Characterization of enzymes involved in ADP-ribose metabolism in trypanosomatids

TEEMU HAIKARAINEN; MARIANA SCHLESINGER; SALOMÉ VILCHEZ LARREA; EZEOGO OBAJI; MOHIT NARWAL; HARIKANTH VENKANNAGARI; MIRTHA M. FLAWIÁ; SILVIA H. FERNÁNDEZ VILLAMIL; LARI LEHTIÖ

Cold Spring Harbor

Congreso; PARP family and ADP-ribosylation; 2016

Cold Spring Harbor Laboratories

Trypanosoma cruzi and Trypanosoma brucei are parasitic protozoa responsible for severe endemichuman and animal diseases. T.cruzi is the causative agent of Chagas disease mainly in Latin America and T.brucei causes sleeping sickness in humans and nagana in cattle in Sub-Saharan Africa.We have studied the enzymes responsible for ADP-ribose metabolism in these parasites and their role in the infection process. In contrast to higher eukaryotes, such as humans, trypanosomatids contain only one poly(ADP-ribose) polymerase (PARP). We have characterized the enzyme from T. cruzi and T. bruceiand shown that their catalytic activity is activated by DNA breaks similar to human DNA-dependent PARPs. Weidentified potent inhibitors for the enzymes in vitroand showed that theyreducepoly-ADP-ribose (PAR) formation in cells. The inhibitors reduced the growth rate ofT. cruziepimastigotesand decreased the amount of amastigotes in cell culture but had no effect on the amount of trypomastigotes. Interestingly,knocking down human PARP1 led to a decrease of the amount of amastigotes and trypomastigotes in cell culture. In contrast to T. cruzi,inhibition ofT. brucei PARPhad no effect on parasite growth. Characterization ofT. brucei PARPrevealed that it binds DNA as a dimer and based on a low-resolution solution structure has a non-symmetrical dimeric DNA-binding mode.The N-terminal domain of the protein was found to be required for nuclear localization but also for high-affinity DNA-binding andDNA-dependent enzymatic activation.In addition to having only one PARP, trypanosomatids also lack most of the proteins required for the hydrolysis of ADP-ribose. The parasites contain only one PAR hydrolyzing enzyme, PARG, and one mono-ADP-ribose hydrolyzing MacroD homolog. We found thatT.brucei and T.cruziMacroD enzymes are capable for hydrolysis of protein linked ADP-ribose as well as O-acetyl-ADP-ribose. Crystal structures of the trypanosomatidmacrodomains revealed a conserved catalytic site with distinct differences to human MacroD1 and MacroD2.The ADP-ribose metabolism in trypanosomatids seems to be highly simplified compared to many higher eukaryotes. The PAR formation and degradation play a role in the infection process of the parasites, although the precise mechanism remains to be elucidated.