RESUMO
Targetable drivers governing 5-fluorouracil and cisplatin (5FU + CDDP) resistance remain elusive due to the paucity of physiologically and therapeutically relevant models. Here, we establish 5FU + CDDP resistant intestinal subtype GC patient-derived organoid lines. JAK/STAT signaling and its downstream, adenosine deaminases acting on RNA 1 (ADAR1), are shown to be concomitantly upregulated in the resistant lines. ADAR1 confers chemoresistance and self-renewal in an RNA editing-dependent manner. WES coupled with RNA-seq identify enrichment of hyper-edited lipid metabolism genes in the resistant lines. Mechanistically, ADAR1-mediated A-to-I editing on 3'UTR of stearoyl-CoA desaturase (SCD1) increases binding of KH domain-containing, RNA-binding, signal transduction-associated 1 (KHDRBS1), thereby augmenting SCD1 mRNA stability. Consequently, SCD1 facilitates lipid droplet formation to alleviate chemotherapy-induced ER stress and enhances self-renewal through increasing ß-catenin expression. Pharmacological inhibition of SCD1 abrogates chemoresistance and tumor-initiating cell frequency. Clinically, high proteomic level of ADAR1 and SCD1, or high SCD1 editing/ADAR1 mRNA signature score predicts a worse prognosis. Together, we unveil a potential target to circumvent chemoresistance.
Assuntos
Neoplasias Gástricas , Humanos , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/genética , Estearoil-CoA Dessaturase/genética , Estearoil-CoA Dessaturase/metabolismo , Edição de RNA , Proteômica , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Cisplatino/metabolismo , Resistência a Medicamentos , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , RNA/metabolismo , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismoRESUMO
BACKGROUND: Adenosine deaminase deficiency (ADA) is an autosomal recessive disorder leading to severe combined immunodeficiency (SCID). It is characterized patho-physiologically by intracellular accumulation of toxic products affecting lymphocytes. Other organ systems are known to be affected causing non-immune abnormalities. We aimed to conduct a cross sectional study to describe liver disease in autosomal recessive ADA-SCID. METHODS: Single center retrospective analysis of genetically confirmed autosomal recessive ADA-SCID was performed. Liver disease was defined as ≥1.5x the gender specific upper limit of normal (ULN; 33 IU/L for males and 25 IU/L for females) alanine aminotransferase (ALT) or moderate and severe increase in liver echogenicity on ultrasound. RESULTS: The cohort included 18 patients with 11 males. The median age was 11.5 (3.5-30.0 years) and median BMI percentile was 75.5 [36.75, 89.5]. All patients received enzyme replacement therapy at the time of evaluation. Seven (38%) and five (27%) patients had gene therapy (GT) and hematopoietic stem cell transplant (HSCT) in the past. Five patients had 1.5x ALT level more than 1.5x the U. Liver echogenicity was mild in 6 (33%), moderate in 2 (11%) and severe in 2 (11%) patients. All patients had normal Fibrosis-4 Index and Non-alcoholic fatty liver disease fibrosis biomarker scores indicating absence of advanced fibrosis in our cohort. Of 5 patients who had liver biopsies, steatohepatitis was noted in 3 patients (NAS score of 3,3,4). DISCUSSION: Non-immunologic manifestations of ADA-SCID have become more apparent in recent years as survival improved. We concluded that steatosis is the most common finding noted in our ADA-SCID cohort.
Assuntos
Doenças do Sistema Digestório , Fígado Gorduroso , Hepatopatias , Imunodeficiência Combinada Severa , Masculino , Feminino , Humanos , Criança , Imunodeficiência Combinada Severa/diagnóstico , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/terapia , Adenosina Desaminase/genética , Estudos Retrospectivos , Estudos Transversais , Hepatopatias/diagnóstico por imagemRESUMO
INTRODUCTION: Deficiency of adenosine deaminase 2 (DADA2) is a rare monogenic autoinflammatory disease, whose clinical phenotype was expanded since the first cases, originally described as mimicker of polyarteritis nodosa, with immunodeficiency and early-onset stroke. METHODS: A systematic review according to PRISMA approach, including all articles published before the 31st of August 2021 in Pubmed and EMBASE database was performed. RESULTS: The search identified 90 publications describing 378 unique patients (55.8% male). To date 95unique mutations have been reported. The mean age at disease onset was 92.15 months (range 0-720 months), 32 (8.5%) showed an onset of the first signs/symptoms after 18 years old and 96 (25.4%) after 10 years old. The most frequent clinical characteristics described were cutaneous (67.9%), haematological manifestations (56.3%), recurrent fever (51.3%), neurological as stroke and polyneuropathy (51%), immunological abnormalities (42.3%), arthralgia/arthritis (35.4%), splenomegaly (30.6%), abdominal involvement (29.8%), hepatomegaly (23.5%), recurrent infections (18.5%), myalgia (17.9%), kidney involvement (17.7%) etc. Patients with skin manifestations were older than the others (101.1 months SD ± 116.5, vs. 75.3 SD ± 88.2, p 0.041), while those with a haematological involvement (64.1 months SD ± 75.6 vs. 133.1 SD ± 133.1, p < 0.001) and immunological involvement (73.03 months SD ± 96.9 vs. 103.2 SD ± 112.9, p 0.05) are younger than the others. We observed different correlations among the different clinical manifestations. The use of anti-TNFα and hematopoietic cell stems transplantation (HCST) has improved the current history of the disease. CONCLUSION: Due to this highly variable phenotype and age of presentation, patients with DADA2 may present to several type of specialists. Given the important morbidity and mortality, early diagnosis and treatment are mandatory.
Assuntos
Adenosina Desaminase , Acidente Vascular Cerebral , Masculino , Feminino , Humanos , Adenosina Desaminase/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Fenótipo , MutaçãoRESUMO
Deficiency of adenosine deaminase 2 (DADA2) is a rare systemic autoinflammatory disease, typically with autosomal recessive inheritance, usually caused by biallelic loss of function mutations in the ADA2 gene. The phenotypic spectrum is broad, generally including fever, early-onset vasculitis, stroke, and hematologic dysfunction. Heterozygous carriers may show related signs and symptoms, usually milder and at an older age. Here we describe the case of two relatives, the proband and his mother, bearing an ADA2 homozygous pathogenic variant, and a heterozygous son. The proband was a 17-year-old boy with intermittent fever, lymphadenopathies, and mild hypogammaglobulinemia. He also had sporadic episodes of aphthosis, livedo reticularis and abdominal pain. Hypogammaglobulinemia was documented when he was 10 years old, and symptoms appeared in his late adolescence. The mother demonstrated mild hypogammaglobulinemia, chronic pericarditis since she was 30 years old and two transient episodes of diplopia without lacunar lesions on MRI. ADA2 (NM_001282225.2) sequencing identified both mother and son as homozygous for the c.1358A>G, p.(Tyr453Cys) variant. ADA2 activity in the proband and the mother was 80-fold lower than in the controls. Clinical features in both patients improved on anti-tumor necrosis factor therapy. An older son was found to be heterozygous for the same mutation post-mortem. He died at the age of 12 years due to a clinical picture of fever, lymphadenitis, skin rash and hypogammaglobulinemia evolving toward fatal multiorgan failure. Biopsies of skin, lymph nodes, and bone marrow excluded lymphomas and vasculitis. Despite being suspected of symptomatic carrier, the contribution of an additional variant in compound heterozygosity, or further genetic could not be ruled out, due to poor quality of DNA samples available. In conclusion, this familiar case demonstrated the wide range of phenotypic variability in DADA2. The search for ADA2 mutations and the assessment of ADA2 activity should be considered also in patients with the association of hypogammaglobulinemia and inflammatory conditions, also with late presentation and in absence of vasculitis. Furthermore, the clinical picture of the deceased carrier suggests a possible contribution of heterozygous pathogenic variants to inflammation.
Assuntos
Agamaglobulinemia , Vasculite , Masculino , Feminino , Adolescente , Humanos , Criança , Adulto , Adenosina Desaminase/genética , Agamaglobulinemia/diagnóstico , Agamaglobulinemia/genética , Agamaglobulinemia/complicações , Peptídeos e Proteínas de Sinalização Intercelular , Vasculite/etiologiaRESUMO
INTRODUCTION: The deficiency of ADA2 (DADA2) is a rare autoinflammatory disease provoked by mutations in the ADA2 gene inherited in a recessive fashion. Up to this moment there is no consensus for the treatment of DADA2 and anti-TNF is the therapy of choice for chronic management whereas bone marrow transplantation is considered for refractory or severe phenotypes. Data from Brazil is scarce and this multicentric study reports 18 patients with DADA2 from Brazil. PATIENTS AND METHODS: This is a multicentric study proposed by the Center for Rare and Immunological Disorders of the Hospital 9 de Julho - DASA, São Paulo - Brazil. Patients of any age with a confirmed diagnosis of DADA2 were eligible for this project and data on clinical, laboratory, genetics and treatment were collected. RESULTS: Eighteen patients from 10 different centers are reported here. All patients had disease onset at the pediatric age (median of 5 years) and most of them from the state of São Paulo. Vasculopathy with recurrent stroke was the most common phenotype but atypical phenotypes compatible with ALPS-like and Common Variable Immunodeficiency (CVID) was also found. All patients carried pathogenic mutations in the ADA2 gene. Acute management of vasculitis was not satisfactory with steroids in many patients and all those who used anti-TNF had favorable responses. CONCLUSION: The low number of patients diagnosed with DADA2 in Brazil reinforces the need for disease awareness for this condition. Moreover, the absence of guidelines for diagnosis and management is also necessary (t).
Assuntos
Adenosina Desaminase , Vasculite , Humanos , Adenosina Desaminase/genética , Brasil , Inibidores do Fator de Necrose Tumoral , Peptídeos e Proteínas de Sinalização Intercelular/genéticaRESUMO
BACKGROUND: Primary Sjögren's syndrome (pSS) is a chronic inflammatory autoimmune disease primarily affecting the exocrine glands, which has a variety of clinical manifestations and unclear pathogenic mechanisms. Adenosine deaminase (ADA) is an enzyme involved in the breakdown of purines, and changes in its activity have been associated with a number of autoimmune diseases. This study aims to investigate the relationship between serum ADA activity and disease activity in patients with pSS. METHODS: In this study, 196 patients with pSS and 196 healthy controls were enrolled. Serum ADA activity and clinical laboratory parameters were collected and analyzed in both groups. Pearson correlation analysis was used to examine the correlation between ADA activity and clinical laboratory parameters, as well as the correlation between ADA activity and the disease activity score. RESULTS: Compared with healthy controls, the activity of ADA in the serum of pSS patients was significantly increased (P < 0.0001), and the ADA activity was significantly decreased after immunosuppressive treatment (P < 0.0001). Correlation analysis revealed that the activity of ADA was significantly positively correlated with erythrocyte sedimentation rate (ESR) (r = 0.3, P < 0.0001) and serum immunoglobulin G (IgG) levels (r = 0.5, P < 0.0001), and significantly negatively correlated with high-density lipoprotein (HDL) (r = -0.4, P < 0.0001). Furthermore, there was a significant positive correlation between ADA activity and the disease activity score as measured by the Sjögren's Syndrome Disease Activity Index (SSDAI) (r = 0.4, P < 0.0001). CONCLUSION: This study found that patients with pSS have higher activity of ADA in serum, which is associated with disease activity as measured by SSDAI. These results suggest that ADA activity may be a potential biomarker for evaluating disease activity and treatment efficacy in pSS patients. Additionally, ADA may be a potential target for the treatment of pSS patients.
Assuntos
Doenças Autoimunes , Síndrome de Sjogren , Humanos , Síndrome de Sjogren/diagnóstico , Adenosina Desaminase , Biomarcadores , Resultado do TratamentoRESUMO
Background and aim: Type I interferons (IFNs) are highly expressed in the gut mucosa of celiac disease (CD) gut mucosa and stimulates immune response prompted by gluten ingestion, but the processes that maintain the production of these inflammatory molecules are not well understood. Adenosine deaminase acting on RNA 1 (ADAR1), an RNA-editing enzyme, plays a crucial role in inhibiting self or viral RNAs from activating auto-immune mediated responses, most notably within the type-I IFN production pathway. The aim of this study was to assess whether ADAR1 could contribute to the induction and/or progression of gut inflammation in patients with celiac disease. Material and methods: ADAR1 expression was assessed by Real time PCR and Western blotting in duodenal biopsy taken from inactive and active celiac disease (CD) patients and normal controls (CTR). To analyze the role of ADAR1 in inflamed CD mucosa, lamina propria mononuclear cells (LPMC) were isolated from inactive CD and ADAR1 was silenced in with a specific antisense oligonucleotide (AS) and then incubated with a synthetic analogue of viral dsRNA (poly I:C). IFN-inducing pathways (IRF3, IRF7) in these cells were evaluated with Western blotting and inflammatory cytokines were evaluated with flow cytometry. Lastly, the role of ADAR1 was investigated in a mouse model of poly I:C-driven small intestine atrophy. Results: Reduced ADAR1 expression was seen in duodenal biopsies compared to inactive CD and normal controls. Ex vivo organ cultures of duodenal mucosal biopsies, taken from inactive CD patients, stimulated with a peptic-tryptic digest of gliadin displayed a decreased expression of ADAR1. ADAR1 silencing in LPMC stimulated with a synthetic analogue of viral dsRNA strongly boosted the activation of IRF3 and IRF7 and the production of type-I IFN, TNF-α and IFN-γ. Administration of ADAR1 antisense but not sense oligonucleotide to mice with poly I:C-induced intestinal atrophy, significantly increased gut damage and inflammatory cytokines production. Conclusions: These data show that ADAR1 is an important regulator of intestinal immune homeostasis and demonstrate that defective ADAR1 expression could provide to amplifying pathogenic responses in CD intestinal mucosa.
Assuntos
Doença Celíaca , Animais , Camundongos , Doença Celíaca/genética , Adenosina Desaminase/genética , Mucosa Intestinal , RNA de Cadeia Dupla , Atrofia , Citocinas , Poli IRESUMO
Immune cell trafficking constitutes a fundamental component of immunological response to tissue injury, but the contribution of intrinsic RNA nucleotide modifications to this response remains elusive. We report that RNA editor ADAR2 exerts a tissue- and stress-specific regulation of endothelial responses to interleukin-6 (IL-6), which tightly controls leukocyte trafficking in IL-6-inflamed and ischemic tissues. Genetic ablation of ADAR2 from vascular endothelial cells diminished myeloid cell rolling and adhesion on vascular walls and reduced immune cell infiltration within ischemic tissues. ADAR2 was required in the endothelium for the expression of the IL-6 receptor subunit, IL-6 signal transducer (IL6ST; gp130), and subsequently, for IL-6 trans-signaling responses. ADAR2-induced adenosine-to-inosine RNA editing suppressed the Drosha-dependent primary microRNA processing, thereby overwriting the default endothelial transcriptional program to safeguard gp130 expression. This work demonstrates a role for ADAR2 epitranscriptional activity as a checkpoint in IL-6 trans-signaling and immune cell trafficking to sites of tissue injury.
Assuntos
Interleucina-6 , RNA , Células Endoteliais/metabolismo , Receptor gp130 de Citocina , Endotélio/metabolismo , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismoRESUMO
Background: A consensus on the diagnostic utility of cerebrospinal fluid adenosine deaminase (ADA) for tuberculous meningitis (TBM) is lacking. Methods: Patients aged ≥12 years admitted with CNS infections were enrolled prospectively. ADA was measured with spectrophotometry. Results: We enrolled 251 TBM and 131 other CNS infections. The optimal cutoff of ADA was calculated at 5.5 U/l against microbiological reference standard with area under curve 0.743, sensitivity 80.7%, specificity 60.3%, positive likelihood ratio 2.03 and negative likelihood ratio 3.12. The widely used cutoff value 10 U/l had specificity 82% and sensitivity 50%. The discriminating power was higher for TBM versus viral meningoencephalitis than bacterial or cryptococcal meningitis. Conclusion: Cerebrospinal fluid ADA has a low-to-modest diagnostic utility.
The diagnosis of tuberculosis (TB) of the brain is mainly made by testing cerebrospinal fluid, a clear liquid that flows in and around the brain and spinal cord. Adenosine deaminase (ADA) is a protein whose production and activity are increased in many diseases, such as TB. ADA testing in cerebrospinal fluid is widely used for the diagnosis of brain TB. However, the experts have split opinions regarding its confirmatory role. This study explores ADA measurement in cerebrospinal fluid for differentiating TB from other brain infections. The report says that this simple and inexpensive test can be helpful, but it cannot make or refute the diagnosis of brain TB and should only be considered along with other tests.
Assuntos
Tuberculose Meníngea , Humanos , Adenosina Desaminase/líquido cefalorraquidiano , Hospitalização , Padrões de Referência , Estudos Retrospectivos , Sensibilidade e Especificidade , Tuberculose Meníngea/diagnóstico , Tuberculose Meníngea/líquido cefalorraquidianoRESUMO
BACKGROUND: Clinical manifestations of Epstein-Barr virus (EBV) infection are diverse. This study aimed to explore the immune response in EBV-related diseases and the correlation between immune cells and adenosine deaminase (ADA) levels. METHODS: This study was conducted at the Children's Hospital of Soochow University. In total, 104 patients with EBV-associated respiratory tract infection (EBV-RTI), 32 patients with atypical EBV infection, 54 patients with EBV-associated infectious mononucleosis (IM1, with normal alanine aminotransferase [ALT] levels), 50 patients with EBV-IM2 (with elevated ALT levels), 50 patients with acute respiratory infection (AURI, with other pathogens), and 30 healthy controls were enrolled in this study. Indicators of ADA, immunoglobulins (Igs), and lymphocyte subsets were analyzed for EBV-related diseases. RESULTS: Differences in the white blood cell, lymphocyte counts, ADA levels, IgA, IgG and IgM titers, percentage of CD3+, CD3+CD4+, CD3+CD8+, CD16+CD56+, CD3-CD19+, and CD19+CD23+ lymphocytes, and CD4+/CD8+ ratio between EBV-related disease groups were all statistically significant (P < 0.01). ADA levels in the EBV-related disease groups were significantly higher than those in the control group (P < 0.01). The lymphocyte count, ADA levels, IgA and IgG titers, and percentage of CD3+ and CD3+CD8 + lymphocytes in the atypical EBV infection, EBV-IM1, and EBV-IM2 groups were significantly higher than those in the EBV-RTI, AUTI, and control groups (P < 0.01), whereas the percentage of CD3+CD4+, CD3-CD19+, and CD19+CD23+ lymphocytes and CD4+/CD8+ ratio showed the opposite trend. ADA levels were consistent with and closely related to the viral load and cellular and humoral immunity in EBV-related diseases. CONCLUSIONS: ADA levels, humoral immunity, and cellular immunity were diverse in EBV-related diseases, and ADA was closely related to Igs and lymphocyte subsets.
Assuntos
Infecções por Vírus Epstein-Barr , Herpesvirus Humano 4 , Criança , Humanos , Infecções por Vírus Epstein-Barr/complicações , Adenosina Desaminase , Subpopulações de Linfócitos , Contagem de Linfócitos , Antígenos CD19 , Imunoglobulina A , Imunoglobulina GRESUMO
Adenosine deaminase acting on RNA ADAR1 promotes A-to-I conversion in double-stranded and structured RNAs. ADAR1 has two isoforms transcribed from different promoters: cytoplasmic ADAR1p150 is interferon-inducible while ADAR1p110 is constitutively expressed and primarily localized in the nucleus. Mutations in ADAR1 cause Aicardi - Goutières syndrome (AGS), a severe autoinflammatory disease associated with aberrant IFN production. In mice, deletion of ADAR1 or the p150 isoform leads to embryonic lethality driven by overexpression of interferon-stimulated genes. This phenotype is rescued by deletion of the cytoplasmic dsRNA-sensor MDA5 indicating that the p150 isoform is indispensable and cannot be rescued by ADAR1p110. Nevertheless, editing sites uniquely targeted by ADAR1p150 remain elusive. Here, by transfection of ADAR1 isoforms into ADAR-less mouse cells we detect isoform-specific editing patterns. Using mutated ADAR variants, we test how intracellular localization and the presence of a Z-DNA binding domain-α affect editing preferences. These data show that ZBDα only minimally contributes to p150 editing-specificity while isoform-specific editing is primarily directed by the intracellular localization of ADAR1 isoforms. Our study is complemented by RIP-seq on human cells ectopically expressing tagged-ADAR1 isoforms. Both datasets reveal enrichment of intronic editing and binding by ADAR1p110 while ADAR1p150 preferentially binds and edits 3'UTRs.
Assuntos
Interferons , RNA de Cadeia Dupla , Humanos , Animais , Camundongos , RNA de Cadeia Dupla/genética , Interferons/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismoRESUMO
Characterizing pollen release and dispersion processes is fundamental for knowledge advancement in ecological, agricultural and public health disciplines. Understanding pollen dispersion from grass communities is especially relevant due to their high species-specific allergenicity and heterogeneously distributed source areas. Here, we aimed to address questions concerning fine level heterogeneity in grass pollen release and dispersion processes, with a focus on characterizing the taxonomic composition of airborne grass pollen over the grass flowering season using eDNA and molecular ecology methods. High resolution grass pollen concentrations were compared between three microscale sites (<300 m apart) in a rural area in Worcestershire, UK. The grass pollen was modelled with local meteorology in a MANOVA (Multivariate ANOVA) approach to investigate factors relevant to pollen release and dispersion. Simultaneously, airborne pollen was sequenced using Illumina MySeq for metabarcoding, analysed against a reference database with all UK grasses using the R packages DADA2 and phyloseq to calculate Shannon's Diversity Index (α-diversity). The flowering phenology of a local Festuca rubra population was observed. We found that grass pollen concentrations varied on a microscale level, likely attributed to local topography and the dispersion distance of pollen from flowering grasses in local source areas. Six genera (Agrostis, Alopecurus, Arrhenatherum, Holcus, Lolium and Poa) dominated the pollen season, comprising on average 77 % of the relative abundance of grass species reads. Temperature, solar radiation, relative humidity, turbulence and wind speeds were found to be relevant for grass pollen release and dispersion processes. An isolated flowering Festuca rubra population contributed almost 40 % of the relative pollen abundance adjacent to the nearby sampler, but only contributed 1 % to samplers situated 300 m away. This suggests that most emitted grass pollen has limited dispersion distance and our results show substantial variation in airborne grass species composition over short geographical scales.
Assuntos
Festuca , Poaceae , Adenosina Desaminase , Peptídeos e Proteínas de Sinalização Intercelular , Pólen/química , Alérgenos/análiseRESUMO
Viruses constantly evolve and adapt to the antiviral defenses of their hosts. The biology of viral circumvention of these selective pressures can often be attributed to the acquisition of novel antagonistic gene products or by rapid genome change that prevents host recognition. To study viral evasion of RNA interference (RNAi)-based defenses, we established a robust antiviral system in mammalian cells using recombinant Sendai virus designed to be targeted by endogenous host microRNAs (miRNAs) with perfect complementarity. Using this system, we previously demonstrated the intrinsic ability of positive-strand RNA viruses to escape this selective pressure via homologous recombination, which was not observed in negative-strand RNA viruses. Here, we show that given extensive time, escape of miRNA-targeted Sendai virus was enabled by host adenosine deaminase acting on RNA 1 (ADAR1). Independent of the viral transcript targeted, ADAR1 editing resulted in disruption of the miRNA-silencing motif, suggesting an intolerance for extensive RNA-RNA interactions necessary for antiviral RNAi. This was further supported in Nicotiana benthamiana, where exogenous expression of ADAR1 interfered with endogenous RNAi. Together, these results suggest that ADAR1 diminishes the effectiveness of RNAi and may explain why it is absent in species that utilize this antiviral defense system. IMPORTANCE All life at the cellular level has the capacity to induce an antiviral response. Here, we examine the result of imposing the antiviral response of one branch of life onto another and find evidence for conflict. To determine the consequences of eliciting an RNAi-like defense in mammals, we applied this pressure to a recombinant Sendai virus in cell culture. We find that ADAR1, a host gene involved in regulation of the mammalian response to virus, prevented RNAi-mediated silencing and subsequently allowed for viral replication. In addition, the expression of ADAR1 in Nicotiana benthamiana, which lacks ADARs and has an endogenous RNAi system, suppresses gene silencing. These data indicate that ADAR1 is disruptive to RNAi biology and provide insight into the evolutionary relationship between ADARs and antiviral defenses in eukaryotic life.
Assuntos
Adenosina Desaminase , Interações entre Hospedeiro e Microrganismos , MicroRNAs , Interferência de RNA , Infecções por Respirovirus , Animais , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Antivirais/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Replicação Viral/genética , Vírus Sendai/classificação , Inativação Gênica , Humanos , Mutação , Fases de Leitura Aberta , Evolução Biológica , Interações entre Hospedeiro e Microrganismos/genética , Infecções por Respirovirus/metabolismo , Infecções por Respirovirus/virologiaRESUMO
A quote attributed to Yogi Berra makes the observation that "It's tough to make predictions, especially about the future," highlighting the difficulties posed to an author writing a manuscript like the present. The history of Z-DNA shows that earlier postulates about its biology have failed the test of time, both those from proponents who were wildly enthusiastic in enunciating roles that till this day still remain elusive to experimental validation and those from skeptics within the larger community who considered the field a folly, presumably because of the limitations in the methods available at that time. If anything, the biological roles we now know for Z-DNA and Z-RNA were not anticipated by anyone, even when those early predictions are interpreted in the most favorable way possible. The breakthroughs in the field were made using a combination of methods, especially those based on human and mouse genetic approaches informed by the biochemical and biophysical characterization of the Zα family of proteins. The first success was with the p150 Zα isoform of ADAR1 (adenosine deaminase RNA specific), with insights into the functions of ZBP1 (Z-DNA-binding protein 1) following soon after from the cell death community. Just as the replacement of mechanical clocks by more accurate designs changed expectations about navigation, the discovery of the roles assigned by nature to alternative conformations like Z-DNA has forever altered our view of how the genome operates. These recent advances have been driven by better methodology and by better analytical approaches. This article will briefly describe the methods that were key to these discoveries and highlight areas where new method development is likely to further advance our knowledge.
Assuntos
DNA Forma Z , Humanos , Animais , Camundongos , RNA/genética , Sítios de Ligação , Isoformas de Proteínas/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Adenosina Desaminase/metabolismoRESUMO
Adenosine deaminase acting on RNA 1 (ADAR1) catalyzes adenosine-to-inosine editing on double-stranded RNA molecules and is involved in regulating cellular responses to endogenous and exogenous RNA. ADAR1 is the primary A-to-I editor of RNA in humans, and the majority of edit sites are found in a class of short interspersed nuclear elements called Alu elements, many of which are located in introns and 3' untranslated regions. Two ADAR1 protein isoforms, p110 (110 kDa) and p150 (150 kDa), are known to be coupled in expression, and decoupling the expression of these isoforms has revealed that the p150 isoform edits a broader range of targets compared to p110. Numerous methods for identification of ADAR1-associated edits have been developed, and we present here a specific method for identification of edit sites associated with individual ADAR1 isoforms.
Assuntos
Adenosina Desaminase , RNA de Cadeia Dupla , Humanos , Adenosina Desaminase/metabolismo , Íntrons , Isoformas de Proteínas/genéticaRESUMO
Adenosine deaminases acting on RNA (ADARs) are RNA editing enzymes that catalyze the hydrolytic deamination of adenosine (A) to inosine (I) in dsRNA. In humans, two catalytically active ADARs, ADAR1 and ADAR2, perform this A-to-I editing event. The growing field of nucleotide base editing has highlighted ADARs as promising therapeutic agents while multiple studies have also identified ADAR1's role in cancer progression. However, the potential for site-directed RNA editing as well as the rational design of inhibitors is being hindered by the lack of detailed molecular understanding of RNA recognition by ADAR1. Here, we designed short RNA duplexes containing the nucleoside analog, 8-azanebularine (8-azaN), to gain insight into molecular recognition by the human ADAR1 catalytic domain. From gel shift and in vitro deamination experiments, we validate ADAR1 catalytic domain's duplex secondary structure requirement and present a minimum duplex length for binding (14 bp, with 5 bp 5' and 8 bp 3' to editing site). These findings concur with predicted RNA-binding contacts from a previous structural model of the ADAR1 catalytic domain. Finally, we establish that neither 8-azaN as a free nucleoside nor a ssRNA bearing 8-azaN inhibits ADAR1 and demonstrate that the 8-azaN-modified RNA duplexes selectively inhibit ADAR1 and not the closely related ADAR2 enzyme.
Assuntos
Ribonucleosídeos , Humanos , Nucleosídeos de Purina , RNA de Cadeia Dupla , Adenosina , Adenosina Desaminase/metabolismoRESUMO
Despite numerous clinically available vaccines and therapeutics, aged patients remain at increased risk for COVID-19 morbidity. Furthermore, various patient populations, including the aged can have suboptimal responses to SARS-CoV-2 vaccine antigens. Here, we characterized vaccine-induced responses to SARS-CoV-2 synthetic DNA vaccine antigens in aged mice. Aged mice exhibited altered cellular responses, including decreased IFNγ secretion and increased TNFα and IL-4 secretion suggestive of TH2-skewed responses. Aged mice exhibited decreased total binding and neutralizing antibodies in their serum but significantly increased TH2-type antigen-specific IgG1 antibody compared to their young counterparts. Strategies to enhance vaccine-induced immune responses are important, especially in aged patient populations. We observed that co-immunization with plasmid-encoded adenosine deaminase (pADA)enhanced immune responses in young animals. Ageing is associated with decreases in ADA function and expression. Here, we report that co-immunization with pADA enhanced IFNγ secretion while decreasing TNFα and IL-4 secretion. pADA expanded the breadth and affinity SARS-CoV-2 spike-specific antibodies while supporting TH1-type humoral responses in aged mice. scRNAseq analysis of aged lymph nodes revealed that pADA co-immunization supported a TH1 gene profile and decreased FoxP3 gene expression. Upon challenge, pADA co-immunization decreased viral loads in aged mice. These data support the use of mice as a model for age-associated decreased vaccine immunogenicity and infection-mediated morbidity and mortality in the context of SARS-CoV-2 vaccines and provide support for the use of adenosine deaminase as a molecular adjuvant in immune-challenged populations.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Animais , Camundongos , Vacinas contra COVID-19 , Fator de Necrose Tumoral alfa , Interleucina-4 , Adenosina Desaminase , Imunização , Anticorpos Antivirais , Modelos Animais de DoençasRESUMO
AIMS: DEAD-box helicase 1 (DDX1) has oncogenic properties in several human cancers. However, the clinical significance and biological role of DDX1 in non-small cell lung cancer (NSCLC) remain elusive. Here, we examined the chemotherapeutic relevance of DDX1 in NSCLC. MAIN METHODS: We used the UALCAN database, Western blot analysis, and immunohistochemical and RT-qPCR assays to assess DDX1 expression in NSCLC cell lines (H1650 and A549) and patient tissues. The role of DDX1 in the chemosensitivity of NSCLC cells and the underlying mechanisms were determined using colony formation, CCK-8, flow cytometry, wound healing, Transwell, tumor sphere formation, and immunostaining assays, together with a xenograft tumor model in nude mice. KEY FINDINGS: Our study revealed that DDX1 was overexpressed in NSCLC cell lines and tissues. We further found that depleting DDX1 increased the sensitivity of NSCLC cells to the chemotherapy drug cisplatin, increased cell apoptosis, and inhibited cell migration and invasion. Co-immunoprecipitation assays revealed that DDX1 bound to ADAR1, and increased ADAR1 protein expression. Furthermore, we found that ADAR1 mediated cancer-promoting effects, independent of deaminase activity, by binding to RAC3 mRNA. Our findings not only show that DDX1 mediates chemosensitivity to cisplatin via the ADAR1/RAC3 axis but also highlight the importance of ADARs as essential RNA-binding proteins for cell homeostasis, as well as cancer progression. SIGNIFICANCE: Our results suggest that DDX1 plays an important role in the development and progression of human NSCLC and that DDX1 may serve as a therapeutic target in NSCLC patients.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , MicroRNAs , Animais , Humanos , Camundongos , Adenosina Desaminase/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Cisplatino/farmacologia , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Neoplasias Pulmonares/patologia , Camundongos Nus , MicroRNAs/genética , Células-Tronco Neoplásicas/metabolismoRESUMO
The Zα domain of ADARp150 is critical for proper Z-RNA substrate binding and is a key factor in the type-I interferon response pathway. Two point-mutations in this domain (N173S and P193A), which cause neurodegenerative disorders, are linked to decreased A-to-I editing in disease models. To understand this phenomenon at the molecular level, we biophysically and structurally characterized these two mutated domains, revealing that they bind Z-RNA with a decreased affinity. Less efficient binding to Z-RNA can be explained by structural changes in beta-wing, part of the Z-RNA-protein interface, and alteration of conformational dynamics of the proteins.
Assuntos
Adenosina Desaminase , Doenças Autoimunes do Sistema Nervoso , Malformações do Sistema Nervoso , Humanos , Adenosina Desaminase/genética , Adenosina Desaminase/química , Adenosina Desaminase/metabolismo , Doenças Autoimunes do Sistema Nervoso/enzimologia , Doenças Autoimunes do Sistema Nervoso/genética , Sítios de Ligação , Malformações do Sistema Nervoso/enzimologia , Malformações do Sistema Nervoso/genética , RNA/química , Domínios Proteicos/genética , Mutação Puntual , Conformação de Ácido NucleicoRESUMO
The most abundant form of RNA editing in metazoa is the deamination of adenosines into inosines (A-to-I), catalyzed by ADAR enzymes. Inosines are read as guanosines by the translation machinery, and thus A-to-I may lead to protein recoding. The ability of ADARs to recode at the mRNA level makes them attractive therapeutic tools. Several approaches for Site-Directed RNA Editing (SDRE) are currently under development. A major challenge in this field is achieving high on-target editing efficiency, and thus it is of much interest to identify highly potent ADARs. To address this, we used the baker yeast Saccharomyces cerevisiae as an editing-naïve system. We exogenously expressed a range of heterologous ADARs and identified the hummingbird and primarily mallard-duck ADARs, which evolved at 40-42°C, as two exceptionally potent editors. ADARs bind to double-stranded RNA structures (dsRNAs), which in turn are temperature sensitive. Our results indicate that species evolved to live with higher core body temperatures have developed ADAR enzymes that target weaker dsRNA structures and would therefore be more effective than other ADARs. Further studies may use this approach to isolate additional ADARs with an editing profile of choice to meet specific requirements, thus broadening the applicability of SDRE.
