Genetic Selection of Peptide Inhibitors of Biological Pathways

Abstract

Genetic selections were used to find peptides that inhibit biological pathways in budding yeast. The peptides were presented inside cells as peptamers, surface loops on a highly expressed and biologically inert carrier protein, a catalytically inactive derivative of staphylococcal nuclease. Peptamers that inhibited the pheromone signaling pathway, transcriptional silencing, and the spindle checkpoint were isolated. Putative targets for the inhibitors were identified by a combination of two-hybrid analysis and genetic dissection of the target pathways. This analysis identified Ydr517w as a component of the spindle checkpoint and reinforced earlier indications that Ste50 has both positive and negative roles in pheromone signaling. Analysis of transcript arrays showed that the peptamers were highly specific in their effects, which suggests that they may be useful reagents in organisms that lack sophisticated genetics as well as for identifying components of existing biological pathways that are potential targets for drug discovery.

Golgi Ribbon Unlinking: An Organelle-Based G2/M Checkpoint

“…50 Interestingly, the S. cerevisiae single homologue of GRASP65/55 (Grh1) was shown to be involved in mitotic progression, since mutant cells failed to be arrested upon pharmacological induction of the spindle checkpoint. 53 Overall, these results indicate that the phosphorylation of GRASP65 may have at least two possible roles on cells progressing in mitosis; one in severing the tubules linking the Golgi ribbon and one in Golgi vesiculation, which could be linked directly or indirectly to mitotic spindle organization (see also below). However, several aspects of the role of GRASP65 in the Golgi-related checkpoint need further clarification.…”

Protein‐protein interactions as a target for drugs in proteomics

“…Using peptides is ideal for research of genetic network complexes and is already applied for studying pheromone action [86,87]. At present alternative methods for estimation of interaction of macromolecules in vitro are used.…”

Isolation of peptide aptamers that inhibit intracellular processes

“…Although similar methods have been developed successfully for use in yeast (3)(4)(5)(6), these methods lacked features that facilitate their use in bacterial systems, such as positively controlled, inducible expression of aptamer production. This property allowed inhibitory and blatantly toxic aptamers to be identified with comparative ease.…”

“…Aptamer targets could also be identified by using powerful genetic methods for detecting protein-protein interactions, termed two-hybrid screens (5,12,21,22). To test this concept, we measured the protein-protein interaction between ThyA and the TrxA101Q aptamer, using an E. coli-based, two-hybrid system (13,23).…”

“…One solution to this problem is to express inhibitory compounds directly inside target cells. For example, a library of random peptides fused to a carrier protein and expressed within yeast cells has been used to screen for protein ''aptamers'' that bind to cyclin-dependent kinase 2 (3) or the retinoblastoma protein (4) or that inhibit signaling through several pathways (5,6). However, application of this approach to the identification of inhibitory peptides or aptamers within bacterial cells has not been reported.…”