title: Evolutionary divergence of Threonine Aspartase1 leads to species-specific substrate recognition.
abstract: Proteases are key regulators of life. Human Threonine Aspartase1 processes substrates, such as the mixed-lineage leukemia (MLL) protein, containing two cleavage sites, CS1 and CS2. Likewise, MLL's Drosophila ortholog trithorax is cleaved by Drosophila Threonine Aspartase1 (dTasp), suggesting a mechanistic coevolution. However, a detailed analysis of dTasp's function was missing so far. Here, active and inactive dTasp mutants allowed to compare substrate recognition and cleavage site selectivity of human and Drosophila enzymes. In contrast to the human protease, our cell-based assay revealed a preferential processing of CS2-like (QLD↓Gx[xD/Dx]) targets for dTasp, whereas cleavage of CS1-like targets (QVD↓Gx[xD/Dx]) was significantly impaired. Systematic mutagenesis of the CS2 sequence defined the motif x[FILMW]D↓Gx[xD/Dx] as the consensus cleavage sequence for dTasp. Substrate species selectivity of the enzymes was uncovered by demonstrating that dTasp cleaves Drosophila TFIIA, but not the human ortholog, suggesting evolutionary divergence of TFIIA downstream networks. Also, Drosophila USF2 was neither predicted nor cleaved by dTasp. Moreover, we found that dTasp cleavage site selectivity is independent of heterocomplex formation, as dTasp exists predominantly as an αβ-monomer. Collectively, we provide novel insights into evolutionary similarities and divergence concerning Threonine Aspartase1 function in different species, which may aid to dissect and better target human Threonine Aspartase1 in malignancies.
authors.forename: Désirée
authors.initials: D
authors.lastname: Wünsch
authors.forename: Angelina
authors.initials: A
authors.lastname: Hahlbrock
authors.forename: Christina
authors.initials: C
authors.lastname: Heiselmayer
authors.forename: Sandra
authors.initials: S
authors.lastname: Bäcker
authors.forename: Christian
authors.initials: C
authors.lastname: Schrenk
authors.forename: Franziska
authors.initials: F
authors.lastname: Benne
authors.forename: Oliver
authors.initials: O
authors.lastname: Schilling
authors.forename: Shirley K
authors.identifier: http://orcid.org/0000-0003-4321-0924
authors.initials: SK
authors.lastname: Knauer
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004330:Drosophila / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D029721:Drosophila Proteins / Q000737:chemistry / Q000378:metabolism*; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D055503:Protein Multimerization; D013045:Species Specificity; D013379:Substrate Specificity; D035561:Transcription Factor TFIIA / Q000378:metabolism; D051720:Upstream Stimulatory Factors / Q000378:metabolism
chemical_list: D029721:Drosophila Proteins; D035561:Transcription Factor TFIIA; D051720:Upstream Stimulatory Factors; D010450:Endopeptidases; C526349:taspase1, Drosophila