LINKING THE OLIGOMERIC STATE TO FUNCTION IN Trypanosoma cruzi HPRT

ROLANDO ROSSI; ALEXANDRA COUSIDO-SIAH; SILVIA FERNANDEZ VILLAMIL; JAVIER SANTOS; MARINA MUÑOZ; EDUARDO HOWARD; WANDA VALSECCHI; JOSE M DELFINO; CAMILE COSTMANN; ALEJANDRO FERRARI

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017

SAIC-SAIB-SAA-SABiol-SAP-SAI-SAFE-SAFIS-SABiofisica-SAH

Hypoxanthine phosphoribosyltransferase (HPRT) is the key enzyme of the purine salvage pathway. It is essential for the survival of trypanosomatids, organisms that -unlike humans- cannot benefit from de novo nucleotide synthesis. As such, this enzyme has been proposed as a potential target for drugs aimed at treating parasitic diseases. This study underscores the effect of different ligands on the structure, stability and function of T. cruzi HPRT (TcHPRT). Light scattering experiments indicate that the protein adopts a tetrameric arrangement. Interestingly, proteolytic removal of the C-terminal region (CTR) yields a well-defined dimer showing somewhat increased activity. Considering that TcHPRT is imported to glycosomes, one can hypothesize that once inside the organelle, the CTR -which includes the import signal- could be excised giving rise to the dimer. The activating effect observed at low concentrations of olpadronate and ibandronate, bisphosphonates (BPs) analogues of the substrate phosphoribosyl pyrophosphate, is more evident for the tetramer than for the dimer. Correspondingly, their inhibitory action at high concentrations is less marked for the former. These facts fall into place in our proposed cooperative model for the activity of this oligomeric enzyme. Strikingly, the inhibitory effect is essentially null when assayed against human HPRT. To assess the action of these small molecules within a cellular environment, we exposed cultures of epimastigotes to selected BPs, demonstrating significant growth inhibition. To visualize the enzyme forms that might be interacting with those ligands, we expressed both proteins in Leishmania tarentolae as fusion constructs with GFP. Western blot analyses reveal that TcHPRT is not processed to a shorter form in this system. This new molecular and cellular knowledge becomes most relevant for the design of innovative parasite-targeted therapeutics.Keywords: T. cruzi, oligomeric state, enzymatic activity, maturation Wanda M. Valsecchi1, Silvia Hebe Fernández Villamil1, Alejandro Ferrari1, Eduardo Howard2, Alexandra Cousido-Siah2, Camille Kostmann2, Marina Cecilia Muñoz1, Rolando Carlos Rossi1, José M. Delfino1*, Javier Santos1*1. Departamento de Química Biológica, IQUIFIB (UBA-CONICET). Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires2. Departamento de Biología Integrativa, IGBMC, CNRS, INSERM, Universidad de Strasbourg, Illkirch, Francia