Progress 11/15/23 to 11/14/24
Outputs
Target Audience:Our primary target audience are researchers working on organisms from the Arthropoda phylum with a special emphasis on researchers working on herbivory mites. Moreover, as significant fraction of our targets is involved in pesticide/acaricide resistance or metabolism we target as well specialist involved in pest management.Our primary target audience are researchers working on organisms from the Arthropoda phylum with a special emphasis on researchers working on herbivory mites. Moreover, as significant fraction of our targets is involved in pesticide/acaricide resistance or metabolism we target as well specialist involved in pest management. In addition, the results of our work are of interest for researchers working in field of structural biology, protein chemistry, biotechnology and enzymology. Changes/Problems:The biggest challeng was related to the move of the Chruszcz group to Michigan State University. Unfortunately, the move affected our productivity at the end of 2022 and the beginning of 2023. However, currently our team greatly benefits from the move to MSU. We have an excellent workking environment, and a large community of researchers interested in plants. Moreover, the Chruszcz group is significantly closer to the groups from Wstern University, and therefore we can have a relatively frequent meeting in person. What opportunities for training and professional development has the project provided?Since May 15th 2023, there have been several graduate and undergraduate students who have worked on this project in the Chruszcz group. The students worked on production of recombinant versions of the target enzymes, and were involved in characterization of these proteins. We were mainly focused on characterization of enzymatic activity and structure of the target proteins, which subsequently was used to identify physiological substrates of the enzyme and their biological roles. Therefore, the students had an opportunity to become familiar with various biochemical and biophysical methods. The methods/techniques used included DNA manipulation, bacterial protein expression protein purification, spectroscopic methods for testing enzyme activity, mass spectrometry, X-ray crystallography, etc. Students from the Chruszcz group working on this project during the past funding period: Brendan Abiskaroon (10 calendar months) - currently 3rd year graduate student at the Department of Biochemistry and Molecular Biology (MSU). Ricardo Hernandez Arriaza (6 calendar months) - successfully defended his dissertation and recently graduated from the Department Chemistry & Biochemistry (USC). Kriti Khatri (1 calendar month) - successfully defended her dissertation, graduated, and is now working as a post-doctoral worker in the Chruszcz laboratory (MSU). Andrea O'Malley (1 calendar month) - successfully defended her dissertation, graduated, and is now working as a post-doctoral worker in the Chruszcz laboratory (MSU). Vishvendra Chouhan (10 months) - post-baccalaureate student and recent graduate in Biochemistry and Molecular Biology (MSU). Matthew Barr (6 months) - undergraduate student (MSU) majoring in Biochemistry and Biotechnology, as well as Molecular Biology and Genomics, in addition to a Pharmacology and Toxicology minor. The results originated from this grant are also used in teaching undergraduate and graduate students. For example, Dr. Chruszcz was teaching BMB 471 (Advanced Molecular Biology Laboratory - undergraduate level class) in Spring 2024. This course has parts dedicated to enzymes; therefore, experimental data generated for proteins focused on in this proposal were used in this class. Dr. Makris teaches BCH 451 (~80 students each semester), which introduces students from diverse majors to protein structure and enzyme mechanisms. Students from the Makris group working on this project during the past funding period: Hannah Gering (12 calendar months) - currently 5th year graduate student that is also completing her dissertation this semester (defense date: 3/4/25) How have the results been disseminated to communities of interest?The Chruszcz group gave two oral presentations titled "TuGSTm12, but not its close homologue TuGSTm06, is required for detoxification of glucosinolate-myrosinase defenses and Tetranychus urticae adaptation to Arabidopsis" and "Intradiol ring-cleavage dioxygenases and their role in the xenobiotic detoxification and host adaptiation in Tetranychidae mites" by Ricardo Hernandez Arriaza and Brendan Abiskaroon, respectively, at the 13th Spider Mite Genome Meeting (Logroño, La Rioja, Spain; September 11th-14th, 2023). Dr. Chruszcz presented seminar titled "Mites affecting humans - from a source of allergens to agricultural pests) at North Carolina State University (Raleigh, NC; April 6th, 2023). Ricardo Hernandez Arriaza from the Chruszcz group, who was applying for postdoctoral positions, has presented his work on TuUGTs at Stanford University and Massachusetts Institute of Technology. Here we would like to mention that he has secured the postdoctoral research associate position at Stanford University, where he will continue his academic career starting from January 2024. The Chruszcz group presented two posters and two oral presentations (Ricardo Hernandez Arriaza and Brendan Abiskaroon presenting one of each type) in the 62nd annual meeting of the Phytochemical Society of North America at Michigan State University (East Lansing, MI; July 16th-20th, 2023). In summary, during the last funding period there were five oral presentations and two poster presentations that were delivered for researchers from different universities. What do you plan to do during the next reporting period to accomplish the goals?We can cuurrently produce approximately 30 recombinant enzymes originating from TSSM. We will continue to work on enzymes that we started to characterize during the previous reporting periods. A special focus will be placed on cytochrome P450 family of proteins. In the next year we hope to add additional 5 recombinant proteins to our list. In addition, we will continue our work on identification of physiological substrates for CYPs, ID-RCDs, GSTs and UGTs. In parallel to the work on recombinant proteins we will perform gene silencing experiments and perform in situ hybridization for these proteins. At his point we work both on Arabidopsis and tomato model systems and focus on these plants defenses against TSSM.
Impacts
What was accomplished under these goals?
We continued the characterization of proteins from our original target list. In addition, we have expanded the list of targets and we also worked on several proteins from the glutathione S-transferases (GSTs) and uridine-diphosphate glycosyltransferases (UGTs). The addition of these targets was prompted by our and literature data showing involvement of the proteins in adaptation to a new host or their involvement during host shift. TuCAS (β-cyanoalanine synthase) TuCAS is required for adaptation to Arabidopsis, thus making it a desirable target for development of acaricides. During this time, we have developed a high-throughput screening assay as a means of discovery of acaricides targeting TuCAS. This high-throughput assay will be tested this year against ~25,000 thousands of compounds at MSU. Intradiol ring-cleavage dioxygenases (ID-RCDs) Previously, two new ID-RCDs, Tetur20g01790 and Tetur20g01790_KSM were produced in recombinant form. Tetur20g01790_KSM originates from T. kanzawai and was identified, two years ago, as required for mite adaptation to tea plants. Interestingly, these two enzymes only differ in two amino acids: Q127A and T203A (Tetur20g01790 compared to Tetur20g01790_KSM). Since the last update, single mutants have been generated for Q127A and T203A and produced in recombinant form in significant quantities. Previous enzymatic data showed that Tetur20g01790, Tetur20g01790_KSM and Tetur07g05930 (required for Arabidopsis adaptation) were able to metabolize four epicatechins originating from tea: (-)-epicatechin, (-)-epigallocatechin, (-)-epicatechin gallate and (-)-epigallocatechin gallate. Current data analysis from the previous experiments showed that Tetur20g01790_KSM was able to be more efficiently than Tetur20g01790, with only two amino acid residues difference. To investigate the role of each mutation, single mutants were generated. Enzymatic assays have been performed with the single mutants, Q127A and T203A, using the epicatechins mentioned above. However, there was no strong correlation to one mutation alone being required for Tetur20g01790_KSM's ability to more efficiently catalyze these substrates. On the other hand, mass-spectrometry studies to identify the product of the reaction suggests that Tetur07g05930 and Tetur20g01790 do not cleave between the same diol groups, in the case of (-)-epicatechin gallate. Ongoing experiments include the determination of the cleavage sites for all of the epicatechins with Tetur07g05930, Tetur20g01790, Tetur20g01790_KSM, and Tetur20g01790 single mutants. Additional experiments to identify natural substrates for these ID-RCDs are also currently being performed. TuGSTs Previously work began focusing on GSTmu enzymes, which are characteristic for vertebrates and not found in insects. We started with two members, TuGSTmu12 and TuGSTmu06 because they share a high sequence similarity but showed different expression patterns. Further studies using mass spectrometry showed that TuGSTmu12 alone was able to metabolize indole-isothiocyanates (indole-ITC) (Fig. 4). Building upon this, we tested several commercially available ITCs with TuGSTmu06 and TuGSTmu12 to look at differences in activity between the two enzymes for this class of compounds. We further expanded our list of GSTmu enzymes, to observe redundancy or novel activities. For this, we chose GSTmu enzymes in pairs based on similar sequence identity. The first pair selected was TuGSTmu05 and TuGSTmu11 (sharing 96.9% identity) and TuGSTmu07 and TuGSTmu09 for the second pair (sharing 96.9% identity). All four of these enzymes are believed to be involved in mite adaptation towards Arabidopsis. These four additional GSTmu have been recombinantly produced and began structural and functional characterization. TSSM Cytochrome P450s (CYPs) As a means to distinguish CYP392 members for potential active-site differences that lead to alternative substrate-binding specificities and metabolic profiles, we developed approaches that leverage the differential binding of small molecule surrogate inhibitors to the heme-iron as a facile spectroscopic handle. The binding of azole (e.g. ketoconazole) to CYPs results in a characteristic shift in the optical spectrum (i.e. type II shift) that necessitates a close approach to the heme-iron for ligation to occur. This shift in the Soret-band is easily monitored and can be applied to a panel of CYPs. As proof-of-concept, this approach was used with a panel of recombinant purified TSSM CYPs on hand (e.g. A13V1, A13V2, and A16). To test whether we could further delineate highly similar CYP variants (e.g. CYP392A13V1 and CYP392A13V2) with this technique, the binding titrations of each were performed. The resulting dissociation constants of ketoconazole (KD) show that while the A13V1 variant binds ketoconazole with a high-affinity (KD~500 nM), the A13V2 shows a ~10x decrease. This approach, augmented with a series of azoles with different molecular properties (e.g. sterics), bodes well as a facile and crystal-free approach to map active-site differences between various TSSM CYPs as a guide to what types of substrates may have access to the distal pocket for ensuing metabolism. Production of Native and functional TSSM Cytochrome P450 reductase (CPR) Cytochrome P450 reductase is an obligate electron donor to CYPs. Due to difficulties in purifying TSSM CPR in high yields, our studies from the prior funding periods relied on a surrogate (rat CPR, or rCPR) that is commonly used as for activity measurements of many membrane-bound CYPs, such as the TSSM CYPs studied here. A more careful examination of the electron transfer efficiency from rCPR to TSSM CYPs revealed extremely sluggish ET rates, and in other cases (e.g. CYP392A16), very little reduction of the heme-iron. Recognizing this limitation for future metabolism studies, we shifted our attention back to the native TSSM CPR. Overproduction of the TSSM CPR in E. coli resulted in active-protein in microsomal fractions (as assayed with cytochrome c as an electron acceptor). When tested against TSSM CYPs (e.g. A13V2 or A16), the TSSM CPR transfers electrons much more efficiently (~40 fold faster for recombinant A13V2) and now restores the ability to reduce A16 for the first time. As the TSSM CPR microsomes are very easily prepared, we are turning our attention to co-expression strategies to generate TSSM CPR:CYP pairs for downstream metabolomics studies. Development of High-Throughput methods to probe Dioxygenase Substrate Scope and Metabolism During the past funding period, we leveraged the obligate substrate-scope (e.g. diol nature) and mechanism (insertion of two oxygens from atmospheric dioxygen) of TSSM dioxygenases to develop mass spectrometry platforms for metabolic studies. After the addition of 16O2 or 18O2 to individual reactions containing an effective dioxygenase/substrate pair, the samples are mixed at a 1:1 ratio. Successful oxidation of a substrate is anticipated to produce a diagnostic m/z = n, n+2 product doublet. We have tested this approach with a handful of TSSM intradiol dioxygenases prepared by the Chruszcz lab with plant-derived flavonoids and it successfully identifies the metabolic profiles of several enzymes. The role of phenylpropanoids and flavonoids in Arabidopsis defenses against T. urticae Non-adapted mites' fecundity was tested on a suite of Arabidopsis mutants deficient in phenylpropanoid and flavonoid biosynthesis to determine branches of the pathway that are contributing to plant defense. This mutant screen determined that sinapic acid derivatives, kaempferol-derived flavanol glycosides, and anthocyanins are acting defensively in Arabidopsis. The role of four ID-RCDs in T. urticae adaptation to Arabidopsis Experiments with use of RNAi on this group of proteins were performed.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2023
Citation:
Arriaza R.H., Abiskaroon B., Patel M., Daneshian L., Kluza A., Snoeck S., Watkins M.B., Hopkins J.B., Van Leeuwen T., Grbic M., Grbic V., Borowski T., Chruszcz M. (2023) Structural and functional studies reveal the molecular basis of substrate promiscuity of a glycosyltransferase originating from a major agricultural pest. Journal of Biological Chemistry 299, 105421. DOI: 10.1016/j.jbc.2023.105421
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Linn C., OMalley A., Khatri K., Wright E.M., Sebagh D., Grbi? M., Kowal K., Chruszcz M. (2024) Microscopic Menaces: The Impact of Mites on Human Health. International Journal of Molecular Sciences 25, 3675. DOI: 10.3390/ijms25073675
|