The variable C‐terminal domain of human type II topoisomerases as a functionally relevant therapeutic target
Authors: Deweese Joseph E. , Hoang Kristine G. , Menzie Renee A. , Fief Cole A. , Ayes Carmen A. , Keck Jacob M. , Wilson James T. , Cordova Salvador , Nelson Chase W. 
Published: June 2021

Topoisomerase II is a critical enzyme involved in unknotting and detangling DNA during replication, transcription, and cell division. Humans have two isoforms of topoisomerase II, α (Top2A) and β (Top2B), originating from genes on separate chromosomes and displaying distinct functional roles. In addition, these enzymes are the target of several successful anticancer therapeutics. Unfortunately, these agents are nonselective and a growing body of evidence implicates Top2B as a mediator of off‐target toxicities, while Top2A is likely a better target for disruption of cancer cell growth. The isoforms share about 65.2% amino acid identity primarily in the N‐terminus and the core regions, which contain the catalytic domains of the enzyme and the regions targeted by all clinically‐relevant anticancer agents. On the other hand, the C‐termini of the human enzymes share only ~30% amino acid identity across ~400 amino acids. The carboxy terminus does not participate in catalysis, but has been hypothesized to have a role in the regulation of topoisomerase II activity, which may explain how these proteins are independently regulated. Since the C‐terminal region has been largely unexplored, we undertook an analysis to identify key differences between the C‐termini that may help explain the differential regulation of the topoisomerase II isoforms. The relative rates of synonymous (dS) and nonsynonymous (dN) DNA substitutions between the isoforms suggests that these sequences are under purifying selection for functional constraint (dN/dS = 0.25; P < 0.0001, Z test, 1,000 bootstrap replicates). The 434 C‐terminal codons, however, had a relatively high dN/dS ratio of 0.59 (P < 0.0001), reflecting elevated amino acid diversity. In addition, bioinformatic evidence from Phosphosite (Phosphosite.org) indicates that nearly half (91/191 for Top2A) of the putative post‐translational modification (PTM) sites are found in the C‐terminus. Of the PTM sites found in the Top2A C‐terminus, over half (~50) are distinct from those found in Top2B. Aside from sequence characteristics, protein‐protein interaction data from the Biogrid database (thebiogrid.org) indicate that ~143 proteins have interaction evidence with either Top2A or TOP2B. Of these proteins, only ~34 are confirmed to interact with both isoforms and several are known to interact with the C‐terminal domain of Top2A or Top2B. Taken together, these data suggest distinct sequence, PTM, and interaction profile characteristics for the C‐termini of the isoforms of Top2, which may provide critical insight into the differential regulation of these enzymes. We hypothesize that these results provide the foundation for topoisomerase II isoform‐specific targeting strategies for anticancer therapeutics.Support or Funding InformationJED, KGH, RAM, CAF, CAA, JMK, and JTW were supported by funds from LUCOPHS. CWN was supported by a Gerstner Scholars Fellowship from the Gerstner Family Foundation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.