Evolutionary divergence of Threonine Aspartase1 leads to species-specific substrate recognition
-
Désirée Wünsch
, Angelina Hahlbrock , Christina Heiselmayer , Sandra Bäcker , Christian Schrenk , Franziska Benne , Oliver Schilling and Shirley K. Knauer
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.
Acknowledgments
We thank Sandra Friedl for her excellent technical assistance.
Conflict of interest: The authors declare that they have no conflict of interest.
Grant support: Fritz Thyssen Stiftung.
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The online version of this article (DOI: 10.1515/hsz-2014-0318) offers supplementary material, available to authorized users.
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Articles in the same Issue
- Frontmatter
- Reviews
- Sortase-mediated backbone cyclization of proteins and peptides
- Making the LINC: SUN and KASH protein interactions
- Enhancers, enhancers – from their discovery to today’s universe of transcription enhancers
- Minireview
- Recent advances and concepts in substrate specificity determination of proteases using tailored libraries of fluorogenic substrates with unnatural amino acids
- Research Articles/Short Communications
- Genes and Nucleic Acids
- microRNA-210 is involved in the regulation of postmenopausal osteoporosis through promotion of VEGF expression and osteoblast differentiation
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- C-terminal truncation of a Tat passenger protein affects its membrane translocation by interfering with receptor binding
- Aspartate 496 from the subsite S2 drives specificity of human dipeptidyl peptidase III
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- Purification and characterisation of recombinant His-tagged RgpB gingipain from Porphymonas gingivalis