Overexpression of the Catalytically Impaired Taspase1T234V or Taspase1D233A Variants Does Not Have a Dominant Negative Effect in T(4;11) Leukemia Cells

Abstract

BackgroundThe chromosomal translocation t(4;11)(q21;q23) is associated with high-risk acute lymphoblastic leukemia of infants. The resulting AF4•MLL oncoprotein becomes activated by Taspase1 hydrolysis and is considered to promote oncogenic transcriptional activation. Hence, Taspase1’s proteolytic activity is a critical step in AF4•MLL pathophysiology. The Taspase1 proenzyme is autoproteolytically processed in its subunits and is assumed to assemble into an αββα-heterodimer, the active protease. Therefore, we investigated here whether overexpression of catalytically inactive Taspase1 variants are able to interfere with the proteolytic activity of the wild type enzyme in AF4•MLL model systems.Methodology/FindingsThe consequences of overexpressing the catalytically dead Taspase1 mutant, Taspase1T234V, or the highly attenuated variant, Taspase1D233A, on Taspase1’s processing of AF4•MLL and of other Taspase1 targets was analyzed in living cancer cells employing an optimized cell-based assay. Notably, even a nine-fold overexpression of the respective Taspase1 mutants neither inhibited Taspase1’s cis- nor trans-cleavage activity in vivo. Likewise, enforced expression of the α- or β-subunits showed no trans-dominant effect against the ectopically or endogenously expressed enzyme. Notably, co-expression of the individual α- and β-subunits did not result in their assembly into an enzymatically active protease complex. Probing Taspase1 multimerization in living cells by a translocation-based protein interaction assay as well as by biochemical methods indicated that the inactive Taspase1 failed to assemble into stable heterocomplexes with the wild type enzyme.ConclusionsCollectively, our results demonstrate that inefficient heterodimerization appears to be the mechanism by which inactive Taspase1 variants fail to inhibit wild type Taspase1’s activity in trans. Our work favours strategies targeting Taspase1’s catalytic activity rather than attempts to block the formation of active Taspase1 dimers to interfere with the pathobiological function of AF4•MLL.

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

“…To experimentally demonstrate that the presumed nucleophile is indeed required for dTaspase1's proteolytic activity, Thr 195 was mutated into Val, in analogy to the catalytically inactive human Taspase1 T134V mutant (10, 13). To investigate protease function in living cells, we first tested whether our translocation biosensor assay could be adapted to Drosophila cells (8, 13) (Fig.…”

“…Hence, hydrophobicity in combination with size is required for productive cleavage. Whereas in both enzymes, position P2′ is highly flexible, specific residues at positions P3′ and P4′ seem to be only required for human Taspase1 (10, 12), explaining why the human Taspase1 degradome is much more restricted. In contrast to previous predictions (9, 22), the identified cleavage motif for dTaspase1 strongly implies an enlarged degradome containing 70 potential substrates in Drosophila in contrast to the rather limited 27 targets predicted for humans (13).…”

“…Thus, the discovery of Taspase1 founded a new class of endopeptidases that use the N‐terminal threonine of its mature β‐subunit as the active site. In humans, this N‐terminal threonine (Thr 234 ) is generated by autoproteolysis of the Taspase1 proenzyme (referred to as cis cleavage) into the 2 subunits α and β, which assemble into an αβ‐monomer representing the active protease (10, 11). Human Taspase1 is accumulating at the nucleus/nucleolus but is exploiting the dynamic importin‐α (IMP‐α)/nucleophosmin (NPM1) axis, allowing the processing of substrates in both the nucleus and the cytoplasm (12).…”

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

“…The nucleophile Thr 234 is generated by autoproteolysis (cis cleavage) of the proenzyme in 2 subunits, α28 and β22, which assemble to form the active protease. Whereas crystal structures of threonine aspartase 1 and other type 2 asparaginases suggest the formation of an asymmetric abba‐heterotetramer (17), in vivo studies revealed that threonine aspartase 1 is already active as αβ‐monomer (18, 19). The essential role of the catalytically active nucleophile Thr 234 for the functionality of threonine aspartase 1 is significant.…”

“…6). As the threonine aspartase 1 proenzyme is autoproteolytically cleaved and assumed to assemble into a heterodimer, overexpression of inactive threonine aspartase 1 mutants would inhibit the formation of active protease dimers (genetic inhibition) (18). However, recent in vivo studies demonstrated that endogenous threonine aspartase 1 predominantly exists as an ab‐monomer, and over‐expression of inactive mutants has no effect on its proteolytic activity (18).…”