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Review 2: "Multiscale Effects of Perturbed Translation Dynamics Inform Antimalarial Design"

The reviewers praised the technical quality of the cryo-electron microscopy imaging and agreed that the data provides important new structural insights, however they questioned some of the interpretations related to the specific role of PfRACK1 in translational regulation.

Published onDec 04, 2023
Review 2: "Multiscale Effects of Perturbed Translation Dynamics Inform Antimalarial Design"
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Multiscale effects of perturbed translation dynamics inform antimalarial design
Multiscale effects of perturbed translation dynamics inform antimalarial design
Description

Abstract Malaria parasites rely heavily on rapid, high fidelity protein synthesis to infect and replicate in human erythrocytes, making translation an attractive target for new antimalarials. Here, we have determined in situ structures of Pf80S ribosomes in thirteen conformational and compositional states from cryoFIB-milled Plasmodium falciparum-infected human erythrocytes across the stages of asexual intraerythrocytic parasite replication. We observe eight active translation intermediates, enabling us to define the native malarial translation elongation cycle, which surprisingly features a bifurcation at the decoding stage of the cycle that has not previously been described. Examination of perturbations in the distribution of ribosomes among these states in the presence of a malaria-specific translation inhibitor suggests that the inhibitor impedes PfeEF2 and PfeEF1α interactions with the ribosome. We integrated our in situ cryoET data with proteomic and ultrastructural data to arrive at a deeper understanding of malarial translation, which will inform development of new therapies.

RR:C19 Evidence Scale rating by reviewer:

  • Strong. The main study claims are very well-justified by the data and analytic methods used. There is little room for doubt that the study produced has very similar results and conclusions as compared with the hypothetical ideal study. The study’s main claims should be considered conclusive and actionable without reservation.

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Review:

There are three main claims of the study. Firstly, the authors claim that their work transforms our understanding of Plasmodium falciparum elongation by providing the first structural evidence of an association between PfRACK1 and the Pf80s ribosome. Secondly, the work provides further understanding of the translation elongation process in P. falciparum through the discovery of a novel bifurcated pathway. Thirdly, the work provides further insights into the mode-of-action of known translation inhibitors. 

This is an elegant piece of work delving into the details of protein translation in Plasmodium falciparum asexual stage parasites. Using CryoET the authors were able to obtain, with 4.1 Å resolution, the structure of the 80s ribosome from in situ trophozoite, schizonts and merozoites stages and ex vivo ring stage parasites. Importantly, this revealed a never-seen-before strong density region in the head of the 40S subunit (4.3 Å resolution). Once thought to be a point of divergence from the human counterpart and a potential drug target, this study is the first to show, structurally, that the conserved binding site for the receptor for activated C kinase 1 (RACK1), a docking site for translation modulators, is also present in P. falcipaum. This corroborates previous Knock-down studies showing the essentially of PfRACK1 and is further supported in the paper by a structural modelling of PfRACK and the 40S ribosome subunit.

The second claim of the study was that pathway of P. falciparum translation elongation is bifurcated. To delineate this, the authors first identified 13 different ribosomal states. After comparison with previous structural studies, they were able to assign 8 of the high-resolution states to distinct steps in translation elongation. States 9-12 were considered hibernating states. Two of which have been described previously. The thirteenth state they refer to as ‘the unloaded state’ as it lacks a dense nascent peptide chain commonly seen in most of the other intermediates. For this reason, they suggest it is likely to be inactive, but its prevalence predicts physiological importance. Of the 8 states considered active in translation elongation only two have been described previously for P. falciparum. This again highlights the novelty of this work. How the authors assign the states involved in the decoding to peptidyl-transfer transition to two alternative pathways is not completely clear to the reader and should be described in more detail.

For the third claim, the authors focussed on drug-induced translation inhibition with a particular focus on cabamiquine (CBQ) and defining its mechanism of action. They used a multidisciplinary approach involving quantitative proteomics, gene ontology and structural analysis (CryoET) to evaluate the parasite responses to CBQ over a 48-hour time course. They showed that after just 1h of drug treatment there is an accumulation of the ribosome states associated with elongation factors and depletion of the eEF1α-bound decoding states. Through quantitative proteomics they show an enrichment of translation related proteins (10-18 h) and an upregulation of ribosome biogenesis (18-36 h) which is further supported by abundance of ribosomes in the nucleus at 18 hours post-treatment. From this data the authors propose that CBQ disrupts protein synthesis by directly or indirectly perturbing interaction of both eEF2 and eEF1α with the Pf80S ribosome.

Overall, this an excellent body of work and the claims of the study are clearly supported by the results presented. The authors highlight the limitations of their own work and areas for further investigation. One criticism is that the focus of the paper when reading the discussion appears different to that told by the abstract and introduction. Also, some results, about other translation inhibitors, are not discussed further. It may be better to streamline the focus of the study for journal submission. 

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