Salmonella mutant strains resistant to herbicides - Acetohydroxyacid synthase inhibitors and their use at the Ames test.

Cytotoxicity of some pesticides is a disadvantage for the Salmonella/microsome assay with regard to the equivalence assessment of pesticide technical grade active ingredients to the original products and detection of low-level impurities. The technical grade active ingredients (TGAIs) of pesticides from certain chemical classes were found to be toxic for Salmonella typhimurium strains. Among the highly cytotoxic compounds were sulfonylureas, which include 20 active ingredients. In addition, this class includes active pharmaceutical ingredients used for the manufacture of antidiabetics drugs. A traditional selection methodology was applied using the cultivation of S. typhimurium TA100 in the presence of high concentrations of thifensulfuronmethyl (TFSM) to obtain a resistant test strain insusceptible to sulfonylurea toxic effect. Two strains resistant not only to sulfonylureas (SFU) but also triazolepyrimidines were received. The first mutant strain (deposited as S. typhimurium VKPM B-14099 in the Russian National Collection of Industrial Microorganisms) demonstrated the TA100 phenotypic characteristics: hisG46, rfa, ΔuvrB-bio, pKM101. The second strain (deposited as S. typhimurium VKPM B-14359) showed the TA1535 phenotypic characteristics and probably lost the R-factor due to the selection using the poor Gm-media with TFSM. Positive controls caused pronounced mutagenic effects (±S9) in both strains, consequently the mutants did not lose the ability to respond to induction of the reverse gene mutations. The maximum non-cytotoxic concentrations of SFUs and triazole-pyrimidines for the Ames test strains did not exceed 0.05-0.125 mg/plate, while no evidence of cytotoxicity was observed for the mutants up to 5.0 mg/plate. Electron microscopy of the ultrathin sections of Salmonella cells grown with and without TFSM showed an obvious difference in the structure of the cell wall and cytoplasm in mutant and parental cultures. The concurrent resistance both to SFU and triazolepyrimidines was assumed to be mediated by the same mechanism of action of the pesticides from these classes - inhibition of acetohydroxyacid synthase. To confirm this hypothesis, the tests in the presence of branched-chain amino acids were carried out. The enrichment of agar with isoleucine prevented the toxic effects of SFU and triazolepyrimidines for all Ames test strains used in the study, while strong cytotoxicity was observed in the presence of valine and leucine. Considering the tolerance of strains both to SFU and triazolpyrimidines and the results with branched-chain amino acids, the modification of target acetohydroxyacid synthase was supposed the key to the acquired resistance. The new strains resistant to sulfonylureas and triazole-pyrimidines expands the possibilities to reveal mutagenic impurities that may occur in TGAIs in small amounts.

Cancer Patients' Preferences and Perceptions of Advantages and Disadvantages of Telehealth Visits During the COVID-19 Pandemic.

PURPOSE: We aimed to ascertain oncology patients' perceptions of telehealth versus in-person (IP) visits for different types of clinical encounters. METHODS: We surveyed adults undergoing cancer treatment at Kaiser Permanente Northern California infusion centers between November 2021 and May 2022 using a self-administered questionnaire. Patients were asked about visit modality preferences (video, phone, and IP) for six types of clinical discussions, overall advantages and disadvantages of telehealth (video or phone) versus IP modalities, and barriers to video visit use. RESULTS: The 839 patients who completed surveys in English were 63% female; median age 63 years; 64% White; and 73% college-educated (45% ≥bachelor's degree). For the first postdiagnosis discussion visit, 83% of patients preferred IP, followed by video (27%) and phone (18%). For follow-up visits, 52% of patients preferred IP, 50% video, and 37% phone. For discussions of bad news and sensitive topics, respectively, 68% and 62% preferred IP, 44% and 48% video, and 32% and 41% phone visits. Delivery of good news was acceptable through IP (49%), video (52%), or phone (49%) visits. Perceived advantages of IP visits were greater feelings of connection with their doctor (58%), confidence in physical examinations (73%), and ease in showing things (67%) and talking (51%) to the doctor. Advantages of telehealth visits included saved time (72%) and money (38%), less infection exposure (64%), less travel concerns (45%), and ability to include more people (28%). Of 24% of patients who felt video visits would be hard, 51% cited poor internet, 41% lack of an adequate device, and 28% difficulty signing on. CONCLUSION: Our results support continued use and reimbursement for telehealth visits with patients with cancer for most types of clinical encounters, including clinical trials.

Spectrum collapse in a 7-core Yb-doped fiber laser with an array of fs-inscribed fiber Bragg gratings.

Femtosecond inscription of fiber Bragg gratings (FBGs) in each core of a cladding-pumped seven-core Yb-doped fiber enables efficient (≈70%) 1064-nm lasing in a robust all-fiber scheme with ≈33 W power, nearly the same for uncoupled and coupled cores. However, the output spectrum is quite different: without coupling, seven individual lines corresponding to the in-core FBG reflection spectra sum up into a broad (0.22 nm) total spectrum, whereas the multiline spectrum collapses into a single narrow line at strong coupling. The developed model shows that the coupled-core laser generates coherent superposition of supermodes at the wavelength corresponding to the geometric mean of the individual FBG spectra, whereas the generated laser line broadens, with a power (0.04-0.12 nm) like the single-core mode of a seven-times larger effective area.

Advance Directives for Patients With Breast Cancer: Applying the Right Info/Right Care/Right Patient/Right Time Oncology Model.

Background Advance directives (AD) are an important component of life care planning for patients undergoing treatment for cancer; however, there are few effective interventions to increase AD rates. In this quality improvement project, the authors integrated AD counseling into a novel right info/right care/right patient/right time (4R) sequence of care oncology delivery intervention for breast cancer patients in an integrated health care delivery system. Methods The authors studied two groups of patients with newly diagnosed breast cancer who attended a multidisciplinary clinic and underwent definitive surgery at a single facility. The usual care (UC) cohort (N = 139) received care from October 1, 2019 to September 30, 2020. The 4R cohort (N = 141) received care from October 1, 2020 to September 30, 2121 that included discussing AD completion with a health educator prior to surgery. The authors used bivariate analyses to assess whether the AD intervention increased AD completion rates and to identify factors influencing AD completion. Results The UC and 4R cohorts were similar in age, gender, race/ethnicity, interpreter need, Elixhauser comorbidity index, National Comprehensive Cancer Network distress score ≥ 5, surgery type, stage, histology, grade, and Estrogen receptor/Progesterone receptor/ human epidermal growth factor receptor 2 (ER/PR/HER2) status. AD completion rates prior to surgery were significantly higher for the 4R vs UC cohort (73.8%, 95% confidence interval [CI] [66.5%-81.0%] vs 15.1%, 95% CI [9.2%-21.1%], p < .01) and did not significantly differ by age, race, need for interpreter, or distress scores. Conclusion Incorporation of a health educator discussion into a 4R care sequence plan significantly increased rates of time-sensitive AD completion.

Therapeutic Targeting of LIF Overcomes Macrophage-mediated Immunosuppression of the Local Tumor Microenvironment.

PURPOSE: Leukemia inhibitory factor (LIF) is a multifunctional cytokine with numerous reported roles in cancer and is thought to drive tumor development and progression. Characterization of LIF and clinical-stage LIF inhibitors would increase our understanding of LIF as a therapeutic target. EXPERIMENTAL DESIGN: We first tested the association of LIF expression with transcript signatures representing multiple processes regulating tumor development and progression. Next, we developed MSC-1, a high-affinity therapeutic antibody that potently inhibits LIF signaling and tested it in immune competent animal models of cancer. RESULTS: LIF was associated with signatures of tumor-associated macrophages (TAM) across 7,769 tumor samples spanning 22 solid tumor indications. In human tumors, LIF receptor was highly expressed within the macrophage compartment and LIF treatment drove macrophages to acquire immunosuppressive capacity. MSC-1 potently inhibited LIF signaling by binding an epitope that overlaps with the gp130 receptor binding site on LIF. MSC-1 showed monotherapy efficacy in vivo and drove TAMs to acquire antitumor and proinflammatory function in syngeneic colon cancer mouse models. Combining MSC-1 with anti-PD1 leads to strong antitumor response and a long-term tumor-free survival in a significant proportion of treated mice. CONCLUSIONS: Overall, our findings highlight LIF as a therapeutic target for cancer immunotherapy.

Inhibition of anti-tumor immunity by melanoma cell-derived Activin-A depends on STING.

The transforming growth factor-ß (TGF-ß) family member activin A (hereafter Activin-A) is overexpressed in many cancer types, often correlating with cancer-associated cachexia and poor prognosis. Activin-A secretion by melanoma cells indirectly impedes CD8+ T cell-mediated anti-tumor immunity and promotes resistance to immunotherapies, even though Activin-A can be proinflammatory in other contexts. To identify underlying mechanisms, we here analyzed the effect of Activin-A on syngeneic grafts of Braf mutant YUMM3.3 mouse melanoma cells and on their microenvironment using single-cell RNA sequencing. We found that the Activin-A-induced immune evasion was accompanied by a proinflammatory interferon signature across multiple cell types, and that the associated increase in tumor growth depended at least in part on pernicious STING activity within the melanoma cells. Besides corroborating a role for proinflammatory signals in facilitating immune evasion, our results suggest that STING holds considerable potential as a therapeutic target to mitigate tumor-promoting Activin-A signaling at least in melanoma.

Activin-A impairs CD8 T cell-mediated immunity and immune checkpoint therapy response in melanoma.

BACKGROUND: Activin-A, a transforming growth factor ß family member, is secreted by many cancer types and is often associated with poor disease prognosis. Previous studies have shown that Activin-A expression can promote cancer progression and reduce the intratumoral frequency of cytotoxic T cells. However, the underlying mechanisms and the significance of Activin-A expression for cancer therapies are unclear. METHODS: We analyzed the expression of the Activin-A encoding gene INHBA in melanoma patients and the influence of its gain- or loss-of-function on the immune infiltration and growth of BRAF-driven YUMM3.3 and iBIP2 mouse melanoma grafts and in B16 models. Using antibody depletion strategies, we investigated the dependence of Activin-A tumor-promoting effect on different immune cells. Immune-regulatory effects of Activin-A were further characterized in vitro and by an adoptive transfer of T cells. Finally, we assessed INHBA expression in melanoma patients who received immune checkpoint therapy and tested whether it impairs the response in preclinical models. RESULTS: We show that Activin-A secretion by melanoma cells inhibits adaptive antitumor immunity irrespective of BRAF status by inhibiting CD8+ T cell infiltration indirectly and even independently of CD4 T cells, at least in part by attenuating the production of CXCL9/10 by myeloid cells. In addition, we show that Activin-A/INHBA expression correlates with anti-PD1 therapy resistance in melanoma patients and impairs the response to dual anti-cytotoxic T-Lymphocyte associated protein 4/anti-PD1 treatment in preclinical models. CONCLUSIONS: Our findings suggest that strategies interfering with Activin-A induced immune-regulation offer new therapeutic opportunities to overcome CD8 T cell exclusion and immunotherapy resistance.

Hinokiflavone Inhibits MDM2 Activity by Targeting the MDM2-MDMX RING Domain.

The proto-oncogene MDM2 is frequently amplified in many human cancers and its overexpression is clinically associated with a poor prognosis. The oncogenic activity of MDM2 is demonstrated by its negative regulation of tumor suppressor p53 and the substrate proteins involved in DNA repair, cell cycle control, and apoptosis pathways. Thus, inhibition of MDM2 activity has been pursued as an attractive direction for the development of anti-cancer therapeutics. Virtual screening was performed using the crystal structure of the MDM2-MDMX RING domain dimer against a natural product library and identified a biflavonoid Hinokiflavone as a promising candidate compound targeting MDM2. Hinokiflavone was shown to bind the MDM2-MDMX RING domain and inhibit MDM2-mediated ubiquitination in vitro. Hinokiflavone treatment resulted in the downregulation of MDM2 and MDMX and induction of apoptosis in various cancer cell lines. Hinokiflavone demonstrated p53-dependent and -independent tumor-suppressive activity. This report provides biochemical and cellular evidence demonstrating the anti-cancer effects of Hinokiflavone through targeting the MDM2-MDMX RING domain.

Multifidelity Statistical Machine Learning for Molecular Crystal Structure Prediction.

The prediction of crystal structures from first-principles requires highly accurate energies for large numbers of putative crystal structures. High accuracy of solid state density functional theory (DFT) calculations is often required, but hundreds or more structures can be present in the low energy region of interest, so that the associated computational costs are prohibitive. Here, we apply statistical machine learning to predict expensive hybrid functional DFT (PBE0) calculations using a multifidelity approach to re-evaluate the energies of crystal structures predicted with an inexpensive force field. The method uses an autoregressive Gaussian process, making use of less expensive GGA DFT (PBE) calculations to bridge the gap between the force field and PBE0 energies. The method is benchmarked on the crystal structure landscapes of three small, hydrogen-bonded organic molecules and shown to produce accurate predictions of energies and crystal structure ranking using small numbers of the most expensive calculations; the PBE0 energies can be predicted with errors of less than 1 kJ mol-1 with between 4.2 and 6.8% of the cost of the full calculations. As the model that we have developed is probabilistic, we discuss how the uncertainties in predicted energies impact the assessment of the energetic ranking of crystal structures.

Mutagenicity evaluation of pesticide analogs using standard and 6-well miniaturized bacterial reverse mutation tests.

The Ames test is widely used in the mutagenicity evaluation of new and existing chemicals as a part of a compound selection strategy, regulatory control, the equivalence assessment, carcinogenic potential measurement etc. Intensification of the chemical industry and synthesis of plenty of new molecules has led to the necessity of tests with a higher throughput capacity. The 6-well miniaturized bacterial reverse mutation test and the standard Ames test were compared using 14 technical grade active ingredients (TGAIs) of pesticides. With some exceptions, the responses obtained in the miniscreen Ames are similar to those seen in the standard method: 4 overall test outcomes were negative and 9 were positive in both test versions, but 1 discordant result between the miniscreen and standard version. Comparison of the standard and the miniscreen Ames test resulted in 98% of concordance across five strains and conditions (±S9). The overall judgment is that the miniscreen Ames test can be used to assess the mutagenicity of pesticide analogs. It has the advantage of decreasing the number of materials and animals (for S9) and keeping a high-test performance.

Advanced distributed feedback lasers based on composite fiber heavily doped with erbium ions.

Specially designed composite heavily Er3+-doped fiber in combination with unique point-by-point inscription technology by femtosecond pulses at 1,026 nm enables formation of distributed-feedback (DFB) laser with ultra-short cavity length of 5.3 mm whose parameters are comparable and even better than those for conventional Er3+-doped fiber DFB lasers having much longer cavity. The composite fiber was fabricated by melting rare-earth doped phosphate glass in silica tube. The ultra-short DFB laser generates single-polarization single-frequency radiation at 1,550 nm with narrow linewidth (3.5 kHz) and 0.5 mW output power at 600 mW 980-nm pumping. The same fiber with conventional CW UV (244 nm) inscription technology using phase mask enables fabrication of 40-mm long DFB laser with > 18 mW output power at 3.3% pump conversion, which is a record efficiency for Er3+-doped fiber DFB lasers. The developed technologies form an advanced platform for Er3+-doped fiber DFB lasers operating around 1.55 µm with excellent output characteristics and unique practical features, in particular, the ultra-short DFB lasers are attractive for sensing applications.

Genotoxicity of mixture of imidacloprid, imazalil and tebuconazole.

Genotoxicity of the mixture of generic pesticides imidacloprid + imazalil + tebuconazole in a ratio of 14.0/1.7/1.0 by weight was assessed using Ames test (Salmonella typhimurium) and micronucleus test in vivo on mammalian bone marrow erythrocytes (CD-1 mice) supporting the data creation for the Real Life Risk Simulation (RLRS) approach. This pesticides' combination is used in the commercial formulation for seed treatment in advance of or immediately before sowing. Tested pesticides' technical grade active ingredients (TGAIs) showed no evidence of genotoxicity upon separate treatments. In combination, the three pesticides demonstrated negative results in the Ames test but induced a statistically significant, dose-depended increase in MN-PCEs in mice bone marrow at doses lower than those used separately. The observed effect may be mediated by the synergistic action of the tested TGAIs, their metabolites or impurities.

Maternal blood folate status during early pregnancy and occurrence of autism spectrum disorder in offspring: a study of 62 serum biomarkers.

BACKGROUND: Autism spectrum disorder (ASD) evolves from an interplay between genetic and environmental factors during prenatal development. Since identifying maternal biomarkers associated with ASD risk in offspring during early pregnancy might result in new strategies for intervention, we investigated maternal metabolic biomarkers in relation to occurrence of ASD in offspring using both univariate logistic regression and multivariate network analysis. METHODS: Serum samples from 100 women with an offspring diagnosed with ASD and 100 matched control women with typically developing offspring were collected at week 14 of pregnancy. Concentrations of 62 metabolic biomarkers were determined, including amino acids, vitamins (A, B, D, E, and K), and biomarkers related to folate (vitamin B9) metabolism, lifestyle factors, as well as C-reactive protein (CRP), the kynurenine-tryptophan ratio (KTR), and neopterin as markers of inflammation and immune activation. RESULTS: We found weak evidence for a positive association between higher maternal serum concentrations of folate and increased occurrence of ASD (OR per 1 SD increase: 1.70, 95% CI 1.22-2.37, FDR adjusted P = 0.07). Multivariate network analysis confirmed expected internal biochemical relations between the biomarkers. Neither inflammation markers nor vitamin D3 levels, all hypothesized to be involved in ASD etiology, displayed associations with ASD occurrence in the offspring. CONCLUSIONS: Our findings suggest that high maternal serum folate status during early pregnancy may be associated with the occurrence of ASD in offspring. No inference about physiological mechanisms behind this observation can be made at the present time because blood folate levels may have complex relations with nutritional intake, the cellular folate status and status of other B-vitamins. Therefore, further investigations, which may clarify the potential role and mechanisms of maternal blood folate status in ASD risk and the interplay with other potential risk factors, in larger materials are warranted.

Mdm2 and MdmX RING Domains Play Distinct Roles in the Regulation of p53 Responses: A Comparative Study of Mdm2 and MdmX RING Domains in U2OS Cells.

Dysfunction of the tumor suppressor p53 occurs in most human cancers. Mdm2 and MdmX are homologous proteins from the Mdm (Murine Double Minute) protein family, which play a critical role in p53 inactivation and degradation. The two proteins interact with one another via the intrinsic RING (Really Interesting New Gene) domains to achieve the negative regulation of p53. The downregulation of p53 is accomplished by Mdm2-mediated p53 ubiquitination and proteasomal degradation through the ubiquitin proteolytic system and by Mdm2 and MdmX mediated inhibition of p53 transactivation. To investigate the role of the RING domain of Mdm2 and MdmX, an analysis of the distinct functionalities of individual RING domains of the Mdm proteins on p53 regulation was conducted in human osteosarcoma (U2OS) cell line. Mdm2 RING domain was observed mainly localized in the cell nucleus, contrasting the localization of MdmX RING domain in the cytoplasm. Mdm2 RING was found to possess an endogenous E3 ligase activity, whereas MdmX RING did not. Both Mdm2 and MdmX RING domains were able to dimerize with endogenous full-length Mdm2 and MdmX protein and affect their cellular function. The results showed that overexpression of the Mdm2 or MdmX RING domains interfered with the endogenous full-length Mdm2 and MdmX activity and resulted in p53 stabilization and p53 target gene activation. However, both Mdm RING domains showed oncogenic activity in a colony formation assay, suggesting that the Mdm RING domains possess p53-independent oncogenic properties. This study highlights the distinct structural and functional traits of the RING domain of Mdm2 and MdmX and characterized their role in cellular responses through interfering with p53 dependent signaling pathway.

In situ antibody phage display yields optimal inhibitors of integrin α11/ß1.

Integrins are transmembrane multi-conformation receptors that mediate interactions with the extracellular matrix. In cancer, integrins influence metastasis, proliferation, and survival. Collagen-binding integrin-α11/ß1, a marker of aggressive tumors that is involved in stroma-tumor crosstalk, may be an attractive target for anti-cancer therapeutic antibodies. We performed selections with phage-displayed synthetic antibody libraries for binding to either purified integrin-α11/ß1 or in situ on live cells. The in-situ strategy yielded many diverse antibodies, and strikingly, most of these antibodies did not recognize purified integrin-α11/ß1. Conversely, none of the antibodies selected for binding to purified integrin-α11/ß1 were able to efficiently recognize native cell-surface antigen. Most importantly, only the in-situ selection yielded functional antibodies that were able to compete with collagen-I for binding to cell-surface integrin-α11/ß1, and thus inhibited cell adhesion. In-depth characterization of a subset of in situ-derived clones as full-length immunoglobulins revealed high affinity cellular binding and inhibitory activities in the single-digit nanomolar range. Moreover, the antibodies showed high selectivity for integrin-α11/ß1 with minimal cross-reactivity for close homologs. Taken together, our findings highlight the advantages of in-situ selections for generation of anti-integrin antibodies optimized for recognition and inhibition of native cell-surface proteins, and our work establishes general methods that could be extended to many other membrane proteins.

Durable shape sensor based on FBG array inscribed in polyimide-coated multicore optical fiber.

The paper presents a novel three-dimensional quasi-continuous shape sensor based on an FBG array inscribed by femtosecond laser pulses into a 7-core optical fiber with a polyimide protective coating. The measured bending sensitivity of individual FBGs ranges from 0.046 nm/m-1 to 0.049 nm/m-1. It is shown that the sensor allows for reconstructing 2- and 3-dimensional shapes with high accuracy. Due to the high value of the core aperture and individual calibration of each FBG we were able to measure the smallest reported bending radii down to 2.6 mm with a record accuracy of ∼1%. Moreover, we investigate the magnitude of the errors of curves reconstruction and errors associated with measurement of curvature radii in the range from 2.6 to 500 mm. The main factors affecting the accuracy of measurements are also discussed. The temperature resistance of both the inscribed FBG structures and of the protective coating, along with the high mechanical strength of the polyimide, makes it possible to use the sensor in harsh environments or in medical and composite material applications.

Limitations of pesticide genotoxicity testing using the bacterial in vitro method.

The results of genotoxicity assessment of 115 technical grade pesticide products (61 names) with the Ames test and mammalian erythrocyte micronucleus test in vivo are presented. In most cases, unidirectional results were found. However, some pesticides belonging to classes of neonicotinoids, derivatives of phenylurea, glycine and phenoxycarboxylic acids showed genotoxic effects only under in vivo conditions. All observed effects were statistically significant and dose-depended but were only slightly beyond the upper limit of the historical negative control. Pendimethalin (dinitroaniline pesticide) showed positive results only in Ames test. Furthermore, the limitations of Ames test related to the high cytotoxicity of pesticides against bacterial cells were shown (e.g. bipyridylium derivatives, chlorothalonils, dithiocarbamates, quinones, phenyl pyridinamines and sulphonylureas). But many pesticides possessing the highest cytotoxic activity against bacterial cells have shown low toxicity in vivo (MTD > 2000 mg/kg b.w.). On the other hand, all of the tested neonicotinoids, organophosphates, pyrethroids, phenoxycarboxylic acid derivatives were highly toxic to mice but non-cytotoxic against microbial cells. Triazole pesticides exerted toxicity to the target organ (bone marrow). Therefore, these findings suggest that genotoxicity assessment using at least two methods is needed for reliable evidence of the safe use of pesticides.

The Legionella pneumophila effector Ceg4 is a phosphotyrosine phosphatase that attenuates activation of eukaryotic MAPK pathways.

Host colonization by Gram-negative pathogens often involves delivery of bacterial proteins called "effectors" into the host cell. The pneumonia-causing pathogen Legionella pneumophila delivers more than 330 effectors into the host cell via its type IVB Dot/Icm secretion system. The collective functions of these proteins are the establishment of a replicative niche from which Legionella can recruit cellular materials to grow while evading lysosomal fusion inhibiting its growth. Using a combination of structural, biochemical, and in vivo approaches, we show that one of these translocated effector proteins, Ceg4, is a phosphotyrosine phosphatase harboring a haloacid dehalogenase-hydrolase domain. Ceg4 could dephosphorylate a broad range of phosphotyrosine-containing peptides in vitro and attenuated activation of MAPK-controlled pathways in both yeast and human cells. Our findings indicate that L. pneumophila's infectious program includes manipulation of phosphorylation cascades in key host pathways. The structural and functional features of the Ceg4 effector unraveled here provide first insight into its function as a phosphotyrosine phosphatase, paving the way to further studies into L. pneumophila pathogenicity.

UbE2E1/UBCH6 Is a Critical in Vivo E2 for the PRC1-catalyzed Ubiquitination of H2A at Lys-119.

UbE2E1/UbcH6 is an E2 ubiquitin-conjugating enzyme that is regulated by USP7. We identified UbE2E1 as a novel component of Polycomb repressive complex 1 (PRC1), the E3 ligase complex responsible for histone H2A ubiquitination and gene silencing. We demonstrate that UbE2E1 is critical for the monoubiquitination of H2A at residue Lys-119 (uH2AK119) through its association with the PRC1 complex. UbE2E1 interacts with PRC1 subunits including Ring1A and Ring1B. Overexpression of UbE2E1 results in increased levels of uH2AK119, whereas overexpression of catalytically inactive UbE2E1_C131A or UbE2E1 knockdown results in decreased levels of uH2AK119. The down-regulation of H2A ubiquitination by loss of function of UbE2E1 is correlated with alleviated p16INK4a promoter repression and induced growth inhibition in HCT116 cells. These results are specific to UbE2E1 as knockdown of UbE2D E2s does not show any effect on uH2AK119. We extended the UbE2E1 regulation of uH2AK119 to USP7 and showed that USP7 is also a key regulator for monoubiquitination at H2A Lys-119 as both knockdown and deletion of USP7 results in decreased levels of uH2AK119. This study reveals that UbE2E1 is an in vivo E2 for the PRC1 ligase complex and thus plays an important role in the regulation of H2A Lys-119 monoubiquitination.

Identification of Kaposi Sarcoma Herpesvirus (KSHV) vIRF1 Protein as a Novel Interaction Partner of Human Deubiquitinase USP7.

Viral interferon regulatory factor 1 (vIRF1), a Kaposi sarcoma herpesvirus protein, destabilizes p53 by inhibiting p53 acetylation and Hdm2 phosphorylation. This leads to increased ubiquitination and degradation of p53 by Hdm2, which cripples the cellular p53-mediated antiviral response. Ubiquitin-specific protease 7 (USP7) deubiquitinates p53 and Hdm2 and regulates their stability. We identified an EGPS consensus sequence in vIRF1, which is identical to that found in Epstein-Barr virus nuclear antigen 1 (EBNA1) that interacts with the N-terminal domain of USP7 (USP7-NTD). GST pulldown assays demonstrated that vIRF1 interacts with USP7-NTD via its EGPS motif. NMR heteronuclear single quantum correlation (HSQC) analysis revealed chemical perturbations after titration of USP7-NTD with vIRF1 (44)SPGEGPSGTG(53) peptide. In contrast, these perturbations were reduced with a mutant vIRF1 peptide, (44)SPGEGPAGTG(53). Fluorescence polarization analysis indicated that the vIRF1 peptide interacted with USP7-NTD with a Kd of 2.0 µm. The crystal structure of the USP7-NTD·vIRF1 peptide complex revealed an identical mode of binding as that of the EBNA1 peptide to USP7-NTD. We also showed that USP7 interacts with vIRF1 in U2OS cells. Decreased levels of p53, but not Hdm2 or ataxia telangiectasia-mutated (ATM), were seen after expression of vIRF1, but not with a vIRF1 mutant protein. Our results support a new role for vIRF1 through deregulation of the deubiquitinating enzyme USP7 to inhibit p53-mediated antiviral responses.