The detailed architecture of a crucial component of a protein linked to the bone development disorder cherubism and involved in a myriad of cellular processes has been identified by the Samuel Lunenfeld Research Institute's Drs. Frank Sicheri, Tony Pawson and Sebastian Guettler, in collaboration with Dr. Robert Rottapel at the Ontario Cancer Institute.
The discovery is the first structural insight into precisely how the enzyme correctly identifies its targets, or substrates. The work provides researchers with a greater understanding of Tankyrase's cellular control processes, and may also lead to the development of new designer drugs to treat cancer.
"Until now, we did not understand, from a structural perspective, how Tankyrase identifies its substrates," said Dr. Sicheri, Lunenfeld Senior Investigator and one of the lead authors of the study. "At atomic resolution, we now have a clearer picture of what these substrates may be, and have new insight into possible novel functions of Tankyrase."
Tankyrase is a poly(ADP-ribose)polymerase (PARP) -- one protein of a family of enzymes that modify other proteins with chains of ADP-ribose and affect many cellular processes. The modification reactions carried out by Tankyrase can directly alter some proteins' functions, bring proteins together in protein complexes, or can mark others for degradation.Initially intrigued by Tankyrase because of its involvement in cherubism (a rare genetic disorder caused by mutations in the signaling protein 3BP2), the researchers built upon the findings of Dr. Rottapel's laboratory. This laboratory found that Tankyrase normally recognizes 3BP2 and targets it for destruction. The amino acids mutated in cherubism coincide with precisely the region in 3BP2 that is recognized by Tankyrase, or the "Tankyrase binding motif." Cherubism mutations in 3BP2 prevent binding of Tankyrase and therefore result in the accumulation of 3BP2 protein in the cell. Dr. Rottapel's findings also appear in the same issue of Cell. The goal of Dr. Sicheri and his team's work was to uncover the exact mechanism by which Tankyrase recruits its substrates, to explain why cherubism mutations in 3BP2 disrupt Tankyrase binding and thereby learn more about how the enzyme works.Using x-ray crystallography, the team determined the structures of the portion of Tankyrase responsible for substrate binding, bound to a range of different substrates including 3BP2. Using a technique known as fluorescence polarization the researchers then determined the essential signature of the Tankyrase binding motif by which Tankyrase identifies its substrates.
With Dr. Evangelia Petsalaki from Dr. Tony Pawson's laboratory, the researchers scanned the entire inventory of human proteins, searching for the signature sequence that is recognized by Tankyrase, correctly predicting many possible new substrates for the enzyme. The result: a deeper understanding of the biology behind Tankyrase's cellular activities.
"Our work provides answers to two big questions. Firstly, we obtained a visual snapshot of how Tankyrase recognizes its substrates and how mutations characteristic of cherubism lead to illness," said Dr. Guettler, a post-doctoral Fellow in Dr. Sicheri's and Dr. Pawson's labs and first author of the study. "Secondly, we learned more about the possible cellular tasks performed by Tankyrase. The apparent abundance of potential Tankyrase targets and the variety of cellular functions they perform suggests that the complexity of Tankyrase's biological functions has been underappreciated to date."Inhibitors of PARPs, and among them Tankyrase, have gained considerable attention recently as potential new anti-cancer agents. Inhibition of Tankyrase function may hold promise for treating certain breast cancers as well as other cancers, and therefore the present study may help refine treatment strategies for blocking Tankyrase.