A picomolar inhibitor of the Plasmodium falciparum IPP pathway.

We identified MMV026468 as a picomolar inhibitor of blood-stage Plasmodium falciparum. Phenotyping assays, including isopentenyl diphosphate rescue of parasite growth inhibition, demonstrated that it targets MEP isoprenoid precursor biosynthesis. MMV026468-treated parasites showed an overall decrease in MEP pathway intermediates, which could result from inhibition of the first MEP enzyme DXS or steps prior to DXS such as regulation of the MEP pathway. Selection of MMV026468-resistant parasites lacking DXS mutations suggested that other targets are possible. The identification of MMV026468 could lead to a new class of antimalarial isoprenoid inhibitors.

A mutagenesis screen for essential plastid biogenesis genes in human malaria parasites.

Endosymbiosis has driven major molecular and cellular innovations. Plasmodium spp. parasites that cause malaria contain an essential, non-photosynthetic plastid-the apicoplast-which originated from a secondary (eukaryote-eukaryote) endosymbiosis. To discover organellar pathways with evolutionary and biomedical significance, we performed a mutagenesis screen for essential genes required for apicoplast biogenesis in Plasmodium falciparum. Apicoplast(-) mutants were isolated using a chemical rescue that permits conditional disruption of the apicoplast and a new fluorescent reporter for organelle loss. Five candidate genes were validated (out of 12 identified), including a triosephosphate isomerase (TIM)-barrel protein that likely derived from a core metabolic enzyme but evolved a new activity. Our results demonstrate, to our knowledge, the first forward genetic screen to assign essential cellular functions to unannotated P. falciparum genes. A putative TIM-barrel enzyme and other newly identified apicoplast biogenesis proteins open opportunities to discover new mechanisms of organelle biogenesis, molecular evolution underlying eukaryotic diversity, and drug targets against multiple parasitic diseases.

Dynamic Glycosylation Governs the Vertebrate COPII Protein Trafficking Pathway.

The COPII coat complex, which mediates secretory cargo trafficking from the endoplasmic reticulum, is a key control point for subcellular protein targeting. Because misdirected proteins cannot function, protein sorting by COPII is critical for establishing and maintaining normal cell and tissue homeostasis. Indeed, mutations in COPII genes cause a range of human pathologies, including cranio-lenticulo-sutural dysplasia (CLSD), which is characterized by collagen trafficking defects, craniofacial abnormalities, and skeletal dysmorphology. Detailed knowledge of the COPII pathway is required to understand its role in normal cell physiology and to devise new treatments for disorders in which it is disrupted. However, little is known about how vertebrates dynamically regulate COPII activity in response to developmental, metabolic, or pathological cues. Several COPII proteins are modified by O-linked ß-N-acetylglucosamine (O-GlcNAc), a dynamic form of intracellular protein glycosylation, but the biochemical and functional effects of these modifications remain unclear. Here, we use a combination of chemical, biochemical, cellular, and genetic approaches to demonstrate that site-specific O-GlcNAcylation of COPII proteins mediates their protein-protein interactions and modulates cargo secretion. In particular, we show that individual O-GlcNAcylation sites of SEC23A, an essential COPII component, are required for its function in human cells and vertebrate development, because mutation of these sites impairs SEC23A-dependent in vivo collagen trafficking and skeletogenesis in a zebrafish model of CLSD. Our results indicate that O-GlcNAc is a conserved and critical regulatory modification in the vertebrate COPII-dependent trafficking pathway.

A fast-acting inhibitor of blood-stage P. falciparum with mechanism distinct from artemisinin and chloroquine.

Artemisinins are first-line treatment for malaria, prized for their extremely fast reduction of parasite load in patients. New fast-acting antimalarial compounds are urgently needed to counter artemisinin resistance, but the fast parasite reduction observed with artemisinins is rare among antimalarial compounds. Here we show that MMV1580853 has a very fast in vitro killing rate, comparable to that of dihydroartemisinin. Near-complete parasite growth inhibition was observed within 1 hour of treatment with MMV1580853 and dihydroartemisinin, while chloroquine, another fast-acting antimalarial, showed partial growth inhibition after 1h. MMV1580853 was reported to inhibit prenyltransferases, but its fast killing rate is inconsistent with this mechanism-of-action and we were unable to validate any of 3 annotated P. falciparum prenyltransferases as MMV1580853 targets. MMV1580853 also did not phenocopy the inhibition phenotype of either chloroquine or dihydroartemisinin. These results indicate that MMV1580853 has a distinct mechanism-of-action leading to a very fast killing rate. MMV1580853 compound development and investigation of its mechanism-of-action will be critical avenues in the search for drugs matching the remarkable clinical efficacy of artemisinin.

Structure-Function Relationship for a Divergent Atg8 Protein Required for a Nonautophagic Function in Apicomplexan Parasites.

Atg8 family proteins are highly conserved eukaryotic proteins with diverse autophagy and nonautophagic functions in eukaryotes. While the structural features required for conserved autophagy functions of Atg8 are well established, little is known about the molecular changes that facilitated acquisition of divergent, nonautophagic functions of Atg8. The malaria parasite Plasmodium falciparum offers a unique opportunity to study nonautophagic functions of Atg8 family proteins because it encodes a single Atg8 homolog whose only essential function is in the inheritance of an unusual secondary plastid called the apicoplast. Here, we used functional complementation to investigate the structure-function relationship for this divergent Atg8 protein. We showed that the LC3-interacting region (LIR) docking site (LDS), the major interaction interface of the Atg8 protein family, is required for P. falciparum Atg8 (PfAtg8) apicoplast localization and function, likely via Atg8 lipidation. On the other hand, another region previously implicated in canonical Atg8 interactions, the N-terminal helix, is not required for apicoplast-specific PfAtg8 function. Finally, our investigations at the cellular level demonstrate that the unique apicomplexan-specific loop, previously implicated in interaction with membrane conjugation machinery in recombinant protein-based in vitro assays, is not required for membrane conjugation nor for the apicoplast-specific effector function of Atg8 in both P. falciparum and related Apicomplexa member Toxoplasma gondii. These results suggest that the effector function of apicomplexan Atg8 is mediated by structural features distinct from those previously identified for macroautophagy and selective autophagy functions. IMPORTANCE The most extensively studied role of Atg8 proteins is in autophagy. However, it is clear that they have other nonautophagic functions critical to cell function and disease pathogenesis that are so far understudied compared to their canonical role in autophagy. Mammalian cells contain multiple Atg8 paralogs that have diverse, specialized functions. Gaining molecular insight into their nonautophagic functions is difficult because of redundancy between the homologs and their role in both autophagy and nonautophagic pathways. Malaria parasites such as Plasmodium falciparum are a unique system to study a novel, nonautophagic function of Atg8 separate from its role in autophagy: they have only one Atg8 protein whose only essential function is in the inheritance of the apicoplast, a unique secondary plastid organelle. Insights into the molecular basis of PfAtg8's function in apicoplast biogenesis will have important implications for the evolution of diverse nonautophagic functions of the Atg8 protein family.

How municipalities support energy cooperatives: survey results from Germany and Switzerland.

BACKGROUND: Energy cooperatives are a prominent and common form of community energy. Community energy has the potential to increase actor diversity and local acceptance of renewable energies and has therefore been highlighted to be conducive to energy transitions. While research has recognized the importance of both the national and the local governance levels for community energy, it remains unclear how these two levels are related. Against this backdrop, this paper investigates how municipalities support energy cooperatives at the local level and how this support is related to national context conditions. METHODS: The study takes a quantitative approach using own survey data from Germany and Switzerland. Based on a typology of municipal support, we compare limiting factors and municipal support for energy cooperatives between the two countries as well as between energy cooperatives with and without municipal membership. By means of this two-tiered comparison, we analyze how municipal support is related to national contexts, specifically regarding national energy policies, and to municipal involvement in the cooperatives. RESULTS: Our analysis shows that municipal support can benefit energy cooperatives as it addresses some of the major limiting factors for energy cooperatives in Germany and Switzerland. However, our data suggest that municipalities only specifically address cooperatives' limitations with support measures if they are a member in the cooperative. This indicates that organizational involvement of a municipality in energy cooperatives leads to a more targeted support compared to non-members and thus is beneficial to meet the specific cooperatives' challenges due to national energy policy. CONCLUSIONS: Cooperatives can benefit from municipal support, especially if the municipality is a cooperative member. Municipal support is likely to become even more important for energy cooperatives in the future, due to reduced national support for renewable energies in Germany and Switzerland. On their part, municipalities can benefit from collaborating with energy cooperatives, as they gain an additional instrument to implement municipal energy policy. Hence, supporting and seeking membership in energy cooperatives appear to be adequate strategies for municipalities to foster a decentralized energy transition.

CaaX-Like Protease of Cyanobacterial Origin Is Required for Complex Plastid Biogenesis in Malaria Parasites.

Plasmodium parasites and related apicomplexans contain an essential "complex plastid" organelle of secondary endosymbiotic origin, the apicoplast. Biogenesis of this complex plastid poses a unique challenge requiring evolution of new cellular machinery. We previously conducted a mutagenesis screen for essential apicoplast biogenesis genes to discover organellar pathways with evolutionary and biomedical significance. Here we validate and characterize a gene candidate from our screen, Pf3D7_0913500. Using a conditional knockdown strain, we show that Pf3D7_0913500 depletion causes growth inhibition that is rescued by the sole essential product of the apicoplast, isopentenyl pyrophosphate (IPP), and results in apicoplast loss. Because Pf3D7_0913500 had no previous functional annotation, we name it apicoplast-minus IPP-rescued 4 (AMR4). AMR4 has an annotated CaaX protease and bacteriocin processing (CPBP) domain, which in eukaryotes typically indicates a role in CaaX postprenylation processing. Indeed, AMR4 is the only putative CaaX-like protease in Plasmodium parasites which are known to require protein prenylation, and we confirm that the conserved catalytic residue of AMR4 (E352) is required for its apicoplast function. However, we unexpectedly find that AMR4 does not act in a CaaX postprenylation processing pathway in Plasmodium falciparum Instead, we find that AMR4 is imported into the apicoplast and is derived from a cyanobacterial CPBP gene which was retained through both primary and secondary endosymbiosis. Our findings suggest that AMR4 is not a true CaaX protease, but instead it performs a conserved, uncharacterized chloroplast function that has been retained for complex plastid biogenesis.IMPORTANCEPlasmodium parasites, which cause malaria, and related apicomplexans are important human and veterinary pathogens. These parasites represent a highly divergent and understudied branch of eukaryotes, and as such often defy the expectations set by model organisms. One striking example of unique apicomplexan biology is the apicoplast, an essential but nonphotosynthetic plastid derived from an unusual secondary (eukaryote-eukaryote) endosymbiosis. Endosymbioses are a major driver of cellular innovation, and apicoplast biogenesis pathways represent a hot spot for molecular evolution. We previously conducted an unbiased screen for apicoplast biogenesis genes in P. falciparum to uncover these essential and innovative pathways. Here, we validate a novel gene candidate from our screen and show that its role in apicoplast biogenesis does not match its functional annotation predicted by model eukaryotes. Our findings suggest that an uncharacterized chloroplast maintenance pathway has been reused for complex plastid biogenesis in this divergent branch of pathogens.