372) in the presence of 5-40 µM Pb(2+) contain more ß-sheet structure than the same amount of fibrils formed by the protein in the absence of Pb(2+). However, unlike wild-type Tau(244-372), the presence of 5-40 µM Pb(2+) has no obvious effects on fibrillization kinetics of single mutants H330A and H362A and double mutant H330A/H362A, and fibrils formed by such mutants in the absence and in the presence of Pb(2+) contain similar amounts of ß-sheet structure. The results from isothermal titration calorimetry show that one Pb(2+) binds to one Tau monomer via interaction with His-330 and His-362, with sub-micromolar affinity. CONCLUSIONS/SIGNIFICANCE: We demonstrate for the first time that the fibrillization of human Tau protein is accelerated by exposure to lead via interaction with His-330 and His-362. Our results suggest the possible involvement of Pb(2+) in the pathogenesis of Alzheimer disease and provide critical insights into the mechanism of lead toxicity.

Quantitative characterization of heparin binding to Tau protein: implication for inducer-mediated Tau filament formation.

Neurofibrillary tangles, principally composed of bundles of filaments formed by the microtubule-associated protein Tau, are a hallmark of a group of neurodegenerative diseases such as Alzheimer disease. Polyanionic cofactors such as heparin can induce Tau filament formation in vitro. Here we quantitatively characterize the interaction between recombinant human Tau fragment Tau(244-372) and heparin (average molecular mass = 7 kDa) as well as heparin-induced fibril formation by using static light scattering, isothermal titration calorimetry, turbidity assays, and transmission electron microscopy. Our data clearly show that at physiological pH, heparin 7K, and human Tau(244-372) form a tight 1:1 complex with an equilibrium association constant exceeding 10(6) m(-1) under reducing conditions, triggering Tau fibrillization. In the absence of dithiothreitol, heparin shows a moderate binding affinity (10(5) m(-1)) to Tau(244-372), similarly triggering Tau fibrillization. Further fibrillization kinetics analyses show that the lag time appears to be approximately invariant up to a molar ratio of 2:1 and then increases at larger ratios of heparin/Tau. The maximum slope representing the apparent rate constant for fibril growth increases sharply with substoichiometric ratios of heparin/Tau and then decreases to some extent with ratios of >1:1. The retarding effect of heparin in excess is attributed to the large increase in ionic strength of the medium arising from free heparin. Together, these results suggest that the formation of the 1:1 complex of Tau monomer and heparin plays an important role in the inducer-mediated Tau filament formation, providing clues to understanding the pathogenesis of neurodegenerative diseases.

Low micromolar zinc accelerates the fibrillization of human tau via bridging of Cys-291 and Cys-322.

A hallmark of a group of neurodegenerative diseases such as Alzheimer disease is the formation of neurofibrillary tangles, which are principally composed of bundles of filaments formed by microtubule-associated protein Tau. Clarifying how natively unstructured Tau protein forms abnormal aggregates is of central importance for elucidating the etiology of these diseases. There is considerable evidence showing that zinc, as an essential element that is highly concentrated in brain, is linked to the development or progression of these diseases. Herein, by using recombinant human Tau fragment Tau(244-372) and its mutants, we have investigated the effect of zinc on the aggregation of Tau. Low micromolar concentrations of Zn(2+) dramatically accelerate fibril formation of wild-type Tau(244-372) under reducing conditions, compared with no Zn(2+). Higher concentrations of Zn(2+), however, induce wild-type Tau(244-372) to form granular aggregates in reducing conditions. Moreover, these non-fibrillar aggregates assemble into mature Tau filaments when Zn(2+) has been chelated by EDTA. Unlike wild-type Tau(244-372), low micromolar concentrations of Zn(2+) have no obvious effects on fibrillization kinetics of single mutants C291A and C322A and double mutant C291A/C322A under reducing conditions. The results from isothermal titration calorimetry show that one Zn(2+) binds to one Tau molecule via tetrahedral coordination to Cys-291 and Cys-322 as well as two histidines, with moderate, micromolar affinity. Our data demonstrate that low micromolar zinc accelerates the fibrillization of human Tau protein via bridging Cys-291 and Cys-322 in physiological reducing conditions, providing clues to understanding the relationship between zinc dyshomeostasis and the etiology of neurodegenerative diseases.

Crowded cell-like environment accelerates the nucleation step of amyloidogenic protein misfolding.

To understand the role of a crowded physiological environment in the pathogenesis of neurodegenerative diseases, we report the following. 1) The formation of fibrous aggregates of the human Tau fragment Tau-(244-441), when hyperphosphorylated by glycogen synthase kinase-3beta, is dramatically facilitated by the addition of crowding agents. 2) Fibril formation of nonphosphorylated Tau-(244-441) is only promoted moderately by macromolecular crowding. 3) Macromolecular crowding dramatically accelerates amyloid formation by human prion protein. A sigmoidal equation has been used to fit these kinetic data, including published data of human alpha-synuclein, yielding lag times and apparent rate constants for the growth of fibrils for these amyloidogenic proteins. These biochemical data indicate that crowded cell-like environments significantly accelerate the nucleation step of fibril formation of human Tau fragment/human prion protein/human alpha-synuclein (a significant decrease in the lag time). These results can in principle be predicted based on some known data concerning protein concentration effects on fibril formation both in vitro and in vivo. Furthermore, macromolecular crowding causes human prion protein to form short fibrils and nonfibrillar particles with lower conformational stability and higher protease resistance activity, compared with those formed in dilute solutions. Our data demonstrate that a crowded physiological environment could play an important role in the pathogenesis of neurodegenerative diseases by accelerating amyloidogenic protein misfolding and inducing human prion fibril fragmentation, which is considered to be an essential step in prion replication.

Oxidative refolding of reduced, denatured lysozyme in AOT reverse micelles.

The refolding kinetics of the reduced, denatured hen egg white lysozyme in sodium bis(2-ethylhexyl)sulfosuccinate (AOT)-isooctane-water reverse micelles at different water-to-surfactant molar ratios has been investigated by fluorescence spectroscopy and UV spectroscopy. The oxidative refolding of the confined lysozyme is biphasic in AOT reverse micelles. When the water-to-surfactant molar ratio (omega 0) is 12.6, the relative activity of encapsulated lysozyme after refolding for 24 h in AOT reverse micelles increases 46% compared with that in bulk water. Furthermore, aggregation of lysozyme at a higher concentration (0.2 mM) in AOT reverse micelles at omega 0 of 6.3 or 12.6 is not observed; in contrast, the oxidative refolding of lysozyme in bulk water must be at a lower protein concentration (5 microM) in order to avoid a serious aggregation of the protein. For comparison, we have also investigated the effect of AOT on lysozyme activity and found that the residual activity of lysozyme decreases with increasing the concentration of AOT from 1 to 5 mM. When AOT concentration is larger than 2 mM, lysozyme is almost completely inactivated by AOT and most of lysozyme activity is lost. Together, our data demonstrate that AOT reverse micelles with suitable water-to-surfactant molar ratios are favorable to the oxidative refolding of reduced, denatured lysozyme at a higher concentration, compared with bulk water.