Phosphotyrosine Substrate Sequence Motifs for Dual Specificity Phosphatases

Protein tyrosine phosphatases dephosphorylate tyrosine residues of proteins, whereas, dual specificity phosphatases (DUSPs) are a subgroup of protein tyrosine phosphatases that dephosphorylate not only Tyr(P) residue, but also the Ser(P) and Thr(P) residues of proteins. The DUSPs are linked to the regulation of many cellular functions and signaling pathways. Though many cellular targets of DUSPs are known, the relationship between catalytic activity and substrate specificity is poorly defined. We investigated the interactions of peptide substrates with select DUSPs of four types: MAP kinases (DUSP1 and DUSP7), atypical (DUSP3, DUSP14, DUSP22 and DUSP27), viral (variola VH1), and Cdc25 (A-C). Phosphatase recognition sites were experimentally determined by measuring dephosphorylation of 6,218 microarrayed Tyr(P) peptides representing confirmed and theoretical phosphorylation motifs from the cellular proteome. A broad continuum of dephosphorylation was observed across the microarrayed peptide substrates for all phosphatases, suggesting a complex relationship between substrate sequence recognition and optimal activity. Further analysis of peptide dephosphorylation by hierarchical clustering indicated that DUSPs could be organized by substrate sequence motifs, and peptide-specificities by phylogenetic relationships among the catalytic domains. The most highly dephosphorylated peptides represented proteins from 29 cell-signaling pathways, greatly expanding the list of potential targets of DUSPs. These newly identified DUSP substrates will be important for examining structure-activity relationships with physiologically relevant targets.     

Structural characterization of inhibitor complexes with checkpoint kinase 2 (Chk2), a drug target for cancer therapy

Chk2 (checkpoint kinase 2) is a serine/threonine kinase that participates in a series of signaling networks responsible for maintaining genomic integrity and responding to DNA damage. The development of selective Chk2 inhibitors has recently attracted much interest as a means of sensitizing cancer cells to current DNA-damaging agents used in the treatment of cancer. Additionally, selective Chk2 inhibitors may reduce p53-mediated apoptosis in normal tissues, thereby helping to mitigate adverse side effects from chemotherapy and radiation. Thus far, relatively few selective inhibitors of Chk2 have been described and none have yet progressed into clinical trials. Here, we report crystal structures of the catalytic domain of Chk2 in complex with a novel series of potent and selective small molecule inhibitors. These compounds exhibit nanomolar potencies and are selective for Chk2 over Chk1. The structures reported here elucidate the binding modes of these inhibitors to Chk2 and provide information that can be exploited for the structure-assisted design of novel chemotherapeutics.     

Association of 25-Hydroxyvitamin D status and genetic variation in the vitamin D metabolic pathway with FEV1 in the Framingham Heart Study

Background

Vitamin D is associated with lung function in cross-sectional studies, and vitamin D inadequacy is hypothesized to play a role in the pathogenesis of chronic obstructive pulmonary disease. Further data are needed to clarify the relation between vitamin D status, genetic variation in vitamin D metabolic genes, and cross-sectional and longitudinal changes in lung function in healthy adults.

Methods

We estimated the association between serum 25-hydroxyvitamin D [25(OH)D] and cross-sectional forced expiratory volume in the first second (FEV₁) in Framingham Heart Study (FHS) Offspring and Third Generation participants and the association between serum 25(OH)D and longitudinal change in FEV₁ in Third Generation participants using linear mixed-effects models. Using a gene-based approach, we investigated the association between 241 SNPs in 6 select vitamin D metabolic genes in relation to longitudinal change in FEV₁ in Offspring participants and pursued replication of these findings in a meta-analyzed set of 4 independent cohorts.

Results

We found a positive cross-sectional association between 25(OH)D and FEV₁ in FHS Offspring and Third Generation participants (P = 0.004). There was little or no association between 25(OH)D and longitudinal change in FEV₁ in Third Generation participants (P = 0.97). In Offspring participants, the CYP2R1 gene, hypothesized to influence usual serum 25(OH)D status, was associated with longitudinal change in FEV₁ (gene-based P < 0.05). The most significantly associated SNP from CYP2R1 had a consistent direction of association with FEV₁ in the meta-analyzed set of replication cohorts, but the association did not reach statistical significance thresholds (P = 0.09).

Conclusions

Serum 25(OH)D status was associated with cross-sectional FEV₁, but not longitudinal change in FEV₁. The inconsistent associations may be driven by differences in the groups studied. CYP2R1 demonstrated a gene-based association with longitudinal change in FEV₁ and is a promising candidate gene for further studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0238-y) contains supplementary material, which is available to authorized users.     

Structure of B-DNA with cations tethered in the major groove

Here, we describe the 1.6-A X-ray structure of the DDD (Dickerson-Drew dodecamer), which has been covalently modified by the tethering of four cationic charges. This modified version of the DDD, called here the DDD(4+), is composed of [d(CGCGAAXXCGCG)](2), where X is effectively a thymine residue linked at the 5 position to an n-propyl-amine. The structure was determined from crystals soaked with thallium(I), which has been broadly used as a mimic of K(+) in X-ray diffraction experiments aimed at determining positions of cations adjacent to nucleic acids. Three of the tethered cations are directed radially out from the DNA. The radially directed tethered cations do not appear to induce structural changes or to displace counterions. One of the tethered cations is directed in the 3' direction, toward a phosphate group near one end of the duplex. This tethered cation appears to interact electrostatically with the DNA. This interaction is accompanied by changes in helical parameters rise, roll, and twist and by a displacement of the backbone relative to a control oligonucleotide. In addition, these interactions appear to be associated with displacement of counterions from the major groove of the DNA.     

Role of Glu312 in binding and positioning of the substrate for the hydride transfer reaction in choline oxidase

Choline oxidase catalyzes the oxidation of choline to glycine betaine, a compatible solute that accumulates in pathogenic bacteria and plants so they can withstand osmotic and temperature stresses. The crystal structure of choline oxidase was determined and refined to a resolution of 1.86 A with data collected at 100 K using synchrotron X-ray radiation. The structure reveals a covalent linkage between His99 Nepsilon2 and FAD C8M atoms, and a 123 A3 solvent-excluded cavity adjacent to the re face of the flavin. A hypothetical model for choline docked into the cavity suggests that several aromatic residues and Glu312 may orient the cationic substrate for efficient catalysis. The role of the negative charge on Glu312 was investigated by engineering variant enzymes in which Glu312 was replaced with alanine, glutamine, or aspartate. The Glu312Ala enzyme was inactive. The Glu312Gln enzyme exhibited a Kd value for choline at least 500 times larger than that of the wild-type enzyme. The Glu312Asp enzyme had a kcat/KO2 value similar to that of the wild-type enzyme but kcat and kcat/Km values that were 230 and 35 times lower, respectively, than in the wild-type enzyme. These data are consistent with the spatial location of the negative charge on residue 312 being important for the oxidation of the alcohol substrate. Solvent viscosity and substrate kinetic isotope effects suggest the presence of an internal equilibrium in the Glu312Asp enzyme prior to the hydride transfer reaction. Altogether, the crystallographic and mechanistic data suggest that Glu312 is important for binding and positioning of the substrate in the active site of choline oxidase.     

Atomic resolution structure of the cytoplasmic domain of Yersinia pestis YscU,a regulatory switch involved in type III secretion

Crystal structures of cleaved and uncleaved forms of the YscU cytoplasmic domain, an essential component of the type III secretion system (T3SS) in Yersinia pestis, have been solved by single‐wavelength anomolous dispersion and refined with X‐ray diffraction data extending up to atomic resolution (1.13 Å). These crystallographic studies provide structural insights into the conformational changes induced upon auto‐cleavage of the cytoplasmic domain of YscU. The structures indicate that the cleaved fragments remain bound to each other. The conserved NPTH sequence that contains the site of the N263‐P264 peptide bond cleavage is found on a β‐turn which, upon cleavage, undergoes a major reorientation of the loop away from the catalytic N263, resulting in altered electrostatic surface features at the site of cleavage. Additionally, a significant conformational change was observed in the N‐terminal linker regions of the cleaved and noncleaved forms of YscU which may correspond to the molecular switch that influences substrate specificity. The YscU structures determined here also are in good agreement with the auto‐cleavage mechanism described for the flagellar homolog FlhB and E. coli EscU.     

Crystal structure of the human dual specificity phosphatase 1 catalytic domain

The dual specificity phosphatase DUSP1 was the first mitogen activated protein kinase phosphatase (MKP) to be identified. It dephosphorylates conserved tyrosine and threonine residues in the activation loops of mitogen activated protein kinases ERK2, JNK1 and p38‐alpha. Here, we report the crystal structure of the human DUSP1 catalytic domain at 2.49 Å resolution. Uniquely, the protein was crystallized as an MBP fusion protein in complex with a monobody that binds to MBP. Sulfate ions occupy the phosphotyrosine and putative phosphothreonine binding sites in the DUSP1 catalytic domain.     

Utility of the white gene in estimating phylogenetic relationships among mosquitoes (Diptera: Culicidae)

The utility of a nuclear protein-coding gene for reconstructing phylogenetic relationships within the family Culicidae was explored. Relationships among 13 species representing three subfamilies and nine genera of Culicidae were analyzed using a 762-bp fragment of coding sequence from the eye color gene, white. Outgroups for the study were two species from the sister group Chaoboridae. Sequences were determined from clone PCR products amplified from genomic DNA, and aligned following conceptual intron splicing and amino acid translation. Third codon positions were characterized by high levels of divergence and biased nucleotide composition, the intensity and direction of which varied among taxa. Equal weighting of all characters resulted in parsimony and neighboring-joining trees at odds with the generally accepted phylogenetic hypothesis based on morphology and rDNA sequences. The application of differential weighting schemes recovered the traditional hypothesis, in which the subfamily Anophelinae formed the basal clade. The subfamily Toxorhynchitinae occupied an intermediate position, and was a sister group to the subfamily Culicinae. Within Culicinae, the genera Sabethes and Tripteroides formed an ancestral clade, while the Culex-Deinocerites and Aedes- Haemagogus clades occupied increasingly derived positions in the molecular phylogeny. An intron present in the Culicinae- Toxorhynchitinae lineage and one outgroup taxon was absent in the basal Anophelinae lineage and the second outgroup taxon, suggesting that intron insertions or deletions may not always be reliable systematic characters.      

Development of antiproliferative phenylmaleimides that activate the unfolded protein response

The current paper presents the synthesis and evaluation of a series of maleimides that were designed to inhibit the Cdc25 phosphatase by alkylation of catalytically essential cysteine residues. Although in HepB3 cell culture assays the analogues did exhibit antiproliferative IC₅₀ values ranging from sub-micromolar to greater than 100 μM, inhibition of Cdc25 through cysteine alkylation could not be demonstrated. It was also found that analysis using fluorescence activated cell sorting (FACS) following treatment with the most potent analogue (1t) did not provide data consistent with inhibition at one specific point in the cell cycle, as would be expected if Cdc25A were inhibited. Further studies with a subset of analogues resulted in a correlation of antiproliferative potencies with activation of the unfolded protein response (UPR). The UPR is a regulatory pathway that temporarily suspends protein production when misfolding of proteins occurs within the endoplastic reticulum (ER). In addition, ER chaperones that promote proper refolding become up-regulated. If cellular damage cannot be resolved by these mechanisms, then the UPR can initiate apoptosis. The current study indicates that these maleimide analogues lead to UPR activation, which is predictive of the selective antiproliferative activity of the series.