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...
| Main Authors: | , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2018-01-01
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| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0209699 |
| _version_ | 1818580808600911872 |
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| author | Rachel L Kooistra Robin David Ana C Ruiz Sean W Powers Kyle J Haselton Kaitlyn Kiernan Andrew M Blagborough Ligin Solamen Kenneth W Olsen Catherine Putonti Stefan M Kanzok |
| author_facet | Rachel L Kooistra Robin David Ana C Ruiz Sean W Powers Kyle J Haselton Kaitlyn Kiernan Andrew M Blagborough Ligin Solamen Kenneth W Olsen Catherine Putonti Stefan M Kanzok |
| author_sort | Rachel L Kooistra |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-16T07:23:29Z |
| format | Article |
| id | doaj.art-21c6b8fb305b4f2f965d0ccb5256fea5 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-16T07:23:29Z |
| publishDate | 2018-01-01 |
| publisher | Public Library of Science (PLoS) |
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| series | PLoS ONE |
| spelling | doaj.art-21c6b8fb305b4f2f965d0ccb5256fea52022-12-21T22:39:34ZengPublic Library of Science (PLoS)PLoS ONE1932-62032018-01-011312e020969910.1371/journal.pone.0209699Characterization of a protozoan Phosducin-like protein-3 (PhLP-3) reveals conserved redox activity.Rachel L KooistraRobin DavidAna C RuizSean W PowersKyle J HaseltonKaitlyn KiernanAndrew M BlagboroughLigin SolamenKenneth W OlsenCatherine PutontiStefan M KanzokWe 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.https://doi.org/10.1371/journal.pone.0209699 |
| spellingShingle | Rachel L Kooistra Robin David Ana C Ruiz Sean W Powers Kyle J Haselton Kaitlyn Kiernan Andrew M Blagborough Ligin Solamen Kenneth W Olsen Catherine Putonti Stefan M Kanzok Characterization of a protozoan Phosducin-like protein-3 (PhLP-3) reveals conserved redox activity. PLoS ONE |
| title | Characterization of a protozoan Phosducin-like protein-3 (PhLP-3) reveals conserved redox activity. |
| title_full | Characterization of a protozoan Phosducin-like protein-3 (PhLP-3) reveals conserved redox activity. |
| title_fullStr | Characterization of a protozoan Phosducin-like protein-3 (PhLP-3) reveals conserved redox activity. |
| title_full_unstemmed | Characterization of a protozoan Phosducin-like protein-3 (PhLP-3) reveals conserved redox activity. |
| title_short | Characterization of a protozoan Phosducin-like protein-3 (PhLP-3) reveals conserved redox activity. |
| title_sort | characterization of a protozoan phosducin like protein 3 phlp 3 reveals conserved redox activity |
| url | https://doi.org/10.1371/journal.pone.0209699 |
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