Characterization of a Protozoan Phosducin-like Protein-3 (PhLP-3) Reveals Conserved Redox Activity
We recently identified three novel thioredoxin-like genes in the genome of the protozoan parasite Plasmodium that belong to the Phosducin-like family of proteins (PhLP). PhLPs are small cytosolic proteins hypothesized to function in G-protein signaling and protein folding. Although PhLPs are highly conserved in eukaryotes from yeast to mammals, only a few representatives have been experimentally characterized to date. In addition, while PhLPs contain a thioredoxin domain, they lack a CXXC motif, a strong indicator for redox activity, and it is unclear whether members of the PhLP family are enzymatically active. Here, we describe PbPhLP-3 as the first phosducin-like protein of a protozoan organism, Plasmodium berghei. Initial transcription analysis revealed continuous low-level expression of pbphlp-3 throughout the complex Plasmodium life cycle. Attempts to knockout pbphlp-3 in P. berghei did not yield live parasites, suggesting an essential role for the gene in Plasmodium. We cloned, expressed and purified PbPhLP-3 and determined that the recombinant protein is redox active in vitro in a thioredoxin-coupled redox assay. It also has the capacity to reduce the organic compound tert-Butyl hydroperoxide (TBHP) in vitro, albeit at low efficiency. Sequence analysis, structural modeling, and site-directed mutagenesis revealed a conserved cysteine in the thioredoxin domain to be the redox active residue. Lastly, we provide evidence that recombinant human PhLP-3 exhibits redox activity similar to that of PbPhLP-3 and suggest that redox activity may be conserved in PhLP-3 homologs of other species. Our data provide new insight into the function of PhLP-3, which is hypothesized to act as co-chaperones in the folding and regulation of cytoskeletal proteins. We discuss the potential implications of PhLP-3 as a thioredoxin-target protein and possible links between the cellular redox network and the eukaryotic protein folding machinery.
Kooistra, Rachel L.; David, Robin; Ruiz, Ana C.; Powers, Sean W.; Haselton, Kyle J.; Kiernan, Kaitlyn; Blagborough, Andrew M.; Solamen, Ligin; Olsen, Kenneth W,; Putonti, Catherine; and Kanzok, Stefan M.. Characterization of a Protozoan Phosducin-like Protein-3 (PhLP-3) Reveals Conserved Redox Activity. PLOS One, , : 21, 2018. Retrieved from Loyola eCommons, Bioinformatics Faculty Publications, http://dx.doi.org/10.1371/journal.pone.0209699
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Author Posting. © The Authors 2018. This article is posted here by permission of the Public Library of Science for personal use, not for redistribution. The article was published in PLOS One, 2018, https://doi.org/10.1371/journal.pone.0209699