ADVANCED TECHNOLOGIES FOR THE GENETIC IMPROVEMENT OF POULTRY (WAS NC-168)

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1008514
• Project No.: FLA-MCS-005482
• Multistate No.: NC-_old1170
• Project Start Date: Nov 1, 2015
• Project End Date: Sep 30, 2018

Project Director
Ferraro, MA, J.

Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611

Performing Department
Microbiology and Cell Science

Non Technical Summary
Chicken is one of the most important sources of animal foods worldwide and the functionalannotation of enzymes and protein modifications is necessary for research on chicken health.Ubiquitination and phosphorylation, controlled by deubiquitnases and kinases respectively, areimportant protein modifications, regulating essential cellular processes, and anomalies in thesemodifications have been implicated in animal diseases. However, ubiquitination andkinases/deubiquitinases have never been systematically studied in chicken. We propose to apply amolecular tool to identify tissue specific expression of this important class of enzymes anddisseminate this information to poultry researchers via a database that can be queried by the user.Our hypothesis is that chicken proteome contains multiple deubiquitinases/kinases, and that theirdistribution in tissues is unique. We will use functional proteomics to annotate catalytically activekinases/deubiquitinases in chicken tissues. The expected outcomes are: (i) identification of activeDUBs and kinases in chicken tissues (ii) Experimental validation of computationally predictedenzymes, (iii) a set of genome-scale experimental DUBs/kinases available in a public database toimprove computational prediction accuracy and inform about the existence of this class of proteinsin other avian species. The positive impact of this project is the ability of animal health researchersto identify drug discovery targets in animal growth, metabolism, or in disease resistance.

Animal Health Component

100%

Research Effort Categories

Basic

70%

Applied

20%

Developmental

10%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
304	3299	1040	100%

Knowledge Area
304 - Animal Genome;

Subject Of Investigation
3299 - Poultry, general/other;

Field Of Science
1040 - Molecular biology;

Keywords

proteomics

poultry

chicken

enzymes

kinases

deubiquitinases

Goals / Objectives
Create and share data and technology to enhance the development and application of genomics and systems biology in poultry.

Project Methods
The following methods will be used:- proteomics methods based on activity profiling and post-translational modification enrichment- mutagenesis/ cloning- functional assays such as viability/toxicity assays, and enzymatic assays- cell culture of avian cells

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Poultry researchers, undergraduate and graduate students Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Dr Edelmann recruited an unpaid undergraduate student to the project who is performing bioinformatics analysis of chicken enzymes as a part of her bioinformatics major. The project provided this student with an opportunity to learn new approaches and contribute to this project. Gary Jones: Master's student at University of Florida, Department of Microbiology and Cell Science was trained in chicken bioinformatics and proteomics. Sedona Koenders: Undergraduate student, at University of Florida, Department of Microbiology and Cell Science completed Independent study work (BSC4913) with Dr. Edelmann focused on poultry genomics and bioinformatics, working towards a minor in Bioinformatics. Winnie Hui: PhD student, has helped with bioinformatics and western blot analysis of the tissues, for identification of DUBs and kinases. She also helped to write and evaluate the manuscript, which was now submitted to Scientific Reports. Alyssa Walker, PhD student, have helped troubleshoot experiments related to chemical tagging of DUBs with the active site-directed probe. She has been trained to work on the activity-based profiling. How have the results been disseminated to communities of interest?publications and conferences; website/database design What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We accomplished the following: We demonstrated that chicken UCH-L5 protein leads to increased pro-inflammatory cell death in chicken macrophages and we showed that chemical inhibition of this enzyme can be used to limit this cell death. This can really help to limit inflammation in chickens suffering from Salmonella if it is taken to the next level. We described the contribution of proteomics in understanding of Listeria monocytogenes pathology, which was done as a collaborative effort and yielded a book published by Elsevier We developed a better method for chemical proteomics for enzyme annotation in animal tissue, and established focused-ultrasonicator-based tissue lysis method as a sample preparation method compatible with activity-based protein assays, and published these results. We identified active kinases in spleen and liver tissues in chicken based on their reactivity with the ATP and ADP desthiobiotin acyl phosphate probes combined with mass spectrometry. We identified 188 chicken kinases and their ATP-binding regions to create a tissue-specific atlas of active kinase expression in chicken. We also determined the possible functions of these kinases by utilizing bioinformatics approach by comparing functional pathways and disease involvement of human, murine and rat orthologs of these kinases. We performed chemical proteomic profiling of active deubiquitinases (DUBs) by utilizing active-site directed ubiquitin (Ub)-vinyl sulfone (VS)-HA probe labeling combined with western blot-based or mass spectrometric identification of DUBs. By using these techniques we identified 29 DUBs in cecum, liver and spleen tissues. We have identified that DUBs such as USP5, USP4, UCH-6, UNP, USP7, UCH-L5, USP9x, USP10, USP19, USP47, OTUD6B, and USP8 are the top 12 DUBs identified in liver while in spleen there were USP5, USP4, UCH-6, UNP, UCH-L5, UCH-L1, USP9x, USP10, USP19, USP16, OTUD6B, and USP8. Cecum overall contained less active DUBs, and the most active DUBs were USP5, USP4, UCH-6, UNP, USP7, UCH-L5, UCH-L1, USP47, USP8, USP10, USP19 and USP9x.

Publications

• Type: Journal Articles Status: Submitted Year Published: 2019 Citation: " Bindu Nanduri, Cathy R. Gresham, Winnie W. Hui, Mark Ou, Richard H. Bailey and Mariola J. Edelmann* Identifying chicken liver and spleen kinome by chemical proteomics. Scientific reports. under review
• Type: Journal Articles Status: Published Year Published: 2018 Citation: " Sheppe, A.E., Kummari, E., Richards, A., Hui, W.W., Lee, J.H., Mangum, L., Borazjani, A., Ross, M. and Edelmann, M.J., 2018. PGE2 augments inflammasome activation and M1 polarization in macrophages infected with Salmonella Typhimurium and Yersinia enterocolitica. Frontiers in Microbiology, 9, p.2447.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Edelmann, MJ, Use of Chemical Proteomics to Identify Novel Chicken Kinases and Deubiquitinases, invited oral presentation at Plant and Animal Genome XXVI Conference, San Diego, 2018
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Bindu Nanduri, Richard H Bailey, Sedona Koenders, Mark S Ou and, Mariola J Edelmann. Distribution of Kinases in Chicken Tissue. USDA Animal genomics PD annual meeting. San Diego, CA Jan 13, 2018

Progress 11/01/15 to 09/30/18

Outputs
Target Audience:Poultry researchers, undergraduate and graduate students Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Dr Edelmann recruited an unpaid undergraduate student to the project who is performing bioinformatics analysis of chicken enzymes as a part of her bioinformatics major. The project provided this student with an opportunity to learn new approaches and contribute to this project. Gary Jones: Master's student at University of Florida, Department of Microbiology and Cell Science was trained in chicken bioinformatics and proteomics. Sedona Koenders: Undergraduate student, at University of Florida, Department of Microbiology and Cell Science completed Independent study work (BSC4913) with Dr. Edelmann focused on poultry genomics and bioinformatics, working towards a minor in Bioinformatics. Winnie Hui: PhD student, has helped with bioinformatics and western blot analysis of the tissues, for identification of DUBs and kinases. She also helped to write and evaluate the manuscript, which was now submitted to Scientific Reports. Alyssa Walker, PhD student, have helped troubleshoot experiments related to chemical tagging of DUBs with the active site-directed probe. She has been trained to work on the activity-based profiling. How have the results been disseminated to communities of interest?Publications, talks and conference presentations What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We accomplished the following: We demonstrated that chicken UCH-L5 protein leads to increased pro-inflammatory cell death in chicken macrophages and we showed that chemical inhibition of this enzyme can be used to limit this cell death. This can really help to limit inflammation in chickens suffering from Salmonella if it is taken to the next level. We described the contribution of proteomics in understanding of Listeria monocytogenes pathology, which was done as a collaborative effort and yielded a book published by Elsevier We developed a better method for chemical proteomics for enzyme annotation in animal tissue, and established focused-ultrasonicator-based tissue lysis method as a sample preparation method compatible with activity-based protein assays, and published these results. We identified active kinases in spleen and liver tissues in chicken based on their reactivity with the ATP and ADP desthiobiotin acyl phosphate probes combined with mass spectrometry. We identified 188 chicken kinases and their ATP-binding regions to create a tissue-specific atlas of active kinase expression in chicken. We also determined the possible functions of these kinases by utilizing bioinformatics approach by comparing functional pathways and disease involvement of human, murine and rat orthologs of these kinases. We performed chemical proteomic profiling of active deubiquitinases (DUBs) by utilizing active-site directed ubiquitin (Ub)-vinyl sulfone (VS)-HA probe labeling combined with western blot-based or mass spectrometric identification of DUBs. By using these techniques we identified 29 DUBs in cecum, liver and spleen tissues. We have identified that DUBs such as USP5, USP4, UCH-6, UNP, USP7, UCH-L5, USP9x, USP10, USP19, USP47, OTUD6B, and USP8 are the top 12 DUBs identified in liver while in spleen there were USP5, USP4, UCH-6, UNP, UCH-L5, UCH-L1, USP9x, USP10, USP19, USP16, OTUD6B, and USP8. Cecum overall contained less active DUBs, and the most active DUBs were USP5, USP4, UCH-6, UNP, USP7, UCH-L5, UCH-L1, USP47, USP8, USP10, USP19 and USP9x.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: " Kummari E, Alugubelly N, Hsu CY, Dong B, Nanduri B, Edelmann MJ. Correction: Activity-Based Proteomic Profiling of Deubiquitinating Enzymes in Salmonella-Infected Macrophages Leads to Identification of Putative Function of UCH-L5 in Inflammasome Regulation. PLoS One. 2015 Sep 15;10(9):e0138635. doi: 10.1371/journal.pone.0138635.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: " Use of focused ultrasonication in activity-based profiling of deubiquitinating enzymes in tissue. Nanduri B, Shack LA, Rai AN, Epperson WB, Baumgartner W, Schmidt TB, Edelmann MJ. Anal Biochem. 2016 Dec 15;515:9-13. doi: 10.1016/j.ab.2016.09.016. Epub 2016 Sep 20. PMID: 27663132
• Type: Journal Articles Status: Published Year Published: 2018 Citation: " Sheppe, A.E., Kummari, E., Richards, A., Hui, W.W., Lee, J.H., Mangum, L., Borazjani, A., Ross, M. and Edelmann, M.J., 2018. PGE2 augments inflammasome activation and M1 polarization in macrophages infected with Salmonella Typhimurium and Yersinia enterocolitica. Frontiers in Microbiology, 9, p.2447.
• Type: Journal Articles Status: Submitted Year Published: 2019 Citation: Bindu Nanduri, Cathy R. Gresham, Winnie W. Hui, Mark Ou, Richard H. Bailey and Mariola J. Edelmann* Identifying chicken liver and spleen kinome by chemical proteomics. Scientific reports. (in review)
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Edelmann MJ, The growing role of functional proteomics in combating infectious diseases, Invited Seminar, University of Florida, May 11th 2015
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: " Kummari E., Alugubelly N., Lee J.H. , Mangum L. , Borazjani A., Ross M., Edelmann, M.J.* Characterization of prostaglandins released from human macrophages infected with enteric bacteria. The 2nd Annual SECIM Metabolomics Workshop and Symposium, SECIM, FL, May 11-14 2015
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: " Evangel Kummari, Jung Hwa Lee, Abdolsamad Borazjani, Mariola Edelmann, Matthew Ross. Characterization of Serine Hydrolases Using Chemoproteomic Profiling Approach in Chicken Macrophages with Salmonella Infection. ASM, New Orleans, May 30-June 2nd 2015
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: " Evangel Kummari, Mariola Edelmann et al. Activity-based proteomic profiling of deubiquitinating enzymes in Salmonella-infected macrophages reveals involvement of UCH-L5 in inflammasome activation. ASM, New Orleans, May 30-June 2nd 2015
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: " Bindu Nanduri, Richard H Bailey, Sedona Koenders, Mark S Ou and, Mariola J Edelmann. Distribution of Kinases in Chicken Tissue, 1. International Plant and Animal Genome XXVI Conference, San Diego, CA Jan 13-17, 2017. (Poster).
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Bindu Nanduri, Richard H Bailey, Sedona Koenders, Mark S Ou and, Mariola J Edelmann. Distribution of Kinases in Chicken Tissue, MidSouth Computational Biology and Bioinformatics Society. Little Rock, AR, Mar 23-25, 2017. (Poster).
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Bindu Nanduri, Richard H Bailey, Sedona Koenders, Mark S Ou and, Mariola J Edelmann. Distribution of Kinases in Chicken Tissue. USDA Animal genomics PD annual meeting. San Diego, CA Jan 13, 2018. (Poster).
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Edelmann, MJ, Use of Chemical Proteomics to Identify Novel Chicken Kinases and Deubiquitinases, invited oral presentation at Plant and Animal Genome XXVI Conference, San Diego, 2018
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Edelmann MJ, Functional analysis of deubiquitinating enzymes in enteric infections. Seminar. COBRE NIH Symposium, 6/28/2015-6/30/2015, Mississippi State University

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:The target audience is scientific community, which include undergraduate and graduate students. Changes/Problems:While conducting experiments to complete specific aim 1, characterization of deubiquitinases in chicken, we had to repeat the experiment to address an unanticipated technical difficulty, which caused considerable delay in progress. What opportunities for training and professional development has the project provided?Gary Jones: Master's student at University of Florida, Department of Microbiology and Cell Science was trained in chicken bioinformatics and proteomics. Sedona Koenders: Undergraduate student, at University of Florida, Department of Microbiology and Cell Science completed Independent study work (BSC4913) with Dr. Edelmann focused on poultry genomics and bioinformatics, working towards a minor in Bioinformatics. How have the results been disseminated to communities of interest?We attended seminars and conferences as listed to present our findings to the interested audience: Seminars/conferences: Bindu Nanduri, Richard H Bailey, Sedona Koenders, Mark S Ou and, Mariola J Edelmann. Distribution of Kinases in Chicken Tissue, 1. International Plant and Animal Genome XXVI Conference, San Diego, CA Jan 13-17, 2017. (Poster), Bindu Nanduri, Richard H Bailey, Sedona Koenders, Mark S Ou and, Mariola J Edelmann. Distribution of Kinases in Chicken Tissue, MidSouth Computational Biology and Bioinformatics Society. Little Rock, AR, Mar 23-25, 2017. (Poster) Bindu Nanduri, Richard H Bailey, Sedona Koenders, Mark S Ou and, Mariola J Edelmann. Distribution of Kinases in Chicken Tissue. USDA Animal genomics PD annual meeting. San Diego, CA Jan 13, 2017. (Poster). What do you plan to do during the next reporting period to accomplish the goals?We will focus on the publication of our data in peer-reviewed journal.

Impacts
What was accomplished under these goals? We accomplished the following: a) We performed chemical proteomics , using active site probes for deubiquitinases and identified ~20 DUBs from chicken spleen, liver and crop tissues. b) We identified approximately 200 kinases in spleen and liver tissues in chicken based on their reactivity with the ATP and ADP desthiobiotin acyl phosphate probe. The assumed functions of these kinases were analyzed by comparing functional pathways and disease involvement of human, murine and rat orthologs of these kinases and a manuscript describing these results is in preparation. c) We helped to add our proteomic data into Chickspress database so these can be available to other researchers

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Bindu Nanduri, Richard H Bailey, Sedona Koenders, Mark S Ou and, Mariola J Edelmann. Distribution of Kinases in Chicken Tissue, MidSouth Computational Biology and Bioinformatics Society. Little Rock, AR, Mar 23-25, 2017
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Bindu Nanduri, Richard H Bailey, Sedona Koenders, Mark S Ou and, Mariola J Edelmann. Distribution of Kinases in Chicken Tissue, 1. International Plant and Animal Genome XXVI Conference, San Diego, CA Jan 13-17, 2017.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Bindu Nanduri, Richard H Bailey, Sedona Koenders, Mark S Ou and, Mariola J Edelmann. Distribution of Kinases in Chicken Tissue. USDA Animal genomics PD annual meeting. San Diego, CA Jan 13, 2017.

Progress 11/01/15 to 09/30/16

Outputs
Target Audience:Scientists, students Changes/Problems:The major problem was to transfer the funding to UF so we can continue the work on timely basis. This caused a delay, otherwise the work has been performed well and with no technical difficulties. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Peer-reviewed publication, regional conferences What do you plan to do during the next reporting period to accomplish the goals?We will continue working on enzyme analysis in tissue, this time performing targeted proteomics approach to quantify the detected kinases in the tissues.

Impacts
What was accomplished under these goals? Objectives Create and share data and technology to enhance the development and application of genomics and systems biology in poultry. Phosphorylation is a key post-translational modification controlled by kinases, and it regulates essential cellular processes, therefore various anomalies in this modification have been implicated in animal diseases. However, kinases have never been systematically studied in chicken. Therefore, annotation of kinases in chicken is necessary for studying chicken health/disease. To map chicken kinases in tissue we used two different approaches. First, we used kinase domain mapping based on published data and Chickspress database (University of Arizona, http://geneatlas.arl.arizona.edu), which incorporates NCBI and Ensembl gene models with the protein expression data from specimens of multiple tissues from both genders. This bioinformatic approach allowed us to detect chicken kinases in various tissues by using peptide evidence, but it was limited to identification of 7 kinases in spleen and 2 in liver. Second, we utilized active-site directed probes combined with mass spectrometry to identify over 200 ATP- and ADP-binding sites in 140 chicken kinases in spleen and liver combined. This experiment led to identification of novel kinases, 71% of which were previously uncharacterized in chicken. By utilizing this chemical proteomics, which is invaluable in novel enzyme identification, we improved existing annotation of these enzymes, which drastically expands current annotations available via Uniprot and other resources. We compared several sequences of detected kinases to known mouse, rat and human orthologues to understand conservation of these ATP-binding domains. Our study confirmed that chicken proteome contains multiple kinases, some of which have not been previously annotated. We also mapped novel nucleotide-binding lysine residues in these kinase and show that these kinases have a unique abundance in chicken tissue. This information will be made publically available in collaboration with AgBase (Arizona Univ. and Mississippi State University).

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Nanduri B, Shack LA, Rai AN, Epperson WB, Baumgartner W, Schmidt TB, Edelmann MJ. Use of focused ultrasonication in activity-based profiling of deubiquitinating enzymes in tissue. Anal Biochem. 2016 Dec 15;515:9-13.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Alugubelly, Navatha, et al. "Analysis of differentially expressed proteins in Yersinia enterocolitica-infected HeLa cells." Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics 1864.5 (2016): 562-569.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Lopez-Castejon, Gloria, and Mariola J. Edelmann. "Deubiquitinases: Novel Therapeutic Targets in Immune Surveillance?." Mediators of Inflammation 2016 (2016).