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.     

CK2 phosphorylation-dependent interaction between aprataxin and MDC1 in the DNA damage response

Aprataxin, defective in the neurodegenerative disorder ataxia oculomotor apraxia type 1, resolves abortive DNA ligation intermediates during DNA repair. Here, we demonstrate that aprataxin localizes at sites of DNA damage induced by high LET radiation and binds to mediator of DNA-damage checkpoint protein 1 (MDC1/NFBD1) through a phosphorylation-dependent interaction. This interaction is mediated via the aprataxin FHA domain and multiple casein kinase 2 di-phosphorylated S-D-T-D motifs in MDC1. X-ray structural and mutagenic analysis of aprataxin FHA domain, combined with modelling of the pSDpTD peptide interaction suggest an unusual FHA binding mechanism mediated by a cluster of basic residues at and around the canonical pT-docking site. Mutation of aprataxin FHA Arg29 prevented its interaction with MDC1 and recruitment to sites of DNA damage. These results indicate that aprataxin is involved not only in single strand break repair but also in the processing of a subset of double strand breaks presumably through its interaction with MDC1.     

Design and evaluation of 3,6-di(hetero)aryl imidazo[1,2-a]pyrazines as inhibitors of checkpoint and other kinases

A range of 3,6-di(hetero)arylimidazo[1,2-a]pyrazine ATP-competitive inhibitors of CHK1 were developed by scaffold hopping from a weakly active screening hit. Efficient synthetic routes for parallel synthesis were developed to prepare analogues with improved potency and ligand efficiency against CHK1. Kinase profiling showed that the imidazo[1,2-a]pyrazines could inhibit other kinases, including CHK2 and ABL, with equivalent or better potency depending on the pendant substitution. These 3,6-di(hetero)aryl imidazo[1,2-a]pyrazines appear to represent a general kinase inhibitor scaffold.     

Farm-scale trials to compare the entomopathogenic fungus Beauveria bassiana with pirimiphos methyl + deltamethrin and essential oil of lemon grass for protection of stored cowpea against Callosobruchus maculatus (Coleoptera: Bruchidae)

In trials conducted in Benin, conidia of Beauveria bassiana were evaluated as a control method against the cowpea weevil, Callosobruchus maculatus , in stored cowpea. In the first trial using a high artificial infestation of C.   maculatus in 8-kg batches of cowpea in jerry cans under typical conditions, concentrations of 1 × 10⁹ and 1 × 10¹¹ conidia kg⁻¹ were compared with lemon grass oil at 2 mL kg⁻¹ and the synthetic pesticide mixture of 1.5% pirimiphos methyl + 0.05% deltamethrin at 0.5 g kg⁻¹. After 2 months of storage, seed losses (SD) were 20.63 (5.3)% in the untreated control, 8.04 (3.2)% in the low-dose B.   bassiana group, 3.12 (1.3)% in the high-dose B.   bassiana group, 2.52 (0.4)% in the lemon grass oil group and 0% in the pirimiphos methyl + deltamethrin group. In a second trial with natural infestations in 50-kg batches of cowpea in 200-L drums, treatment with B.   bassiana 1 × 10¹¹ conidia kg⁻¹ was compared with pirimiphos methyl + deltamethrin at 0.5 g kg⁻¹. After 6 months of storage on six farms, losses reached 30.76 (1.5)% in the control, 1.28 (0.2)% in the pirimiphos methyl + deltamethrin group and 3.69 (0.6)% in the B.   bassiana group.     

A proteomics strategy to discover beta-glucosidases from Aspergillus fumigatus with two-dimensional page in-gel activity assay and tandem mass spectrometry

Economically competitive production of ethanol from lignocellulosic biomass by enzymatic hydrolysis and fermentation is currently limited, in part, by the relatively high cost and low efficiency of the enzymes required to hydrolyze cellulose to fermentable sugars. Discovery of novel cellulases with greater activity could be a critical step in overcoming this cost barrier. beta-Glucosidase catalyzes the final step in conversion of glucose polymers to glucose. Despite the importance, only a few beta-glucosidases are commercially available, and more efficient ones are clearly needed. We developed a proteomics strategy aiming to discover beta-glucosidases present in the secreted proteome of the cellulose-degrading fungus Aspergillus fumigatus. With the use of partial or complete protein denaturing conditions, the secretory proteome was fractionated in a 2DGE format and beta-glucosidase activity was detected in the gel after infusion with a substrate analogue that fluoresces upon hydrolysis. Fluorescing spots were subjected to tryptic-digestion, and identification as beta-glucosidases was confirmed by tandem mass spectrometry. Two novel beta-glucosidases of A. fumigatus were identified by this in situ activity staining method, and the gene coding for a novel beta-glucosidase ( EAL88289 ) was cloned and heterologously expressed. The expressed beta-glucosidase showed far superior heat stability to the previously characterized beta-glucosidases of Aspergillus niger and Aspergillus oryzae. Improved heat stability is important for development of the next generation of saccharifying enzymes capable of performing fast cellulose hydrolysis reactions at elevated temperatures, thereby lowering the cost of bioethanol production. The in situ activity staining approach described here would be a useful tool for cataloguing and assessing the efficiency of beta-glucosidases in a high throughput fashion.     

New covalent capture probes for imaging and therapy, based on a combination of binding affinity and disulfide bond formation

We describe the synthesis and development of new reactive DOTA-metal complexes for covalently targeting engineered receptors in vivo, which have superior tumor uptake and clearance properties for biomedical applications. These probes are found to clear efficiently through the kidneys and minimally through other routes, but bind persistently in the tumor target. We also explore the new technique of Cerenkov luminescence imaging to optically monitor radiolabeled probe distribution and kinetics in vivo. Cerenkov luminescence imaging uniquely enables sensitive noninvasive in vivo imaging of a β(-) emitter such as (90)Y with an optical imager.     

Three-dimensional structure of a macromolecular assembly that regulates type III secretion in Yersinia pestis

Yersinia pestis, the causative agent of plague, utilizes a type III secretion system (T3SS) to inject effector proteins directly into the cytosol of mammalian cells where they interfere with signal transduction pathways that regulate actin cytoskeleton dynamics and inflammation, thereby enabling the bacterium to avoid engulfment and destruction by macrophages. Type III secretion normally does not occur in the absence of close contact with eukaryotic cells. Negative regulation is mediated in part by a multiprotein complex that has been proposed to act as a physical impediment to type III secretion by blocking the entrance to the secretion apparatus prior to contact with mammalian cells. This complex is composed of YopN, its heterodimeric secretion chaperone SycN-YscB, and TyeA. Here, we report two crystal structures of YopN in complex with its heterodimeric secretion chaperone SycN-YscB and the co-regulatory protein TyeA, respectively. By merging these two overlapping structures, it was possible to construct a credible theoretical model of the YopN-SycN-YscB-TyeA complex. The modeled assembly features the secretion signaling elements of YopN at one end of an elongated structure and the secretion regulating TyeA binding site at the other. A patch of highly conserved residues on the surface of the C-terminal alpha-helix of TyeA may mediate its interaction with structural components of the secretion apparatus. Conserved arginine residues that reside inside a prominent cavity at the dimer interface of SycN-YscB were mutated in order to investigate whether they play a role in targeting the YopN-chaperone complex to the type III secretion apparatus. One of the mutants exhibited a phenotype that is consistent with this hypothesis.     

Doubling the cloak of (in)competence in client/therapist interactions

Cultural competence is used (often implicitly) to make decisions in human service settings. When therapists make decisions about whether or not a particular service will be offered, they place themselves in a position where their own competence can be judged. Using narrative data on independence and the elderly, we apply Edgerton's idea of the cloak of competence to demonstrate this doubling effect.