Intrinsic immunity describes the set of recently discovered but poorly understood cellular mechanisms that specifically target viral pathogens. Their discovery derives in large part from intensive ...studies of HIV and SIV that revealed restriction factors acting at various stages of the retroviral life cycle. Recent studies indicate that some factors restrict both retroviruses and retrotransposons but surprisingly in ways that may differ. We screened known interferon-stimulated antiviral proteins previously untested for their effects on cell culture retrotransposition. Several factors, including BST2, ISG20, MAVS, MX2, and ZAP, showed strong L1 inhibition. We focused on ZAP (PARP13/ZC3HAV1), a zinc-finger protein that targets viruses of several families, including Retroviridae, Tiloviridae, and Togaviridae, and show that ZAP expression also strongly restricts retrotransposition in cell culture through loss of L1 RNA and ribonucleoprotein particle integrity. Association of ZAP with the L1 ribonucleoprotein particle is supported by co-immunoprecipitation and co-localization with ORF1p in cytoplasmic stress granules. We also used mass spectrometry to determine the protein components of the ZAP interactome, and identified many proteins that directly interact and colocalize with ZAP, including MOV10, an RNA helicase previously shown to suppress retrotransposons. The detection of a chaperonin complex, RNA degradation proteins, helicases, post-translational modifiers, and components of chromatin modifying complexes suggest mechanisms of ZAP anti-retroelement activity that function in the cytoplasm and perhaps also in the nucleus. The association of the ZAP ribonucleoprotein particle with many interferon-stimulated gene products indicates it may be a key player in the interferon response.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
MOV10 protein, a putative RNA helicase and component of the RNA-induced silencing complex (RISC), inhibits retrovirus replication. We show that MOV10 also severely restricts human LINE1 (L1), Alu, ...and SVA retrotransposons. MOV10 associates with the L1 ribonucleoprotein particle, along with other RNA helicases including DDX5, DHX9, DDX17, DDX21, and DDX39A. However, unlike MOV10, these other helicases do not strongly inhibit retrotransposition, an activity dependent upon intact helicase domains. MOV10 association with retrotransposons is further supported by its colocalization with L1 ORF1 protein in stress granules, by cytoplasmic structures associated with RNA silencing, and by the ability of MOV10 to reduce endogenous and ectopic L1 expression. The majority of the human genome is repetitive DNA, most of which is the detritus of millions of years of accumulated retrotransposition. Retrotransposons remain active mutagens, and their insertion can disrupt gene function. Therefore, the host has evolved defense mechanisms to protect against retrotransposition, an arsenal we are only beginning to understand. With homologs in other vertebrates, insects, and plants, MOV10 may represent an ancient and innate form of immunity against both infective viruses and endogenous retroelements.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Long interspersed elements 1 (LINE-1) occupy at least 17% of the human genome and are its only active autonomous retrotransposons. However, the host factors that regulate LINE-1 retrotransposition ...are not fully understood. Here, we demonstrate that the Aicardi-Goutières syndrome gene product SAMHD1, recently revealed to be an inhibitor of HIV/simian immunodeficiency virus (SIV) infectivity and neutralized by the viral Vpx protein, is also a potent regulator of LINE-1 and LINE-1-mediated Alu/SVA retrotransposition. We also found that mutant SAMHD1s of Aicardi-Goutières syndrome patients are defective in LINE-1 inhibition. Several domains of SAMHD1 are critical for LINE-1 regulation. SAMHD1 inhibits LINE-1 retrotransposition in dividing cells. An enzymatic active site mutant SAMHD1 maintained substantial anti-LINE-1 activity. SAMHD1 inhibits ORF2p-mediated LINE-1 reverse transcription in isolated LINE-1 ribonucleoproteins by reducing ORF2p level. Thus, SAMHD1 may be a cellular regulator of LINE-1 activity that is conserved in mammals.
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•SAMHD1 is as a potent inhibitor of non-LTR retrotransposons in dividing cells•AGS-related SAMHD1 mutants have poor activity against LINE-1 retrotransposition•SAMHD1 suppresses HIV-1 and LINE-1 through distinct mechanisms•SAMHD1 inhibits ORF2p-mediated LINE-1 reverse transcription by reducing ORF2 level
Long interspersed elements 1 (LINE-1) are active autonomous retrotransposons that occupy 17% of the human genome. The host factors that regulate LINE-1 retrotransposition are not fully understood. In this study, Yu and colleagues show that the Aicardi-Goutières syndrome (AGS) gene product, SAMHD1, is a potent regulator of LINE-1 retrotransposition. SAMHD1 mutants linked to AGS are defective in LINE-1 inhibition. Although SAMHD1 has also recently been shown to suppress HIV-1, the authors find that suppression of LINE-1 occurs through a distinct mechanism.
LINE1s occupy 17% of the human genome and are its only active autonomous mobile DNA. L1s are also responsible for genomic insertion of processed pseudogenes and >1 million non-autonomous ...retrotransposons (Alus and SVAs). These elements have significant effects on gene organization and expression. Despite the importance of retrotransposons for genome evolution, much about their biology remains unknown, including cellular factors involved in the complex processes of retrotransposition and forming and transporting L1 ribonucleoprotein particles. By co-immunoprecipitation of tagged L1 constructs and mass spectrometry, we identified proteins associated with the L1 ORF1 protein and its ribonucleoprotein. These include RNA transport proteins, gene expression regulators, post-translational modifiers, helicases and splicing factors. Many cellular proteins co-localize with L1 ORF1 protein in cytoplasmic granules. We also assayed the effects of these proteins on cell culture retrotransposition and found strong inhibiting proteins, including some that control HIV and other retroviruses. These data suggest candidate cofactors that interact with the L1 to modulate its activity and increase our understanding of the means by which the cell coexists with these genomic 'parasites'.
Human retrotransposons generate structural variation and genomic diversity through ongoing retrotransposition and non-allelic homologous recombination. Cell culture retrotransposition assays have ...provided great insight into the genomic impact of retrotransposons, in particular, LINE-1(L1) and Alu elements; however, no such assay exists for the youngest active human retrotransposon, SINE-VNTR-Alu (SVA). Here we report the development of an SVA cell culture retrotransposition assay. We marked several SVAs with either neomycin or EGFP retrotransposition indicator cassettes. Engineered SVAs retrotranspose using L1 proteins supplemented in trans in multiple cell lines, including U2OS osteosarcoma cells where SVA retrotransposition is equal to that of an engineered L1. Engineered SVAs retrotranspose at 1-54 times the frequency of a marked pseudogene in HeLa HA cells. Furthermore, our data suggest a variable requirement for L1 ORF1p for SVA retrotransposition. Recovered engineered SVA insertions display all the hallmarks of LINE-1 retrotransposition and some contain 5′ and 3′ transductions, which are common for genomic SVAs. Of particular interest is the fact that four out of five insertions recovered from one SVA are full-length, with the 5′ end of these insertions beginning within 5 nt of the CMV promoter transcriptional start site. This assay demonstrates that SVA elements are indeed mobilized in trans by L1. Previously intractable questions regarding SVA biology can now be addressed.
The Long interspersed element 1 (LINE1 or L1) retrotransposon constitutes 17% of the human genome. There are currently 80-100 human L1 elements that are thought to be active in any diploid human ...genome. These elements can mobilize into new locations of the genome, resulting in changes in genomic information. Active L1s are thus considered to be a type of endogenous mutagen, and L1 insertions can cause disease. Certain stresses, such as gamma radiation, oxidative stress, and treatment with some agents, can induce transcription and/or mobilization of retrotransposons. In this study, we used a reporter gene assay in HepG2 cells to screen compounds for the potential to enhance the transcription of human L1. We assessed 95 compounds including genotoxic agents, substances that induce cellular stress, and commercially available drugs. Treatment with 15 compounds increased the L1 promoter activity by >1.5-fold (p<0.05) after 6 or 24 hours of treatment. In particular, genotoxic agents (benzoapyrene, camptothecin, cytochalasin D, merbarone, and vinblastine), PPARα agonists (bezafibrate and fenofibrate), and non-steroidal anti-inflammatory drugs (diflunisal, flufenamic acid, salicylamide, and sulindac) induced L1 promoter activity. To examine their effects on L1 retrotransposition, we developed a high-throughput real-time retrotransposition assay using a novel secreted Gaussia luciferase reporter cassette. Three compounds (etomoxir, WY-14643, and salicylamide) produced a significant enhancement in L1 retrotransposition. This is the first study to report the effects of a wide variety of compounds on L1 transcription and retrotransposition. These results suggest that certain chemical- and drug-induced stresses might have the potential to cause genomic mutations by inducing L1 mobilization. Thus, the risk of induced L1 transcription and retrotransposition should be considered during drug safety evaluation and environmental risk assessments of chemicals.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Purpose: Transforming growth factor (TGF)-β blockade has been proposed as an anticancer therapy; however, understanding which tumor
patients might benefit most from such therapy is crucial. An ideal ...target of such inhibitory therapy might be malignant mesothelioma
(MM), a highly lethal, treatment-resistant malignancy of mesothelial cells of the pleura and peritoneum that produces large
amounts of TGF-β. The purpose of this study was to explore the possible therapeutic utility of TGF-β blockade on MM.
Experimental Design: To evaluate this hypothesis, we tested the effects of a soluble TGF-β type II receptor (sTGF-βR) that specifically inhibits
TGF-β1 and TGF-β3 in three different murine MM tumor models, AB12 and AC29 (which produce large amounts of TGF-β) and AB1
(which does not produce TGF-β).
Results: Tumor growth of both established AB12 and AC29 tumors was inhibited by sTGF-βR. In contrast, AB1 tumors showed little response
to sTGF-βR. The mechanism of these antitumor effects was evaluated and determined to be primarily dependent on immune-mediated
responses because ( a ) the antitumor effects were markedly diminished in severe combined immunodeficient mice or mice depleted of CD8 + T cells and ( b ) CD8 + T cells isolated from spleens of mice treated with sTGF-βR showed strong antitumor cytolytic effects, whereas CD8 + T cells isolated from spleens of tumor-bearing mice treated with of control IgG2a showed no antitumor cytolytic effects.
Conclusions: Our data suggest that TGF-β blockade of established TGF-β-secreting MM should be explored as a promising strategy to treat
patients with MM and other tumors that produce TGF-β.
Transforming growth factor beta (TGF-beta) plays a complex role in breast carcinogenesis. Initially functioning as a tumor suppressor, this cytokine later contributes to the progression of malignant ...cells by enhancing their invasive and metastatic potential as well as suppressing antitumor immunity. The purpose of this study was to investigate the efficacy of SM16, a novel small molecule ALK5 kinase inhibitor, to treat a highly metastatic, TGF-beta-producing murine mammary carcinoma (4T1).
Mice bearing established 4T1 tumors were treated with SM16 intraperitoneally (i.p.) or orally, and primary and metastatic tumor growth was assessed.
SM16 inhibited Smad2 phosphorylation in cultured 4T1 tumor cells as well as primary and metastatic 4T1 tumor tissue. Blockade of TGF-beta signal transduction in 4T1 tumor cells by SM16 prevented TGF-beta-induced morphological changes and inhibited TGF-beta-induced invasion in vitro. When delivered via daily i.p. injection or orally through mouse chow, SM16 inhibited the growth of primary and metastatic 4T1 tumors. Splenocytes isolated from mice on the SM16 diet displayed enhanced IFN-gamma production and antitumor CTL activity. Furthermore, SM16 failed to inhibit the growth and metastasis of established 4T1 tumors in immunodeficient SCID mice.
Taken together, the data indicate that the antitumor efficacy of SM16 is dependent on an immune-mediated mechanism and that SM16 may represent a safe and effective treatment for metastatic breast cancer.
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