Systematic genetic interaction profiles can reveal the mechanisms-of-action of bioactive compounds. The imipridone ONC201, which is currently in cancer clinical trials, has been ascribed a variety of different targets. To investigate the genetic dependencies of imipridone action, we screened a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) knockout library in the presence of either ONC201 or its more potent analog ONC212. Loss of the mitochondrial matrix protease CLPP or the mitochondrial intermediate peptidase MIPEP conferred strong resistance to both compounds. Biochemical and surrogate genetic assays showed that impridones directly activate CLPP and that MIPEP is necessary for proteolytic maturation of CLPP into a catalytically competent form. Quantitative proteomic analysis of cells treated with ONC212 revealed degradation of many mitochondrial as well as nonmitochondrial proteins. Prompted by the conservation of ClpP from bacteria to humans, we found that the imipridones also activate ClpP from Escherichia coli, Bacillus subtilis, and Staphylococcus aureus in biochemical and genetic assays. ONC212 and acyldepsipeptide-4 (ADEP4), a known activator of bacterial ClpP, caused similar proteome-wide degradation profiles in S. aureus. ONC212 suppressed the proliferation of a number of Gram-positive (S. aureus, B. subtilis, and Enterococcus faecium) and Gram-negative species (E. coli and Neisseria gonorrhoeae). Moreover, ONC212 enhanced the ability of rifampin to eradicate antibiotic-tolerant S. aureus persister cells. These results reveal the genetic dependencies of imipridone action in human cells and identify the imipridone scaffold as a new entry point for antibiotic development.
Riboswitches are RNA sequences that regulate gene expression by undergoing structural changes upon the specific binding of cellular metabolites. Crystal structures of purine-sensing riboswitches have revealed an intricate network of interactions surrounding the ligand in the bound complex. The mechanistic details about how the aptamer folding pathway is involved in the formation of the metabolite binding site have been previously shown to be highly important for the riboswitch regulatory activity. Here, a combination of single-molecule FRET and SHAPE assays have been used to characterize the folding pathway of the adenine riboswitch from Vibrio vulnificus. Experimental evidences suggest a folding process characterized by the presence of a structural intermediate involved in ligand recognition. This intermediate state acts as an open conformation to ensure ligand accessibility to the aptamer and folds into a structure nearly identical to the ligand-bound complex through a series of structural changes. This study demonstrates that the add riboswitch relies on the folding of a structural intermediate that pre-organizes the aptamer global structure and the ligand binding site to allow efficient metabolite sensing and riboswitch genetic regulation.
Introduction The Bone and Joint Monitor Project was developed to quantify the global burden of musculoskeletal conditions and develop strategies for their prevention. Experts within the Monitor Project have worked previously with officers at the World Health Organization (WHO) to estimate morbidity and mortality associated with rheumatic conditions. The present collaboration seeks means of providing additional and more current burden data. Objective To develop recommendations for performing epidemiological studies in sample populations with musculoskeletal conditions and problems, accounting for determinants and consequences to the individual and society. Methods Recommendations have been developed identifying the most relevant domains for measuring and monitoring the various musculoskeletal conditions by review of epidemiological data on occurrence, determinants and outcomes, and by expert opinion. Instruments that measure these domains were reviewed. Results The domains recommended follow the principles of the WHO International Classification of Functioning, Disability and Health [1,2], and consider: health condition; body function and structure; activity limitation; participation restriction; personal and environmental contextual factors; and, in addition, the resource utilisation and social consequences. The musculoskeletal conditions and problems considered were osteoarthitis, inflammatory arthritis, osteoporosis, spinal problems, musculoskeletal trauma and injuries, and musculoskeletal pain with restricted activity. The selection of indicators for each domain considered the feasibility of their use in a health interview survey (HIS), a health examination survey (HES), a register or a clinical study. Consensus on case definition was reached depending on the study methodology. For example, osteoporosis defined by bone densitometry cannot be ascertained in an HIS, whereas the outcome of osteoporosis (i.e. fragility fracture) can be. Osteoarthitis can be identified as joint pain in an HIS but the preferred definition is pain with X-ray changes and can only be ascertained in an HES. Previously validated generic and disease-specific instruments have been identified that include indicators for all or most of the recommended domains for the consequences of the different conditions and problems. The indicators of the domains for resource utilisation and social consequences and feasibility for collection will vary in different socioeconomic and geographic areas. Guidance on sampling methods is also being developed. Conclusions The comparability of data collected across the globe will improve by the application of agreed upon indicators that consider key domains for the different musculoskeletal conditions and problems in epidemiological studies conducted in different populations.
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