Flyversusman: evolutionary impairment of nucleolar targeting affects the degradome ofDrosophila'sTaspase1

Abstract

Human Taspase1 is essential for development and cancer by processing critical regulators, such as the mixed-lineage leukemia protein. Likewise, its ortholog, trithorax, is cleaved by Drosophila Taspase1 (dTaspase1), implementing a functional coevolution. To uncover novel mechanism regulating protease function, we performed a functional analysis of dTaspase1 and its comparison to the human ortholog. dTaspase1 contains an essential nucleophile threonine 195 , catalyzing cis cleavage into its a-and b-subunits. A cell-based assay combined with alanine scanning mutagenesis demonstrated that the target cleavage motif for dTaspase1 (Q 3

Cleaving for growth: threonine aspartase 1—a protease relevant for development and disease

“…Finally, Zhou et al (38) proved that TFIIA is cleaved by threonine aspartase 1 in vitro and in vivo at the conserved cleavage site (QVDG, aa 272–275). The TFIIA ortholog of Drosophila could also be verified as native substrate of Drosophila threonine aspartase 1 similar to the substrate conservation found for MLL/Trx (20). Whereas RNA interference‐based knockdown of threonine aspartase 1 reduces cleavage of TFIIA, processing is completely abolished in Tasp –/– mouse embryonic fibroblasts.…”

“…Regarding substrate specificity and trans cleavage activity, analyses of the proposed crystal structure of threonine aspartase 1 as well as functional inhibitor studies gave insight into the possible catalytic cleavage mechanism. Because threonine aspartase 1‐mediated cleavage of proteins depends on an aspartate in P1 position, it has been postulated that the protease evolutionarily evolved from hydrolyzing asparagine and glycosylasparagine to recognize a conserved peptide motif containing an aspartate at P1 (11, 20). Based on the evolutionary homology between threonine aspartase 1 and type 2 asparaginases, an unusual proteolysis mechanism involving the formation of a succinimide‐hydrate intermediate is suggested (21).…”

“…These residues are generally conserved among the threonine aspartase 1 homologs, but not in other type 2 asparaginases, pointing to a unique substrate preference of threonine aspartase 1 (17). Additionally, improved consensus recognition sequences for the human [Q 3 (F/I/L/V) 2 D 1 G 19 X 2′ D 3′ D 4′ ] as well as the Drosophila threonine aspartase 1 [Q 3 (F/I/L/M) 2 D 1 G 1′ X 2′ X 3′ X 4′ ] could be defined, underlining the strict cleavage preference at P1 (aspartate) and P1′ (glycine) (20, 22). The fact that threonine aspartase 1 is capable of binding inhibitors that contain reactive warheads on either the main peptide backbone or in place of the P1 side chain suggests that threonine aspartase 1 might be able to cleave substrates that have undergone peptide backbone rearrangement (23).…”

“…MLL, also known as histone‐lysine N‐methyltransferase 2A or acute lymphoblastic leukemia 1, encodes a 3969 aa nuclear protein that is homolog to Drosophila Trx regulating coordinated Hox gene expression (11, 24). Trx is proteolytically cleaved by the Drosophila protease homolog, indicating an evolutionary conservation of the MLL/Trx‐threonine aspartase 1 axis (20, 26).…”

“…5 ). The importin‐α/NPM1 switch has evolved in vertebrates as a mechanism to fine‐tune threonine aspartase 1's proteolytic activity and degradome and is not conserved in invertebrates such as D. melanogaster (20). By controlling the nucleocytoplasmic localization of threonine aspartase 1, cells may be able to process specific substrates in the nucleus and/or in the cytoplasm, thereby achieving a greater flexibility in regulating the degradome of threonine aspartase 1 according to the specific requirements in development or disease.…”