Recipient Organization
PURDUE UNIVERSITY
(N/A)
WEST LAFAYETTE,IN 47907
Performing Department
Biochemistry
Non Technical Summary
Fungal plant pathogens are a major annual cause of crop damage and loss throughout the world and consequently pose a serious threat to global food supply and security 18. Despite the use of disease-resistant varieties and fungicide applications, many common fungal pathogens continue to cause widespread damage to important global crop species, including rice (Magnaporthe oryzae), and cereals (Fusarium graminearum)18. Inevitably, pathogens evolve resistance to fungicides and overcome disease-resistant cultivars7. Thus, additional treatment options, including novel anti-fungal targets are constantly needed to limit crop damage from fungal pathogens. In a recent ranking of the top 10 scientifically and economically important fungal plant pathogens by the journal Molecular Plant Pathology, Magnaporthe oryzae and Fusarium graminearum were ranked first and fourth, respectively7.Cdc14 is an attractive target for anti-fungal drug development for several important reasons. First, genetic deletion of CDC14 severely retards growth and eliminates pathogenicity of F. graminearum and M. oryzae, based on results from our Purdue collaborator, Dr. Jin-Rong Xu. Second, unlike most serine/threonine phosphatases, Cdc14 is a single subunit enzyme whose substrate selectivity appears to be entirely dictated by the structure around its catalytic site, suggesting that design of highly specific competitive inhibitors should be achievable. Third, CDC14 phosphatase genes are conserved in fungi and animals, but are absent from higher plants11. Thus, plants are likely to be unaffected by compounds that specifically inhibit Cdc14. The proposed work will address the problem of crop fungal pathogens by understanding the physiological importance of a protein required for plant infection and by developing inhibitors to test the viability of this protein as a novel anti-fungal drug target.The potential outcomes and benefits to society are the future development of new anti-fungal compounds for treating or preventing plant fungal infections that cause devastating losses to crops around the world each year.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
1. Characterize the enzymatic specificity of Fusarium graminearum and Magnaporthe oryzae Cdc14 phosphatases (FgCdc14 and MoCdc14).2. Use combined bioinformatic (from Objective 1) and biochemical approaches to identify physiological substrates of FgCdc14 and MoCdc14.3. Characterize the phenotypes associated with perturbed phospho-regulation of select substrates from Objective 2.4. Develop specific inhibitors of Cdc14 phosphatases using high throughput screening5. Test effectiveness of Cdc14 inhibitors identified and developed in Objective 4 towards F. graminearum and M. oryzae in vivo and in plant infection assays.
Project Methods
Objective 1. FgCdc14 and MoCdc14 will be characterized to determine if the same determinants of substrate recognition identified in budding yeast and human Cdc14 enzymes exist in these fungal orthologs.Positional scanning phosphopeptide libraries will define the substrate features that promote or inhibit efficient Cdc14 activity. We have executed this approach withSaccharomyces cerevisiaeCdc14 (ScCdc14) but intend to fully characterize the contributions of amino acids at several positions before and after pSer-Pro, which is the essential core sequence of Cdc14 substrates4. We will test all possible amino acids at positions -2, -1, +2, +4, +5, and +6 relative to pSer because these residues are predicted to contact the highly conserved substrate binding channel on Cdc14. Three libraries will be synthesized. All will have pSer-Pro fixed at the 0 and +1 positions. One will have a fixed +3 Lys (optimal), one a fixed +3 Arg (less optimal), and one will leave +3 unfixed. FgCdc14 and MoCdc14 will be compared to ScCdc14. This broader analysis of phosphopeptide substrates has the potential to reveal additional positive and negative determinants of substrate selection and will allow more refined prediction using bioinformatic tools (Objective 2).Screening phosphopeptide libraries is simple8. Purified recombinant Cdc14 is mixed with a fixed low concentration (below Km) of purified synthetic phosphopeptide substrates in 384 well plates and incubated 30 min at 30 °C. A commercially available phosphate detection reagent is added, which stops the reaction and forms a green color when complexed with free phosphate. The absorbance is measured at 620 nm in a plate reader. The rate values are approximations of the catalytic efficiency kcat/Km and provide a useful means to compare the relative ability of collections of substrates to be recognized and dephosphorylated by Cdc14.With Dr. Sergey Savinov, Purdue Center for Cancer Research, we will perform docking and molecular dynamics simulations to model the interactions of optimal substrate sequences identified in the peptide library screen with the substrate binding site, based on the existing crystal structure of human Cdc14B10. We will create a homology model of theF. graminearumandM. oryzaeenzymes using the program Prime (Schrödinger, LLC).Objective 2. Using the initial specificity features identified in ScCdc14 we have demonstrated the ability to accurately predict physiological Cdc14 substrates6,9. This approach will be used to predict substrates inF. graminearumandM. oryzae.In parallel with bioinformatics, we will employ biochemical approaches to identify targets of Cdc14 in these two fungal pathogens. One approach relies on use of a substrate trapping mutant of Cdc14 (Cys to Ser mutation in the active site). This mutation eliminates catalytic activity but allows high affinity substrate binding. It has been used previously in proteomic workflows to globally identify Cdc14 interaction partners and substrates in common model organisms2,5. We have generated the substrate trap mutation in theCDC14genes ofF. graminearumandM. oryzaefor expression and purification of the mutant proteins as GST fusions in E. coli. The mutant fusion proteins will be bound to glutathione agarose to create an affinity column and soluble whole cell extracts ofF. graminearumandM. oryzaecultures will be passed through the column. After extensive washing, proteins will be eluted and subjected to HPLC-coupled tandem mass spectrometry (MS) in the Purdue Proteomics Facility. Dr. Xu will introduce epitope tagged substrate trap alleles into the genomes ofF. graminearumandM. oryzae. The tagged FgCdc14 and MoCdc14 proteins will be affinity-purified from whole cell extracts using commercially available affinity resin and specifically bound proteins identified by MS. Extracts from cells lacking the tagged alleles will serve as a negative control. The list of interaction partners will be filtered using the bioinformatics predictions to find the most promising candidate substrates.To validate top candidates, a series of biochemical and biological experiments will be performed6,9. Synthetic phosphopeptides representing the putative Cdc14 target sites will be analyzed in steady-state kinetic assays with recombinant Cdc14 enzymes and compared to our gold standard set of known substrate peptides from budding yeast. Recombinant substrate proteins will be phosphorylated in vitro with purified Cdk1 from budding yeast and subjected to Cdc14 phosphatase assays in which each individual site can be quantitatively monitored by MS8. The candidate substrate sites will be compared to known high efficiency budding yeast Cdc14 substrates. With Dr. Xu, we will monitor the phosphorylation status of tagged alleles of the candidate substrates in vivo in wild-type andcdc14null strains ofF. graminearumandM. oryzaeusing Phos-tag SDS-PAGE gels and immunoblotting. We expect phosphorylated forms of the substrates to be enhanced in the absence of Cdc14 function. We will introduce additional copies of Cdc14 for overexpression and evaluate phosphorylation status on Phos-tag SDS-PAGE by immublotting. We expect phosphorylation to be reduced upon Cdc14 overexpression. Interaction between the candidate substrate and Cdc14 should be dependent on Cdk phosphorylation sites. We will introduce mutant alleles of the candidate substrates lacking Cdk sites and perform co-IP analysis with the substrate trap variants of Cdc14 to determine if recognition is direct and dependent on Cdk phosphorylation.Objective 3. We will select validated Cdc14 substrates from Objective 2 inF. graminearumfor initial functional characterization of phosphoregulation. The Xu lab will perform genetic replacement of wild-type alleles of the substrate candidates with phosphosite mutants (Ser to Ala to mimic lack of phosphorylation and Ser to Asp to mimic constitutive phosphorylation) to look for phenotypes. Growth rates and plant infectivity will be important for the overall goal of this project, but more specific cell division phenotypes, including DNA damage sensitivity, chromosome mis-segregation, and cytokinesis defects will be of biological interest6,9.Objective 4. We have initiated screening of a diverse small molecule library for Cdc14 inhibitors at the Bindley Bioscience Center. With Dr. Larisa Avrimova we have identified a half dozen lead inhibitors of ScCdc14 thus far (> 75% inhibition using an optimal phosphopeptide substrate and 40 μM each compound), after screening close to 10,000 of the 180,000 compounds at Purdue. We will compare inhibition towards budding yeast Cdc14, FgCdc14, and MoCdc14. We are looking for competitive inhibitors that are highly specific for the Cdc14 family but broadly reactive across fungal species. This will be tested using our standard phosphatase assay and specificity will be assessed using a panel of purified protein phosphatases representing major evolutionary families.The best hits that meet our criteria for specificity and potency will be modeled into the Cdc14 active site by Dr. Savinov. Synthesis of new derivatives of these base compounds will be developed that contribute to substrate recognition. The initial compounds will likely be broadly specific towards many members of the protein tyrosine phosphatase superfamily (of which Cdc14 is member). We expect to generate high affinity specific inhibitors of the Cdc14 sub-family. Rational design and synthesis of derivatives of the base compounds will be conducted with Dr. Antonella Pepe and Dr. Savinov.Objective 5. Practically useful inhibitory compounds must be both bioavailable and bioactive. The most promising inhibitory compounds from objective 4 will be testedin vivoand in plant infection assays to determine if they exhibit these properties. Dr. Xu has expertise in assessing growth rates and phenotypes ofF. graminearumandM. oryzaeand the ability of these species to infect plants.