Summary
Essential gene functions underpin the core reactions required for cell viability, but their contributions and relationships are poorly studied in vivo. Using CRISPR interference, we created knockdowns of every essential gene in Bacillus subtilis and probed their phenotypes. Our high-confidence essential gene network, established using chemical genomics, showed extensive interconnections among distantly related processes and identified modes of action for uncharacterized antibiotics. Importantly, mild knockdown of essential gene functions significantly reduced stationary phase survival without affecting maximal growth rate, suggesting that essential protein levels are set to maximize outgrowth from stationary phase. Finally, high-throughput microscopy indicated that cell morphology is relatively insensitive to mild knockdown but profoundly affected by depletion of gene function, revealing intimate connections between cell growth and shape. Our results provide a framework for systematic investigation of essential gene functions in vivo that is broadly applicable to diverse microorganisms and amenable to comparative analysis.
Sequencing of the human genome provided a wealth of information about the genomic blueprint of a cell. But genes do not tell the entire story of life and living processes; identifying the roles of enzymes and mapping out their interactions is also crucial. Enzymes catalyze virtually every cellular process and metabolic exchange. They not only are instrumental in sustaining life but also are required for its regulation and diversification. Diseases such as cancer can be caused by minor changes in enzyme activities. In addition, the unique enzymes of pathogenic organisms are ripe targets for combating infections. Consequently, nearly one-third of all current drug targets are enzymes. An estimated 18-29% of eukaryotic genes encode enzymes, but only a limited proportion of enzymes have thus far been characterized. Therefore, little is understood about the physiological roles, substrate specificity, and downstream targets of the vast majority of these important proteins. A key step toward the biological characterization of enzymes, as well as their adoption as drug targets, is the development of global solutions that bridge the gap in understanding these proteins and their interactions. We herein present technological advances that facilitate the study of enzymes and their properties in a high-throughput manner. Over the years, our group has introduced and developed a variety of such enabling platforms for many classes of enzymes, including kinases, phosphatases, and proteases. For each of these different types of enzymes, specific design considerations are required to develop the appropriate chemical tools to characterize each class. These tools include activity-based probes and chemical compound libraries, which are rapidly assembled using efficient combinatorial synthesis or "click chemistry" strategies. The resulting molecular assortments may then be screened against the target enzymes in high-throughput using microplates or microarrays. These techniques offer powerful means to study, profile, and discover potent small molecules that can modulate enzyme activity. This Account will describe the concepts involved in designing chemical probes and libraries for comparative enzyme screening and drug discovery applications, as well as highlight how these technologies are changing the way in which enzymes may be rapidly profiled and characterized.
Identifying phosphatase substrates: A peptide microarray has been developed for the high‐throughput study of Ser/Thr phosphatases. Putative peptide substrates, upon immobilization onto a glass slide, could be used to obtain kinetic information and identify the substrate preferences of a Ser/Thr phosphatase (see schematic representation); with this information, new biology of the enzyme could be discovered.
Spezifizieren von 14‐3‐3: Ein fragmentbasierter kombinatorischer Peptid‐Mikroarray erzeugt affinitätsbasierte Fingerprints von sieben Säuger‐14‐3‐3‐Isoformen. Motive mit hoher Affinität für die hoch homologen Isoformen wurden identifiziert. Wahrscheinliche 14‐3‐3σ‐spezifische Peptide wurden zudem durch radiometrisches Zwei‐Farben‐Screening identifiziert (siehe Bild).
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