Kenneth J. Ritchie

UBP43 (USP18) specifically removes ISG15 from conjugated proteins.

UBP43 shows significant homology to well characterized ubiquitin-specific proteases and previously was shown to hydrolyze ubiquitin-β-galactosidase fusions in Escherichia coli. In our assays, the activity of UBP43 toward Ub fusions was undetectable in vitro directing us to investigate the possibility of Ub-like proteins such as SUMO, Nedd8, and ISG15 as probable substrates. We consequently demonstrate that UBP43 can efficiently cleave only ISG15 fusions including native ISG15 conjugates linked via isopeptide bonds. In addition to commonly used methods we introduce a new experimental design featuring ISG15-UBP43 fusion self-processing. Deletion of the UBP43 gene in mouse leads to a massive increase of ISG15 conjugates in tissues indicating that UBP43 is a major ISG15-specific protease. UBP43 is the first bona fide ISG15-specific protease reported. Both ISG15 andUBP43 genes are known to be strongly induced by interferon, genotoxic stress, and viral infection. We postulate that UBP43 is necessary to maintain a critical cellular balance of ISG15-conjugated proteins in both healthy and stressed organisms.

Role of ISG15 protease UBP43 (USP18) in innate immunity to viral infection

Innate immune responses provide the host with an early protection barrier against infectious agents, including viruses, and help shape the nature and quality of the subsequent adaptive immune responses of the host. Expression of ISG15 (UCRP), a ubiquitin-like protein, and protein ISGylation are highly increased upon viral infection. We have identified UBP43 (USP18) as an ISG15 deconjugating protease. Protein ISGylation is enhanced in cells deficient in UBP43 (ref. 6). Here we have examined the role of UBP43, encoded by the gene Usp18, in innate immunity to virus infection. Usp18−/− mice were resistant to the fatal lymphocytic choriomeningitis and myeloencephalitis that developed in wild-type mice after intracerebral inoculation with lymphocytic choriomeningitis virus (LCMV) or vesicular stomatitis virus (VSV), respectively. Survival of Usp18−/− mice after intracerebral LCMV infection correlated with a severe inhibition of LCMV RNA replication and antigen expression in the brain and increased levels of protein ISGylation. Consistent with these findings, mouse embryonic fibroblasts (MEF) and bone marrow–derived macrophages from Usp18−/− mice showed restricted LCMV replication. Moreover, MEF from Usp18−/− mice showed enhanced interferon-mediated resistance to the cytopathic effect caused by VSV and Sindbis virus (SNV). This report provides the first direct evidence that the ISG15 protease UBP43 and possibly protein ISGylation have a role in innate immunity against viral infection.

Glutathione transferase pi plays a critical role in the development of lung carcinogenesis following exposure to tobacco-related carcinogens and urethane.

Human cancer is controlled by a complex interaction between genetic and environmental factors. Such environmental factors are well defined for smoking-induced lung cancer; however, the roles of specific genes have still to be elucidated. Glutathione transferase pi (GSTP) catalyzes the detoxification of electrophilic diol epoxides produced by the metabolism of polycyclic aromatic hydrocarbons such as benzo[a]pyrene (BaP), a common constituent of tobacco smoke. Activity-altering polymorphisms in Gstp have therefore been speculated to be potential risk modifiers in lung cancer development. To clearly establish a role for GSTP in lung tumorigenesis, we investigated whether deletion of the murine Gstp genes (Gstp1 and Gstp2) alters susceptibility to chemically induced lung tumors following exposure to BaP, 3-methylcholanthrene (3-MC), and urethane. Gstp-null mice were found to have substantially increased numbers of adenomas relative to wild-type mice following exposure to all three compounds (8.3-, 4.3-, and 8.7-fold increase for BaP, 3-MC, and urethane, respectively). In Gstp-null mice, the capacity of pulmonary cytosol to catalyze conjugation of the BaP diol epoxide was significantly reduced. Concomitant with this, a significant increase in the level of BaP DNA adducts was measured in the lungs of null animals; however, no increase in DNA adducts was measured in the case of 3-MC exposure, suggesting that an alternative protective pathway exists. Indeed, significant differences in pulmonary gene expression profiles were also noted between wild-type and null mice. This is the first report to establish a clear correlation between Gstp status and lung cancer in vivo.

Markedly enhanced colon tumorigenesis in ApcMin mice lacking glutathione S-transferase Pi

Glutathione transferases are a multigene family of proteins that catalyze the conjugation of toxic electrophiles and carcinogens to glutathione. Glutathione transferase Pi (GSTP) is commonly overexpressed in human tumors and there is emerging evidence that the enzyme has additional cellular functions in addition to its role in drug and carcinogen detoxification. To investigate the unique functions of this enzyme, we have crossed Gstp null mice with an initiated model of colon cancer, the ApcMin mouse. In contrast to the ApcMin/+ Gstp1/p2+/+ (Gstp-wt ApcMin) mice, which rarely develop colonic tumours, ApcMin/+Gstp1/p2−/− (Gstp-null ApcMin) mice had a 6-fold increase in colon adenoma incidence, and a 50-fold increase in colorectal adenoma multiplicity, relative to Gstp-wt ApcMin. This increase was associated with early tumor onset and decreased survival. Analysis of the biochemical changes in the colon tissue of Gstp-null ApcMin mice demonstrated a marked induction of many inflammatory genes, including IL-6, IL-4, IFN-γ, and inducible nitric oxide synthase. In support of the induction of inducible nitric oxide synthase, a profound induction of nitrotyrosine adducts was observed. Gstp therefore appears to play a role in controlling inflammatory responses in the colon, which would explain the change in tumor incidence observed. These data also suggest that individual variation in GSTP levels may be a factor in colon cancer susceptibility.

Ubp43 gene expression is required for normal Isg15 expression and fetal development

BackgroundIsg15 covalently modifies murine endometrial proteins in response to early pregnancy. Isg15 can also be severed from targeted proteins by a specific protease called Ubp43 (Usp18). Mice lacking Ubp43 (null) form increased conjugated Isg15 in response to interferon. The Isg15 system has not been examined in chorioallantoic placenta (CP) or mesometrial (MM) components of implantation sites beyond 9.5 days post coitum (dpc). It was hypothesized that deletion of Ubp43 would cause disregulation of Isg15 in implantation sites, and that this would affect pregnancy rates.MethodsHeterozygous (het) Ubp43 mice were mated and MM and CP implantation sites were collected on 12.5 and 17.5 days post-coitum (dpc).ResultsFree and conjugated Isg15 were greater on 12.5 versus 17.5 dpc in MM. Free and conjugated Isg15 were also present in CP, but did not differ due to genotype on 12.5 dpc. However, null CP had greater free and conjugated Isg15 when compared to het/wt on 17.5 dpc. Null progeny died in utero with fetal genotype ratios (wt:het:null) of 2:5:1 on 12.5 and 2:2:1 on 17.5 dpc. Implantation sites were disrupted within the junctional zone and spongiotrophoblast, contained less vasculature based on lectin B4 staining and contained greater Isg15 mRNA and VEGF protein in Ubp43 null when compared to wt placenta.ConclusionIt is concluded that Isg15 and its conjugates are present in implantation sites during mid to late gestation and that deletion of Ubp43 causes an increase in free and conjugated Isg15 at the feto-maternal interface. Also, under mixed genetic background, deletion of Ubp43 results in fetal death.

Comparative Cytotoxicity of Glycyrrhiza glabra Roots from Different Geographical Origins Against Immortal Human Keratinocyte (HaCaT), Lung Adenocarcinoma (A549) and Liver Carcinoma (HepG2) Cells

Glycyrrhiza glabra L. (Fabaceae), commonly known as ‘liquorice’, is a well‐known medicinal plant. Roots of this plant have long been used as a sweetening and flavouring agent in food and pharmaceutical products, and also as a traditional remedy for cough, upper and lower respiratory ailments, kidney stones, hepatitis C, skin disorder, cardiovascular diseases, diabetes, gastrointestinal ulcers and stomach ache. Previous pharmacological and clinical studies have revealed its antitussive, antiinflammatory, antiviral, antimicrobial, antioxidant, immunomodulatory, hepatoprotective and cardioprotective properties. While glycyrrhizin, a sweet‐tasting triterpene saponin, is the principal bioactive compound, several bioactive flavonoids and isoflavonoids are also present in the roots of this plant. In the present study, the cytotoxicity of the methanol extracts of nine samples of the roots of G. glabra, collected from various geographical origins, was assessed against immortal human keratinocyte (HaCaT), lung adenocarcinoma (A549) and liver carcinoma (HepG2) cell lines using the in vitro 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyl tetrazoliumbromide cell toxicity/viability assay. Considerable variations in levels of cytotoxicity were observed among various samples of G. glabra. Copyright

Increased Skin Papilloma Formation in Mice Lacking Glutathione Transferase GSTP

The glutathione S-transferase GSTP is overexpressed in many human cancers and chemotherapy-resistant cancer cells, where there is evidence that GSTP may have additional functions beyond its known catalytic role. On the basis of evidence that Gstp-deficient mice have a comparatively higher susceptibility to skin carcinogenesis, we investigated whether this phenotype reflected an alteration in carcinogen detoxification or not. For this study, Gstp(-/-) mice were interbred with Tg.AC mice that harbor initiating H-ras mutations in the skin. Gstp(-/-)/Tg.AC mice exposed to the proinflammatory phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) exhibited higher tumor incidence and multiplicity with a significant thickening of skin after treatment, illustrating hyperproliferative growth. Unexpectedly, we observed no difference in cellular proliferation or apoptosis or in markers of oxidative stress, although higher levels of the inflammatory marker nitrotyrosine were found in Gstp(-/-)/Tg.AC mice. Instead, gene set enrichment analysis of microarray expression data obtained from skin revealed a more proapoptotic and proinflammatory environment shortly after TPA treatment. Within 4 weeks of TPA treatment, Gstp(-/-)/Tg.AC mice displayed altered lipid/sterol metabolism and Wnt signaling along with aberrant processes of cytoskeletal control and epidermal morphogenesis at both early and late times. In extending the evidence that GSTP has a vital role in normal homeostatic control and cancer prevention, they also strongly encourage the emerging concept that GSTP is a major determinant of the proinflammatory character of the tumor microenvironment. This study shows that the GSTP plays a major role in carcinogenesis distinct from its role in detoxification and provides evidence that the enzyme is a key determinant of the proinflammatory tumor environment.

ent-Clerodane Diterpenes from the Bark of Croton oligandrus Pierre ex. Hutch. and Assessment of their Cytotoxicity Against Human Cancer Cell Lines

New clerodane diterpenes, 12-epi-megalocarpodolide D (2) and an epimeric mixture of crotonolins A (3) and B (4), were isolated from the bark of Croton oligandrus following a bioassay-guided isolation protocol. Known compounds, megalocarpodolide D (1), 12-epi-crotocorylifuran (5), cluytyl-ferulate (6), hexacosanoyl- ferulate (7), vanillin (8), acetyl-aleuritolic acid (9) and lupeol (10), were also isolated. The structures of the isolated compounds (1–10) were elucidated by spectroscopic means. The cytotoxicity of compounds 1–10 was assessed against A549, MCF7, PC3 and PNT2 cell lines using the MTT assay. Compounds 1 and 2 showed moderate levels of activity against both A549 and MCF7 cells with 1 being the most active with IC50 values of 63.8 ± 13.8 and 136.2 ± 22.7 µM against A549 and MCF7 cells, respectively. The epimeric mixture of 3 and 4 was moderately active against A549 and PC3 cells (IC50 = 128.6 ± 31.0 and 111.2 ± 2.9 µM, respectively).