Purine metabolism regulates DNA repair and therapy resistance in glioblastoma

Intratumoral genomic heterogeneity in glioblastoma (GBM) is a barrier to overcoming therapy resistance. Treatments that are effective independent of genotype are urgently needed. By correlating intracellular metabolite levels with radiation resistance across dozens of genomically-distinct models of GBM, we find that purine metabolites, especially guanylates, strongly correlate with radiation resistance. Inhibiting GTP synthesis radiosensitizes GBM cells and patient-derived neurospheres by impairing DNA repair. Likewise, administration of exogenous purine nucleosides protects sensitive GBM models from radiation by promoting DNA repair. Neither modulating pyrimidine metabolism nor purine salvage has similar effects. An FDA-approved inhibitor of GTP synthesis potentiates the effects of radiation in flank and orthotopic patient-derived xenograft models of GBM. High expression of the rate-limiting enzyme of de novo GTP synthesis is associated with shorter survival in GBM patients. These findings indicate that inhibiting purine synthesis may be a promising strategy to overcome therapy resistance in this genomically heterogeneous disease

Conformational Dynamics and Energy Landscapes of Ligand Binding in RyR1

AbstractUsing experimental single-particle cryo-EM snapshots of ryanodine receptor (RyR1), a Ca2+-channel involved in skeletal muscle excitation/contraction coupling, we present quantitative free-energy landscapes, reaction coordinates, and three-dimensional movies of the continuous conformational changes associated with the binding of activating ligands. Our results show multiple routes to ligand binding with comparable branching ratios. All high-probability routes involve significant conformational changes before and after the binding of ligands. We also present new insights into the local structural changes along the ligand-binding route, including accommodations at the calcium, ATP, and caffeine binding sites. These observations shed new light on the mechanisms and conformational routes to ligand binding.</jats:p

Functional Pathways of Biomolecules Retrieved from Single-particle Snapshots

AbstractWe present a new approach to determining the conformational changes associated with biological function, and demonstrate its capabilities in the context of experimental single-particle cryo-EM snapshots of ryanodine receptor (RyR1), a Ca2+-channel involved in skeletal muscle excitation/contraction coupling. These results include the detailed conformational motions associated with functional paths including transitions between energy landscapes. The functional motions differ substantially from those inferred from discrete structures, shedding new light on the gating mechanism in RyR1. The differences include the conformationally active structural domains, the nature, sequence, and extent of conformational motions involved in function, and the way allosteric signals are transduced within and between domains. The approach is general, and applicable to a wide range of systems and processes.</jats:p

Direct modulation of the outer mitochondrial membrane channel, voltage-dependent anion channel 1 (VDAC1) by cannabidiol: a novel mechanism for cannabinoid-induced cell death

Cannabidiol (CBD) is a non-psychoactive plant cannabinoid that inhibits cell proliferation and induces cell death of cancer cells and activated immune cells. It is not an agonist of the classical CB1/CB2 cannabinoid receptors and the mechanism by which it functions is unknown. Here, we studied the effects of CBD on various mitochondrial functions in BV-2 microglial cells. Our findings indicate that CBD treatment leads to a biphasic increase in intracellular calcium levels and to changes in mitochondrial function and morphology leading to cell death. Density gradient fractionation analysis by mass spectrometry and western blotting showed colocalization of CBD with protein markers of mitochondria. Single-channel recordings of the outer-mitochondrial membrane protein, the voltage-dependent anion channel 1 (VDAC1) functioning in cell energy, metabolic homeostasis and apoptosis revealed that CBD markedly decreases channel conductance. Finally, using microscale thermophoresis, we showed a direct interaction between purified fluorescently labeled VDAC1 and CBD. Thus, VDAC1 seems to serve as a novel mitochondrial target for CBD. The inhibition of VDAC1 by CBD may be responsible for the immunosuppressive and anticancer effects of CBD

Structure of the cell-binding component of the <i>Clostridium difficile</i> binary toxin reveals a di-heptamer macromolecular assembly

Targeting Clostridium difficile infection is challenging because treatment options are limited, and high recurrence rates are common. One reason for this is that hypervirulent C. difficile strains often have a binary toxin termed the C. difficile toxin, in addition to the enterotoxins TsdA and TsdB. The C. difficile toxin has an enzymatic component, termed CDTa, and a pore-forming or delivery subunit termed CDTb. CDTb was characterized here using a combination of single-particle cryoelectron microscopy, X-ray crystallography, NMR, and other biophysical methods. In the absence of CDTa, 2 di-heptamer structures for activated CDTb (1.0 MDa) were solved at atomic resolution, including a symmetric ( Sym CDTb; 3.14 Å) and an asymmetric form ( Asym CDTb; 2.84 Å). Roles played by 2 receptor-binding domains of activated CDTb were of particular interest since the receptor-binding domain 1 lacks sequence homology to any other known toxin, and the receptor-binding domain 2 is completely absent in other well-studied heptameric toxins (i.e., anthrax). For Asym CDTb, a Ca 2+ binding site was discovered in the first receptor-binding domain that is important for its stability, and the second receptor-binding domain was found to be critical for host cell toxicity and the di-heptamer fold for both forms of activated CDTb. Together, these studies represent a starting point for developing structure-based drug-design strategies to target the most severe strains of C. difficile . </jats:p

Structure of the cell-binding component of the<i>Clostridium difficile</i>binary toxin reveals a novel macromolecular assembly

AbstractTargetingClostridium difficileinfection (CDI) is challenging because treatment options are limited, and high recurrence rates are common. One reason for this is that hypervirulent CDI often has a binary toxin termed theC. difficiletoxin (CDT), in addition to the enterotoxins TsdA and TsdB. CDT has an enzymatic component, termed CDTa, and a pore-forming or delivery subunit termed CDTb. CDTb was characterized here using a combination of single particle cryoEM, X-ray crystallography, NMR, and other biophysical methods. In the absence of CDTa, two novel di-heptamer structures foractivated CDTb (aCDTb; 1.0 MDa) were solved at atomic resolution including a symmetric (SymCDTb; 3.14 Å) and an asymmetric form (AsymCDTb; 2.84 Å). Roles played by two receptor-binding domains of aCDTb were of particular interest since RBD1 lacks sequence homology to any other known toxin, and the RBD2 domain is completely absent in other well-studied heptameric toxins (i.e. anthrax). ForAsymCDTb, a novel Ca2+binding site was discovered in RBD1 that is important for its stability, and RBD2 was found to be critical for host cell toxicity and the novel di-heptamer fold for both forms of aCDTb. Together, these studies represent a starting point for structure-based drug-discovery strategies to targeting CDT in the most severe strains of CDI.SIGNIFICANCE STATEMENTThere is a high burden fromC. difficileinfection (CDI) throughout the world, and the Center for Disease Control (CDC) reports more than 500,000 cases annually in the United States, resulting in an estimated 15,000 deaths. In addition to the large clostridial toxins, TcdA/TcdB, a thirdC. difficilebinary toxin (CDT) is associated with the most serious outbreaks of drug resistant CDI in the 21stcentury. Here, structural biology and biophysical approaches were used to characterize the cell binding component of CDT, termed CDTb, at atomic resolution. Surprisingly, two novel structures were solved from a single sample that help to explain the molecular underpinnings ofC. difficiletoxicity. These structures will also be important for targeting this human pathogen via structure-based therapeutic design methods.</jats:sec