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Background and Purpose: Autosomal dominant polycystic kidney disease (ADPKD) is a leading cause of end-stage renal disease with limited treatment options. Drug repurposing offers a promising strategy to find effective treatments.

Experimental Approach: We identified birinapant, bardoxolone methyl and salicylic acid as repurposing candidates for ADPKD and applied thermal proteome profiling to identify disease-relevant targets.

Key Results: Our results uncovered shared effects between the three drugs, including a thermal shift in ADP/ATP translocases (SLC25A4/5/6) caused by reduced ATP production. As expected, salicylic acid displayed a polypharmacological profile but also engaged enzymes along the N-linked glycosylation pathway and inhibited PTK2 (FAK), an activator of the proliferative PI3K/Akt/mTOR pathway. Orthogonal assays and computational modelling confirmed these findings and located the binding site (499–504) of salicylic acid within PTK2's ATP-binding pocket. Engagement of several targets was achieved at clinically relevant concentrations.

Conclusions and Implications: We have identified novel targets of three repurposing candidates that contribute to the cyst growth reducing effects in preclinical ADPKD models and identify salicylic acid, the metabolite of aspirin, as the repurposing candidate with the most promising mechanisms of action.

Glycans play crucial roles in bacteria, such as providing structural integrity or enabling interactions with the ecosystem. They can be linked to lipids, peptides, or proteins. In proteins, they modify either asparagine (N-glycosylation) or serine or threonine (O-glycosylation). Species of the Bacteroidota phylum, a major component of the human microbiome and marine and soil ecosystems, have a unique type of O-glycosylation that modifies multiple noncytoplasmic proteins containing a specific amino acid sequence. Only a small number of species have currently been characterized, but within one species, generally all proteins are modified with the same glycan structure. Most species share a common inner part but differ in the sugar composition and branching of the outer part of their glycan. This suggests that the biosynthesis of the glycan occurs in two separate steps. Both the inner core and the outer glycan are likely assembled from nucleotide-activated monosaccharides on undecaprenyl phosphate on the cytoplasmic side of the inner membrane, prior to being flipped to the periplasm and transferred to the protein. A genomic locus responsible for the biosynthesis of the outer glycan has been identified, containing some conserved genes across species. Despite substantial progress in the characterization of this O-glycosylation system, its function, the overall diversity of glycan structures across the phylum, and the complete biosynthetic pathway remain mostly unknown. Due to the importance of this group of species for the human gut microbiome, elucidating these aspects can open up strategies to modulate the composition of the microbiome community toward a healthy state.

Despite recent advances in our understanding of the structure and function of conjugative Type 4 Secretion Systems (T4SSs), there is still only very scarce data available for the ones from Gram-positive (G+) bacteria. This is a problem, as conjugative T4SSs are main drivers for the spread of antibiotic resistance genes and virulence factors. Here, we aim to increase our understanding of G+ systems, by using bioinformatic approaches to identify proteins that are conserved in all conjugative T4SS machineries and reviewing the current knowledge available for these components. We then combine this information with the most recent advances in structure prediction technologies to propose a structural model for a G+ T4SS from the model system encoded on pCF10. By doing so, we show that conjugative G+ T4SSs likely have more in common with their Gram-negative counterparts than previously expected, and we highlight the potential of predicted structural models to serve as a starting point for experimental design.

Background: Microsporidia are a large taxon of intracellular pathogens characterized by extraordinarily streamlined genomes with unusually high sequence divergence and many species-specific adaptations. These unique factors pose challenges for traditional genome annotation methods based on sequence similarity. As a result, many of the microsporidian genomes sequenced to date contain numerous genes of unknown function. Recent innovations in rapid and accurate structure prediction and comparison, together with the growing amount of data in structural databases, provide new opportunities to assist in the functional annotation of newly sequenced genomes.

Results: In this study, we established a workflow that combines sequence and structure-based functional gene annotation approaches employing a ChimeraX plugin named ANNOTEX (Annotation Extension for ChimeraX), allowing for visual inspection and manual curation. We employed this workflow on a high-quality telomere-to-telomere sequenced tetraploid genome of Vairimorpha necatrix. First, the 3080 predicted protein-coding DNA sequences, of which 89% were confirmed with RNA sequencing data, were used as input. Next, ColabFold was used to create protein structure predictions, followed by a Foldseek search for structural matching to the PDB and AlphaFold databases. The subsequent manual curation, using sequence and structure-based hits, increased the accuracy and quality of the functional genome annotation compared to results using only traditional annotation tools. Our workflow resulted in a comprehensive description of the V. necatrix genome, along with a structural summary of the most prevalent protein groups, such as the ricin B lectin family. In addition, and to test our tool, we identified the functions of several previously uncharacterized Encephalitozoon cuniculi genes.

Conclusion: We provide a new functional annotation tool for divergent organisms and employ it on a newly sequenced, high-quality microsporidian genome to shed light on this uncharacterized intracellular pathogen of Lepidoptera. The addition of a structure-based annotation approach can serve as a valuable template for studying other microsporidian or similarly divergent species.

Sexual reproduction in malaria parasites is essential for their transmission to mosquitoes and offers a divergent eukaryote model to understand the evolution of sex. Through a panel of genetic screens in Plasmodium berghei, we identify 348 sex and transmission-related genes and define roles for unstudied genes as putative targets for transmission-blocking interventions. The functional data provide a deeper understanding of female metabolic reprogramming, meiosis, and the axoneme. We identify a complex of a SUN domain protein (SUN1) and a putative allantoicase (ALLC1) that is essential for male fertility by linking the microtubule organizing center to the nuclear envelope and enabling mitotic spindle formation during male gametogenesis. Both proteins have orthologs in mouse testis, and the data raise the possibility of an ancient role for atypical SUN domain proteins in coupling the nucleus and axoneme. Altogether, our data provide an unbiased picture of the molecular processes that underpin malaria parasite transmission. A record of this paper's transparent peer review process is included in the supplemental information.

Proteasomes play an essential role in the life cycle of intracellular pathogens with extracellular stages by ensuring proteostasis in environments with limited resources. In microsporidia, divergent parasites with extraordinarily streamlined genomes, the proteasome complexity and structure are unknown, which limits our understanding of how these unique pathogens adapt and compact essential eukaryotic complexes. We present cryo-electron microscopy structures of the microsporidian 20S and 26S proteasome isolated from dormant or germinated Vairimorpha necatrix spores. The discovery of PI31-like peptides, known to inhibit proteasome activity, bound simultaneously to all six active sites within the central cavity of the dormant spore proteasome, suggests reduced activity in the environmental stage. In contrast, the absence of the PI31-like peptides and the existence of 26S particles post-germination in the presence of ATP indicates that proteasomes are reactivated in nutrient-rich conditions. Structural and phylogenetic analyses reveal that microsporidian proteasomes have undergone extensive reductive evolution, lost at least two regulatory proteins, and compacted nearly every subunit. The highly derived structure of the microsporidian proteasome, and the minimized version of PI31 presented here, reinforce the feasibility of the development of specific inhibitors and provide insight into the unique evolution and biology of these medically and economically important pathogens.