Characterization of a protozoan Phosducin-like protein-3 (PhLP-3) reveals conserved redox activity

Abstract

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.

The evolutionarily conserved PhLP3 is essential for sperm development inDrosophila melanogaster

“…We determined that the reaction between Trx1 and PhLP3 follows Michaelis Menten kinetics, exhibiting a K m of 2.72 M and a V max of 5.4 M/min (Fig 5C). These values compare well with those of previously tested PhLP3 proteins, P. berghei PhLP3 and human TXNDC9, and support our hypothesis that redox activity is conserved in PhLPs between evolutionarily distant species and may be necessary for its biological function [4].…”

“…To determine whether recombinant PhLP3 is redox-active, we cloned the CDS from D. melanogaster cDNA into a bacterial expression vector. We purified recombinant PhLP3 using a Ni-NTA-based column approach under denaturing conditions as described previously [4] Based on our recent findings that P. berghei PhLP3 and human TXNDC9 exhibit redox activity and have a conserved cysteine at position 95 (Fig 1A ), we hypothesized that D. melanogaster PhLP3 will also be redox-active. To test this, we conducted NADPH reduction assays as outlined in Fig 5B and as described previously [4].…”

“…We purified recombinant PhLP3 using a Ni-NTA-based column approach under denaturing conditions as described previously [4] Based on our recent findings that P. berghei PhLP3 and human TXNDC9 exhibit redox activity and have a conserved cysteine at position 95 (Fig 1A ), we hypothesized that D. melanogaster PhLP3 will also be redox-active. To test this, we conducted NADPH reduction assays as outlined in Fig 5B and as described previously [4]. Briefly, electron donor NADPH was added to a mix of recombinant P. berghei thioredoxin reductase (PbTrxR, PbANKA_0824700, [33]) and D. melanogaster thioredoxin (DmTrx1, CG4193, [34]).…”

“…The copyright holder for this preprint this version posted June 24, 2024. ; https://doi.org/10.1101/2024.06.24.600488 doi: bioRxiv preprint previously described [4]. Briefly, the oxidation of NADPH was followed by an absorption decrease at 340 nm.…”

The malaria parasite chaperonin containing TCP-1 (CCT) complex: Data integration with other CCT proteomes

“…The PfCCT data finds abundant signal for three PhLPs ( Table 1 ). PhLP2 in Plasmodium berghei , a rodent model of the malaria pathogen, can shuttle electrons between proteins in a thioredoxin-coupled redox assay via a highly reactive cysteine residue in the Trx domain and is suggested to regulate CCT folding activity ( Kooistra et al, 2018 ). A comparison of AlphaFold models ( Figure 8 ) of PfPhLPs ( Varadi et al, 2022 ) and yeast PLP1 and PLP2 ( McCormack et al, 2009 ) shows the conserved TWRC motif forms a non-canonical active site that is conserved across several Plasmodium species, including P. falciparum .…”