Understanding the role of RBR-E3 ligase in regulation of cellular
homeostasis in human malaria parasite
Abstract
E3 ligases constitute an important component of proteostasis machinery
which plays a critical role in the survival of malaria parasite through
post-translational modifications of its protein-substrates. In contrast
to humans, parasite E3 ligases have not been extensively studied. Here,
we characterize a unique Plasmodium E3 ligase that has both RING
and HECT-like features with zinc-coordinating domains. Plasmodium
encodes a single RING-between-RING (RBR) E3 ligase that has
evolutionarily diverged from human and other intracellular parasites.
This RBR-E3 ligase is expressed throughout the erythrocytic phase of
P. falciparum lifecycle. Immunoprecipitation experiments showed
that Pf RBR-E3 ligase catalyzes K6, K11, K48 and K63 mediated
polyubiquitination hinting towards its diverse biological roles (DNA
repair, proteasomal degradation, mitochondrial quality control). We
observed that Pf RBR-E3 ligase interacts with UBCH5 and UBC13
family of E2-conjugating enzymes. Through mutational analysis, we
identified residues in RING1 and RING2 domains that are critical for
ubiquitination activity and protein stability of Pf RBR-E3
ligase. Our experiments showed that Pf RBR-E3 ligase participates
in maintenance of organellar homeostasis and exhibits differences in
immunofluorescence profile upon exposure of parasite to different
genotoxic (MMS) and proteotoxic (MG132, FCCP and artemisinin derivative)
stress. Our study opens up avenues for exploring the client substrates
of Pf RBR-E3 ligase and using this knowledge to design
substrate-specific protein degradation based alternative intervention
strategies for malaria.