Plasmodium UIS3 sequesters host LC3 to avoid elimination by autophagy in hepatocytes.

The causative agent of malaria, Plasmodium, replicates inside a membrane-bound parasitophorous vacuole (PV), which shields this intracellular parasite from the cytosol of the host cell 1 . One common threat for intracellular pathogens is the homeostatic process of autophagy, through which cells capture unwanted intracellular material for lysosomal degradation 2 . During the liver stage of a malaria infection, Plasmodium parasites are targeted by the autophagy machinery of the host cell, and the PV membrane (PVM) becomes decorated with several autophagy markers, including LC3 (microtubule-associated protein 1 light chain 3) 3,4 . Here we show that Plasmodium berghei parasites infecting hepatic cells rely on the PVM transmembrane protein UIS3 to avoid elimination by host-cell-mediated autophagy. We found that UIS3 binds host LC3 through a non-canonical interaction with a specialized surface on LC3 where host proteins with essential functions during autophagy also bind. UIS3 acts as a bona fide autophagy inhibitor by competing with host LC3-interacting proteins for LC3 binding. Our work identifies UIS3, one of the most promising candidates for a genetically attenuated vaccine against malaria 5 , as a unique and potent mediator of autophagy evasion in Plasmodium. We propose that the protein-protein interaction between UIS3 and host LC3 represents a target for antimalarial drug development.

Host cell phosphatidylcholine is a key mediator of malaria parasite survival during liver stage infection.

During invasion, Plasmodium, the causative agent of malaria, wraps itself in a parasitophorous vacuole membrane (PVM), which constitutes a critical interface between the parasite and its host cell. Within hepatocytes, each Plasmodium sporozoite generates thousands of new parasites, creating high demand for lipids to support this replication and enlarge the PVM. Here, a global analysis of the total lipid repertoire of Plasmodium-infected hepatocytes reveals an enrichment of neutral lipids and the major membrane phospholipid, phosphatidylcholine (PC). While infection is unaffected in mice deficient in key enzymes involved in neutral lipid synthesis and lipolysis, ablation of rate-limiting enzymes in hepatic PC biosynthetic pathways significantly decreases parasite numbers. Host PC is taken up by both P. berghei and P. falciparum and is necessary for correct localization of parasite proteins to the PVM, which is essential for parasite survival. Thus, Plasmodium relies on the abundance of these lipids within hepatocytes to support infection.

Design and evaluation of primaquine-artemisinin hybrids as a multistage antimalarial strategy.

It is widely accepted that the struggle against malaria depends on the development of new strategies to fight infection. The "magic bullet" thought to be necessary to reach eradication should not only provide treatment for all Plasmodium spp. that infect human red blood cells but should also eliminate the replicative and dormant liver forms of the parasite. Moreover, these goals should ideally be achieved by using different mechanisms of action so as to avoid the development of resistance. To that end, two hybrid molecules with covalently linked primaquine and artemisinin moieties were synthesized, and their effectiveness against the liver and blood stages of infection was compared in vitro and in vivo with those of the parent compounds. Both hybrids displayed enhanced in vitro activities, relative to those of the parent compounds, against Plasmodium berghei liver stages. Both compounds were about as potent as artemisinin against cultured Plasmodium falciparum (50% inhibitory concentration [IC(50)], ∼10 nM). When used to treat a murine P. berghei infection, one of the molecules displayed better efficacy than an equimolar mixture of the parent pharmacophores, leading to improved cure and survival rates. These results reveal a novel approach to the design and evaluation of antimalarials based on the covalent combination of molecules acting on different stages of the parasite life cycle.

Incorporation of basic side chains into cryptolepine scaffold: structure-antimalarial activity relationships and mechanistic studies.

The synthesis of cryptolepine derivatives containing basic side-chains at the C-11 position and their evaluations for antiplasmodial and cytotoxicity properties are reported. Propyl, butyl, and cycloalkyl diamine side chains significantly increased activity against chloroquine-resistant Plasmodium falciparum strains while reducing cytotoxicity when compared with the parent compound. Localization studies inside parasite blood stages by fluorescence microscopy showed that these derivatives accumulate inside the nucleus, indicating that the incorporation of a basic side chain is not sufficient enough to promote selective accumulation in the acidic digestive vacuole of the parasite. Most of the compounds within this series showed the ability to bind to a double-stranded DNA duplex as well to monomeric hematin, suggesting that these are possible targets associated with the observed antimalarial activity. Overall, these novel cryptolepine analogues with substantially improved antiplasmodial activity and selectivity index provide a promising starting point for development of potent and highly selective agents against drug-resistant malaria parasites.

High-resolution statistical mapping reveals gene territories in live yeast.

The nonrandom positioning of genes inside eukaryotic cell nuclei is implicated in central nuclear functions. However, the spatial organization of the genome remains largely uncharted, owing to limited resolution of optical microscopy, paucity of nuclear landmarks and moderate cell sampling. We developed a computational imaging approach that creates high-resolution probabilistic maps of subnuclear domains occupied by individual loci in budding yeast through automated analysis of thousands of living cells. After validation, we applied the technique to genes involved in galactose metabolism and ribosome biogenesis. We found that genomic loci are confined to 'gene territories' much smaller than the nucleus, which can be remodeled during transcriptional activation, and that the nucleolus is an important landmark for gene positioning. The technique can be used to visualize and quantify territory positions relative to each other and to nuclear landmarks, and should advance studies of nuclear architecture and function.

The inner nuclear membrane protein Src1 associates with subtelomeric genes and alters their regulated gene expression.

Inner nuclear membrane proteins containing a LEM (LAP2, emerin, and MAN1) domain participate in different processes, including chromatin organization, gene expression, and nuclear envelope biogenesis. In this study, we identify a robust genetic interaction between transcription export (TREX) factors and yeast Src1, an integral inner nuclear membrane protein that is homologous to vertebrate LEM2. DNA macroarray analysis revealed that the expression of the phosphate-regulated genes PHO11, PHO12, and PHO84 is up-regulated in src1Delta cells. Notably, these PHO genes are located in subtelomeric regions of chromatin and exhibit a perinuclear location in vivo. Src1 spans the nuclear membrane twice and exposes its N and C domains with putative DNA-binding motifs to the nucleoplasm. Genome-wide chromatin immunoprecipitation-on-chip analyses indicated that Src1 is highly enriched at telomeres and subtelomeric regions of the yeast chromosomes. Our data show that the inner nuclear membrane protein Src1 functions at the interface between subtelomeric gene expression and TREX-dependent messenger RNA export through the nuclear pore complexes.

Yeast Ataxin-7 links histone deubiquitination with gene gating and mRNA export.

Targeting of a gene to the nuclear pore complexes (NPCs), known as gene gating, can affect its transcriptional state. However, the mechanism underlying gene gating is poorly understood. Here, we have identified SAGA-associated Sgf73 (ref. 10), the yeast orthologue of human Ataxin-7 (ref. 11), as a regulator of histone H2B ubiquitin levels, a modification linked to both transcription initiation and elongation. Sgf73 is a key component of a minimal histone-deubiquitinating complex. Activation of the H2B deubiquitinating protease, Ubp8, is cooperative and requires complex formation with the amino-terminal zinc-finger-containing domain of Sgf73 and Sgf11-Sus1. Through a separate domain, Sgf73 mediates recruitment of the TREX-2 mRNA export factors Sac3 and Thp1 to SAGA and their stable interaction with Sus1-Cdc31. This latter step is crucial to target TREX-2 to the NPC. Loss of Sgf73 from SAGA abrogates gene gating of GAL1 and causes a GAL1 mRNA export defect. Thus, Sgf73 provides a molecular scaffold to integrate the regulation of H2B ubiquitin levels, tethering of a gene to the NPC and export of mRNA.

SAGA interacting factors confine sub-diffusion of transcribed genes to the nuclear envelope.

Changes in the transcriptional state of genes have been correlated with their repositioning within the nuclear space. Tethering reporter genes to the nuclear envelope alone can impose repression and recent reports have shown that, after activation, certain genes can also be found closer to the nuclear periphery. The molecular mechanisms underlying these phenomena have remained elusive. Here, with the use of dynamic three-dimensional tracking of a single locus in live yeast (Saccharomyces cerevisiae) cells, we show that the activation of GAL genes (GAL7, GAL10 and GAL1) leads to a confinement in dynamic motility. We demonstrate that the GAL locus is subject to sub-diffusive movement, which after activation can become constrained to a two-dimensional sliding motion along the nuclear envelope. RNA-fluorescence in situ hybridization analysis after activation reveals a higher transcriptional activity for the peripherally constrained GAL genes than for loci remaining intranuclear. This confinement was mediated by Sus1 and Ada2, members of the SAGA histone acetyltransferase complex, and Sac3, a messenger RNA export factor, physically linking the activated GAL genes to the nuclear-pore-complex component Nup1. Deleting ADA2 or NUP1 abrogates perinuclear GAL confinement without affecting GAL1 transcription. Accordingly, transcriptional activation is necessary but not sufficient for the confinement of GAL genes at the nuclear periphery. The observed real-time dynamic mooring of active GAL genes to the inner side of the nuclear pore complex is in accordance with the 'gene gating' hypothesis.

Telomere tethering at the nuclear periphery is essential for efficient DNA double strand break repair in subtelomeric region.

In the yeast Saccharomyces cerevisiae that lacks lamins, the nuclear pore complex (NPC) has been proposed to serve a role in chromatin organization. Here, using fluorescence microscopy in living cells, we show that nuclear pore proteins of the Nup84 core complex, Nup84p, Nup145Cp, Nup120p, and Nup133p, serve to anchor telomere XI-L at the nuclear periphery. The integrity of this complex is shown to be required for repression of a URA3 gene inserted in the subtelomeric region of this chromosome end. Furthermore, altering the integrity of this complex decreases the efficiency of repair of a DNA double-strand break (DSB) only when it is generated in the subtelomeric region, even though the repair machinery is functional. These effects are specific to the Nup84 complex. Our observations thus confirm and extend the role played by the NPC, through the Nup84 complex, in the functional organization of chromatin. They also indicate that anchoring of telomeres is essential for efficient repair of DSBs occurring therein and is important for preserving genome integrity.