CHARACTERIZATION OF Toxoplasma gondii TGDJ1 TYPE I HSP40

MUNERA LÓPEZ, JONATHAN; FIGUERAS MARÍA JULIA; FENOY, IGNACIO; ANGEL, SERGIO

Mar del Plata

Congreso; X congreso de protozoología y enfermedades parasitarias; 2014

The HSP40 proteins, many times called the J family of proteins, lead HSP70 to facilitate diverse cellular processes like protein folding, protein translocation across membranes, cellular signal transduction, DNA replication and protein degradation. Little is known about this HSP40 family in Toxoplasma gondii. Data mining of Toxoplasma database has shown that this family is quite large and diverse identifying until now 36 members. Among the Hsp40s, the J protein YDJ1 (Y for yeast) is a cytosolic chaperone able to participate also in the Hsp90-hetercomplex. Ydj1-like Hsp40s present a particular domain organization: the N-terminal J domain, a G/F rich region, a zinc finger domain (ZFD) motif containing region, a DNAJ_C or C-terminal CTD region and a homodimerization region. Based on that, we found only one Hsp40 that has this domain organization and cytosolic localization (TGME49_311240) named TgDJ1. The ORF of TgDJ1 was expressed as a recombinant protein (rTgDJ1) in E. coli, fused to a HIS-tag to enable its purification and posterior polyclonal antibodies generation in mice. The identity of the recombinant protein produced was confirmed by mass spectrometry analysis. The TgDJ1 antiserum was able to recognize a single band of the expected molecular weight (~47-kDa) in T. gondii tachyzoite lysate showing no cross-reaction with host cell protein extract. The subcellular localization of TgDJ1 was consistent with a cytosolic labeling in intracellular tachyzoites and bradyzoites. Based on co-immunoprecipitation (coIP) analysis and Western blot, TgDJ1 may be forming complexes with Hsp90 and Hsp70. CoIP analysis with anti-TgDJ1 on tachyzoites extract followed by mass spect retrieved near 63 proteins which comprise several subcellular localizations and biological functions suggesting a broad biological role for this chaperone in the parasite.