VRK1 is a Paralog Synthetic Lethal Target in VRK2-methylated Glioblastoma

Synthetic lethality - a genetic interaction that results in cell death when two genetic deficiencies co-occur but not when either deficiency occurs alone - can be co-opted for cancer therapeutics. A pair of paralog genes is among the most straightforward synthetic lethal interaction by virtue of their redundant functions. Here we demonstrate a paralog-based synthetic lethality by targeting Vaccinia-Related Kinase 1 (VRK1) in Vaccinia-Related Kinase 2 (VRK2)-methylated glioblastoma (GBM). VRK2 is silenced by promoter methylation in approximately two-thirds of GBM, an aggressive cancer with few available targeted therapies. Genetic knockdown of VRK1 in VRK2-null or VRK2-methylated cells results in decreased activity of the downstream substrate Barrier to Autointegration Factor (BAF), a regulator of post-mitotic nuclear envelope formation. VRK1 knockdown, and thus reduced BAF activity, causes nuclear lobulation, blebbing and micronucleation, which subsequently results in G2/M arrest and DNA damage. The VRK1-VRK2 synthetic lethal interaction is dependent on VRK1 kinase activity and is rescued by ectopic VRK2 expression. Knockdown of VRK1 leads to robust tumor growth inhibition in VRK2-methylated GBM xenografts. These results indicate that inhibiting VRK1 kinase activity could be a viable therapeutic strategy in VRK2-methylated GBM.

Redundant functions of the SLC5A transporters Rumpel, Bumpel, and Kumpel in ensheathing glial cells

Neuronal processing is energy demanding, and relies on sugar metabolism. To nurture the Drosophila nervous system, the blood-brain barrier forming glial cells take up trehalose from the hemolymph and then distribute the metabolic products further to all neurons. This function is provided by glucose and lactate transporters of the solute carrier (SLC) 5A family. Here we identified three SLC5A genes that are specifically expressed in overlapping sets of CNS glial cells, rumpel, bumpel and kumpel. We generated mutants in all genes and all mutants are viable and fertile, lacking discernible phenotypes. Loss of rumpel causes subtle locomotor phenotypes and flies display increased daytime sleep. In addition, in bumpel kumpel double mutants, and to an even greater extent in rumpel bumpel kumpel triple mutants, oogenesis is disrupted at the onset of the vitollegenic phase. This indicates a partially redundant functions between these genes. Rescue experiments exploring this effect indicate that oogenesis can be affected by CNS glial cells. Moreover, expression of heterologous mammalian SLC5A transporters, with known transport properties, suggest that Bumpel and/or Kumpel transport glucose or lactate. Overall, our results imply a redundancy in SLC5A nutrient sensing functions in Drosophila glial cells, affecting ovarian development and behavior.

Mechanism of replication fork reversal and protection by human RAD51 and RAD51 paralogs

SMARCAL1, ZRANB3 and HLTF are all required for the remodeling of replication forks upon stress. Using reconstituted reactions, we show that the motor proteins have unequal biochemical capacities, explaining why they have non-redundant functions. Whereas SMARCAL1 uniquely anneals RPA-coated ssDNA, suggesting an initial function in fork reversal, it becomes comparatively inefficient in subsequent branch migration. We also show that low concentrations of RAD51 and the RAD51 paralog complex, RAD51B-RAD51C-RAD51D-XRCC2 (BCDX2), directly stimulate SMARCAL1 and ZRANB3 but not HLTF, providing a mechanism underlying previous cellular data implicating these factors in fork reversal. Upon reversal, RAD51 protects replication forks from degradation by MRE11, DNA2 and EXO1 nucleases. We show that the protective function of RAD51 unexpectedly depends on its binding to double-stranded DNA, and higher RAD51 concentrations are required for DNA protection compared to reversal. Together, we define the non-canonical functions of RAD51 and its paralogs in replication fork reversal and protection.

Genetic and chemical validation of Plasmodium falciparum aminopeptidase PfA-M17 as a drug target in the hemoglobin digestion pathway

Plasmodium falciparum, a causative agent of malaria, continues to remain a global health threat since these parasites have developed increasing resistance to all anti-malaria drugs used throughout the world. Accordingly, drugs with novel modes of action are desperately required to combat malaria. P. falciparum parasites infect human red blood cells where they digest the hosts main protein constituent, hemoglobin. Leucine aminopeptidase PfA-M17 is one of several aminopeptidases that have been implicated in the last step of this digestive pathway. Here we utilize both reverse genetics and a compound specifically designed to inhibit the activity of PfA-M17 to show that PfA-M17 is essential for P. falciparum survival as it provides parasites with free amino acids for growth, many of which are highly likely to originate from hemoglobin. We further show that our inhibitor is on-target for PfA-M17 and has the ability to kill parasites at nanomolar concentrations. Thus, in contrast to other hemoglobin-degrading proteases that have overlapping redundant functions, we validate PfA-M17 as a potential novel drug target.

Functional Diversity of TonB-Like Proteins in the Heterocyst-Forming Cyanobacterium Anabaena sp. PCC 7120

The genomes of many organisms encode more than one TonB protein, and their number does not necessarily correlate with that of TonB-dependent outer membrane transporters. Consequently, specific as well as redundant functions of the different TonB proteins have been identified.

Physiological Roles of Mammalian Transmembrane Adenylyl Cyclase Isoforms

Adenylyl cyclases (ACs) catalyze the conversion of ATP to the ubiquitous second messenger cAMP. Mammals possess nine isoforms of transmembrane ACs, dubbed AC1-9, that serve as major effector enzymes of G protein-coupled receptors. The transmembrane ACs display varying expression patterns across tissues, giving potential for them having a wide array of physiologic roles. Cells express multiple AC isoforms, implying that ACs have redundant functions. Furthermore, all transmembrane ACs are activated by Gαs so it was long assumed that all ACs are activated by Gαs-coupled GPCRs. AC isoforms partition to different microdomains of the plasma membrane and form prearranged signaling complexes with specific GPCRs that contribute to cAMP signaling compartments. This compartmentation allows for a diversity of cellular and physiological responses by enabling unique signaling events to be triggered by different pools of cAMP. Isoform specific pharmacological activators or inhibitors are lacking for most ACs, making knockdown and overexpression the primary tools for examining the physiological roles of a given isoform. Much progress has been made in understanding the physiological effects mediated through individual transmembrane ACs. GPCR-AC-cAMP signaling pathways play significant roles in regulating functions of every cell and tissue, so understanding each AC isoform's role holds potential for uncovering new approaches for treating a vast array of pathophysiological conditions.

Redundant functions of the SLC5A transporters Rumpel Kumpel and Bumpel in ensheathing glial cells

Neuronal processing is energy demanding, and relies on sugar metabolism as an energy source. To provide a constant metabolite supply neurons and glial cells express many glucose and lactate transporters of the solute carrier (SLC) 5A family. Here we dissect the partially redundant functions of three highly related glia specific Drosophila genes encoding SLC5A proteins, Rumpel, Bumpel and Kumpel. While knockdown of rumpel causes several behavioral phenotypes, they are less prominent in rumpel mutants. bumpel and kumpel mutants are viable and fertile, lacking discernible phenotypes. However, in bumpel kumpel double mutants and to an even greater extent in rumpel bumpel kumpel triple mutants oogenesis is disrupted at the onset of the vitollegenic phase. This indicates at least partially redundant functions between these genes. Rescue experiments exploring this effect indicate that oogenesis can be affected by CNS glial cells. Moreover, expression of heterologous mammalian SLC5A transporter proteins, with known transport properties, suggest that Bumpel and/or Kumpel transport glucose or lactate. Overall, our results imply a redundancy in SLC5A nutrient sensing functions in Drosophila glial cells, affecting ovarian development and behavior.