Delineation of Effectors Impact on The Human Brain Derived Phosphoribosylpyrophosphate Synthetase-1 Activity

Phosphoribosylpyrophosphate synthetase-1 [PRPS-1;
EC=2.7.6.1) catalyzes the phosphoribosylation of ribose 5...


Introduction
Phosphoribosylpyrophosphate synthetase-1 [PRPS-1; EC=2.7.6.1) catalyzes the phosphoribosylation of ribose 5-phosphate to 5-phosphoribosyl-1-pyrophosphate, which is necessary for the salvage pathways of purine and pyrimidine, pyridine nucleotide cofactors NAD and NADP, the amino acids histidine and tryptophan biosynthesis [1,2]. It is regulative enzyme, responsible for the synthesis of purine and pyrimidine. Regulation of this particular enzyme might be dependent on the effectors, including metal ions.
Generally, metal ions are vital for functionality (metallo-enzyme activity, protein stabilization etc.) and maintenance of nervous tissue. The overload of copper lead to neurodegeneration in Menkes and Wilson's disease, increased brain aging, dwindle cognitive and epileptic seizures due to altered brain zinc homeostasis.
Presence of dyshomeostasis of essential and nonessential metals is considered vital in sporadic neurodegeneration. Increased levels of iron and copper in tissue are directly related to increased inflammatory markers and oxidative stress in affected brains. The rich metal (zinc, iron, copper) concentration in degenerated protein aggregates and plaques demonstrates the link between metals and neurodegenerative pathologies. Changes in aggregation properties (a-synuclein and amyloid-b) lead to complex protein formation owing to the presence of aluminium, copper and iron [3].
Super activity of PRPS is an inherited X-chromosome-linked disorder [4] and the excessive enzymatic activity is associated with uric acid overproduction, gout and neurodevelopmental abnormalities [5][6][7][8]. Thus, in case of the genetic pathologies the regulation of PRPS-1 is essential. Three classes of PRSPs have been reported which are divided based on their dependence on phosphate ions for activity, their allosteric regulation mechanism and their diphosphoryl donor specificity [9][10][11][12]. Most PRSPs belong to the class I, which require Mg2+ and phosphate for enzymatic activity, but can be inhibited allosterically by ADP and possibly other nucleotides [13][14][15][16][17][18][19][20]. Class II PRSs are found specifically in plants which are not dependent on phosphate for activity and lack an allosteric site for ADP [9][10]. Recently, a novel class III PRS has been identified from Methanocaldococcus jannaschii which is activated by phosphate and uses ATP and dATP as a diphosphoryl donor, but also lacks an allosteric site for ADP [12]. HPRPSs (human PRP synthetases) have three isoforms that share very high sequence identity (95.0% between hPRPS1 and hPRPS2; 94.3% between hPRPS1 and hPRPS3; and 91.2% between hPRPS2 and hPRPS3 respectively) [21][22][23][24].
Enzyme requires phosphate for activation and uses Mg 2 + [14,[25][26][27]. The crystallization of hPRPS1 has recently been reported [27]. Interestingly, in addition to binding at the R5P binding site and the allosteric site defined previously in bsPRS, an extra SO4 2− ion is found to bind at a new allosteric site at the dimer interface.
Structural and biochemical data together reveal new insights into the allosteric regulatory mechanism of hPRPS1 and possibly other eukaryotic PRPSs (except for class II plant PRSs) [28]. In our previ-ous publications we have proposed that activation of PRPS-1 in the brain might trigger the increase in the rate of the purine and pyrimidine biosynthesis and stimulate the regenerative processes after experimental stroke in rats [29]. The treatment of the animals with the phosphates after experimental stroke reflecting conditions, we have noticed the elevation of the Ki-67 positive neurons in the brain as well as the decrease in the BBB (blood brain barrier) damage process and Evans Blue extravasation [29].
In our current study we were aiming to define non-organic and organic compounds, which are influencing on the activity of the PRPS-1. In addition, we developed the methods for Orotate

Material and Methods
The all reagents were purchased from Sigma/Aldrich or/and Santa Cruz Biotechnology, Inc. We have created the kit for the determination of the PRPS-1 activity. The entire idea of the leaded reactions for the final measurement of the enzyme activity is presented on the ( Figure 1).

(OPT)
The brain tissue, predominantly cortical part of the human brain was obtained from the carcasses of the death bodies. The tissue was desiccated in accordance to the permission of the relatives to use the tissues for the bench-top scientific experiments.
The experimental procedures with the human tissues was Purity of the obtained enzymes was detected by the RP-HPLC.

Purification of the PRPS-1
The enzyme was purified from the human brain cortex. The routine purification procedures were the same as for the OPT.
Semi-affinity chromatography was performed by the utility of the Cibacron Blue gel (Santa Cruz Biotechnology, USA). The elution was performed by the 44% NAD+ (Sigma-Aldrich). Purities of the obtained enzymes were detected by the RP-HPLC.

Statistics
In our calculations we have used t-test (student) for pair comparison as well as ONE-WAYANOVA for the calculation of the significance of the comparable all groups. The results were considered statistically significant when p was lower or equal to 0.05. In some calculations we used t-student test. There were used sigma Plot 10 and Sigma Stat 10 programs. For the creation of the structural formulas there was used the King Draw program.

Results
The Impact of The Activating Metal Ions on the PRPS-1.    A. Impact of the sulfate ions high concentration and adenosine on the activity of PRPS-1. We added 0.04 mg of the sulfate ions into the reactive mixture. Adenosine was added in the same quantity. The inhibition percentile was calculated from the control specific activity, which was accounted as the 100%. Measurements were performed by the Cary 60 spectrophotometer at 730 nm wavelength. The difference between control group and adenosine were statistically significant, p#<0.05, t-student test.

B.
Influence of the low concentrations of the sulfate ions on the activity of PRPS-1. It was applied ONE-WAY-ANOVA for calculation of the significance and p#<0.04. PRPS-1 activity in control (first two colums) vs GB rats (3 th , 4 th columns). B.

Measurement of the PRPS-1, ADA-2 as well as XOR activities in the settings of experimental GB.
ADA activity in control and GB rats. C.
XOR activity in the control and GB rats. D.
D1, D2. Glioblastoma cells culture: D2-light microscopy with 10x objective and D1 is the Methyl Green same culture. D3, D4. Methyle Green stained slices of the brain of the animals suffering from glioblastoma (n=6). It is clearly seen the hippocampus area on the figure D (4X objective, light microscopy with the 1800 magnifications (Boeco, Germany) and accumulation of the chromatin on the figure D3 with 10X objective magnification. Arrows are indicating the areas of tissue damage. The statistical significance was noticed between naïve animals and GB aniamls comparison groups without the inhibitors for the all 3 enzymes (p#<0.05, student t-test, p#<0.03 for PRPS-1 activity evaluation, student t-test) and inside the GB group for XOR activity measurement (p*<0.05, student t-test).

Discussion and Conclusions
Mg 2+ forms a complex with ATP (Mg-ATP) to act as the actual substrate of PRPS-1 although other divalent cations, such as Mn2+, Ni2+, Co 2+ or Cd 2+ can serve as the substitutes for Mg2+ with relatively lower activity [13][14][15]19,20,32]. Phosphate has multiple effects on the activity and structure of the enzyme. It usually acts as an activator for the activity of bacterial and mammalian PRSPs although SO4 2− can mimic the effect of phosphate at approx. 10-fold higher concentrations [11,13,16,19,33]. However, phosphate or This complex has no signifcant influence on its catalytic activity.
ADA*2 exists only as a monomer with molecular weight of 100,000 [35][36]. The main immunological function of ADA is regulation of T, B-cells differentiation as well as B-cells proliferation [35]. At sites of inflammation and tumor growth, the local concentration of extracellular adenosine rapidly increases and plays a role in controlling the immune responses of nearby cells. Adenosine deaminases ADA1 and ADA2 (ADAs) decrease the level of adenosine by converting it to inosine, which serves as a negative feedback mechanism. Mutations in the genes encoding ADAs lead to impaired immune function, which suggests a crucial role for ADAs in immune system regulation [37]. We suggest, ADA activity elevation in GB settings is a marker of the activation of the immune system. Group of scientists from Italy came to the conclusion, tumors with high levels of Xanthine Dehydrogenase (XDH) mRNA are characterized by higher expression of several genes encoding pro-inflammatory and immune cytokines, and increased levels of tumor infiltration with immune cells. The group also underlined the existence of great differences in uricogenesis between different types of human tumors making XDH as the molecular biomarkers of the cancer and cancer types [38].
Similar association between the high uricemia with the metabolic syndrome, diabetes, and cancer was suggested by the numerous other authors [39]. In our experiments we noticed the elevation of XOR activity in GB settings in comparison with the scheme, naïve rats. Thus, we suggest, measurement of 3 above mentioned enzymes activities for the characterization of experimental GB might be informative and serves as the diagnostic tool.