Actins are highly conserved proteins and key players in central processes in all eukaryotic cells. The two actins of the malaria parasite are among the most divergent eukaryotic actins and also differ from each other more than isoforms in any other species. Microfilaments have not been directly observed in Plasmodium and are presumed to be short and highly dynamic. We show that actin I cannot complement actin II in male gametogenesis, suggesting critical structural differences. Cryo-EM reveals that Plasmodium actin I has a unique filament structure, whereas actin II filaments resemble canonical F-actin. Both Plasmodium actins hydrolyze ATP more efficiently than α-actin, and unlike any other actin, both parasite actins rapidly form short oligomers induced by ADP. Crystal structures of both isoforms pinpoint several structural changes in the monomers causing the unique polymerization properties. Inserting the canonical D-loop to Plasmodium actin I leads to the formation of long filaments in vitro. In vivo, this chimera restores gametogenesis in parasites lacking actin II, suggesting that stable filaments are required for exflagellation. Together, these data underline the divergence of eukaryotic actins and demonstrate how structural differences in the monomers translate into filaments with different properties, implying that even eukaryotic actins have faced different evolutionary pressures and followed different paths for developing their polymerization properties.

SummaryThe myelin sheath is a multilamellar plasma membrane extension of highly specialized glial cells laid down in regularly spaced segments along axons. Recent studies indicate that myelin is metabolically active and capable of communicating with the underlying axon. To be functionally connected to the neuron, oligodendrocytes maintain non-compacted myelin as cytoplasmic nanochannels. Here, we used high-pressure freezing for electron microscopy to study these cytoplasmic regions within myelin close to their native state. We identified 2,′3′-cyclic nucleotide 3′-phosphodiesterase (CNP), an oligodendrocyte-specific protein previously implicated in the maintenance of axonal integrity, as an essential factor in generating and maintaining cytoplasm within the myelin compartment. We provide evidence that CNP directly associates with and organizes the actin cytoskeleton, thereby providing an intracellular strut that counteracts membrane compaction by myelin basic protein (MBP). Our study provides a molecular and structural framework for understanding how myelin maintains its cytoplasm to function as an active axon-glial unit.

Tankyrases catalyse poly-ADP-ribosylation of their binding partners and the modification serves as a signal for the subsequent proteasomal degradation of these proteins. tankyrases thereby regulate the turnover of many proteins involved in multiple and diverse cellular processes, such as mitotic spindle formation, telomere homeostasis and Wnt/β-catenin signalling. In recent years, tankyrases have become attractive targets for the development of inhibitors as potential therapeutics against cancer and fibrosis. Further, it has become clear that tankyrases are not only enzymes, but also act as scaffolding proteins forming large cellular signalling complexes. While many potent and selective tankyrase inhibitors of the poly-ADp-ribosylation function exist, the inhibition of tankyrase scaffolding functions remains scarcely explored. In this work we present a robust, simple and costeffective high-throughput screening platform based on FRET for the discovery of small molecule probes targeting the protein-protein interactions of tankyrases. Validatory screening with the platform led to the identification of two compounds with modest binding affinity to the tankyrase 2 ARC4 domain, demonstrating the applicability of this approach. The platform will facilitate identification of small molecules binding to tankyrase ARC or SAM domains and help to advance a structure-guided development of improved chemical probes targeting tankyrase oligomerization and substrate protein interactions. Poly(ADP-ribosyl) polymerases (PARPs) called tankyrases (TNKSs) are key regulators of diverse cellular processes such as mitotic spindle formation, telomere homeostasis, Wnt/β-catenin signalling and glucose metabolism 1-5. In humans, two tankyrases with overlapping functions exist and are termed TNKS1 and TNKS2 6,7. Like other enzymes of the PARP family, tankyrases catalyse the transfer of multiple ADP-ribose units to their protein substrates 8-10 , thus leaving them poly-ADP-ribosylated. In many cases this serves as a signal for subsequent ubiquitination and thereby proteasomal degradation 11,12. Prominent targets of tankyrases include Axin1/2 13,14 , a major regulator of β-catenin levels, TRF1 7,9,15,16 , a telomere binding protein that inhibits telomere extension and NuMA 17-19 , a protein involved in the formation of spindle-poles during mitosis. The major role of tankyrases in the regulation of β-catenin levels has led to the development of multiple TNKS inhibitors 2,20-22. These inhibitors function by binding to the NAD + binding pocket of the catalytic ARTD domain (ADP-ribosyl-transferases diphtheria toxin-like), inhibiting the poly-ADP-ribosylation function. However, it has been shown that tankyrases also contribute through oligomerization and mediation of protein-protein interactions to the Wnt-signalling pathway 23. We refer to these non-catalytic functions of tankyrases as scaffolding functions. By being able to specifically target different domains involved in the scaffolding function with a tool compound, it could be possible to inves...

A structure-guided hybridization approach using two privileged substructures gave instant access to a new series of tankyrase inhibitors. The identified inhibitor 16 displays high target affinity on tankyrase 1 and 2 with biochemical and cellular IC values of 29 nM, 6.3 nM and 19 nM, respectively, and high selectivity toward other poly (ADP-ribose) polymerase enzymes. The identified inhibitor shows a favorable in vitro ADME profile as well as good oral bioavailability in mice, rats, and dogs. Critical for the approach was the utilization of an appropriate linker between 1,2,4-triazole and benzimidazolone moieties, whereby a cyclobutyl linker displayed superior affinity compared to a cyclohexane and phenyl linker.

Rab GDP dissociation inhibitors (GDI)-