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

Bier, Carolin; Hecht, Rouven LSF; Kunst, Lena; Scheiding, Sabine; Wünsch, Desirée; Goesswein, Dorothée; Schneider, Günter; Krämer, Oliver H.; Knauer, Shirley K. LSF; Stauber, Roland H.

Background: The chromosomal translocation t(4;11)(q21;q23) is associated with high-risk acute lymphoblastic leukemia of infants. The resulting AF4NMLL oncoprotein becomes activated by Taspase1 hydrolysis and is considered to promote oncogenic transcriptional activation. Hence, Taspase1’s proteolytic activity is a critical step in AF4NMLL pathophysiology. The Taspase1 proenzyme is autoproteolytically processed in its subunits and is assumed to assemble into an abba-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 AF4NMLL model systems. Methodology/Findings: The consequences of overexpressing the catalytically dead Taspase1 mutant, Taspase1T234V, or the highly attenuated variant, Taspase1D233A, on Taspase1’s processing of AF4NMLL 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 a- or b-subunits showed no trans-dominant effect against the ectopically or endogenously expressed enzyme. Notably, co- expression of the individual a- and b-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. Conclusions: Collectively, 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 AF4NMLL.

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Bier, Carolin / Hecht, Rouven / Kunst, Lena / et al: Overexpression of the Catalytically Impaired Taspase1T234V or Taspase1D233A Variants Does Not Have a Dominant Negative Effect in T(4;11) Leukemia Cells. 2016.

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