Putative Tyrosine Research Articles

Involvement of Protein Tyrosine Phosphatases BcPtpA and BcPtpB in Regulation of Vegetative Development, Virulence and Multi-Stress Tolerance in Botrytis cinerea

Tyrosine phosphorylation and dephosphorylation have emerged as fundamentally important mechanisms of signal transduction and regulation in eukaryotic cells, governing many processes, but little has been known about their functions in filamentous fungi. In this study, we deleted two putative protein tyrosine phosphatase (PTP) genes (BcPTPA and BcPTPB) in Botrytis cinerea, encoding the orthologs of Saccharomyces cerevisiae Ptp2 and Ptp3, respectively. Although BcPtpA and BcPtpB have opposite functions in conidiation, they are essential for sclerotial formation in B. cinerea. BcPTPA and BcPTPB deletion mutants ΔBcPtpA-10 and ΔBcPtpB-4 showed significantly increased sensitivity to osmotic and oxidative stresses, and to cell wall damaging agents. Inoculation tests showed that both mutants exhibited dramatically decreased virulence on tomato leaves, apples and grapes. In S. cerevisiae, it has been shown that Ptp2 and Ptp3 negatively regulate the high-osmolarity glycerol (HOG) pathway and the cell wall integrity (CWI) pathway. Although both BcPtpA and BcPtpB were able to inactive Hog1 and Mpk1 in S. cerevisiae, in contrast to S. cerevisiae, they positively regulate phosphorylation of BcSak1 (the homologue of Hog1) and BcBmp3 (the homologue of Mpk1) in B. cinerea under stress conditions. These results demonstrated that functions of PTPs in B. cinerea are different from those in S. cerevisiae, and BcPtpA and BcPtpB play important roles in regulation of vegetative development, virulence and in adaptation to oxidative, osmotic and cell-wall damage stresses in B. cinerea.

Spontaneous Mutation Reveals Influence of Exopolysaccharide on Lactobacillus johnsonii Surface Characteristics

As a competitive exclusion agent, Lactobacillus johnsonii FI9785 has been shown to prevent the colonization of selected pathogenic bacteria from the chicken gastrointestinal tract. During growth of the bacterium a rare but consistent emergence of an altered phenotype was noted, generating smooth colonies in contrast to the wild type rough form. A smooth colony variant was isolated and two-dimensional gel analysis of both strains revealed a protein spot with different migration properties in the two phenotypes. The spot in both gels was identified as a putative tyrosine kinase (EpsC), associated with a predicted exopolysaccharide gene cluster. Sequencing of the epsC gene from the smooth mutant revealed a single substitution (G to A) in the coding strand, resulting in the amino acid change D88N in the corresponding gene product. A native plasmid of L. johnsonii was engineered to produce a novel vector for constitutive expression and this was used to demonstrate that expression of the wild type epsC gene in the smooth mutant produced a reversion to the rough colony phenotype. Both the mutant and epsC complemented strains had increased levels of exopolysaccharides compared to the wild type strain, indicating that the rough phenotype is not solely associated with the quantity of exopolysaccharide. Another gene in the cluster, epsE, that encoded a putative undecaprenyl-phosphate galactosephosphotransferase, was deleted in order to investigate its role in exopolysaccharide biosynthesis. The ΔepsE strain exhibited a large increase in cell aggregation and a reduction in exopolysaccharide content, while plasmid complementation of epsE restored the wild type phenotype. Flow cytometry showed that the wild type and derivative strains exhibited clear differences in their adhesive ability to HT29 monolayers in tissue culture, demonstrating an impact of EPS on surface properties and bacteria-host interactions.

Novel adaptor protein Shf interacts with ALK receptor and negatively regulates its downstream signals in neuroblastoma

Our neuroblastoma cDNA project previously identified Src homology 2 domain containing F (Shf) as one of the genes expressed at high levels in favorable neuroblastoma. Shf is an adaptor protein containing four putative tyrosine phosphorylation sites and an SH2 domain. In this study, we found that Shf interacted with anaplastic lymphoma kinase (ALK), an oncogenic receptor tyrosine kinase in neuroblastoma. Real-time PCR analysis showed that Shf mRNA is highly expressed in non-metastatic neuroblastomas compared to metastatic tumor samples (P < 0.030, n = 106). Interestingly, patients showing high ALK and low Shf mRNA expressions showed poor prognosis, whereas low ALK and high Shf expressions were related to better prognosis (P < 0.023, n = 38). Overexpression of ALK and siRNA-mediated knockdown of Shf yielded similar results, such as an increase in cellular growth and phosphorylation of ALK, in addition to Erk1/2 and signal transducer and activator of transcription 3 (STAT3) that are downstream signals of the ALK-initiated phospho-transduction pathway. Knockdown of Shf also increased the cellular mobility and invasive capability of neuroblastoma cells. These results suggest that Shf interacts with ALK and negatively regulates the ALK-initiated signal transduction pathway in neuroblastoma. We thus propose that Shf inhibits phospho-transduction signals mediated by ALK, which is one of the major key players on neuroblastoma development, resulting in better prognosis of the tumor.

Interaction of the putative tyrosine recombinases RipX (UU145), XerC (UU222), and CodV (UU529) ofUreaplasma parvumserovar 3 with specific DNA

Phase variation of two loci (‘mba locus’ and ‘UU172 phase-variable element’) in Ureaplasma parvum serovar 3 has been suggested as result of site-specific DNA inversion occurring at short inverted repeats. Three potential tyrosine recombinases (RipX, XerC, and CodV encoded by the genes UU145, UU222, and UU529) have been annotated in the genome of U. parvum serovar 3, which could be mediators in the proposed recombination event. We document that only orthologs of the gene xerC are present in all strains that show phase variation in the two loci. We demonstrate in vitro binding of recombinant maltose-binding protein fusions of XerC to the inverted repeats of the phase-variable loci, of RipX to a direct repeat that flanks a 20-kbp region, which has been proposed as putative pathogenicity island, and of CodV to a putative dif site. Co-transformation of the model organism Mycoplasma pneumoniae M129 with both the ‘mba locus’ and the recombinase gene xerC behind an active promoter region resulted in DNA inversion in the ‘mba locus’. Results suggest that XerC of U. parvum serovar 3 is a mediator in the proposed DNA inversion event of the two phase-variable loci.

Endocytosis of somatodendritic NCKX2 is regulated by Src family kinase-dependent tyrosine phosphorylation

We have previously reported that the surface expression of K+-dependent Na+/Ca2+ exchanger 2 (NCKX2) in the somatodendritic compartment is kept low by constitutive endocytosis, which results in the polarization of surface NCKX2 to the axon. Clathrin-mediated endocytosis is initiated by interaction of the μ subunit of adaptor protein complex 2 (AP-2) with the canonical tyrosine motif (YxxΦ) of a target molecule. We examined whether endocytosis of NCKX2 involves two putative tyrosine motifs (365YGKL and 371YDTM) in the cytoplasmic loop of NCKX2. Coimmunoprecipitation assay revealed that the 365YGKL motif is essential for the interaction with the μ subunit of AP-2 (AP2M1). Consistently, either overexpression of NCKX2-Y365A mutant or knockdown of AP2M1 in cultured hippocampal neurons significantly reduced the internalization of NCKX2 from the somatodendritic surface and thus abolished the axonal polarization of surface NCKX2. Next, we tested whether the interaction between the tyrosine motif and AP2M1 is regulated by phosphorylation of the 365th tyrosine residue (Tyr-365). Tyrosine phosphorylation of heterologously expressed NCKX2-WT, but not NCKX2-Y365A, was increased by carbachol (CCh) in PC-12 cells. The effect of CCh was inhibited by PP2, a Src family kinase (SFK) inhibitor. Moreover, PP2 facilitated the endocytosis of NCKX2 in both the somatodendritic and axonal compartments, suggesting that tyrosine phosphorylation of NCKX2 by SFK negatively regulates its endocytosis. Supporting this idea, activation of SFK enhanced the NCKX activity in the proximal dendrites of dentate granule cells (GCs). These results suggest that endocytosis of somatodendritic NCKX2 is regulated by SFK-dependent phosphorylation of Tyr-365.

Initial Characterization of the Pf-Int Recombinase from the Malaria Parasite Plasmodium falciparum

BackgroundGenetic variation is an essential means of evolution and adaptation in many organisms in response to environmental change. Certain DNA alterations can be carried out by site-specific recombinases (SSRs) that fall into two families: the serine and the tyrosine recombinases. SSRs are seldom found in eukaryotes. A gene homologous to a tyrosine site-specific recombinase has been identified in the genome of Plasmodium falciparum. The sequence is highly conserved among five other members of Plasmodia.Methodology/Principal FindingsThe predicted open reading frame encodes for a ∼57 kDa protein containing a C-terminal domain including the putative tyrosine recombinase conserved active site residues R-H-R-(H/W)-Y. The N-terminus has the typical alpha-helical bundle and potentially a mixed alpha-beta domain resembling that of λ-Int. Pf-Int mRNA is expressed differentially during the P. falciparum erythrocytic life stages, peaking in the schizont stage. Recombinant Pf-Int and affinity chromatography of DNA from genomic or synthetic origin were used to identify potential DNA targets after sequencing or micro-array hybridization. Interestingly, the sequences captured also included highly variable subtelomeric genes such as var, rif, and stevor sequences. Electrophoretic mobility shift assays with DNA were carried out to verify Pf-Int/DNA binding. Finally, Pf-Int knock-out parasites were created in order to investigate the biological role of Pf-Int.Conclusions/SignificanceOur data identify for the first time a malaria parasite gene with structural and functional features of recombinases. Pf-Int may bind to and alter DNA, either in a sequence specific or in a non-specific fashion, and may contribute to programmed or random DNA rearrangements. Pf-Int is the first molecular player identified with a potential role in genome plasticity in this pathogen. Finally, Pf-Int knock-out parasite is viable showing no detectable impact on blood stage development, which is compatible with such function.

Localized Netrins Act as Positional Cues to Control Layer-Specific Targeting of Photoreceptor Axons in Drosophila

SummaryA shared feature of many neural circuits is their organization into synaptic layers. However, the mechanisms that direct neurites to distinct layers remain poorly understood. We identified a central role for Netrins and their receptor Frazzled in mediating layer-specific axon targeting in the Drosophila visual system. Frazzled is expressed and cell autonomously required in R8 photoreceptors for directing their axons to the medulla-neuropil layer M3. Netrin-B is specifically localized in this layer owing to axonal release by lamina neurons L3 and capture by target neuron-associated Frazzled. Ligand expression in L3 is sufficient to rescue R8 axon-targeting defects of Netrin mutants. R8 axons target normally despite replacement of diffusible Netrin-B by membrane-tethered ligands. Finally, Netrin localization is instructive because expression in ectopic layers can retarget R8 axons. We propose that provision of localized chemoattractants by intermediate target neurons represents a highly precise strategy to direct axons to a positionally defined layer.

RNAi of cat-2, a Putative Tyrosine Hydroxylase, Increases Alpha Synuclein Aggregation and Associated Effects in Transgenic C. elegans

Neurodegenerative Parkinson's disease (PD) is a multifactorial disorder; effects like alpha synuclein aggregation, low dopamine levels and dopaminergic neurodegeneration are considered to be hallmarks of the disease. Several recent studies have pointed towards an important role of enzyme tyrosine hydroxylase (TH) in the pathophysiology of PD. We embarked on the present studies to explore the mechanistic role of C. elegans gene cat-2, a putative tyrosine hydroxylase, in PD. Utilizing the powerful genetic model system C. elegans, which has previously provided critical understanding of several human diseases, we employed a reverse genetics approach via RNAi mediated gene silencing of cat-2, to study various disease related effects in three different transgenic strains of the nematode. Knocking-down of cat-2 led to increase in aggregation of alpha synuclein, as was studied via expression of YFP. Similarly the silencing of cat-2 had significant effects on associated endpoints including oxidative stress, lipid content and neurotransmission; exemplifying the role of cat-2, the putative tyrosine hydroxylase, in Parkinsonism of the nematode model. The findings are significant as this model could further be used to study the entire associated pathway in greater detail and with the advantages that the model system C. elegans presents, the knockdown of cat-2 in the alpha synuclein expressing strain, could be employed for screening potential pharmacological agents targeted at TH which could lead to designing of possible therapeutic interventions for the disease.

Two Homologous Putative Protein Tyrosine Phosphatases, OsPFA-DSP2 and AtPFA-DSP4, Negatively Regulate the Pathogen Response in Transgenic Plants

Protein phosphatases, together with protein kinases, regulate protein phosphorylation and dephosphorylation, and play critical roles in plant growth and biotic stress responses. However, little is known about the biological functions of plant protein tyrosine dual-specificity phosphatase (PFA-DSP) in biotic stresses. Here, we found that OsPFA-DSP2 was mainly expressed in calli, seedlings, roots, and young panicles, and localized in cytoplasm and nucleus. Ectopic overexpression of OsPFA-DSP2 in rice increased sensitivity to Magnaporthe grisea (M. grisea Z1 strain), inhibited the accumulation of hydrogen peroxide (H2O2) and suppressed the expression of pathogenesis-related (PR) genes after fungal infection. Interestingly, transgenic Arabidopsis plants overexpressing AtPFA-DSP4, which is homologous to OsPFA-DSP2, also exhibited sensitivity to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), reduced accumulation of H2O2 and decreased photosynthesic capacity after infection compared with Col-0. These results indicate that OsPFA-DSP2 and AtPFA-DSP4 act as negative regulators of the pathogen response in transgenic plants.

One-Step Expression and Tyrosine O-Sulfonation of Ax21 in Escherichia coli

Ax21 (activator of Xa21-mediated immunity), a pathogen-associated molecular pattern secreted by Xanthomonas oryzae pv. oryzae, can be perceived by a membrane-located pattern recognition receptor Xa21 and triggered immune responses in rice. An Ax21-derived peptide (17-amino acid) containing a sulfated tyrosine-22 (axY(S)22) is sufficient for Ax21 activity. Here, we expressed Ax21 and O-sulfated its tyrosine-22 through coexpressing a putative tyrosine sulfotransferase, raxST, and two other genes involved in the synthesis of 3'-phosphoadenosine 5'-phosphosulfate in Escherichia coli BL21 (DE3). The sulfated Ax21 fused with a histidine tag in its N-terminus was extracted and bound onto a Ni-NTA agarose and then cleaved with Factor Xa and CNBr in turn. Δax21Y(S)22, a 36-amino acid peptide covering axY(S)22 in the lysate supernatant, was finally yielded after ultrafiltration. The purified peptide was further verified by Tricine-SDS-PAGE and isoelectrofocusing electrophoresis. Lesion length analysis, reactive oxygen species production, and mitogen-activated protein kinase (MAPK) activation of rice leaves inoculated with Δax21Y(S)22 confirmed the activity of the sulfated peptide. Overall, this study successfully established an efficient system for expression and purification of a sulfated peptide. In addition, the sulfotransferase activity of RaxST was confirmed for the first time.

A putative tyrosine phosphorylation site of the cell surface receptor Golden goal is involved in synaptic layer selection in the visual system

Golden goal (Gogo) is a cell surface protein that is crucial for proper synaptic layer targeting of photoreceptors (R cells) in the Drosophila visual system. In collaboration with the seven-transmembrane cadherin Flamingo (Fmi), Gogo mediates both temporary and final layer targeting of R-cell axons through its cytoplasmic activity. However, it is not known how Gogo activity is regulated. Here, we show that a conserved Tyr-Tyr-Asp (YYD) tripeptide motif in the Gogo cytoplasmic domain is required for photoreceptor axon targeting. Deleting the YYD motif is sufficient to abolish Gogo function. We demonstrate that the YYD motif is a phosphorylation site and that mutations in the YYD tripeptide impair synaptic layer targeting. Gogo phosphorylation results in axon stopping at the temporary targeting layer, and dephosphorylation is crucial for final layer targeting in collaboration with Fmi. Therefore, both temporary and final layer targeting strongly depend on the Gogo phosphorylation status. Drosophila Insulin-like receptor (DInR) has been reported to regulate the wiring of photoreceptors. We show that insulin signaling is a positive regulator, directly or indirectly, of YYD motif phosphorylation. Our findings indicate a novel mechanism for the regulation of Gogo activity by insulin signaling-mediated phosphorylation. We propose the model that a constant phosphorylation signal is antagonized by a presumably temporal dephosphorylation signal, which creates a permissive signal that controls developmental timing in axon targeting.

A Heme Peroxidase with a Functional Role as an l-Tyrosine Hydroxylase in the Biosynthesis of Anthramycin

We report the first characterization and classification of Orf13 (S. refuineus) as a heme-dependent peroxidase catalyzing the ortho-hydroxylation of L-tyrosine to L-DOPA. The putative tyrosine hydroxylase coded by orf13 of the anthramycin biosynthesis gene cluster has been expressed and purified. Heme b has been identified as the required cofactor for catalysis, and maximal L-tyrosine conversion to L-DOPA is observed in the presence of hydrogen peroxide. Preincubation of L-tyrosine with Orf13 prior to the addition of hydrogen peroxide is required for L-DOPA production. However, the enzyme becomes inactivated by hydrogen peroxide during catalysis. Steady-state kinetic analysis of L-tyrosine hydroxylation revealed similar catalytic efficiency for both L-tyrosine and hydrogen peroxide. Spectroscopic data from a reduced-CO(g) UV-vis spectrum of Orf13 and electron paramagnetic resonance of ferric heme Orf13 are consistent with heme peroxidases that have a histidyl-ligated heme iron. Contrary to the classical heme peroxidase oxidation reaction with hydrogen peroxide that produces coupled aromatic products such as o,o'-dityrosine, Orf13 is novel in its ability to catalyze aromatic amino acid hydroxylation with hydrogen peroxide, in the substrate addition order and for its substrate specificity for L-tyrosine. Peroxygenase activity of Orf13 for the ortho-hydroxylation of L-tyrosine to L-DOPA by a molecular oxygen dependent pathway in the presence of dihydroxyfumaric acid is also observed. This reaction behavior is consistent with peroxygenase activity reported with horseradish peroxidase for the hydroxylation of phenol. Overall, the putative function of Orf13 as a tyrosine hydroxylase has been confirmed and establishes the first bacterial class of tyrosine hydroxylases.

Two Sorting Motifs, a Ubiquitination Motif and a Tyrosine Motif, Are Involved in HIV-1 and Simian Immunodeficiency Virus Nef-Mediated Receptor Endocytosis

HIV-1 and SIV Nef proteins downregulate cell surface CD4 and MHC class I (MHC-I) molecules of infected cells, which are necessary for efficient viral replication and pathogenicity. We previously reported that K144 in HIV-1 Nef is di-ubiquitinated, and K144R substitution impairs Nef-mediated CD4 downregulation. In this report, we extend the role of ubiquitination at this lysine residue from Nef-mediated CD4 downregulation to Nef-mediated MHC-I downregulation and from HIV Nef to SIV Nef. All HIV-1 Nef mutants that contain K144R substitution are inactive in MHC-I downregulation. Tested MHC-I alleles include HLA-ABC endogenously expressed and HLA-A2 exogenously expressed in Jurkat T cells. CD4 downregulation by SIV Nef involves K176 that aligns with K144 in HIV-1 Nef, as well as an N-terminal tyrosine motif Y28Y39 not present in HIV-1 Nef. Dual mutation at K176 and Y28Y39 completely impaired SIV Nef-mediated CD4 and MHC-I downregulation, whereas a single mutation at K176 or Y28Y39 did not. The involvement of tyrosine motif in SIV Nef-mediated CD4 and MHC-I downregulation prompted us to investigate a putative tyrosine motif (Y202Y/F203) in HIV-1 Nef that is conserved among HIV-1 species. Single mutation at the tyrosine motif Y202F203 in HIV-1 Nef (NA7) greatly impaired Nef-mediated CD4 downregulation, which is similar to what we observed previously with the single mutation at lysine K144. Thus, our study demonstrated that Nef-mediated receptor endocytosis involves the ubiquitination motif and tyrosine motif.

Structural and Functional Analysis of C2-Type Ketoreductases from Modular Polyketide Synthases

The process by which α-stereocenters of polyketide intermediates are set by modular polyketide synthases (PKSs) when condensation is not immediately followed by reduction is mysterious. However, the reductase-incompetent ketoreductase (KR) from the third module of 6-deoxyerythronolide B synthase has been proposed to operate as a racemase, aiding in the epimerization process that reverses the orientation of the α-methyl group of the polyketide intermediate generated by the ketosynthase to the configuration observed in the 6-deoxyerythronolide B final product. To learn more about the epimerization process, the structure of the C2-type KR from the third module of the pikromycin synthase, analogous to the KR from the third module of 6-deoxyerythronolide B synthase, was determined to 1.88 Å resolution. This first structural analysis of this KR-type reveals differences from reductase-competent KRs such as that the site NADPH binds to reductase-competent KRs is occluded by side chains and the putative catalytic tyrosine possesses more degrees of freedom. The active-site geometry may enable C2-type KRs to align the thioester and β-keto groups of a polyketide intermediate to reduce the p K a of the α-proton and accelerate its abstraction. Results from in vivo assays of engineered PKSs support that C2-type KRs cooperate with epimer-specific ketosynthases to set the configurations of substituent-bearing α-carbons.

Increased Expression of Wild-Type or a Centronuclear Myopathy Mutant of Dynamin 2 in Skeletal Muscle of Adult Mice Leads to Structural Defects and Muscle Weakness

Dynamin 2 (DNM2) is a large GTPase implicated in many cellular functions, including cytoskeleton regulation and endocytosis. Although ubiquitously expressed, DNM2 was found mutated in two genetic disorders affecting different tissues: autosomal dominant centronuclear myopathy (ADCNM; skeletal muscle) and peripheral Charcot-Marie-Tooth neuropathy (peripheral nerve). To gain insight into the function of DNM2 in skeletal muscle and the pathological mechanisms leading to ADCNM, we introduced wild-type DNM2 (WT-DNM2) or R465W DNM2 (RW-DNM2), the most common ADCNM mutation, into adult wild-type mouse skeletal muscle by intramuscular adeno-associated virus injections. We detected altered localization of RW-DNM2 in mouse muscle. Several ADCNM features were present in RW-DNM2 mice: fiber atrophy, nuclear mislocalization, and altered mitochondrial staining, with a corresponding reduction in specific maximal muscle force. The sarcomere and triad structures were also altered. We report similar findings in muscle biopsy specimens from an ADCNM patient with the R465W mutation. In addition, expression of wild-type DNM2 induced some muscle defects, albeit to a lesser extent than RW-DNM2, suggesting that the R465W mutation has enhanced activity in vivo. In conclusion, we show the RW-DNM2 mutation acts in a dominant manner to cause ADCNM in adult muscle, and the disease arises from a primary defect in skeletal muscle rather than secondary to peripheral nerve involvement. Therefore, DNM2 plays important roles in the maintenance of adult muscle fibers.

Abstract 2312: Using multidimensional real-time cell profiling approach to identify novel inhibitors of key targets and pathways involved in cancer maintenance and progression

Abstract A high throughput cell-based phenotypic screening method was developed using real time cell analysis system which is based on impedance sensor technology. Using compounds with known mechanisms, we had previously demonstrated small molecule compounds produce characteristic time-dependent cell response profiles (TCRP) which could be indicative of mechanism of action. The cellular-basis for the anti-mitotic TCRP has been extensively validated using both anti-mitotic compounds and siRNAs targeting mitotic machineries (e.g. Eg5, PLK1 &amp; Mad2). In this study, we used the TCRP approach to screen a 119,595 compound library for novel anti-cancer agents. The screening was performed at a final concentration of 17.8 µM using a human non small cell lung cancer line, H1993. The TCRP was monitored for 48 hours after compound treatment and the data generated was analyzed using in-house developed software to perform clustering analysis. For each screening set, a panel of compounds with known mechanisms of action were used as positive control and also served as reference for TCRP-based clustering to predict mechanisms of action. After multi-dose confirmation, 239 hits with IC50 values less than 8.9 µM were subjected to TCRP analysis. In comparison with reference TCRP patterns, we indentified 26 hits corresponding to tyrosine kinase inhibitors, 9 hits as DNA damaging agents, 125 hits as anti-mitotic agents, and 28 hits as cytostatic agents. Detailed characterization of 117 putative anti-mitotic compounds with traditional p-H3 based methods demonstrates 113 of them (96.6%) mediated mitotic arrest, thus demonstrating the robust predictive nature of the approach. We also have validated the putative tyrosine kinase inhibitors in in vitro kinase assays. TCRP-based phenotypic screening displays a novel drug screening approach with great potentials for discovering new pathway-modulating anticancer agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2312. doi:10.1158/1538-7445.AM2011-2312

Perturbed Heme Binding Is Responsible for the Blistering Phenotype Associated with Mutations in the Caenorhabditis elegans Dual Oxidase 1 (DUOX1) Peroxidase Domain

Dual oxidase (DUOX) enzymes support a wide variety of essential reactions, from cellular signaling to thyroid hormone biosynthesis. In Caenorhabditis elegans, the DUOX system (CeDUOX1/2) plays a crucial role in innate immunity and in stabilizing the cuticle by forming tyrosine cross-links. The current model suggests that superoxide generated by CeDUOX1 at the C-terminal NADPH oxidase domain is rapidly converted to H(2)O(2). The H(2)O(2) is then utilized by the N-terminal peroxidase-like domain to cross-link tyrosines. We have now created a series of mutations in the isolated peroxidase domain, CeDUOX1(1-589). One set of mutations investigate the roles of a putative distal tyrosine (Tyr(105)) and Glu(238), a proposed covalent heme-binding residue. The results confirm that Glu(238) covalently binds to the heme group. A second set of mutations (G246D and D392N) responsible for a C. elegans blistering cuticle phenotype was also investigated. Surprisingly, although not among the catalytic residues, both mutations affected heme co-factor binding. The G246D mutant bound less total heme than the wild type, but a higher fraction of it was covalently bound. In contrast, the D392N mutant appears to fold normally but does not bind heme. Molecular dynamics simulations of a CeDUOX1(1-589) homology model implicate displacements of the proximal histidine residue as the likely cause. Both enzymes are structurally stable and through altered heme interactions exhibit partial or complete loss of tyrosine cross-linking activity, explaining the blistering phenotype. This result argues that the CeDUOX peroxidase domain is primarily responsible for tyrosine cross-linking.

ROR1 Is Expressed on the Surface of Non-Neoplastic Human B-Lymphocyte Precursors (Hematogones) and on a Subset of B Acute Lymphoblastic Leukemia

AbstractAbstract 2735ROR1 is a putative receptor tyrosine kinase that is expressed during embryogenesis, on chronic lymphocytic leukemia (CLL) cells, but not on normal mature lymphocytes, including normal CD5+ B cells (Fukuda et al PNAS 2008; 105:3047). ROR1 binds Wnt5a, and this ligand enhances the survival of CLL cells in vitro, suggesting that ROR1 functionally contributes to leukemogenesis. Additional evidence for the importance of ROR1 function in leukemia is the noted decrease in survival of B acute lymphoblastic leukemia (B-ALL) cells in vitro after RNAi targeting of ROR1 (Tyner et al. PNAS 2009; 106:8695). To further explore the significance of ROR1 in B-ALL, we analyzed 30 cases of B-ALL for surface expression of ROR1 via multiparameter flow cytometry using a fluorochrome-conjugated mAb specific for ROR1. Only two of 30 cases of B-ALL showed uniform surface ROR1 staining with a mean fluorescence intensity (MFI) that was 6.2 or 3.6 times greater than that of B-ALL cells stained with a control mAb. The other 28 B-ALL cases showed a mean MFI ratio of 1.4 (S.D. = 0.4). The two ROR1+ cases shared distinctive immunophenotypic and cytogenetic changes consistent with a more mature B-ALL subtype. We also evaluated marrow from patients recovering from chemotherapy or hematopoietic stem-cell transplantation and found that non-neoplastic B-lymphocyte precursors (BLP, hematogones) also expressed surface ROR1. These BLP were positive for CD10, CD19, CD38, and dim CD45 with variable expression of CD20, CD34, TdT, and cytoplasmic immunoglobulin. While the surface antigen phenotype of B-ALL is similar to BLP, flow cytometry can distinguish B-ALL from BLP because B-ALL shows more uniform antigen expression, lacking the continuum of maturation stages present in BLP. Also, B-ALL usually has aberrant antigen-expression intensity or aberrant antigen co-expression such as terminal deoxynucleotidyl transferase (TdT) plus CD20 on the same cell. Of 22 marrow samples tested with 1–20% hematogones, all had CD10+, CD19+, dim CD45+ cells that expressed ROR1 with a mean MFI ratio of 4.9 (S.D. 2.0). Further study found ROR1 was restricted to a subset of CD10, CD19, and low-level CD45 cells that lack expression of CD34 or TdT. This subset is consistent with an intermediate stage of B-cell maturation, possibly associated with B-lymphoid positive and negative selection. In contrast, CD10+, CD19+ germinal center B cells from reactive lymph nodes were negative for surface ROR1 (N=4). This pattern of ROR1 expression on BLP and a subset of distinctive B-ALL suggests that surface expression of ROR1 occurs during a discrete stage of B-cell maturation in the marrow that may be expanded during recovery from myeloablative therapy and/or hematopoietic stem cell transplantation. Disclosures:No relevant conflicts of interest to declare.

Streptomyces turgidiscabies Car8 contains a modular pathogenicity island that shares virulence genes with other actinobacterial plant pathogens

Streptomyces turgidiscabies Car8 is an actinobacterium that causes the economically important disease potato scab. Pathogenesis in this species is associated with a mobile pathogenicity island (PAISt) that site specifically inserts into the bacA gene in Streptomyces spp. Here we provide the 674,223 bp sequence of PAISt, which consists of two non-overlapping modules of 105,364 and 568,859 bp. These modules are delimited by three copies of an 8 bp palindromic sequence (TTCATGAA), that also is the integration site ( att) of the element. Putative tyrosine recombinase (IntSt) and excisionase (XisSt) proteins are encoded just upstream of att-R. PAISt has regions of synteny to pathogenic, symbiotic and saprophytic actinomycetes. The 105,364 bp PAISt module is identical to a genomic island in Streptomyces scabies 87–22, while the 568,859 bp module contains only a short region of synteny to that genome. However, both modules contain previously characterized and candidate virulence genes.

Identification of structural and molecular determinants of the tyrosine-kinase Wzc and implications in capsular polysaccharide export

Capsular polysaccharides are well-established virulence factors of pathogenic bacteria. Their biosynthesis and export are regulated within the transmembrane polysaccharide assembly machinery by the autophosphorylation of atypical tyrosine-kinases, named BY-kinases. However, the accurate functioning of these tyrosine-kinases remains unknown. Here, we report the crystal structure of the non-phosphorylated cytoplasmic domain of the tyrosine-kinase Wzc from Escherichia coli in complex with ADP showing that it forms a ring-shaped octamer. Mutational analysis demonstrates that a conserved EX(2) RX(2) R motif involved in subunit interactions is essential for polysaccharide export. We also elucidate the role of a putative internal regulatory tyrosine and we show that BY-kinases from proteobacteria autophosphorylate on their C-terminal tyrosine cluster via a single-step intermolecular mechanism. This structure-function analysis also allows us to demonstrate that two different parts of a conserved basic region called the RK-cluster are essential for polysaccharide export and for kinase activity respectively. Based on these data, we revisit the dichotomy made between BY-kinases from proteobacteria and firmicutes and we propose a unique process of oligomerization and phosphorylation. We also reassess the function of BY-kinases in the capsular polysaccharide assembly machinery.