Phosphoribosylaminoimidazole-succinocarboxamide Synthase Research Articles

PAICS/DYRK3 Multienzyme Interactions as Coregulators of Purinosome Formation and Metabolism on Radioresistance in Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is a prevalent type of oral cancer. While therapeutic innovations have made strides, radioresistance persists as a significant hindrance in OSCC treatment. Despite identifying numerous targets that could potentially suppress the oncogenic attributes of OSCC, the exploration of oncogenic protein kinases for cancer therapy remains limited. Consequently, the functions of many kinase proteins in OSCC continue to be largely undetermined. In this research, we aim to disclose protein kinases that target OSCC and elaborate their roles and molecular mechanisms. Through the examination of the kinome library of radiotherapy-resistant/sensitive OSCC cell lines (HN12 and SAS), we identified a key gene, the tyrosine phosphorylation-regulated kinase 3 (DYRK3), a member of the DYRK family. We developed an in vitro cell model, composed of radiation-resistant OSCC, to scrutinize the clinical implications and contributions of DYRK3 and phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthase (PAICS) signaling in OSCC. This investigation involves bioinformatics and human tissue arrays. We seek to comprehend the role of DYRK3 and PAICS signaling in the development of OSCC and its resistance to radiotherapy. Various in vitro assays are utilized to reveal the essential molecular mechanism behind radiotherapy resistance in connection with the DYRK3 and PAICS interaction. In our study, we quantified the concentrations of DYRK3 and PAICS proteins and tracked the expression levels of key pluripotency markers, particularly PPAT. Furthermore, we extended our investigation to include an analysis of Glut-1, a gene recognized for its linkage to radioresistance in oral squamous cell carcinoma (OSCC). Furthermore, we conducted an in vivo study to affirm the impact of DYRK3 and PAICS on tumor growth and radiotherapy resistance, focusing particularly on the role of DYRK3 in the radiotherapy resistance pathway. This focus leads us to identify new therapeutic agents that can combat radiotherapy resistance by inhibiting DYRK3 (GSK-626616). Our in vitro models showed that inhibiting PAICS disrupts purinosome formation and influences the survival rate of radiation-resistant OSCC cell lines. These outcomes underscore the pivotal role of the DYRK3/PAICS axis in directing OSCC radiotherapy resistance pathways and, as a result, influencing OSCC progression or therapy resistance. Our findings also reveal a significant correlation between DYRK3 expression and the PAICS enzyme in OSCC radiotherapy resistance.

Combined Targeted and Untargeted Profiling of HeLa Cells Deficient in Purine De Novo Synthesis.

Three genetically determined enzyme defects of purine de novo synthesis (PDNS) have been identified so far in humans: adenylosuccinate lyase (ADSL) deficiency, 5-amino-4-imidazole carboxamide-ribosiduria (AICA-ribosiduria), and deficiency in bifunctional enzyme phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthase (PAICS). Clinical signs of these defects are mainly neurological, such as seizures, psychomotor retardation, epilepsy, autistic features, etc. This work aims to describe the metabolic changes of CRISPR-Cas9 genome-edited HeLa cells deficient in the individual steps of PDNS to better understand known and potential defects of the pathway in humans. High-performance liquid chromatography coupled with mass spectrometry was used for both targeted and untargeted metabolomic analyses. The statistically significant features from the untargeted study were identified by fragmentation analysis. Data from the targeted analysis were processed in Cytoscape software to visualize the most affected metabolic pathways. Statistical significance of PDNS intermediates preceding deficient enzymes was the highest (p-values 10 × 10−7–10 × 10−15) in comparison with the metabolites from other pathways (p-values of up to 10 × 10−7). Disturbed PDNS resulted in an altered pool of adenine and guanine nucleotides. However, the adenylate energy charge was not different from controls. Different profiles of acylcarnitines observed among deficient cell lines might be associated with a specific enzyme deficiency rather than global changes related to the PDNS pathway. Changes detected in one-carbon metabolism might reduce the methylation activity of the deficient cells, thus affecting the modification state of DNA, RNA, and proteins.

Rational Proteomic Analysis of a New Domesticated Klebsiella pneumoniae x546 Producing 1,3-Propanediol.

In order to improve the capability of Klebsiella pneumoniae to produce an important chemical raw material, 1,3-propanediol (1,3-PDO), a new type of K. pneumoniae x546 was obtained by glycerol acclimation and subsequently was used to produce 1,3-PDO. Under the control of pH value using Na+ pH neutralizer, the 1,3-PDO yield of K. pneumoniae x546 in a 7.5-L fermenter was 69.35 g/L, which was 1.5-fold higher than the original strain (45.91 g/L). After the addition of betaine, the yield of 1,3-PDO reached up to 74.44 g/L at 24 h, which was 40% shorter than the original fermentation time of 40 h. To study the potential mechanism of the production improvement of 1,3-PDO, the Tandem Mass Tags (TMT) technology was applied to investigate the production of 1,3-PDO in K. pneumoniae. Compared with the control group, 170 up-regulated proteins and 291 down-regulated proteins were identified. Through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis, it was found that some proteins [such as homoserine kinase (ThrB), phosphoribosylglycinamide formyltransferase (PurT), phosphoribosylaminoimidazolesuccinocarboxamide synthase (PurC), etc.] were involved in the fermentation process, whereas some other proteins (such as ProX, ProW, ProV, etc.) played a significant role after the addition of betaine. Moreover, combined with the metabolic network of K. pneumoniae during 1,3-PDO, the proteins in the biosynthesis of 1,3-PDO [such as DhaD, DhaK, lactate dehydrogenase (LDH), BudC, etc.] were analyzed. The process of 1,3-PDO production in K. pneumoniae was explained from the perspective of proteome for the first time, which provided a theoretical basis for genetic engineering modification to improve the yield of 1,3-PDO. Because of the use of Na+ pH neutralizer in the fermentation, the subsequent environmental pollution treatment cost was greatly reduced, showing high potential for industry application in the future.

Proteome and transcriptome analysis revealed florfenicol via affected drug metabolism and lipid metabolism induce liver injury of broilers

In order to explore the mechanism of liver injury induced by florfenicol (FFC) in broilers. Sixty broilers were randomly divided into 2 groups: control group: normal drinking water and feed were given every d; FFC group: tap water containing FFC (0.15g/L) was given every d and feed was taken freely; each group was given 5 dd of continuous medication and feed was taken freely. The results showed that compared with the control group, FFC could significantly inhibit the weight gain of broilers (P < 0.05), and significantly inhibit the expression of CYP1A1 and CYP2H1 in liver tissue (P < 0.05). It was found that the expression of genes related to the effect of cytochrome P450 on the metabolism of exogenous substances, the peroxisome proliferators-activated receptors signal pathway, peroxisome pathway and glutathione metabolic pathway in the liver of broilers. The results of qPCR of UDP glucuronosyltransferase family 2A1 (UGT2A1), glutathione S-transferase-like 2 (GSTAL2), hematopoietic prostaglandin D synthase (HPGDS), glutathione S-transferase theta 1(GSTT1), isocitrate dehydrogenase (NADP(+)) 1 (IDH1), acyl-CoA oxidase 2 (ACOX2), fatty acid binding protein 1 (FABP1), adenylosuccinate lyase (ADSL), and phosphoribosyl aminoim idazolesuccino carboxamide synthase (PAICS) genes which were randomly selected from the most significant genes were consistent with those of RNA-seq. The results showed that FFC can affect the drug metabolism and lipid synthesis in the liver of broiler, thus impairing the normal function of liver and the growth and development of broiler.

Paics Inhibition Is a Potential Therapeutic Strategy for MYC-Positive DLBCL

Diffuse large B-cell lymphoma (DLBCL) is among the most common hematological malignancies with varying prognosis. As many as forty percent of patients eventually experience relapsed/refractory disease after combinatorial chemo-immunotherapies, R-CHOP, and prognosis after relapse is dismal. MYC is among the most established prognostic factors and associated with clinically-distinct subsets of DLBCL with poor prognosis: double-expressor lymphoma (DEL) and double-hit lymphoma (DHL). MYC is co-expressed with BCL2 in DEL, which consists of 60% of activated B-cell type DLBCL (ABC-DLBCL) cases, while DHL, defined by coexistence of MYC and BCL2/BCL6 rearrangements, were reportedly observed in 15% of germinal center B-cell like DLBCL (GCB-DLBCL). Considering that MYC-positive DLBCLs exhibit dismal outcomes, pharmacological inhibition of MYC activity is highly demanded; however, direct targeting of MYC has been proven challenging. Here we show that PAICS (phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthase), which catalyzes a critical step in de novo purine synthesis, functions downstream of MYC in DLBCL cells. We further show MRT252040, a newly-developed PAICS inhibitor, effectively suppresses proliferation of MYC-driven DLBCL cells in vitro and in vivo. Through the nCounter-based transcriptome profiling of formalin-fixed paraffin-embedded (FFPE) tissues from 170 untreated DLBCL patients, we found that MYC and PAICS were co-expressed and their mRNA levels were among the most predictive for poor prognosis after standard R-CHOP therapy. Their expression levels were particularly high in a subset of ABC-DLBCL and extranodal DLBCL, namely in DEL and DHL cases. Importantly, these findings were validated using three independent cohorts (Schmitz et al. NEJM, 2018). MYC and PAICS expression levels were high in most DLBCL lines and low in normal B cells in the lymph nodes, while they were variable in primary DLBCL tissues, revealed by nCounter and immunofluorescence. This trend was more evident in PAICS due presumably to active de novo purine biosynthesis in highly-proliferative cell lines and a subset of DLBCLs, including MYC-positive DLBCLs. These findings were also validated using the DepMap, a publicly-available genome-wide CRISPR/Cas9 dropout screen datasets. PAICS was among the top-ranked essential genes for the survival of DLBCL cell lines. Since co-expression of MYC and PAICS in a subset of DLBCL were indicative of a functional relationship between the two factors, we explored publicly-available ChIP-seq datasets to see if MYC directly regulates PAICS expression. As expected, MYC ChIP-seq signals were highly enriched near the PAICS promoter in a series of cancer cell lines. Furthermore, shRNA-mediated MYC knockdown led to reduced levels of PAICS mRNA in MYC-positive DLBCL cells and significantly slowed their growth. Collectively, these data suggest that PAICS is a direct transcriptional target of MYC, playing a key role in proliferation of MYC-positive DLBCL cells. To assess the feasibility of PAICS-inhibition as a therapeutic option for MYC-positive DLBCLs, we tested MRT252040 for its anti-lymphoma activity in vitro and in vivo. To do so, we first assessed cell cycle status and Annexin positivity upon MRT252040 treatment using a series of DLBCL cell lines. As expected, MRT252040-mediated PAICS inhibition induced cell cycle arrest and apoptosis. Furthermore, MRT252040 treatment significantly delayed proliferation of DLBCL cell lines, namely those harboring MYC rearrangements. Finally, to assess anti-lymphoma activity of MRT252040 in vivo, we tested MRT252040 efficacy using patient-derived xenograft DLBCL. After xenotransplantation, proportions of lymphoma cells per total mononuclear cells in peripheral blood were examined over time by FACS, and MRT252040 (or vehicle) treatment was initiated once lymphoma cells constituted &amp;gt;0.1%. MRT252040-treated mice survived significantly longer than vehicle-treated mice, indicative of therapeutic efficacy of MRT252040 monotherapy against DLBCL in vivo. Our data suggest that MYC regulates the de novo purine synthesis pathway via directly transactivating PAICS expression. We propose that MRT252040, a newly-developed PAICS inhibitor, warrants attention as a novel therapeutic approach for MYC-positive DLBCLs, which otherwise exhibit poor clinical outcomes. Disclosures Ohshima: SRL, Inc.: Consultancy; Kyowa Kirin Co., Ltd.: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Celgene Corp.: Honoraria, Research Funding; NEC Corp.: Research Funding. Akashi:Sumitomo Dainippon, Kyowa Kirin: Consultancy; Celgene, Kyowa Kirin, Astellas, Shionogi, Asahi Kasei, Chugai, Bristol-Myers Squibb: Research Funding.

Exploration of the diagnostic value and molecular mechanism of miR‑1 in prostate cancer: A study based on meta‑analyses and bioinformatics.

Prostate cancer (PCa) remains a principal issue to be addressed in male cancer-associated mortality. Therefore, the present study aimed to examine the clinical value and associated molecular mechanism of microRNA (miR)-1 in PCa. A meta-analysis was conducted to evaluate the diagnosis of miR-1 in PCa via Gene Expression Omnibus and ArrayExpress datasets, The Cancer Genome Atlas miR-1 expression data and published literature. It was identified that expression of miR-1 was significantly downregulated in PCa. Decreased miR-1 expression possessed moderate diagnostic value, with area under the curve, sensitivity, specificity and odds ratio values at 0.73, 0.77, 0.57 and 4.60, respectively. Using bioinformatics methods, it was revealed that a number of pathways, including the ‘androgen receptor signaling pathway’, ‘androgen receptor activity’, ‘transcription factor binding’ and ‘protein processing in the endoplasmic reticulum’, were important in PCa. A total of seven hub genes, including phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccin ocarboxamide synthase (PAICS), cadherin 1 (CDH1), SRC proto-oncogene, non-receptor tyrosine kinase, twist family bHLH transcription factor 1 (TWIST1), ZW10 interacting kinetochore protein (ZWINT), PCNA clamp associated factor (KIAA0101) and androgen receptor, among which, five (PAICS, CDH1, TWIST1, ZWINT and KIAA0101) were significantly upregulated and negatively correlated with miR-1, were identified as key miR-1 target genes in PCa. Additionally, it was investigated whether miR-1 and its hub genes were associated with clinical features, including age, tumor status, residual tumor, lymph node metastasis, pathological T stage and prostate specific antigen level. Collectively the results suggest that miR-1 may be involved in the progression of PCa, and consequently be a promising diagnostic marker. The ‘androgen receptor signaling pathway’, ‘androgen receptor activity’, ‘transcription factor binding’ and ‘protein processing in the endoplasmic reticulum’ may be crucial interactive pathways in PCa. Furthermore, PAICS, CDH1, TWIST1, ZWINT and KIAA0101 may serve as crucial miR-1 target genes in PCa.

A marine phytoplankton (Prymnesium parvum) up-regulates ABC transporters and several other proteins to acclimatize with Fe-limitation

Iron (Fe) is one of the vital limiting factors for phytoplankton in vast regions of the contemporary oceans, notably the high nutrient low chlorophyll regions. Therefore, it is apparent to be acquainted with the Fe uptake strategy of marine phytoplankton under Fe-limited condition. In the present study, marine phytoplankton Prymnesium parvum was grown under Fe-deplete (0.0025μM) and Fe-rich (0.05μM) conditions, and proteomic responses of the organism to Fe conditions were compared. In sodium dodecyl sulfate (SDS) gel electrophoresis, 7 proteins (16, 18, 32, 34, 75, 82, and 116kDa) were highly expressed under Fe-deplete condition, while one protein (23kDa) was highly expressed under Fe-rich condition. These proteins were subjected to 2-dimensional gel electrophoresis (2-D DIGE) to differentiate individual proteins, and were identified by matrix-assisted laser desorption–ionization-time of flight-mass spectrometer (MALDI-TOF-MS) analysis. The results showed that under Fe-deplete condition P. parvum increases the biosynthesis of ATP binding cassette (ABC) transporters, flagellar associated protein (FAP), and Phosphoribosylaminoimidazole-succinocarboxamide synthase. These proteins are assumed to be involved in a number of cellular biochemical processes that facilitate Fe acquisition in phytoplankton. Under Fe-deplete condition, P. parvum increases the synthesis of ribulose biphosphate carboxylase (RuBisCo), malate dehydrogenase, and two Fe-independent oxidative stress response proteins, manganese superoxide dismutase (MnSOD) and Serine threonine kinase (STK). Thus, marine phytoplankton may change their Fe acquisition strategy by altering the biosynthesis of several proteins in order to cope with Fe-limitation.

Genetic and Proteomic Analyses of a Xanthomonas campestris pv. campestris purC Mutant Deficient in Purine Biosynthesis and Virulence

Bacterial proliferation in hosts requires activation of a number of housekeeping pathways, including purine de novo biosynthesis. Although inactivation of purine biosynthesis genes can attenuate virulence, it is unclear which biochemical or virulence factors are associated with the purine biosynthesis pathway in vivo. We report that inactivation of purC, a gene encoding phosphoribosylaminoimidazole-succinocarboxamide synthase, caused complete loss of virulence in Xanthomonas campestris pv. campestris, the causal agent of black rot disease of cruciferous plants. The purC mutant was a purine auxotroph; it could not grow on minimal medium, whereas addition of purine derivatives, such as hypoxanthine or adenine plus guanine, restored growth of the mutant. The purC mutation also significantly enhanced the production of an unknown purine synthesis associated pigment and extracellular polysaccharides by the bacterium. In addition, comparative proteomic analyses of bacteria grown on rich and minimal media revealed that the purC mutation affected the expression levels of diverse proteins involved in purine and pyrimidine synthesis, carbon and energy metabolisms, iron uptake, proteolysis, protein secretion, and signal transduction. These results provided clues to understanding the contributions of purine synthesis to bacterial virulence and interactions with host immune systems.

The transcriptome of the novel dinoflagellate Oxyrrhis marina (Alveolata: Dinophyceae): response to salinity examined by 454 sequencing

BackgroundThe heterotrophic dinoflagellate Oxyrrhis marina is increasingly studied in experimental, ecological and evolutionary contexts. Its basal phylogenetic position within the dinoflagellates make O. marina useful for understanding the origin of numerous unusual features of the dinoflagellate lineage; its broad distribution has lent O. marina to the study of protist biogeography; and nutritive flexibility and eurytopy have made it a common lab rat for the investigation of physiological responses of marine heterotrophic flagellates. Nevertheless, genome-scale resources for O. marina are scarce. Here we present a 454-based transcriptome survey for this organism. In addition, we assess sequence read abundance, as a proxy for gene expression, in response to salinity, an environmental factor potentially important in determining O. marina spatial distributions.ResultsSequencing generated ~57 Mbp of data which assembled into 7, 398 contigs. Approximately 24% of contigs were nominally identified by BLAST. A further clustering of contigs (at ≥ 90% identity) revealed 164 transcript variant clusters, the largest of which (Phosphoribosylaminoimidazole-succinocarboxamide synthase) was composed of 28 variants displaying predominately synonymous variation. In a genomic context, a sample of 5 different genes were demonstrated to occur as tandem repeats, separated by short (~200-340 bp) inter-genic regions. For HSP90 several intergenic variants were detected suggesting a potentially complex genomic arrangement. In response to salinity, analysis of 454 read abundance highlighted 9 and 20 genes over or under expressed at 50 PSU, respectively. However, 454 read abundance and subsequent qPCR validation did not correlate well - suggesting that measures of gene expression via ad hoc analysis of sequence read abundance require careful interpretation.ConclusionHere we indicate that tandem gene arrangements and the occurrence of multiple transcribed gene variants are common and indicate potentially complex genomic arrangements in O. marina. Comparison of the reported data set with existing O. marina and other dinoflagellates ESTs indicates little sequence overlap likely as a result of the relatively limited extent of genome scale sequence data currently available for the dinoflagellates. This is one of the first 454-based transcriptome surveys of an ancestral dinoflagellate taxon and will undoubtedly prove useful for future comparative studies aimed at reconstructing the origin of novel features of the dinoflagellates.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of SAICAR synthase fromStreptococcus suisserotype 2

Phosphoribosylaminoimidazole-succinocarboxamide synthase (SAICAR synthase) plays an essential role in the de novo biosynthesis of purine nucleotides. In this study, the SAICAR synthase from Streptococcus suis was cloned and overexpressed in Escherichia coli. The subsequent product was purified and crystallized using the hanging-drop vapour-diffusion method. The crystals diffracted to 2.8 A resolution and belonged to space group P2, with unit-cell parameters a=70.2, b=52.2, c=153.9 A, beta=102.8 degrees.

Identification of new members of the GS ADP-forming family from the de novo purine biosynthesis pathway.

Most living organisms can synthesize isosinate from 5-phosphoribosyl 1-pyrophosphate in the de novo purine biosynthesis pathway, which is basically composed of 10 reaction steps. Phosphoribosylglycinamide synthetase (GARS) catalyzes the second step of the pathway. We found that the enzyme shows weak, but significant, sequence similarity to phosphoribosylglycinamide formyltransferase 2 (GART2) and the ATPase domain of phosphoribosylaminoimidazole carboxylase (AIRCA), which catalyze the third and sixth steps of the pathway, respectively. In addition, the three enzymes were similar in amino acid sequence to biotin carboxylase (BC) and carbamoylphosphate synthetase (CPS), which are the members of the GS ADP-forming family. This family has been identified through a tertiary structure comparison and includes glutathione synthetase, d-alanine:d-alanine ligase, BC, succinyl-CoA synthetase beta-chain, and phosphoribosylaminoimidazole-succinocarboxamide synthase. Molecular phylogenetic analysis based on a multiple alignment of GARS, GART2, AIRCA, BC, and CPS suggests that GART2 is more closely related to AIRCA than to GARS among the three enzymes from the pathway, though the three enzymes are relatively close to each other within the GS ADP-forming family. Moreover, the analysis showed that archaeal GARS had diverged before the speciation between bacteria and eucarya.

The structure of phosphoribosylamino-imidazolesuccinocarboxamide synthase from the yeastSaccharomyces cerevisiaeat 3 Å resolution

The structure of phosphoribosylamino-imidazolesuccinocarboxamide synthase from the yeast<i>Saccharomyces cerevisiae</i>at 3 Å resolution

Crystallization and preliminary X-ray investigation of phosphoribosylaminoimidazolesuccinocarboxamide synthase from the yeast Saccharomyces cerevisiae

Crystals of phosphoribosylaminoimidazolesuccinocarboxamide synthase (EC 6.3.2.6) from the yeast Saccharomyces cerevisiae were grown by the vapor diffusion hanging-drop technique, using ammonium sulfate as the precipitant. The crystals had dimensions up to 1.2 mm. X-ray diffraction experiments indicated a space group of P2 1 2 1 2 1 and unit cell parameters of a = 62.3 A ̊ , b = 63.5 A ̊ and c = 80.9 A ̊ , with one molecule in the asymmetric unit. Native data have been collected to 2.5 Å resolution.