The folate cycle enzyme MTHFD2 induces cancer immune evasion through PD-L1 up-regulation.

Metabolic enzymes and metabolites display non-metabolic functions in immune cell signalling that modulate immune attack ability. However, whether and how a tumour's metabolic remodelling contributes to its immune resistance remain to be clarified. Here we perform a functional screen of metabolic genes that rescue tumour cells from effector T cell cytotoxicity, and identify the embryo- and tumour-specific folate cycle enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2). Mechanistically, MTHFD2 promotes basal and IFN-γ-stimulated PD-L1 expression, which is necessary for tumourigenesis in vivo. Moreover, IFN-γ stimulates MTHFD2 through the AKT-mTORC1 pathway. Meanwhile, MTHFD2 drives the folate cycle to sustain sufficient uridine-related metabolites including UDP-GlcNAc, which promotes the global O-GlcNAcylation of proteins including cMYC, resulting in increased cMYC stability and PD-L1 transcription. Consistently, the O-GlcNAcylation level positively correlates with MTHFD2 and PD-L1 in pancreatic cancer patients. These findings uncover a non-metabolic role for MTHFD2 in cell signalling and cancer biology.

Association between APOBEC3H-Mediated Demethylation and Immune Landscape in Head and Neck Squamous Carcinoma.

Immunotherapy has been demonstrated as a promising strategy in controlling head and neck squamous cell carcinoma (HNSC). The AID/APOBEC family is well characterized as DNA mutator and considered to play critical roles in immune responses in HNSC. However, the expression pattern and deamination-dependent demethylation roles of AID/APOBECs in HNSC are unclear. In this study, the RNA-seq and DNA methylation profiles of HNSC from TCGA database and cell-based experiments were applied to analyze the relationships between AID/APOBEC expression levels, patients' clinical outcomes, methylation alterations, and immune responses. Here, we found that APOBEC3H was abnormally upregulated in HNSC patients. HPV+ patients tended to have higher APOBEC3H levels than HPV- patients. Remarkably, patients with high APOBEC3H levels showed a favorable overall survival. Furthermore, tumors with high APOBEC3H levels exhibited a genome-wide DNA hypomethylation pattern. APOBEC3H was identified to demethylate and upregulate CXCL10 and improve CD8+ T cell tumor infiltration in the tumor microenvironment. Collectively, APOBEC3H plays critical roles in CD8+ T cell immune infiltration and activation in HNSC, which may be a potential biomarker for oncoimmunotherapy in HNSC.

B cells from young and old mice switch isotypes with equal frequencies after ex vivo stimulation.

To determine whether old B cells have the same capacity to switch isotypes as young cells, we purified splenic follicular, marginal zone, and age-associated B cell subsets from C57BL/6 mice. Cells were stimulated in culture with interleukin 4 and either lipopolysaccharide or anti-CD40, and switching to IgG1 was measured by flow cytometry of surface immunoglobulin. The results show that switching was robust in follicular and marginal zone B cells from old mice and was comparable to their young counterparts. However, age-associated B cells from old mice switched poorly relative to the other subsets. Expression of activation-induced deaminase, which initiates switching, was quantified by qPCR of mRNA, and it was equal between young and old follicular B cells. Thus, in this ex vivo system, the follicular and marginal zone cells from young and old mice behaved similarly, showing that the molecular machinery to perform switching is intact in old B cells.

Genetic and mechanistic basis for APOBEC3H alternative splicing, retrovirus restriction, and counteraction by HIV-1 protease.

Human APOBEC3H (A3H) is a single-stranded DNA cytosine deaminase that inhibits HIV-1. Seven haplotypes (I-VII) and four splice variants (SV154/182/183/200) with differing antiviral activities and geographic distributions have been described, but the genetic and mechanistic basis for variant expression and function remains unclear. Using a combined bioinformatic/experimental analysis, we find that SV200 expression is specific to haplotype II, which is primarily found in sub-Saharan Africa. The underlying genetic mechanism for differential mRNA splicing is an ancient intronic deletion [del(ctc)] within A3H haplotype II sequence. We show that SV200 is at least fourfold more HIV-1 restrictive than other A3H splice variants. To counteract this elevated antiviral activity, HIV-1 protease cleaves SV200 into a shorter, less restrictive isoform. Our analyses indicate that, in addition to Vif-mediated degradation, HIV-1 may use protease as a  counter-defense mechanism against A3H in >80% of sub-Saharan African populations.

Der f 34, a Novel Major House Dust Mite Allergen Belonging to a Highly Conserved Rid/YjgF/YER057c/UK114 Family of Imine Deaminases.

The high prevalence of house dust mite (HDM) allergy is a growing health problem worldwide, and the characterization of clinically important HDM allergens is a prerequisite for the development of diagnostic and therapeutic strategies. Here, we report a novel HDM allergen that belongs structurally to the highly conserved Rid/YjgF/YER057c/UK114 family (Rid family) with imine deaminase activity. Isolated HDM cDNA, named der f 34, encodes 128 amino acids homologous to Rid-like proteins. This new protein belongs to the Rid family and has seven conserved residues involved in enamine/imine deaminase activity. Indeed, we demonstrated that purified Der f 34 had imine deaminase activity that preferentially acted on leucine and methionine. Native Der f 34 showed a high IgE binding frequency as revealed by two-dimensional immunoblotting (62.5%) or ELISA (68%), which was comparable with those of a major HDM allergen Der f 2 (77.5 and 79%, respectively). We also found that Der f 34 showed cross-reactivity with another prominent indoor allergen source, Aspergillus fumigatus This is the first report showing that the Rid family imine deaminase represents an additional important pan-allergen that is conserved across organisms.

High Expression of Antiviral Proteins in Mucosa from Individuals Exhibiting Resistance to Human Immunodeficiency Virus.

BACKGROUND: Several soluble factors have been reported to have the capacity of inhibiting HIV replication at different steps of the virus life cycle, without eliminating infected cells and through enhancement of specific cellular mechanisms. Yet, it is unclear if these antiviral factors play a role in the protection from HIV infection or in the control of viral replication. Here we evaluated two cohorts: i) one of 58 HIV-exposed seronegative individuals (HESNs) who were compared with 59 healthy controls (HCs), and ii) another of 13 HIV-controllers who were compared with 20 HIV-progressors. Peripheral blood, oral and genital mucosa and gut-associated lymphoid tissue (GALT) samples were obtained to analyze the mRNA expression of ELAFIN, APOBEC3G, SAMHD1, TRIM5α, RNase 7 and SerpinA1 using real-time PCR. RESULTS: HESNs exhibited higher expression of all antiviral factors in peripheral blood mononuclear cells (PBMCs), oral or genital mucosa when compared with HCs. Furthermore, HIV-controllers exhibited higher levels of SerpinA1 in GALT. CONCLUSIONS: These findings suggest that the activity of these factors is compartmentalized and that these proteins have a predominant role depending on the tissue to avoid the infection, reduce the viral load and modulate the susceptibility to HIV infection.

HIV-1 Vif adaptation to human APOBEC3H haplotypes.

Several human APOBEC3 deaminases can inhibit HIV-1 replication in vitro. HIV-1 Vif counteracts this restriction by targeting APOBEC3 for proteasomal degradation. Human APOBEC3H (A3H) is highly polymorphic, with natural variants differing considerably in anti-HIV-1 activity in vitro. To examine HIV-1 adaptation to variation in A3H activity in a natural infection context, we determined the A3H haplotypes and Vif sequences from 76 recently infected HIV-1 patients. We detected A3H-specific Vif changes suggesting viral adaptation. The patient-derived Vif sequences were used to engineer viruses that specifically differed in their ability to counteract A3H. Replication of these Vif-variant viruses in primary T cells naturally expressing active or inactive A3H haplotypes showed that endogenously expressed A3H restricts HIV-1 replication. Proviral DNA from A3H-restricted viruses showed high levels of G-to-A mutations in an A3H-specific GA dinucleotide context. Taken together, our data validate A3H expressed at endogenous levels as a bona fide HIV-1 restriction factor.

APOBEC3A, APOBEC3B, and APOBEC3H haplotype 2 restrict human T-lymphotropic virus type 1.

The human APOBEC3 family consists of seven cytidine deaminases (A3A to A3H), some of which display potent antiretroviral activity against HIV-1 and other retroviruses. Studies that analyzed the effect of A3G on human T-lymphotropic virus type 1 (HTLV-1) infectivity resulted in conflicting findings, and our knowledge of HTLV-1 restriction by other A3 proteins remains limited. Since HTLV-1, much like HIV, targets CD4(+) T cells, we hypothesized that A3 proteins other than A3G restrict HTLV-1. All seven human A3 proteins were tested in HTLV-1 reporter and HIV-1 infectivity assays. We show that A3A, A3B, and A3H haplotype 2 (A3H hapII) acted as potent inhibitors of HTLV-1. Wild-type HIV-1, in contrast, was restricted by A3B and A3H hapII, but not by A3A. Catalytic site mutants of A3A, A3B, and A3H hapII showed that A3A and A3B restriction of HTLV-1 required deaminase activity. However, A3H hapII acted in a deaminase-independent manner when restricting HTLV-1, while requiring deaminase activity for HIV-1 restriction. We also analyzed A3 editing of HTLV-1 in five T-cell lines obtained from HTLV-1-infected patients. These cell lines contained extensively edited HTLV-1 sequences with G-to-A mutations in dinucleotide contexts suggestive of APOBEC3 mutagenesis. Comparison of the A3-induced mutations from reporter cells and the patient-derived cell lines indicate that A3G but also other A3 members, possibly A3A and A3B, affect HTLV-1 in vivo. Taken together, our data indicate that HTLV-1 is a likely target for multiple A3 proteins.

The localization of APOBEC3H variants in HIV-1 virions determines their antiviral activity.

Several members of the human APOBEC3 family of cytidine deaminases can potently restrict retroviruses such as HIV-1. The single-domain APOBEC3H (A3H) is encoded by four haplotypes, of which only A3H haplotype II-RDD (hapII-RDD) restricts HIV-1 efficiently. The goal of this study was to elucidate the mechanisms underlying the differences in antiviral activity among A3H haplotypes. The naturally occurring A3H hapI-GKE and hapII-RDD variants differ at three amino acid positions. A panel of six site-directed mutants containing combinations of the three variable residues was used to determine A3H protein expression, requirements of A3H virion incorporation, and A3H-Gag interactions. The catalytic activity of each A3H protein was assessed directly by using an Escherichia coli mutator assay. We found that the incorporation efficiencies of A3H variants into HIV-1 virions were comparable despite major differences in cellular expression. An assessment of the enzymes' catalytic activities showed that the deaminase activity of each A3H variant correlated with protein expression, suggesting similar enzymatic efficiencies. Surprisingly, virion incorporation experiments using Gag deletion mutants demonstrated that A3H haplotypes interacted with different Gag regions. A3H hapII-RDD associated with nucleocapsid in an RNA-dependent manner, whereas A3H hapI-GKE associated with the C-terminal part of matrix and the N-terminal capsid domain. Our results show that the A3H hapII-RDD interaction with nucleocapsid is critical for its antiviral activity and that the inability of A3H hapI-GKE to interact with nucleocapsid underlies its limited antiviral potential. Thus, the antiviral activity of A3H haplotypes is determined by its incorporation into the viral core, in proximity to the reverse transcription complex.

Mammalian nitrilase 1 homologue Nit1 is a negative regulator in T cells.

The mammalian Nit1 protein is homologous to plant and bacterial nitrilases. In flies and worms, Nit1 is fused to the 5' end of Fhit, suggesting that Nit1 may functionally interact with the Fhit pathway. Fhit has been shown to play a role of a tumor suppressor. Somatic loss of Fhit in human tissues is associated with a wide variety of cancers. Deletion of Fhit results in a predisposition to induced and spontaneous tumors in mice. It has been suggested that Nit1 collaborates with Fhit in tumor suppression. Similar to mice lacking Fhit, Nit1-deficient mice are more sensitive to carcinogen-induced tumors. It was previously shown that ectopic expression of Nit1 or Fhit led to caspase activation and apoptosis, and that both proteins may play a role in DNA damage-induced apoptosis. In this study, we analyzed the physiological function of Nit1 in T cells using Nit1-knockout mice. Nit1-deficient T cells can undergo apoptosis induced by DNA damage due to irradiation and chemical treatment. However, apoptosis induced by Fas or Ca(++) signals appeared to be compromised. Additionally, Nit1 deficiency resulted in T cell hyperproliferative responses induced by TCR stimulation. The expressions of T cell activation markers were elevated in Nit1(-/-) T cells. There was a spontaneous cell cycle entry and enhanced cell cycle progression in Nit1(-/-) T cells. These data indicate that Nit1 is a novel negative regulator in primary T cells.

Nitrilase of Rhodococcus rhodochrous J1. Purification and characterization.

Nitrilase was purified from an extract of isovaleronitrile-induced cells of Rhodococcus rhodochrous J1 in seven steps. In the last step, the enzyme was crystallized by adding ammonium sulfate. The crystallized enzyme appeared to be homogeneous by polyacrylamide electrophoresis, ampholyte electrofocusing and double immunodiffusion in agarose. The enzyme has a molecular mass of about 78 kDa and consists of two subunits identical in molecular mass. The purified enzyme exhibits a pH optimum of 7.6 and a temperature optimum of 45 degrees C. The enzyme catalyzed stoichiometrically the hydrolysis of benzonitrile to benzoic acid and ammonia, and no formation of amide was detected. The enzyme required thiol compounds such as dithiothreitol, L-cysteine or reduced glutathione to exhibit maximum activity. The enzyme was specific for nitrile groups attached to an aromatic or heteroaromatic ring, e.g. benzonitrile, 3-chlorobenzonitrile, 4-tolunitrile, 2-furonitrile and 2-thiophenecarbonitrile. The comparison of the properties of the enzyme with other nitrilases and nitrile hydratases has been also discussed.

Purification, immunoassay, and tissue distribution of rat C1-tetrahydrofolate synthase.

C1-tetrahydrofolate synthase (C1-THF synthase), a eukaryotic trifunctional enzyme, catalyzes three sequential folate-mediated one-carbon interconversions. These three reactions supply the activated one-carbon units required in the metabolism of purines, thymidylate, and several amino acids. In order to study the regulation of C1-THF synthase expression in mammals, we have purified the enzyme to homogeneity from rat liver, raised polyclonal antisera to it in rabbits, and developed a sensitive solid-phase immunoassay for the enzyme. The enzyme was purified approximately 600-fold to a specific activity of 24.6 U/mg protein based on 10-formyl-THF synthetase activity. Western blot analysis indicated that the antisera is specific for one protein in crude liver extracts which comigrates with purified C1-THF synthase. Using the solid-phase immunoassay, as little as 200 pg of immunoreacting protein can be detected in tissue homogenates. Several rat tissues were examined for the three C1-THF synthase enzymatic activities and immunoreactive protein. The results indicated that the level of C1-THF synthase is regulated in a tissue-specific manner. Enzyme assays revealed that certain tissues differ by more than 100-fold in enzyme activity, with liver and kidney containing the highest levels, and lung and muscle the lowest. However, immunoassay of these same tissues indicated only a 10-fold difference in C1-THF synthase concentration. This apparent masking of enzyme activity was observed in all tissues, but to varying degrees. These results emphasize the advantages of an immunoassay in studying the regulation of C1-THF synthase.

Characterization of antibody against human liver guanase by immunoblotting and immunohistochemical staining of human liver guanase by a direct labeling antibody-enzyme method.

Human liver guanase was purified and a specific antibody against it was raised in rabbits. The antiserum formed a single precipitin line with human liver extract, and also completely inhibited the activity of the liver enzyme. An immunoblotting study showed that the antibody bound specifically to one band of protein with guanase activity and not to other proteins. Therefore, we concluded that this antiserum against the liver enzyme was suitable for use in immunohistochemical demonstration of guanase. In tissue sections, the immunohistochemical reaction with this antibody was positive in the same locations as the histochemical guanase reaction with DAB (3,3'-diaminobenzidine tetrahydrochloride).

Nitrous oxide exposure reduces hepatic C1-tetrahydrofolate synthase expression in rats.

C1-tetrahydrofolate synthase (C1-THF synthase) is a trifunctional enzyme which catalyzes the interconversion of one-carbon units attached to the coenzyme THF. Nitrous oxide (N2O) inhalation is known to inactivate hepatic cobalamin-dependent methionine synthase leading to methionine deficiency and trapping of THF in the methyl-THF form. Liver tissue from rats exposed to N2O for 48 hours exhibited a coordinate decrease in all three activities of C1-THF synthase of approximately 25%. A corresponding 25% decrease in immunoreactive C1-THF synthase was also observed after 48 hours. Thus, the decrease in the concentration of C1-THF synthase accounted entirely for the decreases observed in the three activities. These results suggest that perturbations of hepatic THF pools by N2O affect the level of C1-THF synthase expression at a translational or pretranslational level.

Methylenetetrahydrofolate dehydrogenases in normal and transformed mammalian cells.

Transformed mammalian cells express both the usual NADP-dependent trifunctional methylenetetrahydrofolate dehydrogenase-cyclohydrolase-synthetase as well as the bifunctional NAD-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase. Antisera to these proteins do not crossreact, and Western blots of cell extracts indicate that there is no inactive form of the NAD-dependent enzyme in normal tissues. Immunofluorescence studies suggest a cytosolic location for the NAD-dependent enzyme, although sequence homology seen in the N-terminal 10 residues indicates a closer relationship with the mitochondrial form of the yeast NADP dependent trifunctional enzyme.

The NADP-dependent trifunctional methylenetetrahydrofolate dehydrogenase purified from mouse liver is immunologically distinct from the mouse NAD-dependent [corrected] bifunctional enzyme.

Methylenetetrahydrofolate dehydrogenase - methenyltetrahydrofolate cyclohydrolase - formyltetrahydrofolate synthetase was purified to homogeneity from mouse liver, taking advantage of its very high affinity for 2',5'-ADP-Sepharose. Antibodies raised to this trifunctional enzyme and to the bifunctional NAD-dependent dehydrogenase-cyclohydrolase from mouse Ehrlich ascites tumour cells were found not to cross-react with the purified proteins on Western blots. Each of these polyclonal antibodies detects the appropriate protein in extracts of Ehrlich ascites tumour cells after sodium dodecyl sulfate - polyacrylamide gel electrophoresis and electrophoretic transfer of the proteins to nitrocellulose. The procedure has also been used to obtain a purified preparation of the trifunctional enzyme from human liver obtained at autopsy.

Immunological crossreactivity of eukaryotic C1-tetrahydrofolate synthase and prokaryotic 10-formyltetrahydrofolate synthetase.

Antiserum to yeast C1-tetrahydrofolate (C1-H4folate) synthase reacts with other eukaryotic C1-H4folate synthases and prokaryotic 10-formyltetrahydrofolate (10-CHO-H4folate) synthetases [formate:tetrahydrofolate ligase (ADP-forming), EC 6.3.4.3] even though these enzymes vary in subunit size and function and probably vary widely in sequence. The comigration of the purified enzymes with the immunoreactive material establishes the specificity of the reaction for C1-H4folate synthase proteins. Reciprocal crossreaction of the antibody to Clostridium acidiurici 10-CHO-H4folate synthetase with the eukaryotic proteins indicates that such broad cross-species reactions are not specific to the antisera elicited in response to the yeast C1-H4folate synthase. These specific crossreactions among divergent species have been observed only on an electrophoretic transfer blot of a denaturing polyacrylamide gel. These observations may have been possible because of the sensitivity and specificity of the technique, which differ from more conventional immunochemical methods.