Murine Enzyme Research Articles

Liver lobe and strain differences in the activity of murine cytochrome P450 enzymes

The cytochrome P450 (CYP) enzyme superfamily is the most important enzyme system for phase I biotransformation. For toxico- and pharmacokinetic studies, use of liver-based microsomes, including those of mice, is state-of-the-art to study CYP-dependent metabolism. However, reproducibility and interpretation of these data is still very variable, partly because current testing guidelines do not cover details on organ sampling and potential liver lobe differences. Hence, we analyzed CYP activity, CYP protein content, mRNA expression of CYP1A, CYP2C, CYP2D and CYP3A isozymes, and cytochrome P450 reductase (CPR) activity of the four different liver lobes and processus papillaris of male C57BL/6J mice in comparison to whole liver. Additionally, we used whole liver of Balb/cJ and 129S1/SvImJ for strain comparison.Our data show significant differences in CYP activity, being most prominent in lobus sinister lateralis and lobus medialis, and lowest in processus papillaris. These differences were not caused by varying Cyp gene expression or CYP protein level, but partly correspond with lobe specific CPR activities. We also observed significant strain differences in CYP mRNA expression and activities with overall high activities in 129S1/SvImJ mice and low activities in Balb/cJ mice compared to C57BL/6J mice. In addition, strain specific differences in CYP2C and CYP2D activity seem to be reflected in strain dependent differences in CPR activity.In summary, our results indicate that in mice CYP activity and gene expression are strain dependent and may vary highly between liver lobes. To ensure reproducibility and comparability of different probes and studies, this should be taken into account when liver samples are collected for the analysis of CYP-dependent metabolism.

Homodimer Architecture of QTRT2, the Noncatalytic Subunit of the Eukaryotic tRNA-Guanine Transglycosylase.

The bacterial enzyme tRNA-guanine transglycosylase (TGT) is involved in the biosynthesis of queuosine, a modified nucleoside present in the anticodon wobble position of tRNAHis, tRNATyr, tRNAAsp, and tRNAAsn. Although it forms a stable homodimer endowed with two active sites, it is, for steric reasons, able to bind and convert only one tRNA molecule at a time. In contrast, its mammalian counterpart constitutes a heterodimer consisting of a catalytic and a noncatalytic subunit, termed QTRT1 and QTRT2, respectively. Both subunits are homologous to the bacterial enzyme, yet only QTRT1 possesses all the residues required for substrate binding and catalysis. In mice, genetic inactivation of the TGT results in the uncontrolled oxidation of tetrahydrobiopterin and, accordingly, phenylketonuria-like symptoms. For this reason and because of the recent finding that mammalian TGT may be utilized for the treatment of multiple sclerosis, this enzyme is of potential medical relevance, rendering detailed knowledge of its biochemistry and structural architecture highly desirable. In this study, we performed the kinetic characterization of the murine enzyme, investigated potential quaternary structures of QTRT1 and QTRT2 via noncovalent mass spectrometry, and, finally, determined the crystal structure of the murine noncatalytic TGT subunit, QTRT2. In the crystal, QTRT2 is clearly present as a homodimer that is strikingly similar to that formed by bacterial TGT. In particular, a cluster of four aromatic residues within the interface of the bacterial TGT, which constitutes a "hot spot" for dimer stability, is present in a similar constellation in QTRT2.

Physiological Regulation of Drug Metabolism and Transport: Pregnancy, Microbiome, Inflammation, Infection, and Fasting

This article is a report on a symposium entitled "Physiological Regulation of Drug Metabolism and Transport" sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 2017 meeting in Chicago, IL. The contributions of physiologic and pathophysiological regulation of drug-metabolizing enzymes and transporters to interindividual variability in drug metabolism are increasingly recognized but in many cases are not well understood. The presentations herein discuss the phenomenology, consequences, and mechanism of such regulation. CYP2D6 transgenic mice were used to provide insights into the mechanism of regulation of this enzyme in pregnancy, via hepatocyte nuclear factor 4α, small heterodimer partner, and retinoids. Regulation of intestinal and hepatic drug-processing enzymes by the intestinal microbiota via tryptophan and its metabolites was investigated. The potential impact of parasitic infections on human drug metabolism and clearance was assessed in mice infected with Schistosoma mansoni or Plasmodium chabaudi chabaudi AS, both of which produced widespread and profound effects on murine hepatic drug-metabolizing enzymes. Finally, the induction of Abcc drug efflux transporters by fasting was investigated. This was demonstrated to occur via a cAMP, protein kinase A/nuclear factor-E2-related factor 2/Sirtuin 1 pathway via antioxidant response elements on the Abcc genes.

Human serine racemase is nitrosylated at multiple sites

Serine racemase is a pyridoxal 5′‑phosphate dependent enzyme responsible for the synthesis of d‑serine, a neuromodulator of the NMDA receptors. Its activity is modulated by several ligands, including ATP, divalent cations and protein interactors. The murine orthologue is inhibited by S-nitrosylation at Cys113, a residue adjacent to the ATP binding site. We found that the time course of inhibition of human serine racemase by S-nitrosylation is markedly biphasic, with a fast phase associated with the reaction of Cys113. Unlike the murine enzyme, two additional cysteine residues, Cys269, unique to the human orthologue, and Cys128 were also recognized as S-nitrosylation sites through mass spectrometry and site-directed mutagenesis. The effect of S-nitrosylation on the fluorescence of tryptophan residues and on that of the pyridoxal phosphate cofactor indicated that S-nitrosylation produces a partial interruption of the cross-talk between the ATP binding site and the active site. Overall, it appears that the inhibition results from a conformational change rather than the direct displacement of ATP.

The inhibitory potential of calixarenes against nucleotide pyrophosphatase/phosphodiesterase 1

It has been previously shown that phosphonic acids covalently attached to the macrocyclic platform of calix[4]arenes are capable of inhibiting alkaline phosphatases. In this paper the effects of the upper-rim functionalized calix[4]arenes on the activity of nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) have been examined. Aim. To assess the inhibitory potential of calix[4]arene, thiacalix[4]arene and sulfonylcalix[4]arene derivatives against NPP1. Results and discussion. It has been found that calix[4]arene, thiacalix[4]arene, and sulfonylcalix[4]arene tetrakismethylphosphonic acids inhibit NPP1 with the IC50 values in the micromolar range. The derivatives of sulfonylcalix[4]arene demonstrated the selectivity of inhibition of NPP1 over alkaline phosphatases. In addition, sulfonylcalix[4]arene tetrakismethylphosphonic acid was able to inhibit the nucleotide pyrophosphatase/phosphodiesterase activity of the human serum. The possible mechanism of the inhibition has been discussed. Experimental part. The activity of NPP1 was monitored by spectrophotometry measuring the rate of hydrolysis of bis-p-nitrophenyl phosphate. The phosphodiesterase activity of the human serum was assessed in the presence of p-nitrophenyl ester of thymidine-5-monophosphate as a substrate. The homology model of the human NPP1 was generated based on the crystal structure of the murine enzyme. The molecular docking was performed using AutoDock 4.2. Conclusions. The results obtained have shown the ability of sulfonylcalix[4]arene derivatives to inhibit the activity of NPP1 in vitro, including the nucleotide pyrophosphatase/phosphodiesterase activity in the human blood serum.

Use of the KlADH3 promoter for the quantitative production of the murine PDE5A isoforms in the yeast Kluyveromyces lactis

BackgroundPhosphodiesterases (PDE) are a superfamily of enzymes that hydrolyse cyclic nucleotides (cAMP/cGMP), signal molecules in transduction pathways regulating crucial aspects of cell life. PDEs regulate the intensity and duration of the cyclic nucleotides signal modulating the downstream biological effect. Due to this critical role associated with the extensive distribution and multiplicity of isozymes, the 11 mammalian families (PDE1 to PDE11) constitute key therapeutic targets. PDE5, one of these cGMP-specific hydrolysing families, is the molecular target of several well known drugs used to treat erectile dysfunction and pulmonary hypertension. Kluyveromyces lactis, one of the few yeasts capable of utilizing lactose, is an attractive host alternative to Saccharomyces cerevisiae for heterologous protein production. Here we established K. lactis as a powerful host for the quantitative production of the murine PDE5 isoforms.ResultsUsing the promoter of the highly expressed KlADH3 gene, multicopy plasmids were engineered to produce the native and recombinant Mus musculus PDE5 in K. lactis. Yeast cells produced large amounts of the purified A1, A2 and A3 isoforms displaying Km, Vmax and Sildenafil inhibition values similar to those of the native murine enzymes. PDE5 whose yield was nearly 1 mg/g wet weight biomass for all three isozymes (30 mg/L culture), is well tolerated by K. lactis cells without major growth deficiencies and interferences with the endogenous cAMP/cGMP signal transduction pathways.ConclusionsTo our knowledge, this is the first time that the entire PDE5 isozymes family containing both regulatory and catalytic domains has been produced at high levels in a heterologous eukaryotic organism. K. lactis has been shown to be a very promising host platform for large scale production of mammalian PDEs for biochemical and structural studies and for the development of new specific PDE inhibitors for therapeutic applications in many pathologies.

Branched-Chain Amino Acids and Brain Metabolism

This review aims to provide a historical reference of branched-chain amino acid (BCAA) metabolism and provide a link between peripheral and central nervous system (CNS) metabolism of BCAAs. Leucine, isoleucine, and valine (Leu, Ile, and Val) are unlike most other essential amino acids (AA), being transaminated initially in extrahepatic tissues, and requiring interorgan or intertissue shuttling for complete catabolism. Within the periphery, BCAAs are essential AAs and are required for protein synthesis, and are key nitrogen donors in the form of Glu, Gln, and Ala. Leucine is an activator of the mammalian (or mechanistic) target of rapamycin, the master regulator of cell growth and proliferation. The tissue distribution and activity of the catabolic enzymes in the peripheral tissues as well as neurological effects in Maple Syrup Urine Disease (MSUD) show the BCAAs have a role in the CNS. Interestingly, there are significant differences between murine and human CNS enzyme distribution and activities. In the CNS, BCAAs have roles in neurotransmitter synthesis, protein synthesis, food intake regulation, and are implicated in diseases. MSUD is the most prolific disease associated with BCAA metabolism, affecting the branched-chain α-keto acid dehydrogenase complex (BCKDC). Mutations in the branched-chain aminotransferases(BCATs) and the kinase for BCKDC also result in neurological dysfunction. However, there are many questions of BCAA metabolism in the CNS (as well as the periphery) that remain elusive. We discuss areas of BCAA and BCKA metabolism that have yet to be researched adequately.

The absorption enhancer sodium deoxycholate promotes high gene transfer in skeletal muscles

Gene delivery to skeletal muscle is a promising strategy for the treatment of muscle disorders and for the systemic secretion of therapeutic proteins. In addition, muscle is an attractive target tissue because it is easily accessible. However, very few synthetic vectors proved capable of surpassing naked DNA mediated muscle gene transfer. In fact, only neutral copolymers, in particular poloxamers, demonstrated capacities to increase transgene expression in skeletal muscles. Here, we studied in vitro and in vivo behaviour of different bile salts. We report that sodium deoxycholate (DOC) and derivatives thereof increase after intramuscular injection by more than 100-fold the levels of the reporter gene luciferase compared to naked DNA. Using a LacZ expression cassette, we found that more than 20% of the muscle fibers expressed the reporter gene. Prolonged expression of a secreted reporter gene derived from a natural murine alkaline phosphatase enzyme could be documented. Altogether, our results demonstrate that bile salts belong to the most efficient chemicals identified so far for skeletal muscle gene transfer. Importantly, since these compounds are naturally found in the body, there is no risk of immune response against them and in addition several bile salts are already used in human medicine. Bile salt mediated muscle gene transfer may thus have broad applications in gene therapy.

Murine recombinant angiotensin-converting enzyme 2 attenuates kidney injury in experimental Alport syndrome

Angiotensin-converting enzyme 2 (ACE2) is a monocarboxypeptidase in the renin-angiotensin system that catalyzes the breakdown of angiotensin II to angiotensin 1-7. We have reported that ACE2 expression in the kidney is reduced in experimental Alport syndrome but the impact of this finding on disease progression has not been studied. Accordingly, we evaluated effects of murine recombinant ACE2 treatment in Col4a3 knockout mice, a model of Alport syndrome characterized by proteinuria and progressive renal injury. Murine recombinant ACE2 (0.5 mg/kg/day) was administered from four to seven weeks of age via osmotic mini-pump. Pathological changes were attenuated by murine recombinant ACE2 treatment which ameliorated kidney fibrosis as shown by decreased expression of COL1α1 mRNA, less accumulation of extracellular matrix proteins, and inhibition of transforming growth factor-β signaling. Further, increases in proinflammatory cytokine expression, macrophage infiltration, inflammatory signaling pathway activation, and heme oxygenase-1 levels in Col4a3 knockout mice were also reduced by murine recombinant ACE2 treatment. Lastly, murine recombinant ACE2 influenced the turnover of renal ACE2, as it suppressed the expression of tumor necrosis factor-α converting enzyme, a negative regulator of ACE2. Thus, treatment with exogenous ACE2 alters angiotensin peptide metabolism in the kidneys of Col4a3 knockout mice and attenuates the progression of Alport syndrome nephropathy.

Crosstalk between ABO and Forssman (FORS) blood group systems: FORS1 antigen synthesis by ABO gene-encoded glycosyltransferases

A and B alleles at the ABO genetic locus specify A and B glycosyltransferases that catalyze the biosynthesis of A and B oligosaccharide antigens, respectively, of blood group ABO system which is important in transfusion and transplantation medicine. GBGT1 gene encodes Forssman glycolipid synthase (FS), another glycosyltransferase that produces Forssman antigen (FORS1). Humans are considered to be Forssman antigen-negative species without functional FS. However, rare individuals exhibiting Apae phenotype carry a dominant active GBGT1 gene and express Forssman antigen on RBCs. Accordingly, FORS system was recognized as the 31st blood group system. Mouse ABO gene encodes a cis-AB transferase capable of producing both A and B antigens. This murine enzyme contains the same GlyGlyAla tripeptide sequence as FSs at the position important for the determination of sugar specificity. We, therefore, transfected the expression construct into appropriate recipient cells and examined whether mouse cis-AB transferase may also exhibit FS activity. The result was positive, confirming the crosstalk between the ABO and FORS systems. Further experiments have revealed that the introduction of this tripeptide sequence to human A transferase conferred some, although weak, FS activity, suggesting that it is also involved in the recognition/binding of acceptor substrates, in addition to donor nucleotide-sugars.

Mammalian agmatinases constitute unusual members in the family of Mn2 +-dependent ureahydrolases

Agmatine (1-amino-4-guanidinobutane) plays an important role in a range of metabolic functions, in particular in the brain. Agmatinases (AGMs) are enzymes capable of converting agmatine to the polyamine putrescine and urea. AGMs belong to the family of Mn2+-dependent ureahydrolases. However, no AGM from a mammalian source has yet been extracted in catalytically active form. While in human AGM the six amino acid ligands that coordinate the two Mn2+ ions in the active site are conserved, four mutations are observed in the murine enzyme. Here, we demonstrate that similar to its human counterpart murine AGM does not appear to have in vitro catalytic activity, independent of the presence of Mn2+. However, in presence of agmatine both enzymes are very efficient in promoting cell growth of a yeast strain that is deficient in polyamine biosynthesis (Saccharomyces cerevisiae strain TRY104Δspe1). Furthermore, mutations among the putative Mn2+ binding residues had no effect on the ability of murine AGM to promote growth of the yeast culture. It thus appears that mammalian AGMs form a distinct group within the family of ureahydrolases that (i) either fold in a manner distinct from other members in this family, or (ii) require accessory proteins to bind Mn2+ in a mechanism related to that observed for the Ni2+-dependent urease.

Membrane-dependent Activities of Human 15-LOX-2 and Its Murine Counterpart: IMPLICATIONS FOR MURINE MODELS OF ATHEROSCLEROSIS

The enzyme encoded by the ALOX15B gene has been linked to the development of atherosclerotic plaques in humans and in a mouse model of hypercholesterolemia. In vitro, these enzymes, which share 78% sequence identity, generate distinct products from their substrate arachidonic acid: the human enzyme, a 15-S-hydroperoxy product; and the murine enzyme, an 8-S-product. We probed the activities of these enzymes with nanodiscs as membrane mimics to determine whether they can access substrate esterified in a bilayer and characterized their activities at the membrane interface. We observed that both enzymes transform phospholipid-esterified arachidonic acid to a 15-S-product. Moreover, when expressed in transfected HEK cells, both enzymes result in significant increases in the amounts of 15-hydroxyderivatives of eicosanoids detected. In addition, we show that 15-LOX-2 is distributed at the plasma membrane when the HEK293 cells are stimulated by the addition Ca(2+) ionophore and that cellular localization is dependent upon the presence of a putative membrane insertion loop. We also report that sequence differences between the human and mouse enzymes in this loop appear to confer distinct mechanisms of enzyme-membrane interaction for the homologues.

A Novel Multi-step Virtual Screening for the Identification of Human and Mouse mPGES-1 Inhibitors.

We present here the development of a novel virtual screening protocol combining Structure-based and Ligand-based drug design approaches for the identification of mouse mPGES-1 inhibitors. We used the existing 3D structural data of the murine enzyme to hypothesize the inhibitors binding mode, which was the starting point for docking simulations, shape screening, and pharmacophore hypothesis screening. The protocol allowed the identification of 16 mouse mPGES-1 inhibitors with low micromolar activity, which, notably, also inhibit the human enzyme in the same concentration range. The inhibitors predicted binding mode is expected to be the base for the rational drug design of new potent dual species inhibitors of human and murine mPGES-1.

18F]FMeNER-D2: A systematic in vitro analysis of radio-metabolism

IntroductionThe norepinephrine transporter (NET) presents an important target for therapy and diagnosis of ADHD and other neurodegenerative and psychiatric diseases. Thus, PET is the diagnostic method of choice, using radiolabeled NET-ligands derived from reboxetine. So far, [18F]FMeNER-D2 showed best pharmacokinetic and -dynamic properties. However, the disadvantage of reboxetine derived PET tracers is their high metabolic cleavage—resulting in impeding signals in the PET scans, which hamper a proper quantification of the NET in cortical areas. MethodsMetabolic stability testing was performed in vitro using a plethora of human and murine enzymes. ResultsNo metabolism was observed using monoamine oxidase A and B or catechol-O-methyl transferase. Incubation of [18F]FMeNER-D2 with CYP450-enzymes, predominantly located in the liver, led to a significant and fast metabolism of the tracer. Moreover, the arising three radiometabolites were found to be more polar than [18F]FMeNER-D2. Surprisingly, definitely no formation of free [18F]fluoride was observed. ConclusionAccording to our in vitro data, the interfering uptake in cortical regions might be attributed to these emerging radiometabolites but does not reflect bonding in bone due to defluorination. Further research on these radiometabolites is necessary to elucidate the in vivo situation. This might include an analysis of human blood samples after injection of [18F]FMeNER-D2, to enable a better correction of the PET-input function.

Substrate Specificity of the HEMK2 Protein Glutamine Methyltransferase and Identification of Novel Substrates

Bacterial HEMK2 homologs initially had been proposed to be involved in heme biogenesis or to function as adenine DNA methyltransferase. Later it was shown that this family of enzymes has protein glutamine methyltransferase activity, and they methylate the glutamine residue in the GGQ motif of ribosomal translation termination factors. The murine HEMK2 enzyme methylates Gln(185) of the eukaryotic translation termination factor eRF1. We have employed peptide array libraries to investigate the peptide sequence recognition specificity of murine HEMK2. Our data show that HEMK2 requires a GQX3R motif for methylation activity. In addition, amino acid preferences were observed between the -3 and +7 positions of the peptide substrate (considering the target glutamine as 0), including a preference for Ser, Arg, and Gly at the +1 and a preference for Arg at the +7 position. Based on our specificity profile, we identified several human proteins that contain putative HEMK2 methylation sites and show that HEMK2 methylates 58 novel peptide substrates. After cloning, expression, and purification of the corresponding protein domains, we confirmed methylation for 11 of them at the protein level. Transfected CHD5 (chromodomain helicase DNA-binding protein 5) and NUT (nuclear protein in testis) were also demonstrated to be methylated by HEMK2 in human HEK293 cells. Our data expand the range of proteins potentially subjected to glutamine methylation significantly, but further investigation will be required to understand the function of HEMK2-mediated methylation in proteins other than eRF1.

Algenpantucel-L immunotherapy in pancreatic adenocarcinoma.

Pancreatic adenocarcinoma is the 4th leading cause of cancer death in the USA and the EU. A minority of patients presents with surgically resectable and potentially curable disease, but among these, 80% are destined to relapse and overall survival rates with adjuvant chemotherapy average 24 months. Immunotherapy is a promising therapeutic option and a potential paradigm shift in the treatment of patients with pancreatic cancer, and may be particularly effective when used early in the disease course to prevent metastatic spread. Algenpantucel-L (HyperAcute Pancreas, NewLink Genetics, Ames, IA, USA) is a whole-cell immunotherapy consisting of irradiated allogeneic pancreatic cancer cells genetically engineered to express the murine enzyme α-GT, which results in hyperacute rejection of the tumor cells with complement- and antibody-dependent cytotoxicity. Phase II clinical trial data has been encouraging, particularly for patients who demonstrated humoral immunologic responses. Here, we report preliminary results and biomarkers correlations with clinical activity of algenpantucel-L in pancreatic cancer.

Pancreatic cancer: Update on immunotherapies and algenpantucel-L

ABSTRACTPancreatic adenocarcinoma is notoriously lethal, and despite improvements in systemic chemotherapy approaches bringing survival rates for metastatic disease to almost 1 year, by 2030 it is expected to become the second leading cause of cancer death. Pancreatic cancer (PC) prognosis has been associated with both the presence of intratumoral helper and cytotoxic T lymphocytes, as well as humoral immune responses to tumor associated antigens like mesothelin. It is well described that the PC microenvironment is characterized by a fibroinflammatory and immunosuppressive stroma. On these premises several immune-targeted strategies have been developed to harness the adaptable immune system with a goal of improving survival with little toxicity. Cancer vaccines involve the administration of tumor-associated antigens with the goal of inducing an endogenous anti-tumor response. Among several strategies discussed, we will focus on the algenpantucel-L (HyperAcute™ Pancreas) immunotherapy. Algenpantucel-L is a whole cell immunotherapy consisting of irradiated allogeneic PC cells genetically engineered to express the murine enzyme α(1,3)-galactosyltransferase (αGT), which ultimately leads to hyperacute rejection with complement- and antibody-dependent cytotoxicity. While phase III data in the adjuvant treatment of pancreatic cancer are pending, phase II results have been encouraging, particularly for patients who demonstrated humoral immunologic responses. Novel strategies using immune checkpoint inhibitors, costimulatory antibodies, and combinations with cancer vaccines may overcome immunotolerance and improve treatment success.

Opioid Analgesia in P450 Gene Cluster Knockout Mice: A Search for Analgesia-Relevant Isoforms.

Cytochrome P450 monooxygenases (P450s), which are well-known drug-metabolizing enzymes, are thought to play a signal transduction role in µ opioid analgesia and may serve as high-affinity (3)H-cimetidine ((3)HCIM) binding sites in the brain. (3)HCIM binding sites may also be related to opioid or nonopioid analgesia. However, of the more than 100 murine P450 enzymes, the specific isoform(s) responsible for either function have not been identified. Presently, three lines of constitutive P450 gene cluster knockout (KO) mice with full-length deletions of 14 Cyp2c, 9 Cyp2d, and 7 Cyp3a genes were studied for deficiencies in (3)HCIM binding and for opioid analgesia. Liver and brain homogenates from all three genotypes showed normal (3)HCIM binding values, indicating that gene products of Cyp2d, Cyp3a, and Cyp2c are not (3)HCIM-binding proteins. Cyp2d KO and Cyp3a KO mice showed normal antinociceptive responses to a moderate systemic dose of morphine (20 mg/kg, s.c.), thereby excluding 16 P450 isoforms as mediators of opioid analgesia. In contrast, Cyp2c KO mice showed a 41% reduction in analgesic responses following systemically (s.c.) administered morphine. However, the significance of brain Cyp2c gene products in opioid analgesia is uncertain because little or no analgesic deficits were noted in Cyp2c KO mice following intracerebroventricular or intrathecalmorphine administration, respectively. These results show that the gene products of Cyp2d and Cyp3a do not contribute to µ opioid analgesia in the central nervous system. A possible role for Cyp2c gene products in opioid analgesia requires further consideration.

Manipulating the substrate specificity of murine dihydrofolate reductase enzyme using an expanded set of amino acids

As the number of reactions requiring biotransformation continues to grow, manipulating the enzyme substrate specificity becomes very important. Complementary to conventional enzyme engineering techniques based on only natural amino acids, we hypothesized that the site-specific incorporation of a non-natural amino acid in vivo into an enzyme can be used to re-design the active site for the altered substrate specificity. To test our hypothesis, we introduced the non-natural amino acid L-2-naphthylalanine (2Nal) into the active site of the model enzyme murine dihydrofolate reductase (mDHFR). We explored whether the substrate specificity of the enzyme could be switched from the good substrate dihydrofolate (DHF) to the poor substrate folate (FOL). We used two protein design programs (RosettaLigand and RosettaDesign) to calculate ligand docking and conformational stability, respectively, for the evaluation of multiples sites in the mDHFR. From the calculations, position 31 was predicted as an optimal 2Nal incorporation site. One mDHFR variant containing 2Nal at position 31 (mDHFR2Nal31) was expressed in the Escherichia coli expression host cells equipped with the engineered yeast phenylalanyl-tRNA and phenylalanyl-tRNA synthetase pair. As expected, the kinetic assays of purified mDHFR variant revealed that mDHFR2Nal31 has the enhanced binding affinity toward FOL and also exhibits 7.6-fold enhanced catalytic efficiency of FOL over DHF compared to mDHFRWT.

Aspergillus flavus induces granulomatous cerebral aspergillosis in mice with display of distinct cytokine profile

Aspergillus flavus is one of the leading Aspergillus spp. resulting in invasive aspergillosis of central nervous system (CNS) in human beings. Immunological status in aspergillosis of central nervous system remains elusive in case of both immunocompetent and immunocompromised patients. Since cytokines are the major mediators of host response, evaluation of disease pathology along with cytokine profile in brain may provide snapshots of neuro-immunological response. An intravenous model of A. flavus infection was utilized to determine the pathogenicity of infection and cytokine profile in the brain of male BALB/c mice. Enumeration of colony forming units and histopathological analyses were performed on the brain tissue at distinct time periods. The kinetics of cytokines (TNF-α, IFN-γ, IL-12/IL-23p40, IL-6, IL-23, IL-17A and IL-4) was evaluated at 6, 12, 24, 48, 72 and 96h post infection (hPI) in brain homogenates using murine cytokine specific enzyme linked immunosorbent assay. Histological analysis exhibited the hyphae with leukocyte infiltrations leading to formation of granulomata along with ischemia and pyknosis of neurons in the brain of infected mice. Diseased mice displayed increased secretion of IFN-γ, IL-12p40 and IL-6 with a concomitant reduction in the secretion of Th2 cytokine IL-4, and Th17 promoting cytokine, IL-23 during the late phase of infection. A.flavus induced inflammatory granulomatous cerebral aspergillosis in mice, characterized by a marked increase in the Th1 cytokines and neurons undergoing necrosis. A marked increase in necrosis of neurons with concurrent inflammatory responses might have led to the host mortality during late phase of infection.