Phosphoribosylpyrophosphate Amidotransferase Research Articles

Electrochemistry detection of estrogenic effect: Regulation of de novo purine synthesis and catabolism by gibberellin and fulvestrant

The estrogenic effect of plant growth regulators has been received little attention, which leads to the lack of relevant toxicity data. In this study, the estrogenic effect induced by gibberellin with ERα-dependent manner was found by E-screen and western blot methods, and the electrochemical signals of MCF-7 cells regulated by gibberellin and fulvestrant were investigated. The results showed that the electrochemical signals of MCF-7 cells were increased by gibberellin, while reduced by fulvestrant significantly, and displayed an extremely sensitive response to the effects of estrogenic effect induced by ERα agonist and antagonist. Western blot results showed that the expressions of phosphoribosyl pyrophosphate amidotransferase and hypoxanthine nucleotide dehydrogenase in de novo purine synthesis and adenine deaminase in catabolism were more effective regulated by gibberellin and fulvestrant, resulting in significant changes of the levels of guanine, hypoxanthine and xanthine in cells, and then electrochemical signals. The results provide a theoretical basis for the establishment of new electrochemical detection method of the estrogenic effect of plant regulators.

Uncommon Arrangement of Self-resistance Allows Biosynthesis of de novo Purine Biosynthesis Inhibitor that Acts as an Immunosuppressor.

(-)-FR901483 (1) isolated from the fungus Cladobotryum sp. No.11231 achieves immunosuppression via nucleic acid biosynthesis inhibition rather than IL-2 production inhibition as accomplished by FK506 and cyclosporin A. Recently, we identified the frz gene cluster for the biosynthesis of 1. It contains frzK, a gene homologous to phosphoribosyl pyrophosphate amidotransferase (PPAT)that catalyzes the initial step of de novo purine biosynthesis. We speculated that frzK encodes a PPAT that escapes inhibition by 1 and functions as a self-resistance enzyme (SRE) for the producing host. Nevertheless, details remained elusive. Here, we report the biochemical and structural analyses of FrzK and its Escherichia coli counterpart, PurF. Recombinantly produced FrzK exhibited PPAT activity, albeit weaker than PurF, but evaded strong inhibition by 1. These results confirmed that the target of 1 is PPAT, and FrzK acts as an SRE by maintaining the de novo purine biosynthetic capability in the presence of 1. To understand how FrzK evades inhibition by 1, we determined the crystal structure of PurF in the complex with 1 and constructed a homology model of FrzK. Sequence and structural analyses of various PPATs identified that many residues unique to FrzK occur near the Flexible Loop that remains disordered when inactive but becomes ordered and covers up the active site upon activation by substrate binding. Kinetic characterizations of mutants of the unique residues revealed that the resistance of FrzK against 1 may be conferred by structurally predisposing the Flexible Loop to the active, closed conformation even in the presence of 1.

Phosphoribosyl Pyrophosphate Amido Transferase: A New Prognostic Biomarker for Hepatocellular Carcinoma.

BackgroundPPAT (phosphoribosyl pyrophosphate amido transferase) catalyzes the first committed step of de novo purine biosynthesis and is a key regulatory point in the biosynthesis of nascent purine nucleotides. However, the clinical significance and biologic role of PPAT in hepatocellular carcinoma (HCC) remain unknown.MethodsWe compared the expression of PPAT in carcinomatous and precancerous hepatocellular carcinoma tissues by immunohistochemistry in 90 cases of HCC. Correlation analysis was also made on clinical data, survival, classification, and staging.ResultsThe expression of PPAT in HCC tumor tissues is significantly higher than that in adjacent normal tissues. The results of the Kaplan–Meier analysis showed that HCC patients with high PPAT expression survived shorter than those with low PPAT expression. Moreover, the expression of PPAT was significantly associated with the tumor grade (P=0.014), PD-L1 (P<0.001), and CTLA4 (P=0.003). The later grade of the tumor, the higher the expression of PPAT. In the PD-L1 high expression group, PPAT is also highly expressed.ConclusionOur study demonstrated that PPAT expression might be included in the process of carcinogenesis and prognosis. Hence, PPAT could be served as a new prognostic biomarker for patients of HCC.

Cannabis Sativa: A Remedy for Convulsion by Inhibition of GABAA Receptor and Significantly Delayed Onset of Seizure Latency and Death - An Experimental Validation and Molecular Docking

This study aimed to investigate the anticonvulsant potentials of Cannabis sativa and its possible mechanism using the in vivo and in-silico models. A total of 20 male albino mice was used for this study and were divided randomly into five groups. Group 1 as negative control receiving 0.2ml 0f 10% ethanol only, and groups 2 and 3, received 200mg/kg/ body weight low dose of C. Sativa extract and 400mg/kg bw low dose of C. sativa extract, while groups 4 received 50 mg/kg body weight of phenobarbitone and group 5 received 50 mg/kg body weight of phenobarbitone+ 200 mg/kg body weight of the extract. Administration of C. Sativa extract was done via orogastric feeding; GABA-ergicseizure, was induced by administration of 80 mg/kg/bodyweight of Pentylenetetrazole (PTZ) via the intra-peritoneal route to all the groups. This was done about thirty minutes after the administration of treatment agents. Each animal was observed individually for one hour to determine the onset of muscular jerks and tonic hind limb extensions and death. Animal study results revealed that C. sativa may likely possess’ anticonvulsant potentials as It is significantly seizure latency and death in PTZ treated mice. For the in silico studies, about 49 compounds of C. sativa were obtained from the PubChem library and were docked on various targets site of proteins; Glutaminase, 4-Aminobtyrate Aminotransferase, Perioxisome proliferator Activated receptor (PPAR), Succinic semialdehyde dehydrogenase, Gama amino butyric acid –A receptor (GABA-A), Histone deacetylases (HDAC), Phosphoribosyl pyrophosphate amido transferase and human GAFT-1. Furthermore, the in-silico study revealed that compounds like Cannabisin A and D showed anticonvulsant potential activation of GABA.

Species-Specific Deamidation of RIG-I Reveals Collaborative Action between Viral and Cellular Deamidases in HSV-1 Lytic Replication.

Retinoic acid-inducible gene I (RIG-I) is a sensor that recognizes cytosolic double-stranded RNA derived from microbes to induce host immune response. Viruses, such as herpesviruses, deploy diverse mechanisms to derail RIG-I-dependent innate immune defense. In this study, we discovered that mouse RIG-I is intrinsically resistant to deamidation and evasion by herpes simplex virus 1 (HSV-1). Comparative studies involving human and mouse RIG-I indicate that N495 of human RIG-I dictates species-specific deamidation by HSV-1 UL37. Remarkably, deamidation of the other site, N549, hinges on that of N495, and it is catalyzed by cellular phosphoribosylpyrophosphate amidotransferase (PPAT). Specifically, deamidation of N495 enables RIG-I to interact with PPAT, leading to subsequent deamidation of N549. Collaboration between UL37 and PPAT is required for HSV-1 to evade RIG-I-mediated antiviral immune response. This work identifies an immune regulatory role of PPAT in innate host defense and establishes a sequential deamidation event catalyzed by distinct deamidases in immune evasion.IMPORTANCE Herpesviruses are ubiquitous pathogens in human and establish lifelong persistence despite host immunity. The ability to evade host immune response is pivotal for viral persistence and pathogenesis. In this study, we investigated the evasion, mediated by deamidation, of species-specific RIG-I by herpes simplex virus 1 (HSV-1). Our findings uncovered a collaborative and sequential action between viral deamidase UL37 and a cellular glutamine amidotransferase, phosphoribosylpyrophosphate amidotransferase (PPAT), to inactivate RIG-I and mute antiviral gene expression. PPAT catalyzes the rate-limiting step of the de novo purine synthesis pathway. This work describes a new function of cellular metabolic enzymes in host defense and viral immune evasion.

A metabolism-related gene signature for predicting the prognosis and therapeutic responses in patients with hepatocellular carcinoma.

BackgroundHepatocellular carcinoma (HCC) often has an insidious onset and rapid progression. Often, when the disease is first diagnosed, the opportune time for surgical intervention has already lapsed. In addition, the effects of systemic treatment is relatively unsatisfactory. Metabolic reprogramming is one of the hallmarks of cancer. This study aimed to identify a set of genes related to metabolism to construct a predictive model for the prognosis of HCC.MethodsThe transcriptomic and clinical data of 352 HCC patients were obtained from The Cancer Genome Atlas (TCGA) Liver Hepatocellular Carcinoma (LIHC) dataset and divided into a training cohort (n=212) and a testing cohort (n=140) at a ratio of 6:4. Univariate Cox regression analysis and the LASSO Cox regression model were used to identify 5 genes to establish a risk score for predicting the prognosis of HCC patients. Subsequently, the molecular characteristics of the model were assessed and the ability of the model to predict the tumor immune microenvironment and patient response to immunotherapy and chemotherapy was also examined.ResultsThe risk score model was constructed based on the five genes, methyltransferase-like protein 6 (METTL6), RNA polymerase III subunit G (POLR3G), phosphoribosyl pyrophosphate amidotransferase (PPAT), SET Domain Bifurcated 2 (SETDB2), and suppressor of variegation 3-9 homolog 2 (SUV39H2). The Kaplan-Meier survival analysis and time-dependent receiver operating characteristic (ROC) curves demonstrated that high-risk patients had a poorer overall survival (OS) compared to low-risk patients. he nomogram score had a better predictive ability compared to the common factors. Our results finally showed that high-risk cases were associated with cell proliferation and cell cycle related gene sets, high tumor protein P53 (TP53) mutation rate, suppressive immunity and increased sensitivity to cisplatin, gemcitabine and docetaxel. Meanwhile, low-risk cases were associated with cell cycle and immune response related pathways, low TP53 mutation rate, active immunity and more benefit from immunotherapy.ConclusionsThis study provided novel insights into the role of metabolism-related genes in HCC, and demonstrated that our model could be a promising prognostic biomarker for distinguishing the molecular and immune characteristics and inferring the potential response to chemotherapy and immunotherapy.

Conformational Changes of Glutamine 5'-Phosphoribosylpyrophosphate Amidotransferase for Two Substrates Analogue Binding: Insight from Conventional Molecular Dynamics and Accelerated Molecular Dynamics Simulations.

Glutamine 5′-phosphoribosylpyrophosphate amidotransferase (GPATase) catalyzes the synthesis of phosphoribosylamine, pyrophosphate, and glutamate from phosphoribosylpyrophosphate, as well as glutamine at two sites (i.e., glutaminase and phosphoribosylpyrophosphate sites), through a 20 Å NH3 channel. In this study, conventional molecular dynamics (cMD) simulations and enhanced sampling accelerated molecular dynamics (aMD) simulations were integrated to characterize the mechanism for coordination catalysis at two separate active sites in the enzyme. Results of cMD simulations illustrated the mechanism by which two substrate analogues, namely, DON and cPRPP, affect the structural stability of GPATase from the perspective of dynamic behavior. aMD simulations obtained several key findings. First, a comparison of protein conformational changes in the complexes of GPATase–DON and GPATase–DON–cPRPP showed that binding cPRPP to the PRTase flexible loop (K326 to L350) substantially effected the formation of the R73-DON salt bridge. Moreover, only the PRTase flexible loop in the GPATase–DON–cPRPP complex could remain closed and had sufficient space for cPRPP binding, indicating that binding of DON to the glutamine loop had an impact on the PRTase flexible loop. Finally, both DON and cPRPP tightly bonded to the two domains, thereby inducing the glutamine loop and the PRTase flexible loop to move close to each other. This movement facilitated the transfer of NH3 via the NH3 channel. These theoretical results are useful to the ongoing research on efficient inhibitors related to GPATase.

Genome-wide CRISPR screening reveals nucleotide synthesis negatively regulates autophagy

Macroautophagy (hereafter, autophagy) is a process that directs the degradation of cytoplasmic material in lysosomes. In addition to its homeostatic roles, autophagy undergoes dynamic positive and negative regulation in response to multiple forms of cellular stress, thus enabling the survival of cells. However, the precise mechanisms of autophagy regulation are not fully understood. To identify potential negative regulators of autophagy, we performed a genome-wide CRISPR screen using the quantitative autophagic flux reporter GFP-LC3-RFP. We identified phosphoribosylformylglycinamidine synthase, a component of the de novo purine synthesis pathway, as one such negative regulator of autophagy. Autophagy was activated in cells lacking phosphoribosylformylglycinamidine synthase or phosphoribosyl pyrophosphate amidotransferase, another de novo purine synthesis enzyme, or treated with methotrexate when exogenous levels of purines were insufficient. Purine starvation-induced autophagy activation was concomitant with mammalian target of rapamycin complex 1 (mTORC1) suppression and was profoundly suppressed in cells deficient for tuberous sclerosis complex 2, which negatively regulates mTORC1 through inhibition of Ras homolog enriched in brain, suggesting that purines regulate autophagy through the tuberous sclerosis complex-Ras homolog enriched in brain-mTORC1 signaling axis. Moreover, depletion of the pyrimidine synthesis enzymes carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase and dihydroorotate dehydrogenase activated autophagy as well, although mTORC1 activity was not altered by pyrimidine shortage. These results suggest a different mechanism of autophagy induction between purine and pyrimidine starvation. These findings provide novel insights into the regulation of autophagy by nucleotides and possibly the role of autophagy in nucleotide metabolism, leading to further developing anticancer strategies involving nucleotide synthesis and autophagy.

Recent Advances in Construction of the Efficient Producers of Riboflavin and Flavin Nucleotides (FMN, FAD) in the Yeast Candida famata.

The approaches used by the authors to design the Candida famata strains capable to overproduce riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) are described. The metabolic engineering approaches include overexpression of SEF1 gene encoding positive regulator of riboflavin biosynthesis, IMH3 (coding for IMP dehydrogenase) orthologs from another species of flavinogenic yeast Debaryomyces hansenii, and the homologous genes RIB1 and RIB7 encoding GTP cyclohydrolase II and riboflavin synthase, the first and the last enzymes of riboflavin biosynthesis pathway, respectively. Overexpression of the abovementioned genes in the genetically stable riboflavin overproducer AF-4 obtained by classical selection resulted in fourfold increase of riboflavin production in shake flask experiments.Overexpression of engineered enzymes phosphoribosyl pyrophosphate synthetase and phosphoribosyl pyrophosphate amidotransferase catalyzing the initial steps of purine nucleotide biosynthesis enhances riboflavin synthesis in the flavinogenic yeast C. famata even more.Recombinant strains of C. famata containing FMN1 gene from D. hansenii encoding riboflavin kinase under control of the strong constitutive TEF1 promoter were constructed. Overexpression of the FMN1 gene in the riboflavin-producing mutant led to the 30-fold increase of the riboflavin kinase activity and 400-fold increase of FMN production in the resulting recombinant strains which reached maximally 318.2mg/L.FAD overproducing strains of C. famata were also constructed. This was achieved by overexpression of FAD1 gene from D. hansenii in C. famata FMN overproducing strain. The 7- to 15-fold increase in FAD synthetase activity as compared to the wild-type strain and FAD accumulation into cultural medium were observed. The maximal FAD titer 451.5mg/L was achieved.

Identification of Clock Genes Related to Hypertension in Kidney From Spontaneously Hypertensive Rats.

BackgroundThere is a diurnal variation in the blood pressure fluctuation of hypertension, and blood pressure fluctuation abnormality is considered to be an independent risk factor for organ damage including cardiovascular complications. In the current study, we tried to identify molecules responsible for blood pressure circadian rhythm formation under the control of the kidney biological clock in hypertension.MethodsDNA microarray analysis was performed in kidneys from 5-week-old spontaneously hypertensive rats (SHRs)/Izm, stroke-prone SHR rats (SHRSP)/Izm, and Wistar Kyoto (WKY)/Izm rats. To detect variation, mouse tubular epithelial cells (TCMK-1) were stimulated with dexamethasone. We performed immunostaining and western blot analysis in the renal medulla of kidney from 5-week-old WKY rats and SHRs.ResultsWe extracted 1,032 genes with E-box, a binding sequence for BMAL1 and CLOCK using a Gene Set Enrichment Analysis. In a microarray analysis, we identified 12 genes increased as more than 2-fold in the kidneys of SHRs and SHRSP in comparison to WKY rats. In a periodic regression analysis, phosphoribosyl pyrophosphate amidotransferase (Ppat) and fragile X mental retardation, autosomal homolog 1 (Fxr1) showed circadian rhythm. Immunocytochemistry revealed PPAT-positivity in nuclei and cytoplasm in the tubules, and FXR1-positivity in the cytoplasm of TCMK-1. In 5-week-old WKY rat and SHR kidneys, PPAT was localized in the nucleus and cytoplasm of the proximal and distal tubules, and FXR1 was localized to the cytoplasm of the proximal and distal tubules.ConclusionsPPAT and FXR1 are pivotal molecules in the control of blood pressure circadian rhythm by the kidney in hypertension.

Phosphoribosyl Pyrophosphate Amidotransferase Promotes the Progression of Thyroid Cancer via Regulating Pyruvate Kinase M2.

BackgroundPyruvate kinase is an enzyme that catalyzes the conversion of phosphoenolpyruvate and ADP to pyruvate and ATP in glycolysis and plays a role in regulating cell metabolism. It is reported that the activity of pyruvate kinase is increased in cancers. Phosphoribosyl amidotransferase (PPAT) is reported to be a crucial regulator for pyruvate kinase activity in lung cancer. However, its role in thyroid cancer remains largely unknown.Materials and MethodsImmunohistochemical analysis and qRT-PCR were used to detect the expression of PPAT in thyroid cancer samples. Both gain-of-function and loss-of-function models were constructed in thyroid cancer cell lines and the biological functions of PPAT on cellular phenotypes were studied using CCK-8 assay and transwell assay in vitro, respectively. Then, Western blot was used to evaluate the change of PKM2 and downstream signal pathways after PPAT was overexpressed or knocked down.ResultsImmunohistochemical analysis showed increased expression of PPAT in thyroid cancer tissues, and it was associated with unfavorable pathological characteristics. Knockdown and overexpression assays suggested that altering PPAT expression modulated cell proliferation, migration, and invasion. In terms of mechanism, PPAT could positively regulate the expression of PKM2 and activate ERK and STAT3 signaling pathways.ConclusionPPAT plays crucial roles in regulating proliferation, migration, and invasion of thyroid cancer cells via activating PKM2, ERK, and STAT3.

Nwd1 Regulates Neuronal Differentiation and Migration through Purinosome Formation in the Developing Cerebral Cortex.

SummaryEngagement of neural stem/progenitor cells (NSPCs) into proper neuronal differentiation requires the spatiotemporally regulated generation of metabolites. Purines are essential building blocks for many signaling molecules. Enzymes that catalyze de novo purine synthesis are assembled as a huge multienzyme complex called “purinosome.” However, there is no evidence of the formation or physiological function of the purinosome in the brain. Here, we showed that a signal transduction ATPases with numerous domains (STAND) protein, NACHT and WD repeat domain-containing 1 (Nwd1), interacted with Paics, a purine-synthesizing enzyme, to regulate purinosome assembly in NSPCs. Altered Nwd1 expression affected purinosome formation and induced the mitotic exit and premature differentiation of NSPCs, repressing neuronal migration and periventricular heterotopia. Overexpression/knockdown of Paics or Fgams, other purinosome enzymes, in the developing brain resulted in a phenocopy of Nwd1 defects. These findings indicate that strict regulation of purinosome assembly/disassembly is crucial for maintaining NSPCs and corticogenesis.

A shift in glutamine nitrogen metabolism contributes to the malignant progression of cancer

Glucose metabolism is remodeled in cancer, but the global pattern of cancer-specific metabolic changes remains unclear. Here we show, using the comprehensive measurement of metabolic enzymes by large-scale targeted proteomics, that the metabolism both carbon and nitrogen is altered during the malignant progression of cancer. The fate of glutamine nitrogen is shifted from the anaplerotic pathway into the TCA cycle to nucleotide biosynthesis, with this shift being controlled by glutaminase (GLS1) and phosphoribosyl pyrophosphate amidotransferase (PPAT). Interventions to reduce the PPAT/GLS1 ratio suppresses tumor growth of many types of cancer. A meta-analysis reveals that PPAT shows the strongest correlation with malignancy among all metabolic enzymes, in particular in neuroendocrine cancer including small cell lung cancer (SCLC). PPAT depletion suppresses the growth of SCLC lines. A shift in glutamine fate may thus be required for malignant progression of cancer, with modulation of nitrogen metabolism being a potential approach to SCLC treatment.

Modulation of the Purine Pathway for Riboflavin Production in Flavinogenic Recombinant Strain of the Yeast Candida famata.

Riboflavin (vitamin B2 ) is an indispensable nutrient for humans and animals, since it is the precursor of the essential coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), involved in variety of metabolic reactions. Riboflavin is produced on commercial scale and is used for feed and food fortification purposes, and in medicine. Until recently, the mutant strains of the flavinogenic yeast Candida famata were used in industry for riboflavin production. Guanosine triphosphate is the immediate precursor of riboflavin synthesis. Therefore, the activation of metabolic flux toward purine nucleotide biosynthesis is a promising approach to improve riboflavin production. The phosphoribosyl pyrophosphate synthetase and phosphoribosyl pyrophosphate amidotransferase are the rate limiting enzymes in purine biosynthesis. Corresponding genes PRS3 and ADE4 from yeast Debaryomyces hansenii are modified to avoid feedback inhibition and cooverexpressed on the background of a previously constructed riboflavin overproducing strain of C. famata. Constructed strain accumulates twofold more riboflavin when compared to the parental strain.

Goose astrovirus infection affects uric acid production and excretion in goslings

In 2018, a new goose astrovirus (GAstrV) was reported in China, which causes 2 to 20% deaths in 4- to 16-day-old goslings causing great damages to the livestock industry. Gout is the typical feature of GAstrV infection in goslings. However, the mechanism of gout formation remains unclear. In the present study, 2-day-old goslings were infected intramuscularly with GAstrV for 14 D. One quarter of the infected goslings died, and typical gout pathological changes were found in the dead infected goslings. Pathological changes were observed in the morphology of the kidney and liver, such as degeneration, necrosis, and inflammatory cell infiltration. Accordingly, a high virus load was found in both organs. The serum level of uric acid in the inoculated goslings was higher, whereas no differences were found in levels of creatinine, calcium, and phosphorus. Moreover, the xanthine dehydrogenase (XOD) and adenosine deaminase (ADA) activities and the mRNA levels of xanthine dehydrogenase, adenosine deaminase, phosphoribosyl pyrophosphate amidotransferase, and phosphoribosyl pyrophosphate synthetase 1 in livers increased, wheres the multidrug resistance–associated protein 4 mRNA level and Na-K-ATPase activity in the kidneys decreased. These results showed that GAstrV infection could cause lesions on the liver and kidney and then increase the expression or activity of enzymes related to uric acid production in the liver and decrease renal excretion function, which contribute to hyperuricemia and gout formation.

Crystal Structure of the Chloroplastic Glutamine Phosphoribosylpyrophosphate Amidotransferase GPRAT2 From Arabidopsis thaliana.

Chloroplastic glutamine phosphoribosylpyrophosphate amidotransferase (GPRATase) catalyzes the first committed step of de novo purine biosynthesis in Arabidopsis thaliana, and DAS734 is a direct and specific inhibitor of AtGPRAT, with phytotoxic effects similar to the leaf beaching phenotypes of known AtGPRAT genetic mutants, especially cia1 and atd2. However, the structure of AtGPRAT and the inhibition mode of DAS734 still remain poorly understood. In this study, we solved the structure of AtGPRAT2, which revealed structural differences between AtGPRAT2 and bacterial enzymes. Kinetics assay demonstrated that DAS734 behaves as a competitive inhibitor for the substrate phosphoribosyl pyrophosphate (PRPP) of AtGPRAT2. Docking studies showed that DAS734 forms electrostatic interactions with R264 and hydrophobic interactions with several residues, which was verified by binding assays. Collectively, our study provides important insights into the inhibition mechanism of DAS734 to AtGPRAT2 and sheds light on future studies into further development of more potent herbicides targeting Arabidopsis GPRATases.

Phosphorus-mediated alleviation of aluminum toxicity revealed by the iTRAQ technique in Citrus grandis roots.

Citrus grandis seedlings were irrigated with nutrient solutions with four Al-P combinations [two Al levels (0 mM and 1.2 mM AlCl3·6H2O) × two P levels (0 μM and 200 μM KH2PO4)] for 18 weeks. Al dramatically inhibited the growth of C. grandis seedlings, as revealed by a decreased dry weight of roots and shoots. Elevating P level could ameliorate the Al-induced growth inhibition and organic acid (malate and citrate) secretion in C. grandis. Using a comparative proteomic approach revealed by the isobaric tags for relative and absolute quantification (iTRAQ) technique, 318 differentially abundant proteins (DAPs) were successfully identified and quantified in this study. The possible mechanisms underlying P-induced alleviation of Al toxicity in C. grandis were proposed. Furthermore, some DAPs, such as GLN phosphoribosyl pyrophosphate amidotransferase 2, ATP-dependent caseinolytic (Clp) protease/crotonase family protein, methionine-S-oxide reductase B2, ABC transporter I family member 17 and pyridoxal phosphate phosphatase, were reported for the first time to respond to Al stress in Citrus plants. Our study provides some proteomic details about the alleviative effects of P on Al toxicity in C. grandis, however, the exact function of the DAPs identified herein in response to Al tolerance in plants must be further investigated.

In Silico Structure Modeling and Molecular Docking Analysis of Phosphoribosyl Pyrophosphate Amidotransferase (PPAT) with Antifolate Inhibitors.

Cancer remains one of the most serious disease worldwide. Robust metabolism is the hallmark of cancer. PPAT (phosphoribosyl pyrophosphate amidotransferase) catalyzes the first committed step of de novo purine biosynthesis. Hence PPAT, the key regulatory spot in De novo purine nucleotide biosynthesis, is an attractive and credible drug target for leukemia and other cancer therapeutics. In the present study, detailed computational analysis has been performed for PPAT protein, the key enzyme in de novo purine biosynthesis which is inhibited by many folate derivatives, hence we aimed to investigate and gauge the inhibitory effect of antifolate derivatives; lomexterol (LTX) methotrexate (LTX), and pipretixin (PTX) with human PPAT to effectively capture and inhibit De novo purine biosynthesis pathway. The sequence to structure computational approaches followed by molecular docking experiments was performed to gain insight into the inhibitory mode, binding orientation and binding affinities of selected antifolate derivatives against important structural features of PPAT. Results indicated a strong affinity of antifolate inhibitors for the conserved active site of PPAT molecule encompassing a number of hydrophobic, hydrogen bonding, Vander Waals and electrostatic interactions. Conclusively, the strong physical interaction of selected antifolate inhibitors with human PPAT suggests the selective inhibition of De novo purine biosynthesis pathway by antifolate derivatives towards cancer therapeutics.

Molecular Tunnels in Enzymes and Thermophily: A Case Study on the Relationship to Growth Temperature.

Developments in protein expression, analysis and computational capabilities are decisively contributing to a better understanding of the structure of proteins and their relationship to function. Proteins are known to be adapted to the growth rate of microorganisms and some microorganisms (named (hyper)thermophiles) thrive optimally at high temperatures, even above 100 °C. Nevertheless, some biomolecules show great instability at high temperatures and some of them are universal and required substrates and cofactors in multiple enzymatic reactions for all (both mesophiles and thermophiles) living cells. Only a few possibilities have been pointed out to explain the mechanisms that thermophiles use to successfully thrive under high temperatures. As one of these alternatives, the role of molecular tunnels or channels in enzymes has been suggested but remains to be elucidated. This study presents an analysis of channels in proteins (i.e., substrate tunnels), comparing two different protein types, glutamate dehydrogenase and glutamine phosphoribosylpyrophosphate amidotransferase, which are supposed to present a different strategy on the requirement for substrate tunnels with low and high needs for tunneling, respectively. The search and comparison of molecular tunnels in these proteins from microorganisms thriving optimally from 15 °C to 100 °C suggested that those tunnels in (hyper)thermophiles are required and optimized to specific dimensions at high temperatures for the enzyme glutamine phosphoribosylpyrophosphate amidotransferase. For the enzyme glutamate dehydrogenase, a reduction of empty spaces within the protein could explain the optimization at increasing temperatures. This analysis provides further evidence on molecular channeling as a feasible mechanism in hyperthermophiles with multiple relevant consequences contributing to better understand how they live under those extreme conditions.

Proteomic Analysis of the Function of a Novel Cold-Regulated Multispanning Transmembrane Protein COR413-PM1 in Arabidopsis.

The plasma membrane is the first subcellular organ that senses low temperature, and it includes some spanning transmembrane proteins that play important roles in cold regulation. COR413-PM1 is a novel multispanning transmembrane cold-regulated protein; however, the related functions are not clear in Arabidopsis. We found the tolerance to freezing stress of cor413-pm1 was lower than wild-type (WT). A proteomics method was used to analyze the differentially abundant proteins (DAPs) between cor413-pm1 and WT. A total of 4143 protein groups were identified and 3139 were accurately quantitated. The DAPs associated with COR413-PM1 and freezing treatment were mainly involved in the metabolism of fatty acids, sugars, and purine. Quantitative real-time PCR (qRT-PCR) confirmed the proteomic analysis results of four proteins: fatty acid biosynthesis 1 (FAB1) is involved in fatty acid metabolism and might affect the plasma membrane structure; fructokinase 3 (FRK3) and sucrose phosphate synthase A1 (SPSA1) play roles in sugar metabolism and may influence the ability of osmotic adjustment under freezing stress; and GLN phosphoribosyl pyrophosphate amidotransferase 2 (ASE2) affects freezing tolerance through purine metabolism pathways. In short, our results demonstrate that the multispanning transmembrane protein COR413-PM1 regulates plant tolerance to freezing stress by affecting the metabolism of fatty acids, sugars, and purine in Arabidopsis.