Inosine-5'-monophosphate Dehydrogenase 2 Research Articles

SARS-CoV-2 Nsp14 protein associates with IMPDH2 and activates NF-κB signaling.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leads to NF-κB activation and induction of pro-inflammatory cytokines, though the underlying mechanism for this activation is not fully understood. Our results reveal that the SARS-CoV-2 Nsp14 protein contributes to the viral activation of NF-κB signaling. Nsp14 caused the nuclear translocation of NF-κB p65. Nsp14 induced the upregulation of IL-6 and IL-8, which also occurred in SARS-CoV-2 infected cells. IL-8 upregulation was further confirmed in lung tissue samples from COVID-19 patients. A previous proteomic screen identified the putative interaction of Nsp14 with host Inosine-5’-monophosphate dehydrogenase 2 (IMPDH2), which is known to regulate NF-κB signaling. We confirmed the Nsp14-IMPDH2 protein interaction and identified that IMPDH2 knockdown or chemical inhibition using ribavirin (RIB) and mycophenolic acid (MPA) abolishes Nsp14- mediated NF-κB activation and cytokine induction. Furthermore, IMPDH2 inhibitors (RIB, MPA) or NF-κB inhibitors (bortezomib, BAY 11-7082) restricted SARS-CoV-2 infection, indicating that IMPDH2-mediated activation of NF-κB signaling is beneficial to viral replication. Overall, our results identify a novel role of SARS-CoV-2 Nsp14 in inducing NF-κB activation through IMPDH2 to promote viral infection.

Quantitative Chemoproteomic Profiling of Protein Cross-Links Induced by Methylglyoxal.

Methylglyoxal (MGO) is a highly reactive metabolite mainly formed as a byproduct of glycolysis. Elevated MGO has been considered as a risk factor for several diseases including diabetes and neurodegeneration. While MGO modifications on proteins were globally profiled, the cross-links between proteins induced by MGO in proteomes are unexplored to date. Here, we reported a quantitative chemoproteomic platform based on mass shifts that enables identification of events of protein cross-links induced by MGO in proteomes. A total of 66 cross-linked targets were identified from the profiling experiments when cells were treated with MGO, among which the components of functional complexes such as spliceosomes and ribosomes were enriched. We found that inosine-5'-monophosphate dehydrogenase 2 (IMPDH2) was homocross-linked by MGO and the active-site Cys331 was critical for mediating the cross-link, which in turn affected IMPDH2's activity. Our study has provided new clues for the functional impact in proteomes by MGO, and the methodology can be, in principle, applied to profile protein cross-links induced by other reactive metabolites.

Enantioselective Synthesis and Biological Evaluation of Sanglifehrin A and B and Analogs.

Sanglifehrin A and B are immunosuppressive macrocyclic natural products endowed with and differentiated by a unique spirocyclic lactam. Herein, we report an enantioselective total synthesis and biological evaluation of sanglifehrin A and B and analogs. Access to the spirocyclic lactam was achieved through convergent assembly of a key pyranone intermediate followed by a stereo-controlled spirocyclization. The 22-membered macrocyclic core was synthesized by ring-closing metathesis in the presence of 2,6-bis(trifluoromethyl) benzeneboronic acid (BFBB). The spirocyclic lactam and macrocycle fragments were united by a Stille coupling to furnish sanglifehrin A and B. Additional sanglifehrin B analogs with variation at the C40 position were additionally prepared. Biological evaluation revealed that the 2-CF3 analog of sanglifehrin B exhibited higher anti-proliferative activity than the natural products sanglifehrin A and B in Jurkat cells. Both natural products induced higher-order homodimerization of cyclophilin A (CypA), but only sanglifehrin A promoted CypA complexation with inosine-5'-monophosphate dehydrogenase 2 (IMPDH2). The synthesis reported herein will enable further evaluation of the spirolactam and its contribution to sanglifehrin-dependent immunosuppressive activity.

Enantioselective Synthesis and Biological Evaluation of Sanglifehrin A and B and Analogs

AbstractSanglifehrin A and B are immunosuppressive macrocyclic natural products endowed with and differentiated by a unique spirocyclic lactam. Herein, we report an enantioselective total synthesis and biological evaluation of sanglifehrin A and B and analogs. Access to the spirocyclic lactam was achieved through convergent assembly of a key pyranone intermediate followed by a stereo‐controlled spirocyclization. The 22‐membered macrocyclic core was synthesized by ring‐closing metathesis in the presence of 2,6‐bis(trifluoromethyl) benzeneboronic acid (BFBB). The spirocyclic lactam and macrocycle fragments were united by a Stille coupling to furnish sanglifehrin A and B. Additional sanglifehrin B analogs with variation at the C40 position were additionally prepared. Biological evaluation revealed that the 2‐CF3 analog of sanglifehrin B exhibited higher anti‐proliferative activity than the natural products sanglifehrin A and B in Jurkat cells. Both natural products induced higher‐order homodimerization of cyclophilin A (CypA), but only sanglifehrin A promoted CypA complexation with inosine‐5′‐monophosphate dehydrogenase 2 (IMPDH2). The synthesis reported herein will enable further evaluation of the spirolactam and its contribution to sanglifehrin‐dependent immunosuppressive activity.

De novo purine biosynthesis is a major driver of chemoresistance in glioblastoma.

Glioblastoma is a primary brain cancer with a near 100% recurrence rate. Upon recurrence, the tumour is resistant to all conventional therapies, and because of this, 5-year survival is dismal. One of the major drivers of this high recurrence rate is the ability of glioblastoma cells to adapt to complex changes within the tumour microenvironment. To elucidate this adaptation's molecular mechanisms, specifically during temozolomide chemotherapy, we used chromatin immunoprecipitation followed by sequencing and gene expression analysis. We identified a molecular circuit in which the expression of ciliary protein ADP-ribosylation factor-like protein 13B (ARL13B) is epigenetically regulated to promote adaptation to chemotherapy. Immuno-precipitation combined with liquid chromatography-mass spectrometry binding partner analysis revealed that that ARL13B interacts with the purine biosynthetic enzyme inosine-5'-monophosphate dehydrogenase 2 (IMPDH2). Further, radioisotope tracing revealed that this interaction functions as a negative regulator for purine salvaging. Inhibition of the ARL13B-IMPDH2 interaction enhances temozolomide-induced DNA damage by forcing glioblastoma cells to rely on the purine salvage pathway. Targeting the ARLI3B-IMPDH2 circuit can be achieved using the Food and Drug Administration-approved drug, mycophenolate mofetil, which can block IMPDH2 activity and enhance the therapeutic efficacy of temozolomide. Our results suggest and support clinical evaluation of MMF in combination with temozolomide treatment in glioma patients.

DDRE-14. DE-NOVO PURINE BIOSYNTHESIS IS A MAJOR DRIVER OF CHEMORESISTANCE IN GLIOBLASTOMA

Glioblastoma is a primary brain cancer with a near 100% recurrence rate. Upon recurrence, the tumor is resistant to all conventional therapies, and because of this, 5-year survival is dismal. One of the major drivers of this high recurrence rate is glioblastoma cells’ ability to adapt to complex changes within the tumor microenvironment. To elucidate this adaptation’s molecular mechanisms, specifically during chemotherapy temozolomide, we employed chromatin immunoprecipitation followed by sequencing and gene expression analysis. We identified a molecular circuit in which the expression of ciliary protein ADP-ribosylation factor-like protein 13B (ALR13B) is epigenetically regulated to promote adaptation to chemotherapy. Immuno-precipitation combined with Liquid Chromatography-Mass Spectrometry binding partner analysis revealed that that ARL13B interacts with the purine biosynthetic enzyme inosine-5’-monophosphate dehydrogenase 2 (IMPDH2). Further, radioisotope tracing revealed that this interaction function as a negative regulator for purine salvaging. Inhibition of ARL13B-IMPDH2 interaction enhances temozolomide-induced DNA damage by forcing glioblastoma cells to rely on the purine salvage pathway. Targeting the ARLI3B-IMPDH2 circuit can be achieved using a Food and Drug Administration-approved drug, Mycophenolate Mofetil, that can block the IMPDH2 activity and enhance the therapeutic efficacy of TMZ. Our results suggest and support clinical evaluation of MMF in combination with TMZ treatment in glioma patients.

SARS-CoV-2 infection remodels the host protein thermal stability landscape.

The severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is a global threat to human health and has compromised economic stability. In addition to the development of an effective vaccine, it is imperative to understand how SARS‐CoV‐2 hijacks host cellular machineries on a system‐wide scale so that potential host‐directed therapies can be developed. In situ proteome‐wide abundance and thermal stability measurements using thermal proteome profiling (TPP) can inform on global changes in protein activity. Here we adapted TPP to high biosafety conditions amenable to SARS‐CoV‐2 handling. We discovered pronounced temporal alterations in host protein thermostability during infection, which converged on cellular processes including cell cycle, microtubule and RNA splicing regulation. Pharmacological inhibition of host proteins displaying altered thermal stability or abundance during infection suppressed SARS‐CoV‐2 replication. Overall, this work serves as a framework for expanding TPP workflows to globally important human pathogens that require high biosafety containment and provides deeper resolution into the molecular changes induced by SARS‐CoV‐2 infection.

Shikonin is a novel and selective IMPDH2 inhibitor that target triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is heterogeneous disease with a poor prognosis. It is therefore important to explore novel therapeutic agents to improve the clinical efficacy for TNBC. The inosine 5'-monophosphate dehydrogenase 2 (IMPDH2) is a rate-limiting enzyme in the de novo synthesis of guanine nucleotides. It is always overexpressed in many types of tumors, including TNBC and regarded as a potential target for cancer therapy. Through screening a library of natural products, we identified shikonin, a natural bioactive component of Lithospermum erythrorhizon, is a novel and selective IMPDH2 inhibitor. Enzymatic analysis using Lineweaver-Burk plot indicates that shikonin is a competitive inhibitor of IMPDH2. The interaction between shikonin and IMDPH2 was further investigated by thermal shift assay, fluorescence quenching, and molecular docking simulation. Shikonin treatment effectively inhibits the growth of human TNBC cell line MDA-MB-231, and murine TNBC cell line, 4T1 in a dose-dependent manner, which is impaired by exogenous supplementation of guanosine, a salvage pathway of purine nucleotides. Most importantly, IMPDH2 knockdown significantly reduced cell proliferation and conferred resistance to shikonin in TNBC. Collectively, our findings showed the natural product shikonin as a selective inhibitor of IMPDH2 with anti-TNBC activity, impelling its further study in clinical trials.

CLOCK Acetylates ASS1 to Drive Circadian Rhythm of Ureagenesis

In addition to responding to environmental entrainment with diurnal variation, metabolism is also tightly controlled by cell-autonomous circadian clock. Extensive studies have revealed key roles of transcription in circadian control. Post-transcriptional regulation for the rhythmic gating of metabolic enzymes remains elusive. Here, we show that arginine biosynthesis and subsequent ureagenesis are collectively regulated by CLOCK (circadian locomotor output cycles kaput) in circadian rhythms. Facilitated by BMAL1 (brain and muscle Arnt-like protein), CLOCK directly acetylates K165 and K176 of argininosuccinate synthase (ASS1) to inactivate ASS1, which catalyzes the rate-limiting step of arginine biosynthesis. ASS1 acetylation by CLOCK exhibits circadian oscillation in human cells and mouse liver, possibly caused by rhythmic interaction between CLOCK and ASS1, leading to the circadian regulation of ASS1 and ureagenesis. Furthermore, we also identified NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9 (NDUFA9) and inosine-5'-monophosphate dehydrogenase 2 (IMPDH2) as acetylation substrates of CLOCK. Taken together, CLOCK modulates metabolic rhythmicity by acting as a rhythmic acetyl-transferase for metabolic enzymes.

Serum depletion induces changes in protein expression in the trophoblast-derived cell line HTR-8/SVneo.

BackgroundHow nutrition and growth factor restriction due to serum depletion affect trophoblast function remains poorly understood. We performed a proteomic differential study of the effects of serum depletion on a first trimester human immortalized trophoblast cell line.MethodsThe viability of HTR-8/SVneo trophoblast cells in culture with 0, 0.5 and 10 % fetal bovine serum (FBS) were assayed via MTT at 24, 48 and 64 h. A comparative proteomic analysis of the cells grown with those FBS levels for 24 h was performed using two-dimensional electrophoresis (2DE), followed by mass spectrometry for protein spot identification, and a database search and bioinformatics analysis of the expressed proteins. Differential spots were identified using the Kolmogorov-Smirnov test (n = 3, significance level 0.10, D > 0.642) and/or ANOVA (n = 3, p < 0.05).ResultsThe results showed that low serum doses or serum depletion differentially affect cell growth and protein expression. Differential expression was seen in 25 % of the protein spots grown with 0.5 % FBS and in 84 % of those grown with 0 % FBS, using 10 % serum as the physiological control. In 0.5 % FBS, this difference was related with biological processes typically affected by the serum, such as cell cycle, regulation of apoptosis and proliferation. In addition to these changes, in the serum-depleted proteome we observed downregulation of keratin 8, and upregulation of vimentin, the glycolytic enzymes enolase and pyruvate kinase (PKM2) and tumor progression-related inosine-5’-monophosphate dehydrogenase 2 (IMPDH2) enzyme. The proteins regulated by total serum depletion, but not affected by growth in 0.5 % serum, are members of the glycolytic and nucleotide metabolic pathways and the epithelial-to-mesenchymal transition (EMT), suggesting an adaptive switch characteristic of malignant cells.ConclusionsThis comparative proteomic analysis and the identified proteins are the first evidence of a protein expression response to serum depletion in a trophoblast cell model. Our results show that serum depletion induces specific changes in protein expression concordant with main cell metabolic adaptations and EMT, resembling the progression to a malignant phenotype.Electronic supplementary materialThe online version of this article (doi:10.1186/s11658-016-0018-9) contains supplementary material, which is available to authorized users.

MP85-04 DE NOVO GTP SYNTHESIS IS CRITICAL FOR CELL MIGRATION THROUGH IMPDH2 IN RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology II1 Apr 2016MP85-04 DE NOVO GTP SYNTHESIS IS CRITICAL FOR CELL MIGRATION THROUGH IMPDH2 IN RENAL CELL CARCINOMA Hirofumi Yoshino, Hideki Hideki, Masayuki Nakagawa, and Atsuo Sasaki Hirofumi YoshinoHirofumi Yoshino More articles by this author , Hideki HidekiHideki Hideki More articles by this author , Masayuki NakagawaMasayuki Nakagawa More articles by this author , and Atsuo SasakiAtsuo Sasaki More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2270AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES In the strategy of cancer treatment, it is important to target cancer specific pathway on survival and proliferation of cancer cells. In cells, there are two distinct pathways to generate Guanosine-5'-triphosphate (GTP): salvage and de novo pathway. The salvage pathway is an energy efficient pathway that utilizes assembled parts of nucleotides, which are formed during degradation of DNA and RNA. On the other hand, de novo pathway is an energy consuming, multi-step pathway that uses glucose as the starting material for the formation of GTP. Interestingly, the energy consuming de novo pathway is mainly employed by rapidly proliferating cancer cells, while the energy-efficient salvage pathway is predominantly used by non-cancerous cells. Then we focused on inosine 5'-monophosphate dehydrogenase 2 (IMPDH2) which is a enzyme on GTP de novo pathway. The aim of the present study is to find a novel molecular network involved in renal cell carcinoma (RCC) invasion and metastasis through investigating the function of GTP synthesis. METHODS Expression levels of IMPDH2 were examined by immunohistochemistry (IHC) in clinical RCC tissue array (70 RCCs and 10 normal renal tissues). GTP levels were measured by high performance liquid chromatography (HPLC) in RCC cell lines (786-o and A498) and normal mouse primary cells treated by IMPDH2 inhibitor (mycophenolic acid (MPA)). The functional studies of IMPDH2 were performed to investigate cell migration by RCC cell lines. Fluorescence resonance energy transfer (FRET) analyses were performed to monitor RhoA activity with RhoA biosensor (W47). GTP pull down assay was performed to monitor GTP- bound form of Rho. RESULTS The expression levels of IMPDH2 were significantly up-regulated in RCC tissues in IHC. GTP levels were significantly reduced by MPA in RCC cell lines, on the other hand those in primary cell were not effected. GTP reduction inhibited cell migration in RCC cell lines. FRET analysis showed that Rho activation signals were significantly reduced by MPA. In addition, GTP pull down assay showed that active form of Rho was inhibited by MPA. CONCLUSIONS IMPDH2 was up-regulated in RCCs, and reduction of IMPDH2 inhibited cell migration through GTP down-regulation. FRET and GTP pull down assay indicated that Rho inactivation might be associated with inhibition of cell migration. Our data suggest that IMPDH2 could be a new molecular maker for RCC. In addition, control of GTP could be a new therapeutic target for RCC metastasis and invasion through IMPDH2 regulation. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1099 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Hirofumi Yoshino More articles by this author Hideki Hideki More articles by this author Masayuki Nakagawa More articles by this author Atsuo Sasaki More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

P38α Activates Purine Metabolism to Initiate Hematopoietic Stem Cell Cycling

Hematopoietic stem cells (HSCs) maintain quiescence by activating specific metabolic pathways, including glycolysis. However, how stress hematopoiesis, including bone marrow transplantation (BMT), induces metabolic changes in HSCs remains unclear. Here, we report a critical role for the p38MAPK family isoform p38α in initiating HSC proliferation during stress hematopoiesis in mouse. First, we identified p38α as the major p38MAPK isozyme highly expressed in HSCs and we also performed conditional knockout of p38α in mice. This mouse showed no overt difference relative to wild type mouse. However, treatment of p38α-deficient mice with 5-FU exhibited defective recovery of hematopoiesis, and the survival rate were lower in p38α-deficient mice than wild-type mice (42.9%, N=7, p38α-deficient mice, vs 100%, wild-type mice, N=6, p=0.03) and loss of p38α in HSCs showed a defective transplantation capacity in primary and secondary transplantation. To gain further insight into p38MAPK function during hematological stress, we evaluated the time course of p38MAPK activation in stressful contexts by intracellular flow cytometry. We found that p38MAPK was immediately phosphorylated in HSCs after hematological stress and returned to normal in a short period, suggesting that p38α functions rapidly after hematological stresses to activate downstream events. To identify events downstream of p38α after hematological stress, we initially evaluated mechanisms such as homing, apoptosis, and ROS generation immediately after BMT. However, defects seen in p38α-deficient HSCs after hematological stress could not be explained by these mechanisms. Therefore we next focused on cell cycle. In CFSE assay, p38α loss resulted in defective recovery from hematological stress and a delay in initiating cycling of HSCs. In addition, p38α-deficient HSCs showed lower BrdU incorporation in vivo (p=0.045) and EdU incorporation in vitro (p=0.003). Transcriptome analysis of transplanted wild-type or p38α-deficient HSCs suggested that p38α-deficient HSCs showed lower enrichment of genes related to HSC-related markers and proliferation. Taken together, loss of p38α resulted in defective HSC cell cycle progression in stressed settings such as transplantation. Given that altered metabolic activities can change cell cycle status, we asked whether p38α regulation of a particular metabolic pathway could initiate HSC cycling under stress conditions. To do so, we collected p38α-deficient or wild-type LSK cells either at steady state or after BMT and extracted metabolites for metabolome analysis using mass spectrometry. Among metabolites surveyed, we focused on changes in glycine and aspartic acid, which are required for purine biosynthesis. Levels of both increased in p38α-deficient as compared with wild-type LSK cells after BMT. Also, mice transplanted with p38α-deficient compared with wild-type LSK cells showed lower levels of allantoin, a product of purine catabolism. These findings suggest that p38α loss suppresses purine metabolism during stress hematopoiesis. Next, we evaluated mRNAs encoding key enzymes functioning in purine metabolism by qPCR. Expression of both inosine-5'-monophosphate dehydrogenase 2 (IMPDH2), and guanosine monophosphate synthetase (GMPS) was significantly decreased in p38α-deficient HSCs relative to wild-type HSCs on day 1 after BMT. To assess how changes in purine metabolism could affect the HSC response to stress, we treated HSCs with cytokines in the presence or absence of mycophenolic acid (MPA), an IMPDH2 inhibitor. MPA treatment significantly suppressed colony formation capacity of HSCs in a dose-dependent manner. Also, EdU incorporation into HSCs was reduced by MPA dose-dependently. Finally, isolated HSCs were cultured with or without MPA for 3 days and then transplanted into recipients along with competitor cells. PB chimerism was dose-dependently decreased in recipients of MPA-treated cells. These findings suggest that purine metabolism directly maintains proliferation capacity of HSCs in stress conditions. In summary, expression of purine-synthesizing enzymes decreased in p38α-deficient HSCs after transplantation, an activity correlated with defective cell cycle progression in vitro and in vivo. Overall, this is the first report of p38α-regulated changes in purine metabolism associated with HSC stress and cell cycle initiation. DisclosuresNo relevant conflicts of interest to declare.

Mycobacterium tuberculosis IMPDH in Complexes with Substrates, Products and Antitubercular Compounds.

Tuberculosis (TB) remains a worldwide problem and the need for new drugs is increasingly more urgent with the emergence of multidrug- and extensively-drug resistant TB. Inosine 5’-monophosphate dehydrogenase 2 (IMPDH2) from Mycobacterium tuberculosis (Mtb) is an attractive drug target. The enzyme catalyzes the conversion of inosine 5’-monophosphate into xanthosine 5’-monophosphate with the concomitant reduction of NAD+ to NADH. This reaction controls flux into the guanine nucleotide pool. We report seventeen selective IMPDH inhibitors with antitubercular activity. The crystal structures of a deletion mutant of MtbIMPDH2 in the apo form and in complex with the product XMP and substrate NAD+ are determined. We also report the structures of complexes with IMP and three structurally distinct inhibitors, including two with antitubercular activity. These structures will greatly facilitate the development of MtbIMPDH2-targeted antibiotics.

Assembly of IMPDH2-Based, CTPS-Based, and Mixed Rod/Ring Structures Is Dependent on Cell Type and Conditions of Induction

Inhibition of guanosine triphosphate (GTP) and cytidine triphosphate (CTP) biosynthetic pathways induces cells to assemble rod/ring (RR) structures, also named cytoophidia, which consist of the enzymes cytidine triphosphate synthase (CTPS) and inosine-5′-monophosphate dehydrogenase 2 (IMPDH2). We aim to explore the interaction of CTPS and IMPDH2 in the generation of RR structures. HeLa and COS-7 cells were cultured in normal conditions or in the presence of 6-diazo-5-oxo-L-norleucine (DON), ribavirin, or mycophenolic acid (MPA). Over 90% of DON-treated cells presented RR structures. In HeLa cells, 35% of the RR structures were positive for IMPDH2 alone, 26% were CTPS alone, and 31% were IMPDH2/CTPS mixed, while in COS-7 cells, 42% of RR were IMPDH2 alone, 41% were CTPS alone, and 10% were IMPDH2/CTPS mixed. Ribavirin and MPA treatments induced only IMPDH2-based RR. Cells were also transfected with an N-terminal hemagglutinin (NHA)-tagged CTPS1 construct. Over 95% of NHA-CTPS1 transfected cells with DON treatment presented IMPDH2-based RR and almost 100% presented CTPS1-based RR; when treated with ribavirin, over 94% of transfected cells presented IMPDH2-based RR and 37% presented CTPS1-based RR, whereas 2% of untreated transfected cells presented IMPDH2-based RR and 28% presented CTPS1-based RR. These results may help in understanding the relationship between CTP and GTP biosynthetic pathways, especially concerning the formation of filamentous RR structures.

Microinjection of specific anti-IMPDH2 antibodies induces disassembly of cytoplasmic rods/rings that are primarily stationary and stable structures.

BackgroundOur laboratory previously reported interesting rods 3–10 μm long and rings 2–5 μm diameter (RR) in the cytoplasm of mammalian cells. Experimental evidence show that both inosine-5'-monophosphate dehydrogenase 2 (IMPDH2) and cytidine triphosphate synthetase (CTPS) are components of RR structures. Several cell types, including mouse embryonic stem cells, and cell lines, such as mouse 3 T3 and rat NRK, naturally present RR structures, while other cells can present RR when treated with compounds interfering with GTP/CTP biosynthetic pathways. In this study, we aimed to investigate the dynamic behavior of these RR in live cells.ResultsRR were detected in >90% of COS-7 and HeLa cells treated with 1 mM ribavirin or 6-Diazo-5-oxo-L-norleucine (DON) for 24 h, and in 75% of COS-7 cells treated with 1 mM mycophenolic acid (MPA) for the same period of time. Microinjection of affinity-purified anti-IMPDH2 antibodies in live COS-7 cells treated with ribavirin, DON, or MPA showed mature forms of RR presented as stable and stationary structures in 71% of cells. In the remaining 29% of cells, RR acquired erratic movement and progressively disassembled into fragments and disappeared within 10 min. The specific stationary state and antibody-dependent disassembling of RR structures was independently confirmed in COS-7 and HeLa cells transfected with GFP-tagged IMPDH2.ConclusionsThis is the first demonstration of disassembly of RR structures upon microinjection of anti-IMPDH2 antibodies that led to the disappearance of the molecular aggregates. The disassembly of RR after microinjection of anti-IMPDH2 antibody further strengthens the notion that IMPDH2 are major building blocks of RR. Using two independent methods, this study demonstrated that the induced RR are primarily stationary structures in live cells and that IMPDH2 is a key component of RR.Electronic supplementary materialThe online version of this article (doi:10.1186/2045-3701-5-1) contains supplementary material, which is available to authorized users.

Mechanistic studies of a novel mycophenolic acid-glucosamine conjugate that attenuates renal ischemia/reperfusion injury in rat.

Renal ischemia/reperfusion (I/R) injury causes high mortality and morbidity during renal procedures, yet current drugs should be used at high doses or for long periods due to lack of tissue specificity. In previous work we described a novel mycophenolic acid-glucosamine conjugate (MGC) that targets the proximal tubule epithelium, where it efficiently reduces renal I/R injury in rats and promotes recovery from reperfusion. Here we perform mechanistic studies of MGC in rats that suggest that the conjugate works by repressing the activation of renal inosine-5'-monophosphate dehydrogenase 2 (IMPDH2), thereby inhibiting the proliferation and accumulation of lympholeukocytes in the proximal tubules. In addition, MGC appears to inhibit inflammation through various pathways, including inhibition of free oxygen radical production, upregulation of bone morphogenetic protein-7, and downregulation of complement protein 3, TLR 4, intracellular adhesion molecules in the endothelium, proinflammatory cytokines (e.g., TNF-α, IL-6, IL-1, TGF-β), and chemotactic cytokines [e.g., monocyte chemoattractant protein-1 (MCP-1) and IL-8]. These findings suggest that MGC specifically targets the proximal tubules and acts through numerous mechanisms to substantially mitigate I/R injury in rats; this conjugate may provide a more effective alternative to current combination therapy.

T Cells Engineered For Resistance To Mycophenolate Mofetil Demonstrate Enhanced Expansion and Improved Tumour Control In Immunosuppressed Hosts

Background Adoptive transfer of therapeutic T cells targeting viral infection or tumour following allogeneic solid organ or hematopoietic stem cell transplantation is hindered by the requirement for immunosuppression. Recent reports have suggested the strategy of using genes encoding cyclophilin or FK506 mutants to confer resistance of T cells to calcineurin inhibitors (Brewin et al. Blood 2009;114:4792 &amp; De Angelis et al. Blood 2009;114:4784). Mycophenolate mofetil (MMF) is another widely used immunosuppressant that acts as a non-competitive inhibitor of inosine-5’-monophosphate dehydrogenase 2 (IMPDH2), an inducible enzyme that generates guanine nucleotides for DNA and RNA synthesis in T cells. In this study, we used T cells transduced with mutated IMPDH2 that confers &gt;2000-fold resistance to MMF (IMPDH2R; T333I, S351Y). Methods Genes encoding human IMPDH2R and IMPDH2CS(containing a catalytic site mutation, C331A) were cloned into SFG retroviral vectors as fusions to eGFP reporter sequences. T cells were transduced with either vector, transferred to recipient mice and evaluated in the presence or absence of MMF. Results IMPDH2R-transduced (td) murine and human T cells demonstrated a selective advantage to MMF in vitro by overcoming drug-induced G1-S phase cell cycle blockade (p=0.02, 55.0% IMPDH2R-td vs 25.6% IMPDH2CS-td cells in S phase) and preventing apoptosis (p=0.01, 22.5% vs. 39.3% Annexin V positive). Overexpression of the wild type IMPDH2 gene conferred intermediate protection against vector alone controls. To test for selection of IMPDH2R-td cells in vivo, sub-lethally irradiated (1.5-2Gy) B6.PL (Thy1.1) mice were injected with a 1:1 mix of OT-I TCR transgenic CD8 T cells transduced with IMPDH2R (CD45.1, Thy1.2) or IMPDH2CS (CD45.2, Thy1.2). Host mice received cognate peptide (SIINFEKL) in adjuvant or adjuvant alone, and no drug or MMF (200 μg/g/day) by daily ip injection. Compared to mice given adjuvant alone (spleen ratio IMPDH2R:IMPDH2CS= 1.1), selection of IMPDH2R-td T cells occurred with antigenic stimulation even in the absence of drug, indicating a moderate increase in overall proliferative/survival potential of these cells (ratio IMPDH2R:IMPDH2CS = 2.5, p=0.04). As predicted, in vivo selection of IMPDH2R-td T cells was significantly greater in the presence of MMF (ratio=7.8, drug v no drug, p=0.01). Unexpectedly, however, absolute numbers of IMPDH2R-td T cells in the spleen were actually greater in the presence of MMF rather than its absence indicating that drug resistance per se was not the sole explanation underlying the capacity of gene-modified cells to expand in vivo (27.3 x 105 MMF vs. 19.4 x 105 no MMF, p=0.01). Similarly, not only did IMPDH2R-td OT-I cells control EL4.OVA tumor in vivo better than IMPDH2CS-td OT-I cells in the presence of MMF (log rank p&lt;0.0001), but they also improved tumour control to a greater extent in the presence of drug than in its absence (log rank p&lt;0.0001). Improved tumor control was linked to higher frequencies and absolute numbers of IMPDH2R-td T cells in the spleen of MMF-treated versus non-treated recipients, and was independent of any direct effects of MMF upon tumor growth. Higher IMPDH2R-td T cell numbers in the presence rather than the absence of drug occurred in the context of strong MMF-mediated suppression of endogenous T and B cell compartments. Conclusions Synergy between clinically relevant doses of MMF and transfer of IMPDH2R-td T cells is a potential means for enhancing in vivo expansion and performance of therapeutic T cells beyond that observed in the absence of drug. Future studies will evaluate the potential for this strategy used alone or in combination with other approaches designed to overcome sensitivity of therapeutic T cells to immunosuppressive drugs. Disclosures: No relevant conflicts of interest to declare.

Development of a recombinant cell-based indirect immunofluorescence assay (RC-IFA) for the determination of autoantibodies against “rings and rods”-associated inosine-5′-monophosphate dehydrogenase 2 in viral hepatitis C

Background and aimsAutoantibodies against so-called “rings and rods” structures, as determined by indirect immunofluorescence assay (IFA) using the human cell line HEp-2, have been described in chronic hepatitis C virus (HCV) infected patients treated with interferon/ribavirin. Recently, cytidine triphosphate synthase (CTPS) and inosine-5′-monophosphate dehydrogenase 2 (IMPDH2), the enzyme inhibited by ribavirin, were proposed as the target antigens. We wanted to confirm the identification and setup a robust system for autoantibody testing in routine laboratories. MethodsCTPS and IMPDH2 were individually expressed in HEK293 cells and the recombinant cells were used in IFA (RC-IFA) to analyze sera from 33 anti-“rings and rods” antibody positive individuals with unknown diagnosis, 50 patients with chronic HCV infection, 100 with autoimmune hepatitis, 50 with primary biliary cirrhosis and 50 healthy blood donors. ResultsWe found that all sera with anti-“rings and rods” reacted with recombinant IMPDH2 but none with CTPS. In western blot or ELISA, anti-IMPDH2 positive sera reacted only weakly, if at all, with Escherichia coli derived recombinant IMPDH2 indicating that the autoantibody reaction probably depends on the 3-dimensional conformation of the antigen. A rabbit hyperimmune serum raised against bacterially expressed IMPDH2 produced the ring/rods pattern in IFA using HEp-2. ConclusionsWe conclude, that IMPDH2 is indeed the main target of anti-“rings and rods” while CTPS is an unlikely target. Moreover, the novel RC-IFA test system allows a standardized semi-quantitative determination of anti-IMPDH2 basing on a defined recombinant antigen.

In Vivo Selection of Mycophenolate Mofetil-Resistant T Cells for Adoptive Immunotherapy.

AbstractAbstract 3008In vivo selection of mycophenolate mofetil-resistant T cells for adoptive immunotherapy. BackgroundFollowing allogeneic solid organ or hematopoietic stem cell transplantation, adoptive transfer of therapeutic T cells may be hindered by the requirement for immune suppressive drugs to prevent rejection or graft-versus-host disease. Mycophenolate mofetil (MMF) is a non-competitive inhibitor of inosine-5’-monophosphate dehydrogenase 2 (IMPDH2), an inducible enzyme that generate guanine nucleotides for DNA and RNA synthesis in T cells. In this study, we have evaluated the potential for gene transfer to T cells of a mutated IMPDH2 that confers >2000-fold resistance to MMF (IMPDH2R; T333I, S351Y). MethodsWild type IMPDH2WT, IMPDH2R and IMPDH2 with a catalytic site mutation (IMPDH2CS; C331A) were cloned into SFG retroviral vectors as fusions to eGFP reporter sequences. Murine thymoma (BW 5147) or CD8 T cells were transduced with each vector and their phenotype and function evaluated in the presence or absence of mycophenolic acid (MPA), the active metabolite of MMF. ResultsBW thymoma cells transduced with IMPDH2R exhibited less apoptosis than cells transduced with IMPDH2CS in response to MPA (ratio % Annexin V+ MPA: no MPA- IMPDH2R = 1.2; IMPDH2CS = 2.9, p=0.01). Cells transduced with IMPDH2R were also able to overcome the G1 cell-cycle arrest induced by MPA when compared to control IMPDH2CS cells (Ratio %cells in S-G2/M phases MPA: no MPA- IMPDH2R = 1.0; IMPDH2CS = 0.3, p=0.03). This led to selective enrichment of IMPDH2R transduced cells in the presence of MPA. At low dose MPA (450nM), IMPDH2R transduced cells enriched compared to IMPDH2CS but not IMPDH2WT (ratio % GFP MPA: no MPA- IMPDH2R = 1.6 vs IMPDH2CS = 1.1, p=0.04 and IMPDH2WT = 1.5 p= 0.14). However, at high dose (4500nM) IMPDH2R exhibited enhanced enrichment (ratio % GFP MPA: no MPA- IMPDH2R = 2.8 vs IMPDH2CS = 1.0 p=0.03 and vs IMPDH2WT= 1.5 p=0.03).Gene transfer of IMPDH2R into murine CD8 T cells also led to selective enrichment compared to controls in the presence of MPA when cultured with proliferation-inducing common gamma-chain cytokines (ratio MPA: no MPA IMPDH2R = 4.4 vs. IMPDH2CS = 1.10, p=0.02). Strong selection for IMPDH2R-tranduced CD8 OT-1 T cells in the presence of MPA was also observed under conditions of antigen-induced proliferation (ratio IMPDH2R= 7.1, control = 0.8, p=0.002).To assess in vivo selection, sub-lethally irradiated (2Gy) B6.PL (Thy1.1) mice were injected with a 1:1 mix of OT1 TCR transgenic Thy1.2 CD8 T cells transduced with IMPDH2R or IMPDH2CS that could be differentiated by the congenic markers CD45.1 and CD45.2. Transferred cells were stimulated by s.c. injection with cognate peptide (SIINFEKL) in IFA and MMF (200mg/kg/day) was given by daily ip injection. As in vitro, IMPDH2R-transduced OT1 cells were preferentially selected over IMPDH2CS-transduced cells following MMF treatment (day 14 ratio IMPDH2R to IMPDHCSwas 19.3 vs 1.2 in the absence of MMF). ConclusionsT cells transduced with IMPDH2R are resistant to the anti-proliferative and apoptotic effects of MPA in vitro and demonstrate strong selection in vivo compared to controls at therapeutic levels of MMF. These data support the potential of conferring MMF resistance as a strategy to permit the survival of therapeutic T cells in immunosuppressed allograft recipients. Disclosures:Stauss:Cell Medica: Scientific Advisor Other.

T-12 Rods and rings autoantibodies developing in chronic hepatitis C patients treated with peginterferon and ribavirin target inosine-5’-monophosphate dehydrogenase 2 (IMPDH2)

T-12 Rods and rings autoantibodies developing in chronic hepatitis C patients treated with peginterferon and ribavirin target inosine-5’-monophosphate dehydrogenase 2 (IMPDH2)