In response to environmental and other stresses, the σ subunit of bacterial RNA polymerase (RNAP) controls expression of several genes that play a significant role in the virulence of both plant and animal pathogens. Recruitment of σ to RNAP initiates promoter-specific transcription via the double-stranded DNA denaturation mechanism of the cofactor. The RpoN box, a recognition helix found in the C-terminal region of σ, has been identified as the component necessary for major groove insertion at the -24 position of the promoter. We employed the hydrocarbon stapled peptide methodology to design and synthesize stapled σ peptides capable of penetrating Gram-negative bacteria, binding the σ promoter, and blocking the interaction between endogenous σ and its target DNA sequence, thereby reducing transcription and activation of σ response genes.

No abstract

Linear ubiquitylation, in which ubiquitin units are covalently linked through N- and C-terminal amino acids, is a unique cellular signaling mechanism. This process is controlled by a single E3 ubiquitin ligase, the linear ubiquitin chain assembly complex (LUBAC), which is composed of three proteins - HOIL-1L, HOIP and SHARPIN. LUBAC is involved in the activation of the canonical NF-κB pathway and has been linked to NF-κB dependent malignancies. In this work, we present HOIP-based stapled alpha-helical peptides designed to inhibit LUBAC through the disruption of the HOIL-1L-HOIP interaction and loss of the functional complex. We find our HOIP peptides to be active LUBAC ubiquitylation inhibitors in vitro, though through interaction with HOIP rather than HOIL. Active peptides were shown to have inhibitory effects on cell viability, reduced NF-κB activity and decreased production of NF-κB related gene products. This work further demonstrates the potential of LUBAC as a therapeutic target and of the use of stapled peptides as inhibitors of protein-protein interactions.