Structural Analysis of Membrane-associated Forms of Helicobacter pylori VacA Toxin.

Helicobacter pylori colonizes the stomach in about half of the human population, leading to an increased risk of peptic ulcer disease and gastric cancer. H. pylori secretes an 88 kDa VacA toxin that contributes to pathogenesis. VacA assembles into oligomeric complexes in solution and forms anion-selective channels in cell membranes. Cryo-electron microscopy (cryo-EM) analyses of VacA oligomers in solution provided insights into VacA oligomerization but failed to reveal the structure of the hydrophobic N-terminal region predicted to be a pore-forming domain. In this study, we incubated VacA with liposomes and used single particle cryo-EM to analyze detergent-extracted VacA oligomers. A 3D structure of detergent-solubilized VacA hexamers revealed the presence of six α-helices extending from the center of the oligomers, a feature not observed in previous studies of water-soluble VacA oligomers. Cryo-electron tomography analysis and 2D averages of VacA associated with liposomes confirmed that central regions of the membrane-associated VacA oligomers can insert into the lipid bilayer. However, insertion is heterogenous, with some membrane-associated oligomers appearing only partially inserted and others sitting on top of the bilayer. These studies indicate that VacA undergoes a conformational change when contacting the membrane and reveal an α-helical region positioned to extend into the membrane. Although the reported VacA 3D structure does not represent a selective anion channel, our combined single particle 3D analysis, cryo-electron tomography, and modeling allow us to propose a model for the structural organization of the VacA N-terminus in the context of a hexamer as it inserts into the membrane.

Comparative Biochemical Studies of Disease-Associated Human Dicer Mutations on Processing of a Pre-microRNA and snoRNA.

Dicer is an RNase III enzyme that is responsible for the maturation of small RNAs such as microRNAs. As Dicer's cleavage products play key roles in promoting cellular homeostasis through the fine-tuning of gene expression, dysregulation of Dicer activity can lead to several human diseases, including cancers. Mutations in Dicer have been found to induce tumorigenesis and lead to the development of a rare pleiotropic tumor predisposition syndrome found in children and young adults called DICER1 syndrome. These patients harbor germline and somatic mutations in Dicer that lead to defective microRNA processing and activity. While most mutations occur within Dicer's catalytic RNase III domains, alterations within the Platform-PAZ (Piwi-Argonaute-Zwille) domain also cause loss of microRNA production. Using a combination of in vitro biochemical and cellular studies, we characterized the effect of disease-relevant Platform-PAZ-associated mutations on the processing of a well-studied oncogenic microRNA, pre-microRNA-21. We then compared these results to those of a representative from another Dicer substrate class, the small nucleolar RNA, snord37. From this analysis, we provide evidence that mutations within the Platform-PAZ domain result in differential impacts on RNA binding and processing, adding new insights into the complexities of Dicer processing of small RNA substrates.

Discovery of Surfactins as Inhibitors of MicroRNA Processing Using Cat-ELCCA.

MicroRNAs (miRNAs) are a family of small noncoding RNAs that regulate gene expression. Due to their important activity in the fine-tuning of protein translation, abnormal expression of miRNAs has been linked to many human diseases, making the targeting of miRNAs attractive as a novel therapeutic strategy. Accordingly, researchers have been heavily engaged in the discovery of small molecule modulators of miRNAs. With an interest in the identification of new chemical space for targeting miRNAs, we developed a high-throughput screening (HTS) technology, catalytic enzyme-linked click chemistry assay (cat-ELCCA), aimed at the discovery of small molecule ligands for pre-miR-21, a miRNA that is frequently overexpressed in human cancers. From our HTS campaign, we found that natural products, a source of many impactful human medicines, may be a promising source of potential pre-miR-21-selective maturation inhibitors. Herein we describe our first efforts in natural product inhibitor discovery leading to the identification of a depsipeptide class of natural products as RNA-binding inhibitors of Dicer-mediated miRNA processing.