In vivo formation of Plasmodium falciparum ribosomal stalk — A unique mode of assembly without stable heterodimeric intermediates

Wawiórka, Leszek; Krokowski, Dawid; Gordiyenko, Yuliya; Krowarsch, Daniel; Robinson, Carol V.; Adam, Ishag; Grankowski, Nikodem; Tchórzewski, Marek

doi:10.1016/j.bbagen.2014.10.015

Cited by 5 publications

(7 citation statements)

References 59 publications

(84 reference statements)

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“…To evaluate the binding of the pentameric stalk complex with RTA we have assembled the human pentameric complex in vivo in E. coli using a previously developed poly-cistronic expression system 52 , where three genes for the truncated ΔuL10 and P1/P2 proteins were placed in one row for simultaneous expression of all stalk elements. The uL10/P1/P2 gene array was expressed in E. coli cells with ΔuL10 lacking the rRNA binding domain as hybrid proteins with an N-terminal GST-tag.…”

Section: Resultsmentioning

confidence: 99%

Human ribosomal P1-P2 heterodimer represents an optimal docking site for ricin A chain with a prominent role for P1 C-terminus

Grela

Horbowicz

et al. 2017

Sci Rep

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The eukaryotic P-stalk contains two P1-P2 protein dimers with a conserved C- terminal domain (CTD) critical for the interaction with external factors. To understand the role of the individual CTD of human P1/P2 proteins, we examined the interaction of reconstituted human P-protein complexes and C-terminally truncated forms with ricin A chain (RTA), which binds to the stalk to depurinate the sarcin/ricin loop (SRL). The interaction between P-protein complexes and RTA was examined by surface plasmon resonance, isothermal titration calorimetry, microscale thermophoresis and bio-layer interferometry. The P1-P2 heterodimer missing a CTD on P2 was able to bind RTA. In contrast, the P1-P2 heterodimer missing the CTD of P1 protein displayed almost no binding toward RTA. Very low interaction was detected between RTA and the non-truncated P2-P2 homodimer, suggesting that the structural architecture of the P1-P2 heterodimer is critical for binding RTA. The reconstituted pentameric human stalk complex had higher affinity for RTA than the P1-P2 dimer. Deletion of P1 CTD, but not P2 CTD reduced the affinity of the pentamer for RTA. These results highlight the importance of the heterodimeric organization of P1-P2 in the human stalk pentamer and functional non-equivalence of the individual P-protein CTDs in the interaction with RTA.

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Section: Resultsmentioning

confidence: 99%

Human ribosomal P1-P2 heterodimer represents an optimal docking site for ricin A chain with a prominent role for P1 C-terminus

Grela

Horbowicz

et al. 2017

Sci Rep

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“…All tested P1, P2, P0, and Msp1 19 proteins displayed a purity level exceeding 95%, as quantified by SDS-PAGE (Figure 1). The in vivo assembled complex was purified by single-step affinity chromatography, in native conditions, using glutathione-affinity chromatography [29]. The complex was analyzed using the size-exclusion chromatography approach (SEC) and showed a single symmetrical peak indicating monodispersity of the sample; the SEC fractions used for subsequent immunization were analyzed with SDS-PAGE, and protein fraction displayed 95% purity ().…”

Section: Resultsmentioning

confidence: 99%

“…All genetic constructs were verified by sequencing. In the case of the pentameric P0-(P1-P2) 2 complex, a tricistronic expression cassette was used together with the pGEX4T-1 expression vector, as described previously [29].…”

Section: Methodsmentioning

confidence: 99%

“…Using a heterologous expression system in E. coli , several antigens were prepared, such as individual P1, P2, and P0 proteins. A P0-(P1-P2) 2 pentameric complex was also produced, assembled in bacterial cells, and purified in a form reflecting the native features of the P-protein complex [29]. As a parasite-specific antigen, the Msp-1 protein was included as a reference.…”

Section: Introductionmentioning

confidence: 99%

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Immunogenic Evaluation of Ribosomal P-Protein Antigen P0, P1, and P2 and Pentameric Protein Complex P0-(P1-P2)₂ofPlasmodium falciparumin a Mouse Model

Szuster‐Ciesielska

Wawiórka

Krokowski

et al. 2019

Journal of Immunology Research

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Malaria remains one the most infectious and destructive protozoan diseases worldwide. Plasmodium falciparum, a protozoan parasite with a complex life cycle and high genetic variability responsible for the difficulties in vaccine development, is implicated in most malaria-related deaths. In the course of study, we prepared a set of antigens based on P-proteins from P. falciparum and determined their immunogenicity in an in vivo assay on a mouse model. The pentameric complex P0-(P1-P2)2 was prepared along with individual P1, P2, and P0 antigens. We determined the level of cellular- and humoral-type immunological response followed by development of specific immunological memory. We have shown that the number of Tc cells increased significantly after the first immunization with P2 and after the second immunization with P1 and P0-(P1-P2)2, which highly correlated with the number of Th1 cells. P0 appeared as a poor inducer of cellular response. After the third boost with P1, P2, or P0-(P1-P2)2, the initially high cellular response dropped to the control level accompanied by elevation of the number of activated Treg cells and a high level of suppressive TGF-β. Subsequently, the humoral response against the examined antigens was activated. Although the titers of specific IgG were increasing during the course of immunization for all antigens used, P2 and P0-(P1-P2)2 were found to be significantly stronger than P1 and P0. A positive correlation between the Th2 cell abundance and the level of IL-10 was observed exclusively after immunization with P0-(P1-P2)2. An in vitro exposure of spleen lymphocytes from the immunized mice especially to the P1, P2, and P0-(P1-P2)2 protein caused 2-3-fold higher cell proliferation than that in the case of lymphocytes from the nonimmunized animals, suggesting development of immune memory. Our results demonstrate for the first time that the native-like P-protein pentameric complex represents much stronger immune potential than individual P-antigens.

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“…However, interestingly the core structure in both these homo and hetero-dimers is identical [4] , [5] . A very recent report on the Plasmodium P-protein assembly demonstrated a lack of stable hetero-dimeric complex of P1/P2 proteins, indicating a distinct behavior of these Plasmodium proteins [6] . In this study it was observed that PfP1 and PfP2 proteins do not form hetero-dimers, and that in the presence of all three P-proteins, the assembly of pentameric P0-(P1/P2) 2 occurred directly, bypassing the stable hetero-dimeric complex.…”

Section: Introductionmentioning

confidence: 95%

Molten globule nature of Plasmodium falciparum P2 homo-tetramer

Mishra

Choudhary

Mukherjee

et al. 2015

Biochemistry and Biophysics Reports

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The P2 protein in Plasmodium falciparum has a high tendency to oligomerize, which seems to drive many of its non-ribosomal functions. During nuclear division of the parasite inside RBC, P2 translocates to the RBC surface as a tetramer. From a systematic study using variety of biophysical techniques, NMR spectral characteristics and relaxation dispersion measurements under different conditions of pH and/or urea concentrations, we deduce that (i) PfP2, an almost entirely helical protein, forms a molten globule monomer at low pH, (ii) at physiological pH, and at micro-molar concentrations, PfP2 is a stable tetramer wherein two dimmers associate sideways with close packing of helices at the interface, and (iii) the molten globule characteristic of the monomer is preserved in the tetramer. This dynamism in the structure of PfP2 may have functional implications since it is known that different kinds of oligomers are transiently formed in the parasite.

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In vivo formation of Plasmodium falciparum ribosomal stalk — A unique mode of assembly without stable heterodimeric intermediates

Cited by 5 publications

References 59 publications

Human ribosomal P1-P2 heterodimer represents an optimal docking site for ricin A chain with a prominent role for P1 C-terminus

Human ribosomal P1-P2 heterodimer represents an optimal docking site for ricin A chain with a prominent role for P1 C-terminus

Immunogenic Evaluation of Ribosomal P-Protein Antigen P0, P1, and P2 and Pentameric Protein Complex P0-(P1-P2)₂ofPlasmodium falciparumin a Mouse Model

Molten globule nature of Plasmodium falciparum P2 homo-tetramer

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In vivo formation of Plasmodium falciparum ribosomal stalk — A unique mode of assembly without stable heterodimeric intermediates

Cited by 5 publications

References 59 publications

Human ribosomal P1-P2 heterodimer represents an optimal docking site for ricin A chain with a prominent role for P1 C-terminus

Human ribosomal P1-P2 heterodimer represents an optimal docking site for ricin A chain with a prominent role for P1 C-terminus

Immunogenic Evaluation of Ribosomal P-Protein Antigen P0, P1, and P2 and Pentameric Protein Complex P0-(P1-P2)2ofPlasmodium falciparumin a Mouse Model

Molten globule nature of Plasmodium falciparum P2 homo-tetramer

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Immunogenic Evaluation of Ribosomal P-Protein Antigen P0, P1, and P2 and Pentameric Protein Complex P0-(P1-P2)₂ofPlasmodium falciparumin a Mouse Model