TY - JOUR
T1 - Characterization of a Protozoan Phosducin-like Protein-3 (PhLP-3) Reveals Conserved Redox Activity
AU - Kooistra, Rachel L.
AU - David, Robin
AU - Ruiz, Ana C.
AU - Powers, Sean W.
AU - Haselton, Kyle J.
AU - Kiernan, Kaitlyn
AU - Blagborough, Andrew M.
AU - Solamen, Ligin
AU - Olsen, Kenneth W,
AU - Putonti, Catherine
AU - Kanzok, Stefan M.
N1 - 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.
PY - 2018/12/31
Y1 - 2018/12/31
N2 - 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 . berghe i 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.
AB - 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 . berghe i 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.
KW - plasmodium
KW - oxidation-reduction reactions
KW - malarial parasites
KW - blood
KW - cysteine
KW - mosquitoes
KW - sequence alignment
KW - parasitic protozoans
KW - Protein folding
UR - https://ecommons.luc.edu/bioinformatics_facpub/46
UR - https://doi.org/10.1371/journal.pone.0209699
UR - https://ecommons.luc.edu/biology_facpubs/99
UR - https://ecommons.luc.edu/chemistry_facpubs/107
U2 - 10.1371/journal.pone.0209699
DO - 10.1371/journal.pone.0209699
M3 - Article
VL - 13
JO - Bioinformatics Faculty Publications
JF - Bioinformatics Faculty Publications
IS - 12
ER -