Multiple Weak Linear Motifs Enhance Recruitment and Processivity in SPOP-Mediated Substrate Ubiquitination

AbstractPrimary sequence motifs, with millimolar affinities for binding partners, are abundant in disordered protein regions. In multivalent interactions, such weak linear motifs can cooperate to recruit binding partners via avidity effects. If linear motifs recruit modifying enzymes, optimal placement of weak motifs may regulate access to modification sites. Weak motifs may thus exert physiological relevance stronger than that suggested by their affinities, but molecular mechanisms of their function are still poorly understood. Herein, we use the N-terminal disordered region of the Hedgehog transcriptional regulator Gli3 (Gli31-90) to determine the role of weak motifs encoded in its primary sequence for the recruitment of its ubiquitin ligase CRL3SPOP and the subsequent effect on ubiquitination efficiency. The substrate adaptor SPOP binds linear motifs through its MATH (meprin and TRAF homology) domain and forms higher-order oligomers through its oligomerization domains, rendering SPOP multivalent for its substrates. Gli3 has multiple weak SPOP binding motifs. We map three such motifs in Gli31-90, the weakest of which has a millimolar dissociation constant. Multivalency of ligase and substrate for each other facilitates enhanced ligase recruitment and stimulates Gli31-90 ubiquitination in in vitro ubiquitination assays. We speculate that the weak motifs enable processivity through avidity effects and by providing steric access to lysine residues that are otherwise not prioritized for polyubiquitination. Weak motifs may generally be employed in multivalent systems to act as gatekeepers regulating post-translational modification

Catestatin, an endogenous Chromogranin A-derived peptide, inhibits in vitro growth of Plasmodium falciparum

Catestatin, an endogenous peptide derived from bovine chromogranin A, and its active domain cateslytin display powerful antimicrobial activities. We have tested the activities of catestatin and other related peptides on the growth of Plasmodium falciparum in vitro. Catestatin inhibits growth of the chloroquine-sensitive strain of P. falciparum 3D7, exhibiting 88% inhibition at 20 lM. A similar partial inhibition of parasite growth was observed for the chloroquine-resistant strain, 7G8 (64%,) and the multidrug-resistant strain, W2 (62%). In the presence of parasite-specific lactate dehydrogenase, a specific protein– protein interaction between catestatin and plasmepsin II precursor was demonstrated. In addition, catestatin partially inhibited the parasite-specific proteases plasmepsin in vitro. A specific interaction between catestatin and plasmepsins II and IV from P. falciparum and plasmepsin IV from the three remaining species of Plasmodium known to infect man was observed, suggesting a catestatininduced reduction in availability of nutrients for protein synthesis in the parasite

Enzymatic Characterization of Recombinant Food Vacuole Plasmepsin 4 from the Rodent Malaria Parasite Plasmodium berghei.

The rodent malaria parasite Plasmodium berghei is a practical model organism for experimental studies of human malaria. Plasmepsins are a class of aspartic proteinase isoforms that exert multiple pathological effects in malaria parasites. Plasmepsins residing in the food vacuole (FV) of the parasite hydrolyze hemoglobin in red blood cells. In this study, we cloned PbPM4, the FV plasmepsin gene of P. berghei that encoded an N-terminally truncated pro-segment and the mature enzyme from genomic DNA. We over-expressed this PbPM4 zymogen as inclusion bodies (IB) in Escherichia coli, and purified the protein following in vitro IB refolding. Auto-maturation of the PbPM4 zymogen to mature enzyme was carried out at pH 4.5, 5.0, and 5.5. Interestingly, we found that the PbPM4 zymogen exhibited catalytic activity regardless of the presence of the pro-segment. We determined the optimal catalytic conditions for PbPM4 and studied enzyme kinetics on substrates and inhibitors of aspartic proteinases. Using combinatorial chemistry-based peptide libraries, we studied the active site preferences of PbPM4 at subsites S1, S2, S3, S1', S2' and S3'. Based on these results, we designed and synthesized a selective peptidomimetic compound and tested its inhibition of PbPM4, seven FV plasmepsins from human malaria parasites, and human cathepsin D (hcatD). We showed that this compound exhibited a >10-fold selectivity to PbPM4 and human malaria parasite plasmepsin 4 orthologs versus hcatD. Data from this study furthesr our understanding of enzymatic characteristics of the plasmepsin family and provides leads for anti-malarial drug design

The inhibition of peptidomimetic compounds on FV plasmepsins and hcatD.

<p>^aΨ = -CH2-NH-, nL = norleucine.</p><p>^b<i>Pf</i>PM1, <i>Pf</i>PM2 and <i>Pf</i>PM4, plasmepsins 1, 2 and 4 from <i>Plasmodium falciparum</i>, respectively; <i>Pv</i>PM4, plasmepsin 4 from <i>P</i>. <i>vivax</i>; <i>Po</i>PM4, plasmepsin 4 from <i>P</i>. <i>ovalae</i>; and <i>Pm</i>PM4, plasmepsin 4 from <i>P</i>. <i>malariae</i>.</p><p>^cThese dissociation constants were reported in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141758#pone.0141758.ref041" target="_blank">41</a>].</p><p>^dThese dissociation constants were reported in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141758#pone.0141758.ref042" target="_blank">42</a>].</p><p>The inhibition of peptidomimetic compounds on FV plasmepsins and hcatD.</p