Effect on cell survival and cytoophidium assembly of the adRP-10-related IMPDH1 missense mutation Asp226Asn

Introduction: Inosine monophosphate dehydrogenase 1 (IMPDH1) is a critical enzyme in the retina, essential for the correct functioning of photoreceptor cells. Mutations in IMPDH1 have been linked to autosomal dominant retinitis pigmentosa subtype 10 (adRP-10), a genetic eye disorder. Some of these mutations such as the Asp226Asn (D226N) lead to the assembly of large filamentous structures termed cytoophidia. D226N also gives IMPDH1 resistance to feedback inhibition by GDP/GTP. This study aims to emulate the adRP-10 condition with a long-term expression of IMPDH1-D226N in vitro and explore cytoophidium assembly and cell survival. We also assessed whether the introduction of an additional mutation (Y12C) to disrupt the cytoophidium has an attenuating effect on the toxicity caused by the D226N mutation.Results: Expression of IMPDH1-D226N in HEp-2 cells resulted in cytoophidium assembly in ∼70% of the cells, but the presence of the Y12C mutation disrupted the filaments. Long-term cell survival was significantly affected by the presence of the D226N mutation, with a decrease of ∼40% in the cells expressing IMPDH1-D226N when compared to IMPDH1-WT; however, survival was significantly recovered in IMPDH1-Y12C/D226N, with only a ∼10% decrease when compared to IMPDH1-WT. On the other hand, the IMPDH1 expression level in the D226N-positive cells was <30% of that of the IMPDH1-WT-positive cells and only slightly higher in the Y12C/D226N, suggesting that although cell survival in Y12C/D226N was recovered, higher expression levels of the mutated IMPDH1 were not tolerated by the cells in the long term.Conclusion: The IMPDH1-D226N effect on photoreceptor cell survival may be the result of a sum of problems: nucleotide unbalance plus a toxic long-life cytoophidium, supported by the observation that by introducing Y12C in IMPDH1 the cytoophidium was disrupted and cell survival significantly recovered, but not the sensibility to GDP/GTP regulation since higher expression levels of IMPDH1-D226N were not tolerated

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

Background: Our laboratory previously reported interesting rods 3-10 mu m long and rings 2-5 mu m 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.Results: RR 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.Conclusions: This 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.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Univ Florida, Dept Oral Biol, Gainesville, FL 32610 USAUniversidade Federal de São Paulo, Div Rheumatol, BR-04023062 São Paulo, BrazilFleury Med & Hlth Labs, Div Immunol, BR-04102050 São Paulo, BrazilUniv Idaho, Dept Biol Sci, Moscow, ID 83844 USAUniversidade Federal de São Paulo, Div Rheumatol, BR-04023062 São Paulo, BrazilCAPES: 9028-11-0FAPESP: 2011/12448-0Web of Scienc

Longitudinal Study of a Human Drug-Induced Model of Autoantibody to Cytoplasmic Rods/Rings following HCV Therapy with Ribavirin and Interferon-alpha

Background: A novel pattern in the indirect immunofluorescence antinuclear antibody assay on HEp-2 cells (IIF-HEp-2) characterized by cytoplasmic rods and rings (RR) was reported in HCV patients, but stringent disease specificity studies and longitudinal analysis are lacking. We investigated the clinical significance of anti-RR in an HCV cohort with up to a 12-month treatment follow up.Methodology/Results: 597 patients (342 HCV, 55 HCV/HIV, 200 non-HCV) were screened and titered for anti-RR. Serial samples were available from 78 of 176 treated and 27 of 166 untreated patients. Anti-RR was detected in 14.1% of 342 HCV patients, 9.1% of 55 HCV/HIV, 3.4% of 29 Hepatitis B, and none of 171 non-HCV (p47% tested positive for anti-RR. the anti-RR titer generally increased with sustained treatment and remained high in 53% of patients. After treatment, anti-RR titer was negative in 41%. Non-responders to HCV therapy were 77% in anti-RR-positive versus 64% in anti-RR-negative patients. Response to treatment was not associated with anti-RR titer or the dynamics of anti-RR reactivity during and after treatment.Conclusions: the exquisite association of anti-RR reactivity with combined interferon-a/ribavirin therapy in HCV patients represents a unique model for drug-induced autoantibody generation in humans as demonstrated by the fact that a significant fraction of patients who have anti-RR during therapy becomes anti-RR-negative after completion of therapy.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Brazilian National Council for Research and Technological DevelopmentUniversidade Federal de São Paulo, Div Rheumatol, São Paulo, BrazilUniversidade Federal de São Paulo, Div Gastroenterol, São Paulo, BrazilUniversidade Federal de São Paulo, Div Infect Dis, São Paulo, BrazilFleury Med & Hlth Labs, Div Immunol, São Paulo, BrazilUniv Florida, Dept Oral Biol, Gainesville, FL 32610 USAUniversidade Federal de São Paulo, Div Rheumatol, São Paulo, BrazilUniversidade Federal de São Paulo, Div Gastroenterol, São Paulo, BrazilUniversidade Federal de São Paulo, Div Infect Dis, São Paulo, BrazilFAPESP: 2010/50710-6Brazilian National Council for Research and Technological Development: 305064/2011-8Web of Scienc

Caracterização da resposta autoimune contra impdh2 e aspectos da biologia celular das estruturas “rods and rings” reconhecidas pelos anticorpos anti-impdh2

Enzyme aggregation into non-membrane bound large bodies is a common feature among eukaryotic cells. In the last five years, many reports have described the ability of Cytidine Triphosphate Synthase (CTPS) and Inosine Monophosphate Dehydrogenase (IMPDH2), enzymes that are critical in the cytidine and guanine nucleotide biosynthesis pathways, respectively, to form aggregates named rods and rings (RR) or cytoophidia (?cellular snakes?). Interestingly, a considerable fraction of hepatitis C (HCV) patients treated with IFN-? plus ribavirin, an inhibitor of IMPDH2, develops autoantibodies against the RR structures. We study aspects of the formation of RR structures in vitro and in vivo, as well as explore the temporal evolution of anti-RR autoantibodies production. By studying the process of aggregation of IMPDH2 and CTPS into RR structures in the presence of their respective inhibitors, we demonstrated the independent in vitro formation of IMPDH2-based and CTPS-based RR structures in cell lines of two mammalian species, and reported that both enzymes can interact in the formation of mixed RR structures as a mosaic of IMPDH2 and CTPS aggregates. By studying the temporal behavior of RR IMPDH2-based structures in living cells in vitro, we show the disassembly of RR structures in the presence of an antibody targeting IMPDH2, a molecular constituent of RR. We also demonstrate that the IMPDH2-inhibitory drugs ribavirin and MPA generate IMPDH2-rich RR structures in vivo in PBMC from HCV and SLE patients, respectively, but only ribavirin-treated HCV patients present anti-RR autoantibodies. The autoantibodies that elicit the anti-RR indirect immunofluorescence pattern recognized predominantly the IMPDH2 enzyme. By analyzing the temporal behavior of the anti-RR/IMPDH2 autoimmune response, we report that such autoantibodies were induced at 3 to 6 months of IFN-?+ribavirin treatment, reached a plateau around the twelfth month, and decreased/disappeared after treatment conclusion. The above described scenario characterizes a human model of immune tolerance breakdown and represents a unique opportunity to study various aspects of the autoimmune response development in humans.Agregação de enzimas em corpos grandes e sem membrana é uma característica comum entre as células eucarióticas. Nos últimos cinco anos, várias publicações têm descrito a capacidade de Citidina Trifosphato Sintase (CTPS) e Inosina Monofosfato Desidorgenase 2 (IMPDH2), enzimas que são críticas nas vias de biossíntese dos nucleotídeos citidina e guanina, respectivamente, de formar agregados chamados de anéis e bastões (RR) ou cytoophidia ("cobras celulares"). Curiosamente, uma fração considerável de pacientes com hepatite C (HCV) tratados com IFN-? e ribavirina, um inibidor da IMPDH2, desenvolvem autoanticorpos contra as estruturas de RR. Neste trabalho, estudamos aspectos da formação de estruturas RR in vitro e in vivo, bem como a evolução temporal da produção dos autoanticorpos anti-RR. Ao estudar o processo de agregação da IMPDH2 e CTPS em RR, na presença dos respectivos inibidores, demonstramos a formação de estruturas RR independentes compostas por IMPDH2 ou CTPS em duas linhagens celulares de duas espécies de mamíferos. Também relatamos que ambas as enzimas podem interagir na formação de estruturas RR mistas, como um mosaico de IMPDH2 e CTPS. Ao estudar o comportamento temporal das estruturas RR, ricas em IMPDH2, em células vivas in vitro, observamos que estas se desmontam na presença de um anticorpo contra IMPDH2, um componente molecular do RR. Também demonstramos que as drogas ribavirina e MPA, ambas inibidoras da IMPDH2, produzem estruturas RR ricas em IMPDH2 in vivo em PBMC de pacientes HCV e lúpicos, respectivamente, mas apenas os pacientes HCV apresentam os autoanticorpos anti-RR. Os autoanticorpos que geram o padrão de imunofluorescência indireta RR reconhecem predominantemente a enzima IMPDH2 como alvo. Ao analisar o comportamento temporal da resposta autoimune anti-RR/IMPDH2, observamos que tais anticorpos foram induzidos após 3 a 6 meses de tratamento com IFN-?+ribavirina, com um pico em torno do décimo segundo mês, e diminuição ou desaparecimento após a conclusão do tratamento. O cenário acima descrito caracteriza um modelo humano de quebra de tolerância imunológica e representa uma oportunidade única para estudar vários aspectos do desenvolvimento de uma resposta autoimune.Dados abertos - Sucupira - Teses e dissertações (2013 a 2016

Temporal evolution of human autoantibody response to cytoplasmic rods and rings structure during anti-HCV therapy with ribavirin and interferon-alpha

Autoantibodies to inosine monophosphate dehydrogenase-2 (IMPDH2), an enzyme involved in de novo biosynthesis of guanine nucleotides, are observed in a subset of hepatitis C virus (HCV) patients receiving interferon alpha (IFN-alpha) plus ribavirin. Anti-IMPDH2 antibodies display a peculiar cytoplasmic rod/ring (RR) pattern in IIF-HEp-2. We examined the dynamics of anti-RR autoimmune response with respect to immunoglobulin isotypes, titer, avidity, and protein targets in 80 sequential samples from 15 HCV patients (plus 12 randomly selected anti-RR-positive, totalizing 92 samples) collected over an 18-month period, including samples collected before, during, and after IFN-alpha + ribavirin treatment. Immunoprecipitation showed reactivity with the 55 kDa IMPDH2 protein in 12/15 patients (80 %) and 11/15 (73 %) reacted with IMPDH2 in a sandwich ELISA. During treatment, anti-IMPDH2 autoantibodies hit their highest levels after 6-12 months of treatment and decreased post-treatment, while anti-HCV antibodies levels were stable over time. Anti-IMPDH2 IgM levels increased up until the sixth month of treatment and remained stable thereafter, while IgG levels increased steadily up to the twelfth month. Both IgG and IgM decreased during the post-treatment period. IgG avidity increased steadily up to the twelfth month of treatment. in conclusion, this study showed that the temporal kinetics of IFN-alpha + ribavirin-induced humoral autoimmune response to IMPDH2 exhibited a considerably delayed pace of increase in antibody levels and avidity as well as in isotype class switch in comparison with a conventional humoral response to infectious agents. These unique findings uncover intriguing differences between the autoimmune response and the immune response to exogenous agents in humans.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Div Rheumatol, BR-04023062 São Paulo, BrazilUniv Florida, Dept Oral Biol, Gainesville, FL 32610 USAUniv Occupat & Environm Hlth, Sch Hlth Sci, Dept Clin Nursing, Yahata Nishi Ku, Kitakyushu, Fukuoka 8078555, JapanUniv Florida, Dept Med, Div Rheumatol & Clin Immunol, Gainesville, FL 32610 USAUniversidade Federal de São Paulo, Div Gastroenterol, BR-04023062 São Paulo, BrazilFleury Med & Hlth Labs, Div Immunol, BR-04102050 São Paulo, BrazilUniversidade Federal de São Paulo, Div Rheumatol, BR-04023062 São Paulo, BrazilUniversidade Federal de São Paulo, Div Gastroenterol, BR-04023062 São Paulo, BrazilFAPESP: 2010/50710-6FAPESP: 2011/12448-0CAPES: 9028-11-0CNPq: 305064/2011-8Web of Scienc

CTPS forms the cytoophidium in zebrafish

Cytidine triphosphate synthase (CTPS) catalyzes the rate-limiting step of de novo CTP biosynthesis. An intracellular structure of CTPS, the cytoophidium, has been found in many organisms including prokaryotes and eukaryotes. Formation of the cytoophidium has been suggested to regulate the activity and stability of CTPS and may participate in certain physiological events. Herein, we demonstrate that both CTPS1a and CTPS1b in zebrafish are able to form the cytoophidium in cultured cells. A point mutation, H355A, abrogates cytoophidium assembly of zebrafish CTPS1a and CTPS1b. In addition, we show the presence of CTPS cytoophidia in multiple tissues of larval and adult fish under normal conditions, while treatment with a CTPS inhibitor 6-diazo-5-oxo-l-norleucine (DON) can induce more cytoophidia in some tissues. Our findings reveal that forming the CTPS cytoophidium is a natural phenomenon of zebrafish and provide valuable information for future research on the physiological importance of this intracellular structure in vertebrates.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)2017/20745-