Search

Spinocerebellar ataxia type 12 (SCA12) is caused by a CAG expansion mutation in PPP2R2B, a gene encoding brain-specific regulatory units of protein phosphatase 2A (PP2A); while normal alleles carry 4 to 31 triplets, the disease alleles carry 43 to 78 triplets. Here, by CRISPR/Cas9n genome editing, we have generated a human heterozygous SCA12 iPSC line with 73 triplets for the mutant allele. The heterozygous SCA12 iPSCs have normal karyotype, express pluripotency markers and are able to differentiate into the three germ layers.

The apicoplast of Plasmodium falciparum parasites is believed to rely on the import of three-carbon phosphate compounds for use in organelle anabolic pathways, in addition to the generation of energy and reducing power within the organelle. We generated a series of genetic deletions in an apicoplast metabolic bypass line to determine which genes involved in apicoplast carbon metabolism are required for blood-stage parasite survival and organelle maintenance. We found that pyruvate kinase II (PyrKII) is essential for organelle maintenance, but that production of pyruvate by PyrKII is not responsible for this phenomenon. Enzymatic characterization of PyrKII revealed activity against all NDPs and dNDPs tested, suggesting that it may be capable of generating a broad range of nucleotide triphosphates. Conditional mislocalization of PyrKII resulted in decreased transcript levels within the apicoplast that preceded organelle disruption, suggesting that PyrKII is required for organelle maintenance due to its role in nucleotide triphosphate generation.

Iron-sulfur clusters (FeS) are ancient and ubiquitous protein cofactors that play fundamental roles in many aspects of cell biology. These cofactors cannot be scavenged or trafficked within a cell and thus must be synthesized in any subcellular compartment where they are required. We examined the FeS synthesis proteins found in the relict plastid organelle, called the apicoplast, of the human malaria parasite Plasmodium falciparum. Using a chemical bypass method, we deleted four of the FeS pathway proteins involved in sulfur acquisition and cluster assembly and demonstrated that they are all essential for parasite survival. However, the effect that these deletions had on the apicoplast organelle differed. Deletion of the cysteine desulfurase SufS led to disruption of the apicoplast organelle and loss of the organellar genome, whereas the other deletions did not affect organelle maintenance. Ultimately, we discovered that the requirement of SufS for organelle maintenance is not driven by its role in FeS biosynthesis, but rather, by its function in generating sulfur for use by MnmA, a tRNA modifying enzyme that we localized to the apicoplast. Complementation of MnmA and SufS activity with a bacterial MnmA and its cognate cysteine desulfurase strongly suggests that the parasite SufS provides sulfur for both FeS biosynthesis and tRNA modification in the apicoplast. The dual role of parasite SufS is likely to be found in other plastid-containing organisms and highlights the central role of this enzyme in plastid biology.

Malaria parasites rely on a plastid organelle for survival during the blood stages of infection. However, the entire organelle is dispensable as long as the isoprenoid precursor, isopentenyl pyrophosphate (IPP), is supplemented in the culture medium. We engineered parasites to produce isoprenoid precursors from a mevalonate-dependent pathway, creating a parasite line that replicates normally after the loss of the apicoplast organelle. We show that carbon-labeled mevalonate is specifically incorporated into isoprenoid products, opening new avenues for researching this essential class of metabolites in malaria parasites. We also show that essential apicoplast proteins, such as the enzyme target of the drug fosmidomycin, can be deleted in this mevalonate bypass parasite line, providing a new method to determine the roles of other important apicoplast-resident proteins. Several antibacterial drugs kill malaria parasites by targeting basic processes, such as transcription, in the organelle. We used metabolomic and transcriptomic methods to characterize parasite metabolism after azithromycin treatment triggered loss of the apicoplast and found that parasite metabolism and the production of apicoplast proteins is largely unaltered. These results provide insight into the effects of apicoplast-disrupting drugs, several of which have been used to treat malaria infections in humans. Overall, the mevalonate bypass system provides a way to probe essential aspects of apicoplast biology and study the effects of drugs that target apicoplast processes.