ABSTRACT
Purine salvage enzymes have been of significant interest in anti-Leishmanial drug development due to the parasite's critical dependence on this pathway for the supply of nucleotides in the absence of a de novo purine synthesis pathway. Adenylosuccinate lyase (ADSL) one of the key enzymes in this pathway is a homo-tetramer, where the active site is formed by residues from three distinct subunits. Analysis of the subunit interfaces of LdADSL, revealed a conserved Arg40 forming critical inter-subunit interactions and also involved in substrate binding. We hypothesized that mutating this residue can affect both the structural stability and activity of the enzyme. In our study, we used biochemical, biophysical, and computational simulation approaches to understand the structural and functional role of Arg40 in LdADSL. We have replaced Arg40 with an Ala and Glu using site directed mutagenesis. The mutant enzymes were similar to wild-type enzyme in secondary structure and subunit association. Thermal shift assays indicated that the mutations affected the protein stability. Both mutants showed decreased specific activities in both forward and reverse directions with significantly weakened affinities towards succinyl-adenosine monophosphate (SAMP). The mutations resulted in changes in C3 loop conformation and D3 domain rotation. Consequently, the orientation of the active site amino acid residues changed resulting in compromised activity and stability. Studies so far have majorly focused on the ADSL active site for designing drugs against it. Our work indicates that an alternative inhibitory mechanism for the enzyme can be designed by targeting the inter-subunit interface.
Subject(s)
Adenylosuccinate Lyase , Arginine , Enzyme Stability , Leishmania donovani , Adenylosuccinate Lyase/genetics , Adenylosuccinate Lyase/chemistry , Adenylosuccinate Lyase/metabolism , Leishmania donovani/enzymology , Leishmania donovani/genetics , Arginine/metabolism , Arginine/chemistry , Purines/metabolism , Purines/chemistry , Protozoan Proteins/genetics , Protozoan Proteins/chemistry , Protozoan Proteins/metabolism , Mutagenesis, Site-Directed , Catalytic Domain , Molecular Dynamics SimulationABSTRACT
High levels of purine and uric acid, which are associated with health issues such as gout and cardiovascular disease, are found in the meat of fast-growing broiler chickens, which raises concerns about the quality of chicken meat and the health of the consumers who consume it. High genetic homogeneity and uniformity, particularly in genes involved in the synthesis of inosine monophosphate (IMP) and subsequent process of purine synthesis, which are associated with the meat quality, are exhibited in commercial broiler chickens owing to intensive inbreeding programs. Adenosine succinate lyase (ADSL) is a key enzyme involved in de novo purine biosynthetic pathway and its genetic polymorphisms affect IMP metabolism and purine content. In this study, we investigated the polymorphism of the ADSL gene in indigenous and local chicken breeds and red junglefowl in Thailand, using metabarcoding and genetic diversity analyses. Five alleles with 73 single nucleotide polymorphisms in exon 2, including missense and silent mutations, which may act on the synthesis efficiency of IMP and purine. Their protein structures revealed changes in amino acid composition that may affect ADSL enzyme activity. Weak purifying selection in these ADSL alleles was observed in the chicken population studied, implying that the variants have minor fitness impacts and a greater probability of fixation of beneficial mutations than strong purifying selection. A potential selective sweep was observed in Mae Hong Son chickens, whose purine content was lower than that in other breeds. This suggests a potential correlation between variations of the ADSL gene and reduced purine content and an impact of ADSL expression on the quality of chicken meat. However, further studies are required to validate its potential availability as a genetic marker for selecting useful traits that are beneficial to human health and well-being.
Subject(s)
Adenylosuccinate Lyase , Chickens , Polymorphism, Single Nucleotide , Selection, Genetic , Animals , Chickens/genetics , Adenylosuccinate Lyase/genetics , Thailand , Alleles , Inosine Monophosphate/metabolism , Breeding , Meat , Genetic Variation , Purines/metabolism , Purines/biosynthesisABSTRACT
OBJECTIVE: Adenylosuccinate lyase (ADSL) deficiency is a rare inherited metabolic disorder with a wide phenotypic presentation, classically grouped into three types (neonatal, type I, and type II). We aim to better delineate the pathological spectrum, focusing on the electroclinical characteristics and phenotypic differences of patients with ADSL deficiency. PATIENTS AND METHODS: Seven patients, from four different families, underwent serial electroencephalogram (EEG), clinical assessment, and neuroimaging. We also performed a systematic review of the cases published in the literature, summarizing the available clinical, neurophysiological, and genetic data. RESULTS: We report seven previously unreported ADSL deficiency patients with long-term follow-up (10-34 years). From the literature review, we collected 81 previously reported cases. Of the included patient population, 58 % (51/88) were classified as having ADSL deficiency type I, 28% (25/88) as having type II, and 14% (12/88) as having neonatal. The most frequently reported pathogenic variants are p.R426H homozygous (19 patients), p.Y114H in compound heterozygosity (13 patients), and p.D430N homozygous (6 patients). In the majority (89.2%), disease onset was within the first year of life. Epilepsy is present in 81.8% of the patients, with polymorphic and often intractable seizures. EEG features seem to display common patterns and developmental trajectories: (i) poor general background organization with theta-delta activity; (ii) hypsarrhythmia with spasms, usually adrenocorticotropic hormone-responsive; (iii) generalized epileptic discharges with frontal or frontal temporal predominance; and (iv) epileptic discharge activation in sleep with an altered sleep structure. Imaging features present consistent findings of cerebral atrophy with frontal predominance, cerebellar atrophy, and white matter abnormalities among the three types. SIGNIFICANCE: ADSL deficiency presents variable phenotypic expression, whose severity could be partially attributed to residual activity of the mutant protein. Although a precise phenotype-genotype correlation was not yet feasible, we delineated a common pattern of clinical, neuroradiological, and neurophysiological features.
Subject(s)
Adenylosuccinate Lyase , Autistic Disorder , Epilepsy , Purine-Pyrimidine Metabolism, Inborn Errors , Infant, Newborn , Humans , Adenylosuccinate Lyase/genetics , Adenylosuccinate Lyase/chemistry , Follow-Up Studies , Autistic Disorder/genetics , AtrophyABSTRACT
In recent years, strategic plans for poultry production have emphasized quantitative traits, particularly body weight and carcass traits (meat yield), in response to overpopulation challenges. Candidate genes such as adenylosuccinate lyase (ADSL), melanocortin-4-receptor (MC4R), and calpain 1 (CAPN1) have played vital roles in this context due to their associations with muscle growth and body composition. This study aims to investigate the influence of polymorphisms and gene expressions of the aforementioned genes on body weight (BW), growth rate (GR), breast weight (BrW), and thigh weight (TW) across four distinct chicken breeds: Fayoumi, Matrouh, Mamourah, and Leghorn. The use of PCR-SSCP analysis revealed genetic polymorphisms through the identification of various patterns (genotypes) within the three examined genes. The ADSL, MC4R, and CAPN1 genes exhibited five, three, and two different genotypes, respectively. These polymorphisms displayed promising connections with enhancing economically significant production traits, particularly BW, BrW and TW. Furthermore, gene expression analyses were conducted on breast and thigh tissues obtained from the chicken breeds at 60 days of age, where ADSL and MC4R exhibited a noteworthy up-regulation in Fayoumi and Matrouh breeds, and down-regulation in Mamourah and Leghorn. In contrast, CAPN1 expression decreased across most breeds with a slight increase noted in Fayoumi breed. In conclusion, this investigation underscores the substantial impact of ADSL, MC4R, and CAPN1 genes on economically important production traits within Egyptian domestic chicken breeds. Consequently, these genes emerge as significant molecular markers, holding potential utility in avian selection and breeding programs aimed at enhancing productive performance.
Subject(s)
Adenylosuccinate Lyase , Chickens , Animals , Chickens/metabolism , Adenylosuccinate Lyase/genetics , Adenylosuccinate Lyase/metabolism , Egypt , Polymorphism, Single Nucleotide/genetics , Genotype , Meat , Body WeightABSTRACT
Dacarbazine (DTIC) is the primary first-line treatment for advanced-stage metastatic melanoma; thus, DTIC resistance is poses a major challenge. Therefore, investigating the mechanism underlying DTIC resistance must be investigated. Dicer, a type III cytoplasmic endoribonuclease, plays a pivotal role in the maturation of miRNAs. Aberrant Dicer expression may contribute to tumor progression, clinical aggressiveness, and poor prognosis in various tumors. Dicer inhibition led to a reduction in DTIC sensitivity and an augmentation in stemness in melanoma cells. Clinical analyses indicated a low Dicer expression level as a predictor of poor prognosis factor. Metabolic alterations in tumor cells may interfere with drug response. Adenylosuccinate lyase (ADSL) is a crucial enzyme in the purine metabolism pathway. An imbalance in ADSL may interfere with the therapeutic efficacy of drugs. We discovered that DTIC treatment enhanced ADSL expression and that Dicer silencing significantly reduced ADSL expression in melanoma cells. Furthermore, ADSL overexpression reversed Dicer silencing induced DTIC resistance and cancer stemness. These findings indicate that Dicer-mediated ADSL regulation influences DTIC sensitivity and stemness in melanoma cells.
Subject(s)
Adenylosuccinate Lyase , Melanoma , Humans , Dacarbazine/therapeutic use , Melanoma/drug therapy , Melanoma/genetics , Melanoma/metabolismABSTRACT
Adenylosuccinate lyase (PurB) catalyzes two distinct reactions in the purine nucleotide biosynthetic pathway using the same active site. The ability to recognize two different sets of substrates is of structural and evolutionary interest. In the present study, the crystal structure of PurB from the thermophilic bacterium Thermus thermophilus HB8 (TtPurB) was determined at a resolution of 2.38â Å by molecular replacement using a structure predicted by AlphaFold2 as a template. The asymmetric unit of the TtPurB crystal contained two TtPurB molecules, and some regions were disordered in the crystal structure. The disordered regions were the substrate-binding site and domain 3. TtPurB forms a homotetramer and the monomer is composed of three domains (domains 1, 2 and 3), which is a typical structure for the aspartase/fumarase superfamily. Molecular dynamics simulations with and without substrate/product were performed using a full-length model of TtPurB which was obtained before deletion of the disordered regions. The substrates and products were bound to the model structures during the MD simulations. The fluctuations of amino-acid residues were greater in the disordered regions and became smaller upon the binding of substrate or product. These results demonstrate that the full-length model obtained using AlphaFold2 can be used to generate the coordinates of disordered regions within the crystal structure.
Subject(s)
Adenylosuccinate Lyase , Adenylosuccinate Lyase/genetics , Adenylosuccinate Lyase/chemistry , Adenylosuccinate Lyase/metabolism , Amino Acid Sequence , Thermus thermophilus , Sequence Homology, Amino Acid , Crystallography, X-RayABSTRACT
Adenylosuccinate lyase deficiency is an ultrarare congenital metabolic disorder associated with muscle weakness and neurobehavioral dysfunction. Adenylosuccinate lyase is required for de novo purine biosynthesis, acting twice in the pathway at non-sequential steps. Genetic models can contribute to our understanding of the etiology of disease phenotypes and pave the way for development of therapeutic treatments. Here, we establish the first model to specifically study neurobehavioral aspects of adenylosuccinate lyase deficiency. We show that reduction of adsl-1 function in C. elegans is associated with a novel learning phenotype in a gustatory plasticity assay. The animals maintain capacity for gustatory plasticity, evidenced by a change in their behavior in response to cue pairing. However, their behavioral output is distinct from that of control animals. We link substrate accumulation that occurs upon adsl-1 deficiency to an unexpected perturbation in tyrosine metabolism and show that a lack of tyramine mediates the behavioral changes through action on the metabotropic TYRA-2 tyramine receptor. Our studies reveal a potential for wider metabolic perturbations, beyond biosynthesis of purines, to impact behavior under conditions of adenylosuccinate lyase deficiency.
Subject(s)
Adenylosuccinate Lyase , Adenylosuccinate Lyase/deficiency , Autistic Disorder , Caenorhabditis elegans Proteins , Purine-Pyrimidine Metabolism, Inborn Errors , Receptors, Biogenic Amine , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Adenylosuccinate Lyase/genetics , Adenylosuccinate Lyase/metabolism , Purine-Pyrimidine Metabolism, Inborn Errors/genetics , Caenorhabditis elegans Proteins/geneticsABSTRACT
Inborn errors of purine metabolism are rare syndromes with an array of complex phenotypes in humans. One such disorder, adenylosuccinate lyase deficiency (ASLD), is caused by a decrease in the activity of the bi-functional purine biosynthetic enzyme adenylosuccinate lyase (ADSL). Mutations in human ADSL cause epilepsy, muscle ataxia, and autistic-like symptoms. Although the genetic basis of ASLD is known, the molecular mechanisms driving phenotypic outcome are not. Here, we characterize neuromuscular and reproductive phenotypes associated with a deficiency of adsl-1 in Caenorhabditis elegans. We demonstrate that adsl-1 function contributes to regulation of spontaneous locomotion, that adsl-1 functions acutely for proper mobility, and that aspects of adsl-1-related dysfunction are reversible. Using pharmacological supplementation, we correlate phenotypes with distinct metabolic perturbations. The neuromuscular defect correlates with accumulation of a purine biosynthetic intermediate whereas reproductive deficiencies can be ameliorated by purine supplementation, indicating differing molecular mechanisms behind the phenotypes. Because purine metabolism is highly conserved in metazoans, we suggest that similar separable metabolic perturbations result in the varied symptoms in the human disorder and that a dual-approach therapeutic strategy may be beneficial.
Subject(s)
Adenylosuccinate Lyase , Autistic Disorder , Purine-Pyrimidine Metabolism, Inborn Errors , Animals , Humans , Autistic Disorder/genetics , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Adenylosuccinate Lyase/genetics , Adenylosuccinate Lyase/metabolism , Purine-Pyrimidine Metabolism, Inborn Errors/genetics , Purine-Pyrimidine Metabolism, Inborn Errors/diagnosis , Phenotype , PurinesABSTRACT
Adenylosuccinase deficiency is a rare inborn error of metabolism. We present a newborn who died at 52 days of age with clinical features suggestive of severe epileptic encephalopathy and leukodystrophy of unknown cause. Post-mortem examination showed an unusual vacuolar appearance of the brain. A molecular autopsy performed via singleton clinical exome analysis revealed a known pathogenic and a variant of uncertain significance in ADSL that encodes adenylosuccinase. Tests on previously stored plasma samples showed elevated succinyladenosine and succinylaminoimidazole carboxamide riboside levels. Adenylosuccinase activity in stored fibroblasts was only ~5% of control confirming the diagnosis of adenylosuccinase deficiency in the child. The parents opted for a chorionic villus biopsy in a subsequent pregnancy and had a child unaffected by adenylosuccinase deficiency. This report adds vacuolating leukodystrophy as a novel feature of adenylosuccinase deficiency and shows the power of biochemical investigations directed by genomic studies to achieve accurate diagnosis. Importantly, this case demonstrates the importance of anticipatory banking of biological samples for reverse biochemical phenotyping in individuals with undiagnosed disorders who may not survive.
Subject(s)
Adenylosuccinate Lyase , Autistic Disorder , Purine-Pyrimidine Metabolism, Inborn Errors , Child , Infant, Newborn , Infant , Humans , Autopsy , Adenylosuccinate Lyase/genetics , Purine-Pyrimidine Metabolism, Inborn Errors/geneticsABSTRACT
In EGFR-mutant lung cancer, drug-tolerant persister cells (DTPCs) show prolonged survival when receiving EGFR tyrosine kinase inhibitor (TKI) treatments. They are a likely source of drug resistance, but little is known about how these cells tolerate drugs. Ribonucleic acids (RNAs) molecules control cell growth and stress responses. Nucleic acid metabolism provides metabolites, such as purines, supporting RNA synthesis and downstream functions. Recently, noncoding RNAs (ncRNAs), such as microRNAs (miRNAs), have received attention due to their capacity to repress gene expression via inhibitory binding to downstream messenger RNAs (mRNAs). Here, our study links miRNA expression to purine metabolism and drug tolerance. MiR-21-5p (guide strand) is a commonly upregulated miRNA in disease states, including cancer and drug resistance. However, the expression and function of miR-21-3p (passenger strand) are not well understood. We found that upregulation of miR-21-5p and miR-21-3p tune purine metabolism leading to increased drug tolerance. Metabolomics data demonstrated that purine metabolism was the top pathway in the DTPCs compared with the parental cells. The changes in purine metabolites in the DTPCs were partially rescued by targeting miR-21. Analysis of protein levels in the DTPCs showed that reduced expression of adenylosuccinate lyase (ADSL) was reversed after the miR-21 knockdown. ADSL is an essential enzyme in the de novo purine biosynthesis pathway by converting succino-5-aminoimidazole-4-carboxamide riboside (succino-AICAR or SAICAR) to AICAR (or acadesine) as well as adenylosuccinate to adenosine monophosphate (AMP). In the DTPCs, miR-21-5p and miR-21-3p repress ADSL expression. The levels of top decreased metabolite in the DTPCs, AICAR was reversed when miR-21 was blocked. AICAR induced oxidative stress, evidenced by increased reactive oxygen species (ROS) and reduced expression of nuclear factor erythroid-2-related factor 2 (NRF2). Concurrently, miR-21 knockdown induced ROS generation. Therapeutically, a combination of AICAR and osimertinib increased ROS levels and decreased osimertinib-induced NRF2 expression. In a MIR21 knockout mouse model, MIR21 loss-of-function led to increased purine metabolites but reduced ROS scavenging capacity in lung tissues in physiological conditions. Our data has established a link between ncRNAs, purine metabolism, and the redox imbalance pathway. This discovery will increase knowledge of the complexity of the regulatory RNA network and potentially enable novel therapeutic options for drug-resistant patients.
Subject(s)
Adenylosuccinate Lyase , MicroRNAs , Mice , Animals , Adenylosuccinate Lyase/chemistry , Adenylosuccinate Lyase/genetics , Adenylosuccinate Lyase/metabolism , NF-E2-Related Factor 2 , Reactive Oxygen Species , MicroRNAs/genetics , Purines , RNA, Messenger/chemistry , ErbB Receptors/geneticsABSTRACT
Adenylosuccinate lyase (ADSL) functions in de novo purine synthesis (DNPS) and the purine nucleotide cycle. ADSL deficiency (ADSLD) causes numerous neurodevelopmental pathologies, including microcephaly and autism spectrum disorder. ADSLD patients have normal serum purine nucleotide levels but exhibit accumulation of dephosphorylated ADSL substrates, S-Ado, and SAICAr, the latter being implicated in neurotoxic effects through unknown mechanisms. We examined the phenotypic effects of ADSL depletion in human cells and their relation to phenotypic outcomes. Using specific interventions to compensate for reduced purine levels or modulate SAICAr accumulation, we found that diminished AMP levels resulted in increased DNA damage signaling and cell cycle delays, while primary ciliogenesis was impaired specifically by loss of ADSL or administration of SAICAr. ADSL-deficient chicken and zebrafish embryos displayed impaired neurogenesis and microcephaly. Neuroprogenitor attrition in zebrafish embryos was rescued by pharmacological inhibition of DNPS, but not increased nucleotide concentration. Zebrafish also displayed phenotypes commonly linked to ciliopathies. Our results suggest that both reduced purine levels and impaired DNPS contribute to neurodevelopmental pathology in ADSLD and that defective ciliogenesis may influence the ADSLD phenotypic spectrum.
Subject(s)
Adenylosuccinate Lyase/deficiency , Adenylosuccinate Lyase/metabolism , Autistic Disorder/metabolism , Neurogenesis , Purine-Pyrimidine Metabolism, Inborn Errors/metabolism , Aminoimidazole Carboxamide/analogs & derivatives , Aminoimidazole Carboxamide/metabolism , Animals , Autism Spectrum Disorder/metabolism , Cell Cycle , Cell Cycle Proteins/metabolism , Cell Line , Chickens/metabolism , Ciliopathies/metabolism , DNA Damage , Humans , Microcephaly/metabolism , Microtubule-Associated Proteins/metabolism , Phenotype , Phosphoproteins/metabolism , Purines/metabolism , Ribonucleotides/metabolism , Zebrafish/metabolismABSTRACT
Purines are essential molecules that are components of vital biomolecules, such as nucleic acids, coenzymes, signaling molecules, as well as energy transfer molecules. The de novo biosynthesis pathway starts from phosphoribosylpyrophosphate (PRPP) and eventually leads to the synthesis of inosine monophosphate (IMP) by means of 10 sequential steps catalyzed by six different enzymes, three of which are bi-or tri-functional in nature. IMP is then converted into guanosine monophosphate (GMP) or adenosine monophosphate (AMP), which are further phosphorylated into nucleoside di- or tri-phosphates, such as GDP, GTP, ADP and ATP. This review provides an overview of inborn errors of metabolism pertaining to purine synthesis in humans, including either phosphoribosylpyrophosphate synthetase (PRS) overactivity or deficiency, as well as adenylosuccinate lyase (ADSL), 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC), phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS), and adenylosuccinate synthetase (ADSS) deficiencies. ITPase deficiency is being described as well. The clinical spectrum of these disorders is broad, including neurological impairment, such as psychomotor retardation, epilepsy, hypotonia, or microcephaly; sensory involvement, such as deafness and visual disturbances; multiple malformations, as well as muscle presentations or consequences of hyperuricemia, such as gouty arthritis or kidney stones. Clinical signs are often nonspecific and, thus, overlooked. It is to be hoped that this is likely to be gradually overcome by using sensitive biochemical investigations and next-generation sequencing technologies.
Subject(s)
Adenylosuccinate Lyase , Purine-Pyrimidine Metabolism, Inborn Errors , Adenylosuccinate Lyase/deficiency , Adenylosuccinate Lyase/genetics , Adenylosuccinate Lyase/metabolism , Autistic Disorder , Humans , Inosine Monophosphate , Purine-Pyrimidine Metabolism, Inborn Errors/genetics , Purine-Pyrimidine Metabolism, Inborn Errors/metabolism , PurinesABSTRACT
DNA modifications vary in form and function but generally do not alter Watson-Crick base pairing. Diaminopurine (Z) is an exception because it completely replaces adenine and forms three hydrogen bonds with thymine in cyanophage S-2L genomic DNA. However, the biosynthesis, prevalence, and importance of Z genomes remain unexplored. Here, we report a multienzyme system that supports Z-genome synthesis. We identified dozens of globally widespread phages harboring such enzymes, and we further verified the Z genome in one of these phages, Acinetobacter phage SH-Ab 15497, by using liquid chromatography with ultraviolet and mass spectrometry. The Z genome endows phages with evolutionary advantages for evading the attack of host restriction enzymes, and the characterization of its biosynthetic pathway enables Z-DNA production on a large scale for a diverse range of applications.
Subject(s)
2-Aminopurine/metabolism , Adenylosuccinate Synthase/chemistry , Bacteriophages/chemistry , Bacteriophages/enzymology , DNA, Viral/chemistry , DNA, Z-Form/chemistry , Viral Nonstructural Proteins/chemistry , 2-Aminopurine/chemistry , Adenylosuccinate Lyase/chemistry , Adenylosuccinate Lyase/genetics , Adenylosuccinate Lyase/metabolism , Adenylosuccinate Synthase/genetics , Adenylosuccinate Synthase/metabolism , Bacteriophages/genetics , Base Pairing , Biosynthetic Pathways , DNA, Viral/biosynthesis , DNA, Viral/genetics , DNA, Z-Form/biosynthesis , DNA, Z-Form/genetics , Genome, Viral , Hydrogen Bonding , Protein Domains , Substrate Specificity , Thymine/chemistry , Thymine/metabolism , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolismABSTRACT
BACKGROUND: Adenylosuccinate lyase deficiency (ADSLD) is an ultrarare neurometabolic recessive disorder caused by loss-of-function mutations in the ADSL gene. The disease is characterized by wide clinical variability. Here we provide an updated clinical profiling of the disorder and discuss genotype-phenotype correlations. RESULTS: Data were collected through "Our Journey with ADSL deficiency Association" by using a dedicated web survey filled-in by parents. Clinical and molecular data were collected from 18 patients (12 males, median age 10.9 years ± 7.3), from 13 unrelated families. The age at onset ranged from birth to the first three years (median age 0.63 years ± 0.84 SD), and age at diagnosis varied from 2 months to 17 years, (median age 6.4 years ± 6.1 SD). The first sign was a psychomotor delay in 8/18 patients, epilepsy in 3/18, psychomotor delay and epilepsy in 3/18, and apneas, hypotonia, nystagmus in single cases. One patient (sibling of a previously diagnosed child) had a presymptomatic diagnosis. The diagnosis was made by exome sequencing in 7/18 patients. All patients were definitively diagnosed with ADSL deficiency based on pathogenic variants and/or biochemical assessment. One patient had a fatal neonatal form of ADSL deficiency, seven showed features fitting type I, and nine were characterized by a milder condition (type II), with two showing a very mild phenotype. Eighteen different variants were distributed along the entire ADSL coding sequence and were predicted to have a variable structural impact by impairing proper homotetramerization or catalytic activity of the enzyme. Six variants had not previously been reported. All but two variants were missense. CONCLUSIONS: The study adds more details on the spectrum of ADSLD patients' phenotypes and molecular data.
Subject(s)
Adenylosuccinate Lyase , Autistic Disorder , Purine-Pyrimidine Metabolism, Inborn Errors , Adenylosuccinate Lyase/deficiency , Adenylosuccinate Lyase/genetics , Adolescent , Child , Child, Preschool , Female , Humans , Infant , Infant, Newborn , Male , Phenotype , Purine-Pyrimidine Metabolism, Inborn Errors/diagnosis , Purine-Pyrimidine Metabolism, Inborn Errors/geneticsABSTRACT
Rationale: Adenylosuccinate lyase (ADSL) is an essential enzyme for de novo purine biosynthesis. Here we sought to investigate the putative role of ADSL in colorectal carcinoma (CRC) carcinogenesis and response to antimetabolites. Methods: ADSL expression levels were assessed by immunohistochemistry or retrieved from The Cancer Genome Atlas (TCGA) dataset. The effects of ADSL silencing or overexpression were evaluated on CRC cell proliferation, cell migration and cell-cycle. In vivo tumor growth was assessed by the chicken chorioallantoic membrane (CAM). Transfected cell lines or patient-derived organoids (PDO) were treated with 5-fluorouracil (5-FU) and 6-mercaptopurine (6-MP) and drug response was correlated with ADSL expression levels. Metabolomic and transcriptomic profiling were performed to identify dysregulated pathways and ADSL downstream effectors. Mitochondrial respiration and glycolytic capacity were measured using Seahorse; mitochondrial membrane potential and the accumulation of ROS were measured by FACS using MitoTracker Red and MitoSOX staining, respectively. Activation of canonical pathways was assessed by immunohistochemistry and immunoblotting. Results: ADSL expression is significantly increased in CRC tumors compared to non-tumor tissue. ADSL-high CRCs show upregulation of genes involved in DNA synthesis, DNA repair and cell cycle. Accordingly, ADSL overexpression accelerated progression through the cell cycle and significantly increased proliferation and migration in CRC cell lines. Additionally, ADSL expression increased tumor growth in vivo and sensitized CRCs to 6-MP in vitro, ex vivo (PDOs) and in vivo (CAM model). ADSL exerts its oncogenic function by affecting mitochondrial function via alteration of the TCA cycle and impairment of mitochondrial respiration. The KEAP1-NRF2 and mTORC1-cMyc axis are independently activated upon ADSL overexpression and may favor the survival and proliferation of ROS-accumulating cells, favoring DNA damage and tumorigenesis. Conclusions: Our results suggest that ADSL is a novel oncogene in CRC, modulating mitochondrial function, metabolism and oxidative stress, thus promoting cell cycle progression, proliferation and migration. Our results also suggest that ADSL is a predictive biomarker of response to 6-mercaptopurine in the pre-clinical setting.
Subject(s)
Adenylosuccinate Lyase/genetics , Colorectal Neoplasms/genetics , Mitochondria/genetics , NF-E2-Related Factor 2/genetics , Oncogenes/genetics , Proto-Oncogene Proteins c-myc/genetics , TOR Serine-Threonine Kinases/genetics , Caco-2 Cells , Carcinogenesis/genetics , Carcinogenesis/pathology , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Cell Respiration/genetics , Colorectal Neoplasms/pathology , Gene Expression Regulation, Neoplastic/genetics , HT29 Cells , Humans , Mitochondria/pathologySubject(s)
Adenylosuccinate Lyase/deficiency , Adenylosuccinate Lyase/genetics , Agenesis of Corpus Callosum/physiopathology , Autistic Disorder/genetics , Developmental Disabilities/physiopathology , Epilepsy/physiopathology , Lissencephaly/physiopathology , Muscle Hypotonia/physiopathology , Purine-Pyrimidine Metabolism, Inborn Errors/genetics , Respiratory Distress Syndrome, Newborn/physiopathology , Agenesis of Corpus Callosum/diagnostic imaging , Autistic Disorder/diagnosis , Autistic Disorder/physiopathology , Cerebellar Vermis/abnormalities , Cerebellar Vermis/diagnostic imaging , Cerebral Cortex/abnormalities , Cerebral Cortex/diagnostic imaging , Electroencephalography , Heterozygote , Humans , Infant, Newborn , Lissencephaly/diagnostic imaging , Magnetic Resonance Imaging , Male , Mutation , Organ Size , Purine-Pyrimidine Metabolism, Inborn Errors/diagnosis , Purine-Pyrimidine Metabolism, Inborn Errors/physiopathology , Exome SequencingABSTRACT
Adenylosuccinate lyase deficiency is a rare inherited disorder of purine metabolism causing severe neurological impairment ranging from early-onset neonatal epileptic encephalopathy to progressive psychomotor retardation and autism in later life. Diagnostic workup involves the measurement of toxic succinyl purines in body fluids and gene sequencing. The authors describe a 13-mo-old girl with compound heterozygous variants in the ADSL gene, presenting as early-onset seizures, severe neurological impairment, development delay, and hypotonia. Neuroimaging revealed cerebral atrophy, delayed myelination and diffusion restriction in bilateral basal ganglia, thalamus and periventricular white matter. The present case highlights ADSL deficiency as a rare cause of metabolic epilepsy that needs timely recognition and prevention of unnecessary investigations.
Subject(s)
Adenylosuccinate Lyase , Autistic Disorder , Epilepsy , Purine-Pyrimidine Metabolism, Inborn Errors , Adenylosuccinate Lyase/deficiency , Epilepsy/diagnosis , Epilepsy/etiology , Female , Humans , Infant , Infant, Newborn , Purine-Pyrimidine Metabolism, Inborn Errors/diagnosis , Purine-Pyrimidine Metabolism, Inborn Errors/geneticsABSTRACT
The dreadful disease malaria is one among the infectious diseases that comes in third number after the tuberculosis and HIV. This disease is spread by female Anopheles mosquito and caused by the malarial parasite sp notably Plasmodium falciparum. In this, the organism has several enzymes for processing the infection and growth mechanism and among that, the adenylosuccinate lyase is an enzyme that plays a critical role in metabolism and cellular replication via its action in the de novo purine biosynthetic pathway. Adenylosuccinate has been studied for two reaction mechanisms, and in that, the adenylosuccinate to AMP and fumarate is core important. As of now, there have been several studies indicating the reaction mechanism of adenylosuccinate lyase, this study projects the conformations of the reactant and product changes through molecular docking and molecular dynamic simulations. Adenylosuccinate bound complex involves His role in the product than the reactant complex, and the complex shows high flexibility due to fumarate. Thus, identifying the core inhibitor that binds to His rings could be a standard adenylosuccinate lyase inhibitor, that can block the malarial diseases in humans. In addition to the competitive inhibition site, we also predicted the uncompetitive ligand binding site, which suggest the alternate region to be targeted. Thus, from this work, we suggest both competitive and uncompetitive binding regions for the purpose identifying the malarial inhibitors.
Subject(s)
Adenylosuccinate Lyase/chemistry , Malaria, Falciparum/parasitology , Molecular Dynamics Simulation , Plasmodium falciparum/enzymology , Receptors, Cell Surface/metabolism , Adenylosuccinate Lyase/metabolism , Animals , Female , Malaria, Falciparum/metabolism , Protein ConformationABSTRACT
BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) is among the most common cancer types worldwide, yet patients with HCC have limited treatment options. There is an urgent need to identify drug targets that specifically inhibit the growth of HCC cells. APPROACH AND RESULTS: We used a CRISPR library targeting ~2,000 druggable genes to perform a high-throughput screen and identified adenylosuccinate lyase (ADSL), a key enzyme involved in the de novo purine synthesis pathway, as a potential drug target for HCC. ADSL has been implicated as a potential oncogenic driver in some cancers, but its role in liver cancer progression remains unknown. CRISPR-mediated knockout of ADSL impaired colony formation of liver cancer cells by affecting AMP production. In the absence of ADSL, the growth of liver tumors is retarded in vivo. Mechanistically, we found that ADSL knockout caused S-phase cell cycle arrest not by inducing DNA damage but by impairing mitochondrial function. Using data from patients with HCC, we also revealed that high ADSL expression occurs during tumorigenesis and is linked to poor survival rate. CONCLUSIONS: Our findings uncover the role of ADSL-mediated de novo purine synthesis in fueling mitochondrial ATP production to promote liver cancer cell growth. Targeting ADSL may be a therapeutic approach for patients with HCC.
Subject(s)
Adenylosuccinate Lyase/antagonists & inhibitors , Carcinoma, Hepatocellular/drug therapy , Liver Neoplasms/drug therapy , Purines/biosynthesis , Adenosine Triphosphate/biosynthesis , Adenylosuccinate Lyase/genetics , Adenylosuccinate Lyase/metabolism , Animals , Carcinogenesis/drug effects , Carcinogenesis/metabolism , Carcinoma, Hepatocellular/mortality , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Disease Models, Animal , Gene Knockout Techniques , Humans , Liver Neoplasms/mortality , Liver Neoplasms/pathology , Mice , Mitochondria/drug effects , Mitochondria/metabolism , Survival RateABSTRACT
Schistosomiasis, caused by Schistosoma mansoni trematode worm, affects more than 1.5 million people in Brazil. The current treatment consists in the administration of Praziquantel, the only medicine used for treatment for more than 40 years. Some of the limitations of this drug consist in its inactivity against schistosomula and parasite eggs, the appearance of resistant strains and non-prevention against reinfection. Thus, the objective of this study was to evaluate the effect of immunization with recombinant functional enzymes of the purine salvage pathway of S. mansoni, Nucleoside Diphosphate Kinase (NDPK) and Adenylosuccinate Lyase (ADSL), to evaluate the host immune response, as well as the parasite load after vaccination. For this, Balb/c mice were divided into 5 groups: control (uninfected and untreated), non-immunized/infected, NDPK infected, ADSL infected, and NDPK + ADSL infected. Immunized groups received three enzyme dosages, with a 15-day interval between each dose, and after 15 days of the last application the animals were infected with 80 cercariae of S. mansoni. On the 47th day after the infection, fecal eggs were counted and, on the 48th day after the infection, the evaluation of leukocyte response, parasite load, antibody production, cytokines quantification, and histopathological analysis were performed. The results showed that immunizations with NDPK, ADSL or NDPK + ADSL promoted a discreet reduction in eosinophil counts in lavage of peritoneal cavity. All immunized animals showed increased production and secretion of IgG1, IgG2a, and IgE antibodies. Increased production of IL-4 was observed in the group immunized with the combination of both enzymes (NDPK + ADSL). In addition, in all immunized groups there were reductions in egg counts in the liver and intestine, such as reductions in liver granulomas. Thus, we suggest that immunizations with these enzymes could contribute to the reduction of schistosomiasis transmission, besides being important in immunopathogenesis control of the disease.