Two main Arabidopsis thimet oligopeptidases (AtTOP) involved in stress responses are: (1) thimet metalloendopeptidase 1 (TOP1), found in the mitochondria and chloroplasts, annotated as At5g65620, and (2) thimet metalloendopeptidase 2 (TOP2), found in the cytosol annotated as At5g10540. Both AtTOP1 and AtTOP2 are located on chromosome 5 and share high homology. AtTOP1 and AtTOP2 are zincin-like metalloendopeptidases with the characteristic HEXXH active motif of the M3 clan. Their peptidase activity is related to the oxidative stress triggered by plant immune responses. AtTOPs are involved in plant immune responses through a mechanism regulated by Salicylic Acid (SA); both AtTOP1 and AtTOP2 bind plant SA, which inhibits their peptidase activities. However, we engineered a series of mutations to identify which cysteines are responsible for TOPs dimerization and other oxidative, structureunction related events. Each of the cysteine in TOPs (i.e., six cysteines in TOP1, and four cysteines in TOP2) were independently mutated to alanine, as a single mutant. The dynamics of the oxidative dimerization processes were measured using gel filtration and native gel methods to quantify the dimerization process of both native and mutant TOPs under variable redox potentials ex vivo and in vitro at various GSH/GSSG and DTTox/DTTred ratios, with the underlying hypothesis that the TOP dimerization and enzymatic activities are regulated by changes in the disulfide bond formation that is linked to cellular redox environments. Overall our results indicate that TOP1 is sensitive to changes in the redox environment, while TOP2 is not. The monomer/dimer ratio of TOP1 in solution is higher under highly reducing conditions compared to mildly and highly oxidative environments. Two TOP1 cysteines control the formation of dimers, one located in its N-terminal signal peptide (C52) and the other located in the peptidase domain (C611). These findings bring a mechanistic understanding of TOP1 and TOP2 functions in the plant immune response.
| Identifer | oai:union.ndltd.org:MSSTATE/oai:scholarsjunction.msstate.edu:td-1875 |
| Date | 13 December 2019 |
| Creators | Almohanna, Thualfeqar |
| Publisher | Scholars Junction |
| Source Sets | Mississippi State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
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